Nuoi Cay Mo Va Te Bao Thuc Vat

TRNG I HC CN TH
KHOA NNG NGHIP

GIO TRNH
N
N
U
U
I
I
C
C
Y
Y
M
M
V
V
T
T
B
B
O
O
T
T
H
H
C
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V
V
T
Tc gi: NGUYN BO TON
Tin s, Ging Vin Chnh
-2004-
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MC LC
Trang
Chng M U 1
Chng 1 LCH S CA NUI CY M
1.1 LCH S PHT TRIN CA NUI CY M TRN TH GII
1.1.1 L thuyt t bo ca Schleiden v Schwann
1.1.2 Ngi tin phong trong lnh vc nui cy m
1.1.3 Nhng ngi ng gp ln cho lnh vc nui cy m
1.1.4 Nghin cu chuyn gen thong qua Agrobacterium
1.2 HIN TRNG NUI CY M VIT NAM
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Chng 2 CC NGUYN TC CA NUI CY M
V T BO THC VT
2.1 NUI CY M V T BO
2.1.1 nh ngha
2.1.2 Kin thc sinh hc thc vt cn thit cho nui cy m v t bo
2.1.2.1 S sinh sn v sinh trng ca t bo
2.1.2.2 Chu k ca t bo thc vt
2.2.3 Cc kiu nui cy
2.2.3.1 Nui cy c quan (organ culture)
2.2.3.2 S pht sinh hnh thi (morphogenesis)
2.2 THC VT V MU CY
2.2.1 Thc vt
2.2.2 Mu nui cy
2.3 S V TRNG
2.3.1 Kh trng b mt mu cy
2.3.1.1 Ho cht kh trng
2.3.1.2 Thi gian kh trng
2.3.2 Kh trng mi trng v dng c nui cy
2.3.2.1 Cc kiu ni hp kh trng
2.3.2.2 Tit trng bng lc
2.3.2.3 T cy v trng
2.3.2.4 Kh trng nhit kh
2.4 MI TRNG NUI CY
2.4.1 Nc:
2.4.2 Cc nguyn t khong
2.4.2.1Cc nguyn t khong a lng
2.4.2.2 Cc nguyn t khong vi lng
2.4.3 Ngun carbohydrate, ng sucrose
2.4.4 Vitamin
2.4.5 Cht to gel
2.4.6 Cht iu ha sinh trng
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2.4.7 Chelates
2.4.8 Tc nhn thm thu
2.4.9 Than hot tnh
2.4.10 pH
2.4.11 Acid hu c
2.4.12 Thnh phn b sung khng xc nh
2.5 CC KIU BNH CHA V NP Y S DNG TRONG NUI CY
M V T BO
2.6 CHUN B MI TRNG NUI CY
2.6.1 Chun b ha cht:
2.6.2 Chun b dung dch gc
2.6.3 Nu mi trng
2.6.4 o pH
2.7 IU KIN NUI CY
2.7.1 Nhit
2.7.2 nh sng
2.7.3 Quang k
2.7.4 Cng nh sng
2.7.5 m
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Chng 3 THIT K PHNG TH NGHIM NUI CY M
3.1 NGUYN TC THIT K PHNG TH NGHIM NUI CY M
3.2 C S H TNG
3.3 TRANG THIT B V HA CHT PHNG TH NGHIM NUI
CY M
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Chng 4 CC NG DNG CA K THUT NUI CY M V T
BO TRONG NHN GING
4.1 NHN GING HU TNH
4.1.1 Chun b mi trng gieo ht
4.1.2-Kh trng tri Lan
4.1.3 Gieo ht Lan vo trong bnh
4.2 NHN GING V TNH VI NHN GING
4.2.1 Mc ch ca vi nhn ging
4.2.2 Cc giai on (Stages)
4.2.3 Mt s k thut gip gia tng s sinh trng v gim gi thnh
4.3 NHN GING T PHI V TNH
4.3.1 Phi v tnh
4.3.2 S thnh lp phi v tnh
4.3.2.1 S thnh lp gin tip
4.3.2.2 S thnh lp trc tip
4.3.2.3 Cc giai on ca s pht trin phi
4.3.3 Cc yu t nh hng n qu trnh hnh thnh phi v tnh
4.3.3.1 Cht iu ho sinh trng
4.3.3.2 Cc thnh phn khc
4.3.4 ng dng ca phi v tnh trong s to ht tng hp
4.3.5 Phng php gia tng sinh khi bng cch s dng bnh phn ng sinh hc
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BO TRONG TO GING
5.1 RA HOA TRONG NG NGHIM
5.2 NUI CY TH GIAO T (gametophytes culture)
5.2.1 Th bi (ploidy) v gen
5.2.2 Cy t bo n bi (Haploid cell culture)
5.2.2.1 Mc ch ca cy t bo n bi
5.2.2.2 K thut cy ti phn v ht phn (anther and pollen culture)
5.2.3 Li v bt li ca nui cy ht phn
5.3 S CU PHI
5.4 TH TINH TRONG NG NGHIM
5.5 CHN LC IN VITRO CC T BO THC VT C CC C TNH
KHNG
5.6 NUI CY T BO TRN
5.6.1 Vch t bo (cell walls)
5.6.2 L do nui cy t bo trn
5.6.3 Chun b t bo trn
5.6.4 Cc phng php tch t bo trn
5.6.5 Lm tinh khit cc t bo trn c tch
5.6.6 Th sc sng ca t bo trn
5.6.7 Mi trng v cch nui cy t bo trn
5.6.8 m mt s t bo trn
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5.6.9 Bin i t bo trn thnh cy
5.6.10 ng dng ca t bo trn trong thao tc di truyn
5.7 NUI CY PHI NH
5.7.1 Phi nh
5.7.2 Nui cy phi nh
5.8 CNG NGH DI TRUYN
5.8.1 K thut chuyn np gen
5.8.1.1 Chuyn np gen bng phng php gin tip thng qua
5.8.1.2 Chuyn np gen bng phng php trc tip
5.8.1.3 Chn cc marker cho s chuyn gen
5.8.2 Hiu qu ca kiu gen thay i trn qun th xy ra t nhin ca thc vt.
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Chng 6 CC NG DNG KHC CA K THUT
NUI CY M V T BO
6.1 TO CY SCH BNH V PHC HI GING NHIM VIRUS
6.1.1 Phng php phc hi cy nhim virus
6.1.1.1 X l nhit:
6.1.1.2 Cy nh sinh trng:
6.1.1.3 Vi ghp
6.2 CY T BO, CALLUS
6.2.1 To Callus
6.2.2 Cy t bo treo (Nui cy trong mi trng lng)
6.3 SN XUT CC CHT BIN DNG TH CP BNG PHNG
PHP NUI CY T BO
6.3.2 Cht bin dng th cp
6.3.2 Sn phm ca cht bin dng th cp
6.4 TR LNH THEO CNG NGH SINH HC
6.4.1 Mc ch
6.4.2 K thut tr c bn
6.4.2.1 Tr mu vt thc vt trong mt thi k ngn
6.4.2.2 Tr mu vt thc vt trong mt thi k lu di
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Chng 7 S THUN DNG
7.1 CU TRC HNH THI V GII PHU CA CY CON CY M
7.1.1. Lp cutin
7.1.2. Kh Khu (stomata)
7.1.3. L
7.1.4.Thn
7.1.5 R
7.2. QUANG HP
7.3 K THUT THUN DNG:
7.3.1 Trong bnh nui cy.
7.3.2 Trong mi trng t nhin hoc nh li
7.3.3 C s h tng cho s thun dng
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7.3.4.T ng ha
7.4 TM TT CC THNG S V S DNG HO CHT TRONG QU
TRNH THUN DNG
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Chng 8 CC S C V CCH X L
8.1. NHIM VI SINH VT
8.1.1 Nhim do virus
8.1.2 Vi khun:
8.1.3 Nm
8.1.4 Mycoplasma
8.1.5 Cc con nhn v b tr
8.2. SINH L SINH THI
8.2.1 Kh nng gi nc:
8.2.2 S vn chuyn nc
8.2.3 Thnh phn kh trong bnh nui cy
8.2.3 1. Cc yu t xc nh thnh phn kh ca bnh cha nui cy m
8.2.3 2. Hiu qu ca s trao i kh trong bnh nui cy m:
8.3. S HA NU
8.3.1 Triu chng
8.3.2 Nguyn nhn
8.3.3 Cc bin php cha tr
8.4. S BIN D T BO SO MA
8.5 S THA NC
8.5.1 Triu chng
8.5.2 Nguyn nhn
8.5.3 Cha tr
8.6 S CHT CHI
8.7 CU TRC BT THNG
8.8 CC DNG TH KHM
TI LIU THAM KHO
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i
MC LC
Trang
Chng M U 1
1.1.4 Nghin cu chuyn gen thong qua Agrobacterium
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V T BO THC VT
2.1 NUI CY M V T BO
2.1.1 nh ngha
2.2.3 Cc kiu nui cy
2.2 THC VT V MU CY
2.2.1 Thc vt
2.2.2 Mu nui cy
2.3 S V TRNG
2.3.2.2 Tit trng bng lc
2.3.2.3 T cy v trng
2.4 MI TRNG NUI CY
2.4.1 Nc:
2.4.4 Vitamin
2.4.5 Cht to gel
2.4.7 Chelates
2.4.8 Tc nhn thm thu
2.4.9 Than hot tnh
2.4.10 pH
2.4.11 Acid hu c
2.5 CC KIU BNH CHA V NP Y S DNG TRONG NUI CY
M V T BO
2.6.2 Chun b dung dch gc
2.6.3 Nu mi trng
2.6.4 o pH
2.7 IU KIN NUI CY
2.7.1 Nhit
2.7.2 nh sng
2.7.3 Quang k
2.7.4 Cng nh sng
2.7.5 m
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3.2 C S H TNG
3.3 TRANG THIT B V HA CHT PHNG TH NGHIM NUI
CY M
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BO TRONG NHN GING
4.1 NHN GING HU TNH
4.1.1 Chun b mi trng gieo ht
4.3 NHN GING T PHI V TNH
4.3.1 Phi v tnh
4.3.3.1 Cht iu ho sinh trng
4.3.4 ng dng ca phi v tnh trong s to ht tng hp
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BO TRONG TO GING
5.3 S CU PHI
KHNG
5.6 NUI CY T BO TRN
5.7 NUI CY PHI NH
5.7.1 Phi nh
5.7.2 Nui cy phi nh
5.8 CNG NGH DI TRUYN
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NUI CY M V T BO
6.1.1.1 X l nhit:
6.1.1.3 Vi ghp
6.2 CY T BO, CALLUS
6.2.1 To Callus
6.3 SN XUT CC CHT BIN DNG TH CP BNG PHNG
PHP NUI CY T BO
6.4 TR LNH TRONG CNG NGH SINH HC
6.4.1 Mc ch
6.4.2 K thut tr c bn
6.4.2.1 Tr mu vt thc vt trong mt thi k ngn
6.4.2.2 Tr mu vt thc vt trong mt thi k lu di (tr ng lnh)
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Chng 7 S THUN DNG
7.1.1. Lp cutin
7.1.2. Kh Khu (stomata)
7.1.3. L
7.1.4.Thn
7.1.5 R
7.2. QUANG HP
7.3.1 Trong bnh nui cy.
7.3.4.T ng ha
TRNH THUN DNG
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8.1.1 Nhim do virus
8.1.2 Vi khun:
8.1.3 Nm
8.1.4 Mycoplasma
8.1.5 Cc cn trng c chn kch thc nh
8.2. SINH L SINH THI
8.2.1 Kh nng gi nc:
8.2.2 S vn chuyn nc
8.3. S HA NU
8.3.1 Triu chng
8.3.2 Nguyn nhn
8.3.3 Cc bin php cha tr
8.4. S BIN D T BO SO MA
8.5 S THA NC
8.5.1 Triu chng
8.5.2 Nguyn nhn
8.5.3 Cha tr
8.6 S CHT CHI
8.7 CU TRC BT THNG
8.8 CC DNG TH KHM (Chimeras)
TI LIU THAM KHO
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1
Chng M U
Nui cy m v t bo thc vt c Haberlandt 1902 nghin cu u tin. T
cho n nay lnh vc ny pht trin nhanh chnh v ng gp rt ln vo s pht trin
ca sinh hc v nng nghip hin i. Ngy nay, hu ht cc phng php hin i trong
to, chn ging, nhn ging, gi ging, phc hi cy nhim virus....tt c u thng qua
k thut nui cy m.
Nui cy m v t bo thc vt khng nhng ng gp cho nng nghip m cn
ng gp cho cc lnh vc khc nh to chn nhn ging cy lm nghip; nui cy to
cc cht bin dng th cp phc v cho ngnh dc, nguyn liu cho m phm...
Hin nay, nui cy m v t bo thc vt ang c nghin cu pht trin cc k
thut cao rt ngn thi gian to ging, gim chi ph gi thnh nh s dng robot, chuyn
np gen.
Gio trnh ny c chia thnh 8 chng
Chng 1 l im li qu trnh lch s pht trin ca nui cy m v t bo thc vt.
Chng 2 cp n cc nguyn tc ca nui cy m t bo
Chng 3 cp n thit k phng th nghim nui cy m
Chng 4 cp n cc ng dng ca k thut nui cy m t bo trong nhn ging
Chng 5 cp n cc ng dng ca k thut nui cy m t bo trong to ging
Chng 6 cp n cc ng dng khc ca k thut nui cy m t bo
Chng 7 cp n s thun dng
Chng 8 cp n cc s c v cch x l
Gio trnh ny c s dng ging dy cho sinh vin ngnh Cng Ngh Sinh
Hc, Trng Trt, Nng hc v Sinh hc. N cng c th dc s dng nh l ti liu
tham kho cho nhng ai quan tm n lnh vc Cng Ngh Sinh Hc Thc Vt.
2

Matthias Schleiden Thodore Schwann
(1804-1881) (1810-1882)
Schleiden v Schwann l nhng ngi tin phong a ra l thuyt t bo v tnh
ton nng (totipotency) ca thc vt nm 1839. V c bn, l thuyt ny c ni dung
chnh l t bo ca sinh vt c kh nng bin i thnh mt c th hon chnh. Nm 1839,
Schwann pht biu "Ngi ta c th xy dng hai gi thuyt lin quan n ngun gc
ca cc hin tng hu c nh s sinh trng l mt chc nng ton th sinh vt; hoc s
sinh trng khng xy ra bng phng tin ca bt c lc ang c tr trong ton th sinh
vt, nhng mi phn c s c mt lc ring, mt i sng tch bit. Trong cc thc vt
bc thp, bt c t bo no cng c th c tch ra v tip tc sinh trng. V vy, ton
th thc vt bao gm cc t bo, m n c kh nng cho thy i sng c lp.
C hai tc gi, Schleiden v Schawnn, cng b khi nim cc t bo ca mt
sinh vt v c bn th ging nhau, trong mi t bo c kh nng tn ti c lp v c
th pht sinh mt sinh vt mi hon ton. Mc d Schleiden v Schawnn khng th
nghim l thuyt ca ho, nhng n lm c s cho khi nim tnh ton nng (Debergh,
2003).
Haberlandt, mt nh thc vt hc ngi c, cha ca lnh vc nui cy m thc
vt, ng a ra khi nim v nui cy t bo nm 1902. ng cho rng :"Theo nh ti
bit n nay khng c th nghim c t ra cy cc t bo thc vt bc cao trong mi
trng dinh dng thch hp. Tuy nhin, cc kt qu ca cc th nghim nh th s mang
nhiu minh ha l th v tnh cht k l v kh nng m t bo c. Chng ta thy rng c
mi quan h qua li v nh hng ln nhau c t bo trong sinh vt a bo.
3
Haberlandt 1902
Trong kt lun ti tin rng ngi ta c th nui cy thnh cng phi nhn to t t
bo thc vt".
Khi Haberlandt bt tay vo nghin cu cy t bo thc vt trong iu kin v
trng v trong dung dch dinh dng, Haberlandt khng nhn bit l cc t bo quang
hp, tng i c chuyn ha. Phn sinh m ca n khng biu hin mt cch d dng
v ng ta cng khng bit rng iu ny rt cn cc cht kch thch sinh trng hat
ha (cht ny khng c bit vo lc ). V vy, ng ta chn th nghim vi cc t bo
giu (palisade cells), t bo li (pith cells), lng ca nh hoa (stamen hairs) v t bo khu
(stomatal cells). Haberlandt cy cc t bo ny vo trong mi trng hu c n gin
cha glucose di cc iu kin v trng, v tt c cc trng hp u khng thnh cng.
Cc t bo nui cy ca ng ta khng phn chia, nhng vn tn ti trong vi tun. S tht
bi ca Haberlandt cho thy rng, cc t bo phn sinh m ca c th thc vt c bn l d
dng v ng ta khng bit rng lm mt s chuyn ha t bo cn c s hin din ca
cht iu ha sinh trng.
Debergh (2003) im qua nhng ngi c cng ng gp ln trong lnh vc
nui cy m t bo theo thi gian c lit k di y
Kotte (1922), nh khoa hc ngi c, v Robbins (1922) nh khoa hc ngi
M, c hai hot ng c lp v c cng tng l s dng cc phn sinh m lm mu
cy. Kotte th nghim vi u r tch ri ca cy u ln cy Bp. Cc mu ny c t
vo mt loi mi trng dinh dng cha dung dch mui Knop, glucose v mt s hn
hp nitrogen nh asparagine, alanine v cht trch t thit. Kotte t c s sinh trng
ca u r ko di n hai tun. Tri li, Robbin duy tr r Bp trong mi trng lu hn.
White (1934) kch thch s sinh trng ko di ca u r C chua trong dung dch
mui v c c ng v vitamin. ng ta chng minh phn sinh m ca cy nhim virus
th sch bnh.
4
Went v Thimann (1934) xc inh c indole-acetic acid (IAA) nh l mt cht
iu ha sinh trng.
Gautheret v Nobecourt (1934-1939) tin hnh nui cy in vitro m thc vt trong
cc thi k khng xc nh. Gautheret v Nobercourt (1939) thnh cng nui cy trn cy
c ci (carrot). Cy callus t c bi Gautheret (1934) bao gm ch yu l cc t bo
nhu m c thu th gia v cc cm nh ca t bo phn sinh m hot ng phn tn. Cc
bc k tip ca Gautheret l cy thnh cng m b thng v callus t cc m tng tng
tch ri ca cy Salix. ng ta s dng mi trng dinh dng c c agar, nhng kt qu
cho thy l sau 6 thng hot ng nui cy ngng sinh trng.
Nm 1937, Bonner khm ph thnh phn quan trng trong yeast extract l thiamin
(Vit B1).
Snow (1935) chng minh s kch thch hot ng ca tng tng bng cch thm
IAA v Viatmine B trong mi trng nui cy.
Van Overbeck (1941) s dng nc da cy phi cy Datura.
Ball (1946) m t chi tit v s hnh thnh chi t phn sinh m v v th cng vic
vi nhn ging c bt u
LaRue (1947) cy cy mt l mm t phi nh Bp
Caplin v Steward (1948) chng minh nc da kch thch s nui cy carrot
nhanh hn auxin.
Limaset v Cornuet (1949) chng minh phn sinh m chi ca cc cy nhim virus
th sch bnh.
Morel v Martin (1952) phc hi cc cy khe mnh t nui cy phn sinh m ca
cy hoa Thc dc.
Muir v cng s (1953) to ra phng php cy t bo treo trong mi trng lng
Tellecke (1953) t c cc t bo nui cy ht phn cy Ginkgo biloba pollen
trong mi trng vi amino acid v vitamin.
White (1943, 1954), Heller (1953), Gautheret (1959) v Nitsch (1951) l nhng
ngi tin phong pht trin cc cng thc mi trng dinh dng thch hp cho tng loi
m khc nhau.
Skoog, 1954 thc hin mt khm ph ni ting khi ng ta s dng mt mu
Deoxyribonucleic acid c t tinh trng c Trch (herring sperm) th v kch thch s
sinh trng hot ng ca callus vt thng trn cy thuc l v t c hiu qu kch
thch. Thnh phn hot ng ch hin din trong cc DNA c hoc trong cc mu ti
c hp kh trng v cho ra thnh phn hot ng l sn phm phn hy ca DNA.
Skoog (1955) xc nh cht ny nh l kinetin v sau c cho l cc cht ging
kinetin hin din trong cy nh cht iu ha sinh trng thc vt t nhin.
Skoog v cng s (1955) xc nh kinetin trong dch trch yeast extracts hp kh
trng c cng ht tnh nh nc da trong nui cy callus thn cy thuc l.
Muir (1957) pht trin mt k thut dng giy nui cy t bo n.
Folke Skoog v Carlos Millar (1957) chng minh s hnh thnh c quan nui cy
cy thuc l c kim sot bi s cn bng auxin v cytokinins
Reinert v Steward (1958) to phi v tnh t cy carrot t nui cy treo.
Letham (1963) xc nh cytokinin t nhin trong phi nh cy bp
Murashige v Skoog (1962), Gamborg (1968) v Chu (1978) xut cc mi
trng dinh dng b sung cho cy m cy trng; trong mi trng Murashige v
5
Skoog (1962) gi tt l mi trng MS cung cp mt phng tin cho cc k thut nui
cy in vitro, mt phng php thc hnh sng ng nhn ging cc loi hoa king. Tn
mi trng MS tr nn quen thuc vi k thut nui cy m.
Maheshwari (1964,1967) chng minh rng c th cy ht phn v c kh nng to
ra nhiu phi n bi (haploid).
Cocking trong thp nin 1960, pht trin cc enzyme phn hu vch t bo v nui
cy t bo trn. Phng php bao gm vic ly i vch t bo vi cc enzyme cellulase v
pectinase tinh khit. Trong khi iu ha s pht trin nguyn sinh cht vi s thm thu
bn ngoi. Nguyn sinh cht c nui cy t to ra vch mi, thnh lp cc cm t bo
v cui cng thnh lp cy con.
Melchers v cng s (1978) to ra mt cy lai t s dung hp t bo trn ca
Khoai ty v Thuc l, cho php s chuyn thng tin di truyn t hai loi khc nhau.
Phng php cung cp c hi to ra cc cy lai gia cc loi khng tng thch v gii
tnh.
Vasil v Hildebrandt (1965) pht trin mt phng php cy t bo tch ri trong
mt git treo v chng minh c tnh ton nng ca t bo v tnh.
Carlson (1970) chn lc cc dng t bin sinh ha in vitro.
Takebe v cc ngi khc (1970) to ra cy u tin t nui cy t bo trn.
Carlson (1972) lai v tnh thng qua dung hp t bo trn ca hai loi thuc l
Nicotiana.
Nallin v cng s (1973); Kao v cng s (1973) m t mt phng php hiu qu
cho s dung hp t bo trn trn c s thm polyethylen glycol (PEG) vo trong dung dch
cha t bo trn
Seibert (1976) kch thch s ti sinh chi t chi hoa Cm chng c tr lnh.
Zenk v cng s (1977); Akasu v cng s (1977) chng minh rng cc t bo nui
cy c th cha nhiu cht bin dng th cp hn cc m cy bnh thng.
Melchers (1978) t c cy lai gia Khoai ty v C chua bng dung hp t bo
cht
Davey v cng s (1980), Kreus v cng s (1980) chng minh s hp thu trc
tip plasmid ca vi khun Agrobacterium.
Pelletier v cng s (1983) to ra cy lai t t bo cht bng dung hp t bo trn.
Fujimura v cng s (1985) to ra cy la t t bo trn
Vasil v cng s (1990) s ti sinh cy La m hon chnh t nui cy t bo trn.
Vasil v cng s (1991) bo co v to cy La m chuyn gen bng sng bn gen vo cc
phi non
1.1.4 Nghin cu chuyn gen thng qua Agrobacterium
Paul (2003) dim li nhng thnh cng trong lnh vc chuyn gen thng qua vi
khun Agrobacterium nh sau
Smith v Townshend (1907) xc inh vi khun Agrobacterium tumefaciens nh l
tc nhn gy bnh khi u trn thc vt.
Braun (1970) thnh cng trong vic nui cy m khi u khng c vi khun trn mi
trng cha ng v khong v c. ng ta ngh rng vi khun xm nhp vo t bo
thc vt to ra khi u gi l nguyn l kch thch khi u (tumor-inducing principle =TIP).
6
Morel v cng s thp nin 1960 chng minh rng cc t bo to ra opine l
do TIP (DNA) iu hnh
Schell v Van Montagu (1974) tm thy cc plasmid rt ln tt c cc dng gy
c khng c trong cc dng khng gy c.
Nestor, Gordon v Chilton (1977) chng minh mt mnh nh ca plasmid DNA
c chuyn vo trong t bo thc vt (T-DNA) v chng minh Agrobacterium c hiu
qu trong vic chuyn an ny.
1983 Cy chuyn gen u tin c to ra thng qua vi khun Agrobacterium.
1994 Cy C chua Favr-savr chuyn gen c chp nhn cho bun bn Hoa k.
1995-1996 Chuyn gen ca cy La v cy Bp thng qua vi khun Agrobacterium
c bo co.
1996-2003 - 90% ca cy Canola v khong 30% ca cy Bp v u nnh l cy
chuyn gen Hoa k.
Vit Nam, cng vic nghin cu nui cy m t bo bt u thc hin trong
khong nm 1977 Phn Vin Khoa hc Thnh ph H Ch Minh. Hin nay, c rt nhiu
phng th nghim nui cy m cc Trng ai hc, cc Vin nghin cu, cc S Khoa
hc v Cng ngh cc Tnh, Thnh ph. Lt l ni c nhiu phng th nghim nui
cy m ca t nhn phc v cho cng tc nhn ging hoa cnh v rau c.
7
V T BO THC VT
2.1 NUI CY M V T BO
2.1.1 nh ngha
Nui cy m v t bo thc vt l s nui cy v trng cc c quan, m, t bo
thc vt trn mi trng nui cy c xc nh r; vic nui cy c duy tr di cc
iu kin c kim sot.
Trong lnh vc nui cy m v t bo thc vt, s hiu bit cc kin thc c bn
v thc vt s gip ch rt nhiu cho nhng ngi nghin cu v lnh vc ny.
T bo ca sinh vt n bo cng nh t bo c trong c th a bo lun sinh sn
bng cch phn i, to nn hai t bo con mang c tnh sinh l ging t bo m.
Bng con ng trao i cht, t bo con tng trng khi lng t bo cht v nhn p
ng cc hot ng sng. T bo tng trng n mc no th t bo phn chia thnh
cc t bo mi.
Thi k t bo trao i cht, sinh trng c gi l gian k (interphase), thi k
tip theo phn thnh cc t bo mi c gi l thi k phn bo (cell division). Ngi ta
phn bit hai kiu phn bo ch yu l phn bo nguyn nhim hay nguyn phn (mitosis)
v phn bo gim nhim (meiosis) hay gim phn.
Chu k sng ca t bo l thi gian din ra t thi im t bo c hnh thnh
nh phn bo ca t bo m v kt thc s phn bo hnh thnh t bo con. Ngi ta
chia chu k t bo ra hai thi k chnh:
- Thi k gia hai ln phn chia c gi l gian k (interphase), y l thi gian t
bo trao i cht, sinh trng v chun b cho phn bo.
- Thi gian tip theo l thi k nguyn phn (mitosis) c k hiu l M, y l thi
k t bo m phn i ra hai t bo con.
a. Gian k (interphase)
Trong gian k, t bo thc hin chc nng trao i cht, hot ng sng khc nhau
nh tng hp ARN v ADN, protein, enzyme v chun b cho t bo phn bo. Ty
theo c im chc nng ngi ta chia gian k ra 3 giai on lin tip nhau: giai on G1
(gap 1), giai on S (synthesis) v giai on G2 (gap 2) (hnh 2.1). Thi gian ko di ca
gian k ty thuc vo thi gian ca ba giai on G1+S+G2, c bit ty thuc vo G1 v
cc loi t bo khc nhau th thi gian G1 rt khc nhau, cn giai on S v G2 tng
i n nh.
8
Hnh 2.1 S i din cho chu k phn bo ca t bo thc vt
Pha G1
Pha G1 c tip ngay sau s phn bo
Thi gian ca G1 ko di t ngay sau khi t bo c to thnh do phn bo, cho
n khi bt u pha S (pha tng hp ADN). Thi gian ca G1 ty thuc vo chc nng
sinh l ca t bo.
Khi kt thc G1, t bo i vo pha S v G2 vo thi k phn bo v ty thuc
vo iu kin mi trng. Vo cui pha G1 c mt thi im c gi l im kim sot
(control point).
Nu t bo vt qua im kim sot, n s tip tc i vo pha S. Nhn t iu
chnh im kim sot l protein khng bn vng c tc dng km hm. Pha G1 l pha sinh
trng ca t bo, v trong pha ny xy ra s tng hp cc ARN v protein. i vi cc t
bo chuyn ha th t bo khng vt qua im kim sot m i vo qu trnh chuyn ho
t bo.
Tng hp cht trong pha G1: trong pha G1 khng c s tng hp ADN. ADN
c tng hp trong pha S tip theo, v vy ngi ta cn gi G1 l pha trc ti bn.
Pha S
Pha S l pha tip theo ca pha G1 nu t bo vt qua im kim sot. Trong pha
G1 t bo chun b iu kin cho pha S, vo cui pha G1 t bo tng hp mt loi
protein c trng l cyclin A v nhanh chng tch ly trong nhn t bo. Protein cyclin A
cng vi protein kinase s xc tin s ti bn ADN v nhn i nhim sc th.
Protein cyclin A (nhn t hot ha tng hp ADN) tc ng cho ti cui pha S th
bin mt. Thi gian ko di ca pha S tng i c nh (t 6 - 8gi). S tng hp ADN
mi c cu trc v c tnh ging vi DNA c nn c gi l s ti bn ADN
(replication). Qua pha S, hm lng ADN c tng gp i.
9
Pha G2
Tip theo pha S l pha G2, thi gian ca G2 ngn t 4-5 gi. Trong pha G2 cc
ARN v protein c tng hp chun b cho phn bo. Cui pha G2 mt protein c
tng hp l cyclin B v c tch ly trong nhn cho n tin k phn bo. Cyclin B hot
ha enzyme kinase v ng vai tr quan trng trong vic thc hin qu trnh phn bo
nh s to thnh cc vi ng tubulin to thnh thoi phn bo.
b. Phn bo
Tip theo pha G2 l thi k t bo m phn chia thnh 2 t bo con. S phn bo l
phng thc sinh sn ca t bo, ng thi qua phng thc t bo m truyn thng
tin di truyn cha trong ADN ( c nhn i qua pha S) cho hai t bo con. S phn
bo cng vi s tng hp cc cht ni bo l c s ca s sinh trng ca cc m, cc c
quan v c th a bo.
Phn bo nguyn nhim (Mitosis)
Cn gi l nguyn phn l dng phn bo chun, ph bin cho tt c cc dng t
bo, qua cc t bo c nguyn b nhim sc th nh t bo m (2n).
Nguyn phn l pha M ca chu k t bo, tip ngay sau pha G2. pha M, t bo
m sau khi i qua pha S (trong b my di truyn c nhn i) s to ra 2 t bo
con, mi t bo con mang b my di truyn ging ht t bo m. Hin tng nguyn phn
l hin tng phn bo phc tp c trng cho cc t bo Eucaryota, c pht hin ln
u tin bi Strasburger v Flemming t nhng nm 1882.
Bn cht ca hin tng phn bo l phng thc t bo m phn i ADN cho 2
t bo con. t bo Eucaryota, do b m di truyn c cu trc phc tp, phn t ADN
lin kt vi histon v cc protein khc histon to thnh cc nhim sc th c phn tch
vi t bo cht bi mng nhn, cho nn tin trnh phn bo din ra phc tp, i hi phi
c b my phn bo (mitotic apparatus) l h thng si to nn thoi phn bo (mitotic
spindle) cng vi s xut hin cc cu trc nhim sc th.
c im ca phn bo nguyn nhim
- Phn bo nguyn nhim l dng phn bo ph bin Eucaryota.
- Kt qu ca phn bo hnh thnh 2 t bo con c s lng nhim sc th gi
nguyn nh t bo m.
- Xut hin nhim sc th v phn chia nhim sc th v 2 t bo con.
- Xut hin trong t bo cht b my phn bo c vai tr hng dn cc nhim sc
th con di chuyn v 2 cc t bo.
- Trong tin trnh phn bo, mng nhn bin mt v li c ti to hai t bo
con.
Cc k ca nguyn phn
Qu trnh phn bo din ra theo 6 k lin tip nhau bt u thi gian tip theo pha
G2 ca gian k v kt thc khi hnh thnh 2 t bo con.
Thc t, trong t bo sng rt kh phn bit gii hn chuyn tip gia cc k. Mi
k c c trng bi cu trc, tp tnh ca nhim sc th v b my phn bo.
10
- Gian k (interphase)
Cc t bo ang thc hin hot ng sng bnh thng. Nhim sc th c nhn
i
- Tin k (prophase)
Tin k c tip theo sau pha G2 ca gian k. Rt kh phn bit mt cch chnh
xc im chuyn tip ny, cc hin tng c trng cho tin k l:
+ Hnh thnh nhim sc th: Cht nhim sc gian k bao gm cc si nhim
sc c nhn i qua pha S, tr nn xon v c c li, hnh thnh cc nhim sc th
thy r di knh hin vi thng. Mi mt nhim sc th gm 2 nhim sc t ch em
(sister chromere). Hai nhim sc t ch em trong mt nhim sc th chng t rng nhim
sc th c nhn i qua pha S.
+ Mng nhn v hch nhn c nhiu thay i: Hch nhn gim th tch, phn r v
bin mt. Mng nhn t ra thnh nhiu on v bin thnh cc bng khng bo b phn
tn trong t bo cht.
+ Hnh thnh b my phn bo: a s t bo c trung tm t chc vi ng
(microtubule organizing center, MTOC) l ni thnh lp vi ng gm 2 trung t (centriole)
v vng quanh trung t (pericentriole). Qua pha S, trung t c nhn i to thnh hai
i trung t con. Mi i trung t thnh mt MTOC mi. Do s hot ha ca cht trung
t cc n hp tubulin trong t bo cht trng hp ha thnh cc vi ng tubulin. Cc ng
sp xp quanh trung t mi to thnh sao phn bo (aster). Hai sao di chuyn v hai cc
t bo. gia hai sao cc vi ng pht trin sp xp thnh h thng c dng hnh thoi c
gi l thoi phn bo. Cu to nn thoi 2 dng vi ng chy t sao ca cc ny n cc kia.
Cc vi ng cc (hay si cc) chy lin tc t cc ny n cc kia, cn cc vi ng tm
ng (hay si tm ng) l cc si ni vi tm ng ca nhim sc th vng xch o
ca t bo. n cui tin k khi mng nhn bin mt th b my thoi c hai sao c
hnh thnh.
Nh ta bit, t bo thc vt bc cao khng quan st thy trung t, nhng
vng cnh nhn vn c vng m c tng t vng trung t v vai tr ca chng l hot
ha s trng hp tulubin to thnh thoi phn bo t bo thc vt
- Trung k (Metaphase)
Nhim sc th trung k xon, c c v co ngn ti a. Mi nhim sc th nh
vi si thoi qua tm ng v do tc ng ca cc si thoi v sc cc nhim sc th sp
xp cng trn mt phng xch o to nn ci gi l tm trung k. Tm trung k nm
thng gc vi trc dc ca thoi. Hai tm ng nh vi cc thoi v sc c hai pha i
vi sao. Ngoi cc si tm ng l si dnh tm ng mt phng xch o v ko di ti
vng quanh sao nhng khng dnh vi trung t, thoi cn c cc si cc - si cc ca thoi
khng dnh vi tm ng, si cc c hai loi: 1 loi lin tc chy t cc ny n cc kia,
1 loi ch chy t cc n min xch o.
11
Hnh 2.2 Cc k ca phn bo
Hu k (anaphase).
c im ca hu k l s tch i ca hai nhim sc t ch em khi nhau v tr
thnh nhim sc th con c lp. Mi nhim sc th con mang mt tm ng ring nh
vi si tm ng pha i mt vi sao. Tt c cc nhim sc th con cng tch khi nhau
v cng thi gian di chuyn v 2 cc nh s co ngn ca si tm ng (do s gii trng
hp vi vi ng tulubin) phi hp vi s ko di ca cc si cc v hp li ca thoi. Ngi
ta tnh c tc di chuyn v cc ca nhim sc th khong 1m trong 1 pht.
12
Mt k (telophase)
Trong k ny, cc nhim sc th con di chuyn ti hai cc, dn xon, di ra v
bin dng tr thnh cht nhim sc. Thoi phn bo bin mt, ng thi hnh thnh mng
nhn bao quanh cht nhim sc. Hch nhn c ti to, hnh thnh 2 nhn con trong khi
t bo cht chung.
S phn bo (cytokinesis )
S phn t bo cht c bt u t cui hu k hoc u mt k v din ra sut
mt k. T bo thc vt c bao bi lp v cellulose lm cho t bo khng vn ng
c. S phn t bo cht t bo thc vt c th tc ng ln s ti bn ADN, ln s to
thnh thoi, ln nhim sc th hoc ln s phn t bo cht. Cc cht khng sinh nh
actinomycin D, daunomycin, nogalomycin c tc dng lin kt vi DNA, do c ch s
tng hp ADN. Cc cht cycloheximid, puromycin c ch t bo pha G2. Cc cht
chuyn chng chuyn ha (antimetabolite) cht alkylant, cc thuc nhum u c tc
ng c ch hoc lm sai lch s ti bn ADN dn n c ch phn bo.
Cc cht c ngun gc thc vt nh colchichine, colcemid, podophylin, vinblastin
v.v. u c tc dng c ch s to thnh thoi phn bo, t bo dng li trung k v to
thnh cc nhn a bi. Nhiu cht c tc ng ln nhim sc th lm t gy nhim sc
th hoc phn ly khng chnh xc v 2 cc.
Lithium, cysteamin v cytochalasin c ch s phn t bo cht dn n to thnh t
bo a nhn.
2.2.3 Cc kiu nui cy
- Cc c quan xc nh (determinate organs) l nhng c quan c cu trc, hnh
dng v kch thc r rng v d nh l, hoa, tri...
- Cc c quan cha xc nh (indeterminate organs) l nhng c quan cha hnh
thnh cu trc, hnh dng r rng nh phn sinh m chi, phn sinh m r.
Trc y ngi ta cho rng, cc c quan cha xc nh v d cc phn sinh m th
cha l mt c quan pht trin. Tuy nhin, hin nay ngi ta cho rng cc c quan cha
pht trin tht s l tin c quan xc nh (primordia of determinate organs).
- Cy cc t bo cha c t chc (unorganised cells): callus (m so) l mt khi t
bo v nh hnh cha c t chc
S pht sinh hnh thi c ngha l bin i cc cu trc ca m cy thnh mt cy
hon chnh vi y cu trc.
- Tnh cht v s kch thch
Cc c quan mi nh chi, r c th c kch thch thnh lp t cc m c
nui cy. Cc c quan mi nh th c gi l cc m hoc chi bt nh (adventitious).
n nay ngi ta c th bin i cc b phn nui cy to ra chi (caulogenesis) v r
(rhizogenesis) hoc to phi v tnh c cu trc tng t nh phi hu tnh. Cc cu trc
thnh lp chi r hoc phi v tnh rt quan trong trong cng vic nhn ging v tnh.
- S pht sinh hnh thi c th trc tip hoc gin tip
13
S to thnh trc tip l s hnh thnh cc c quan mi nh chi r t mu cy
khng thng qua giai on callus. S hnh thnh gin tip l s to thnh cc c quan mi
thng qua giai on callus.
2.2 THC VT V MU CY
2.2.1 Thc vt
Cy s dng ly mu cy c gi l cy m hay cy gc l cy c s dng
nh mt ngun cung cp mu nui cy v c chn theo cc kiu hnh mong mun. Cy
ly mu nui cy mun t c kt qu cn lu n cc iu kin sau:
- Ch dinh dng ca cy m: mu cy thch hp cho nui cy m l cc mu
nui cy c ly trn cc cy c cung cp dinh dng.
- Tnh trng sch bnh ca cy m: cy m c chn ly mu nui cy phi
hon ton khng b nhim vi bt c mm bnh no. Thng thng nhng cy b
nhim bnh thng to ra chi hoc phn sinh m chi c kch thc nh.
- Thi gian ly mu: cc yu t bn ngoi nh nhit , di ngy, tnh trng nc
trong cy.v.v. nh hng rt nhiu n hm lng carbohydrate, protein d tr v
cc cht iu ho sinh trng trong m cy. Ty thuc cy m c trng t nhin
ngoi ng hay c chm sc c bit trong nh li m c th ly mu nui cy
theo ma hoc c th ly mu quanh nm.
2.2.2 Mu nui cy (explants)
Mu vt nui cy l mt mnh vt liu thc vt c tch t cy m c
nui cy (hnh 2.3)
Hnh 2.3 V tr trn cy c th c ly mu nui cy (George, 1996)
14
Phm cht ca mu cy s quyt nh s thnh cng ca mu vt c nui cy vo mi
trng. i vi cc mu nui cy cn lu mt s c tnh sau
Cc v tr thc vt c s dng lm mu nui cy trn mt t sch hn cc v
tr thc vt di t
Mu nui cy cng nh cng d trnh cc vn v nhim bnh thc vt (virus,
microplasma, bacteria) nhng gim mc sng st sau khi kh trng
M bn trong t nhim hn m bn ngoi
Tui ca mu nui cy tu thuc vo tui ca cnh c ly mu:
V tr tui cnh pha di thng non hn v tr tui cnh pha trn (hnh 2.4).
Hnh 2.4 V tr ca tui cnh trn cc phn ca cy trng thnh. J: chi non; A:
chi gc; B: chi vt (George, 1996)
15
nh hng ca v tr trn cy m (topophysis, cyclophysis v periphysis)
Hnh 2.5 V tr ca cnh trn cc phn ca cy trng thnh (Debergh, 2003)
Topophysis nh l s khc nhau ca cc phn ng sinh l cc v tr khc nhau.
Cyclophysis l cc thay i ca cc phn ng sinh l do tui.
Periphysis cc c tnh phn bit c cho thy cc phn c cng tui v cng v
tr m chu nh hng ca cc iu kin vt l khc, v d: nh sng.
S bin i thnh cy ca cc mu nui cy c th b nh hng ca v tr ly mu
cy (explant) trn cy.
Phn non mt dng cy hnh nn: non (tr ha) ca mt phn sinh m th t
l vi khong cch (dc theo thn v nhnh) gia s lin kt chi v r (A) ti phn sinh
m. Cc phn sinh m c khang cch xa chi v r th cng gi. Chng c ngun gc t
khong cch AB>AC>AD>AE>AF v do phn sinh m B th gi nht v phn sinh
m F th non nht.
Hnh 2.6. Phn non mt dng cy hnh nn (Debergh, 2003)
S tr ha v s nhn ging
Nhn ging In vitro ca cy hnh chp ch c th vi cc phn ca phi (A) hoc
phn ny mm non (B). V th, kh nng nhn ging in vitro b mt cc cy con (C),
16
nhng vn c th c nhn ging bi k thut nhn ging In vitro ca cy hnh chp v
to r theo phng php c in. Cnh gim c ly t cy con to r ny v thng to
ra s nhn ging ng kiu. Cnh gim t mt cy hi gi hn (D) c mt kh nng to r
gim v cc cnh gim cho thy s thay i sinh trng hng nghing (plagiotropic
growth). Cnh gim t nhng cy gi (E) khng to r.
Hnh 2.7. Nhn ging In vitro ca cy hnh chp (Bonga v Durzan, 1982)
2.3 S V TRNG
Nui cy m l mt k thut nui cy cn iu kin v trng. Mt s t chuyn
mn cn c lm r.
S v trng (asepsis): Trnh nhim bng cch s dng phng php tit trng.
Lm sch quan h vi cc sinh vt sng khc (Axenic).
Tit trng (sterilization): Git hoc loi vi sinh vt hoc bo t ca n bng nhit,
lc, ha cht hoc cc cht tit trng khc.
Kh trng b mt mu cy thng c thc hin vi cc cht dit nm bnh. Sn
phm dng kh trng tt nht l cc sn phm r tin, khng c vi mu vt cng nh
i vi ngi thao tc v hiu qu lai cc vi sinh vt trong mt phm vi rng . Cc bc
thao tc kh trng mu c trnh by hnh 2.8
17
Hnh 2.8. Phng php kh trng mu cy. T giai on 4 n giai on 7 c
thc hin trong t cy v trng (George, 1993)
2.3.1.1 Ho cht kh trng: C nhiu loi ho cht c s dng kh trng b mt.
Dung dch hypochloride
Ion hypochloride c trong sodium hypochloride (NaOCl) hoc Calcium
hypochloride [Ca(OCl)
2
]. Sodium hypochloride ho tan trong nc dng lng. Dung
dch ny c trong cc sn phm ty ra nh nc Javel, Clorox. Nng cui cng cn
thit c th thay i t 0,25-2% trng lng/th tch tu thuc vo vt liu th nghim v
thi gian ngm mu. Dung dch ny thng bng vi 5-20% th tch/th tch ca cc dung
dch Javel hoc Clorox. Tuy nhin, nn xem k liu lng ca dung dch ty ra thng
mi.
Tc dng dit khun ca dung dch hypochloride l c hypochlorous acid (HOCl)
v ion OCl
-
. Ngi ta cho rng HOCl hiu qu hn OCl
-
do hiu qu dit khun chlorine
tt nht dung dch hypochloride acid nh. Dung dch hypochloride nn c s dng
pH 6-7. C sodium v calcium hypochloride c quan h n kh nng oxy ho.
Calcium hypochloride c bn dng bt v thng r hn sodium
hypochloride. V mt l thuyt calcium hypochloride [Ca(OCl)
2
] cha 99,2 % Chloride,
nhng trong thc t th chim 65-70% tinh khit.
Dung dch kh calcium hypochloride c chun b bng cch ho tan bt calcium
hypochloride vo trong nc sau lc qua giy lc v s dng ngay. Calcium
hypochloride s phng thch chloride khi n c ho tan vo trong nc. iu ny c
ngha l chloride s b mt nu bt calcium hypochloride b m, trong khi sodium
hypochloride s b mt hot tnh theo thi gian v nu t ni nh sng mnh. Nng s
dng t 5-10% ngm mu trong 20 pht.
18
Cc hn hp chloride v halogen khc
- Nc Chloride
- Nc Bromide
- Iodine
- Hn hp Chloramine hu c
Cn
Trong s cc loi cn c s dng kh trng, cn ethanol c s dng rng
ri nht. Cn khng nhng git khun m cn ly i cc cht sp t m mu cy. Cn
kh trng thng s dng nng t 70-95%. Cn Methanol t hiu qu hn cn
ethanol. Tuy nhin, mt s trng hp cn isopropanol rt c hiu qu nhng thi gian
kh trng tng i ngn.0.5-1 pht nng 100%.
Ion kim loi nng
- Trong cc kim loi nng th Mercuric Chloride (HgCl) l cht kh trng thng dng
nht. S dng ho cht ny phi ht sc cn thn v c cho thc vt ln ng vt,
cng nh cht thi sau khi kh trng c nh hng n mi trng. cc nc pht
trin, cc cht thi sau khi kh trng bng HgCl s c thu hi v ti s dng li. Cc
cht ny khng c thi ra mi trng t nhin nu cha x l.
- Cc ion Ag v Cu i khi cng c s dng nh l cc cht dit khun.
Cc cht kh trng khc
Hai cht oxy ho mnh l hydrogen peroxide (H
2
O
2
) v potassium
permanganate (KMnO
4
) cng c bo co c hiu qu cao trong kh trng b mt. Tuy
nhin, cc cht ny thng c kt hp trc hoc sau khi kh trng vi cht kh trng
khc.
Cht kh nm
Nhiu cht kh nm c ngun gc l thuc bo v thc vt cng c s dng
trong kh trng. V d Benomyl, Carbendazim, Fenbendazol l cc cht kh nm ca
thuc bo v thc vt c s dng trong kh trng b mt. Nng v liu lng thay
i theo loi mu thc vt. Ngi ta cng cho cc cht ny vo trong mi trng nui
cy.
Cht khng sinh (antibiotic)
Mt s cht khng sinh cng c s dng cho kh trng b mt loi trc tip
cc vi khun trong mu cy. Cc cht khng sinh thng c s dng l :
- 0,15% streptomycin 0,01% merthiolate thm vo trong 4% NaOCl cho kh trng b
mt tri Euterpe trong 12 gi.
- 100g/l ca hn hp penicilin/streptomycin trong 15 pht ca chi cy o.
- 17% Alcide trong 1 gi sau trong rifampicin (50g/l) kh trng nt thn cy
Nauclea.
2.3.1.2 Thi gian kh trng:
Nng ho cht v thi gian kh trng mu cc k quan trng. Nu nng qu
cao v thi gian kh trng kh di mu c th b tn thng v cht. Cn nu nng qu
19
thp v thi gian kh trng qu ngn th khng git c vi sinh vt. Nng thp v
thi gian kh trng di cng c hiu qu nh nng cao v thi gian kh trng ngn.
Bng 1 Cc ha cht, nng v thi gian p dng c Pauls (2003) ngh

Ha cht kh trng b mt
Ha cht Nng p dng Thi gian p
dng (pht)
Chlorin [Ca(OCl)
2
] 9-10% 5-30 .
Nc Javel hoc Clorox
(NaOCl)
2% of dung dch ty ra (20%
NaOCl)
50-30
Oxi gi (H
2
O
2
) 10-12% 5-15
Nc Brome (Bromide Water) 1-2% 2-10
Nitrat bc (AgNO
3
) 1% 5-30
Clorua thy ngn ( HgCl
2
) 0,1-1% 2-10
Khng sinh (Antibiotic) 4-50 mg/l 30-60
Mt s th d v cch kh trng b mt
* on cnh ca cy trng ngoi ng Pseudotsuga v Pinus (Gupta v Durzan, 1985).
- Ra nc my trong 3-4 pht.
- Ngm trong bt git 0,1% trong 5-10 pht, sau ra li bng nc my.
Thc hin trong t v trng.
- Ngm trong hydrogen peroxide 30% trong 15 pht, ra li bng nc ct v trng
3 ln
- Ngm trong sodium hypochloride 0,53% trong 10 pht, ra li nc ct kh trng
3 ln
- Ngm trong HgCl 0,53 % trong 10 pht, ra li bng nc ct kh trng 5-6 ln
* Mt phng php khc (Debergh, 2003).
Ngm mu v ra mu bng nc v bt git.
- Lc trong alcohol trong vi giy
- HgCl (0,1 - 1%) + Teepol 2 git /100ml: 3-5 pht
- Ra trong nc ct tit trng
- Cht ty thng mi 7-15% + Teepol 2 git/100ml: 10-30 pht
- Ra vi ln trong nc ct tit trng
Ch thch:
- Nc my v bt git ly cc phn nh bm dnh trn b mt.
- Ethanol 70% hu nh hiu qu lm bin tnh protein. i vi ethanol 95% c
s dng ha tan cc lp sp b mt.
- NaOCl (cht ty thng mi nh javel, clorox) hoc Ca(OCl)
2
hot ng trn c
s c ion Cl
-
v cc phn ng oxy ha khc.
- HgCl hot ng trn c s ion Cl
-
v kim loi nng lm bin tnh protein, cht
thm (wetting agent) (v d. Teepol) c th c thm vo tng s bm dnh b
mt.
20
Thnh phn chu nhit th c hp kh trng. Thnh phn khng chu nhit c
lc.
Hp kh trng: Mi trng c hp kh trng cn mt nhit chn sn v
mt p sut tng ng trong mt thi gian n nh. V d 50 kPa tng ng vi
112
o
C v 100 kPa tng ng vi 120
o
C.

a b

c d e
Hnh 2.9 Cc kiu ni hp kh trng. kiu bn (a); kiu phng th nghim bn t
ng (b); kiu phng th nghim hon ton t ng (c, d); kiu s dng cho sn xut
ln (e).
21
2.3.2.2 Tit trng bng lc: mng lc mt ln trc ht c hp kh trng; kch thc
l khng ln hn 0.22m; dung dch tit trng c lc v cha trong mt cha tit
trng
Hnh 2.10 B vi lc, cc phn cu thnh v cc thao tc lc dung dch
Tit trng keo l nui cy m, dng c lm vic v mi trng lm vic
- Dng c thy tinh: hp kh trng, nhit kh (180
o
C mt vi gi), bc x
- Dng c cha s dng mt ln: bc x
- Giy: nhit kh
- Dng c lm vic: n cn hoc nhit (la, ht thy tinh)
- B mt t nui cy: lau bng cn
22
2.3.2.3 T cy v trng:
T cy n gin t ch:
y l t cy n gin c th t thit k v ng ly. Nguyn tc thit k t cy
ny l to mt khng gian tng i kn, t c s di chuyn ca khng kh, cc dng c
thao tc cy bn trong n gin gm kim cy, kp cy, n cn v cn dng kh trng
b mt t cy. mt s ni, ngi ta c th gn thm n cc tm tit trng bo t vi
sinh vt trong khng kh. Thao tc trong t cy ny phi tht kho lo v k lng, nu
khng mu cy s d nhim trng.
Hnh bn di l mt kiu t cy v trng n gin, mt s t cy v trng n
gin c th to thm mt l thng hi trn nc t vi ng knh 5 cm. Khi khng s
dng l thng hi ny c th c ng li.

Hnh 2.11. S t cy n gin t ch
T cy v trng c mng vi lc (HEPA-filtered)
Hnh 2.12. S m t chi tit mt t cy v trng (George, 1993)
T cy v trng hin nay c s dng cho nhiu phng th nghim nui cy m
cng nh nui cy vi sinh vt. u im ca ca t cy v trng l tin li v thoi mi
cho ngi thao tc trong iu kin v trng. T cy v trng c hai kiu l lung khng
23
kh v trng i ngang v lung khng kh v trng i theo chiu ng t trn xung.
Lung khng kh c ly vo v thi ra xuyn qua mt mng lc HEPA nh mt qut
gi cc mnh. Mng lc ny c kch thc l rt nh c kh nng ngn cn cc bo t vi
khun xuyn qua mng.
Hnh 2.13 T cy v trng
T sy cao : c s dng tit trng dng c cy, chai l v giy (hnh 2.14)
Hnh 2.14 T sy cao
24
Tit trng dng c cy: c th s dng n cn hoc hp nng c ht thu tinh (hnh
2.15). Hin nay, nhiu phng th nghim nui cy m s dng hp nng v n c nhiu li
ch nh trnh c s c do dng n cn.
Hnh 2.15 Hp nng c ht thu tinh
2.4 MI TRNG NUI CY
Thnh phn (Ingredients)
CC THNH PHN C BN CC THNH PHN T CHN
Nc
Cc nguyn t khong
Ngun carbohydrate
Vitamins
Cht to gel
Cht iu ha sinh trng
Chelates
Cht thm thu
Than
Amino acid
m hu c
2.4.1 Nc:
Phm cht nc l iu kin quan trng trong nui cy. Nc s dng trong cy
m thng l nc ct mt ln. Trong mt s trng hp ngi ta cng s dng nc ct
hai ln hoc nc kh khong.
Nc kh khong l nc ct mt hoc hai ln i qua cc ct kh khong hoc cc
ct trao i ion.
25
Thit b kh khong Ct trao i ion
Hnh 2.16 Dng c kh khong v trao i ion
C nhiu nguyn t khong c s dng trong mi trng nui cy, mi nguyn
t c mt vai tr ring. Cc nguyn t khong c chia thnh hai nhm ring: cc
nguyn t khong a lng v cc nguyn t khong vi lng
Cc nguyn t khong a lng
(Thm vo lng mM)
Cc nguyn t khong vi lng
(Thm vo lng M)
Nitrogen (dng ammonium v
nitrate)
Ln (P)
Potassium (K)
Magnesium (Mg)
Calcium (Ca)
Lu hunh (S)
Sodium (Na)
Chloride (Cl)
Iodine (I)
Bo (B)
Mangan (Mn)
Km (Zn)
Molybdenum (Mo)
ng (Cu)
Cobalt (Co)
Aluminium (Al)
Nickel (Ni)
St (Fe)
Nitrogen (N)
Nitrogen l thnh phn cu to ca nhiu hp cht hu c cha N nh protein
amino acid, chlorophyll...
26
Nitrogen c s dng trong mi trng nui cy m thng hai dng l
nitrogen v c v nitrogen hu c. Nitrogen v c thng hai dng NO
3
-
v NH
4
+
.
Nitrogen hu c thng l cc amino acid v cc polyamine.
Ln (P)
Ln l thnh phn cu to ca cc thnh phn quan trng trong cy nh acid nhn
(DNA, ARN), mng t bo (phospholipid), ATP, NADPH v.v. M cy hp thu ln cc
hnh thc khc nhau H
2
PO
4
-
, HPO
4
2-
, PO4
3-
Potassium (K)
Potassium l thnh phn xc tc ca nhiu enzyme. Ngi ta thy c hn 50
enzyme c xc tc bi K. Vai tr ca potassium lin quan nhiu n qu trnh tng hp
carbohydrate. M cy hp thu potassium dng ion K
+
Magnesium (Mg)
Magnesium l thnh phn ca chlorophyll. Ngoi ra, magnesium cn l thnh phn
ca nhiu enzyme v cng cn thit cho qu trnh bin dng nng lng trong s tng
hp ATP. Cy hp thu Mg dng Mg
2+
.
Calcium
Calcium l thnh phn ca vch t bo, mng t bo v hot tnh ca mt s
enzyme. M cy hp thu calcium dng Ca
2+
Lu hunh (S)
Lu hunh l thnh phn ca mt s amino acid nh cystein, methionin v vi
vitamine. M cy hp thu lu hunh dng SO
4
2-
.
Sodium (Na)
Ion Na
+
c hp thu vo trong cy, tuy nhin chc nng ca Na i vi cy trng
thc s cha r. Ngi ta cho rng Na c chc nng n nh thm thu ca cc cy sng
trong vng mn.
Chloride (Cl)
Chlorine c vai tr trong quang hp ch yu h thng quang II trong qu trng quang phn ly
nc. Ngoi ra, ion Cl cn iu ho s ng v m ca khu. M cy hp thu Chlorine dng
Cl
-
Iodine (I)
Iodine khng c cng nhn nh mt s nguyn t dinh dng cho thc vt bc
cao, mc d n c th cn thit cho mt s to v mt lng rt nh ca thc vt bc cao.
Bo (B)
Vai tr ca Bo trong cy cng cha thc s r. Ngi ta cng thy l Bo c lin
quan n s iu ho hot ng ca enzyme phenolase, bin dng acid phenolic v s
tng hp lignin.
Mangan (Mn)
27
Mangan l mt trong cc nguyn t vi lng quan trong trong mi trng nui
cy, l thnh phn quan trng trong quang phn ly nc v cng l thnh phn hot ho
cho nhiu enzyme. M cy hp thu Mangan dng Mn
2+
Km (Zn)
Vai tr ca km lin quan n cc phn ng tng hp Indol acetic acid (IAA).
Km cng l thnh phn hot ho ca mt s enzyme. M cy hp thu km dng Zn
2+
Molybden (Mo)
M cy hp thu molybden dng ion molybdate (MoO
4
2-
).
ng (Cu)
ng l nguyn t vi lng tham gia vo s hot ho ca cc enzyme cytocrome
oxydase trong h hp, trong cc cht vn chuyn in t nh plastocyanin. ng cng l
thnh phn ca ascorbic acid oxydase.
Cobalt (Co)
Co l thnh phn kim loi ca vitamine B13 lin quan ti s tng hp acid nhn.
M cy hp thu Co dng Co
2+
.
St (Fe)
St (Fe) l nguyn t cn thit bi v n hnh thnh b phn ca vi enzyme hay
ca nhiu protein vn chuyn in t trong qu trnh quang hp v h hp. N tri qua s
oxy ho v s kh lun phin gia trng thi Fe
2+
v Fe
3+
khi n ng vai tr nh cha
kha ca cc h thng enzyme. Cc enzyme ny bao gm catalase, peroxidase v mt s
cytocrom. Cytocrom hot ng trong c ch h hp ca cc t bo sng. Mt s cc
enzyme u phn ng oxy ha kh trong quang hp. St khng phi l thnh phn ca
dip lc t, nhng rt cn cho s sinh tng hp ca dip lc t.
Aluminium, Nickel (Al, Ni)
Aluminium v Nickel l cc nguyn t vi lng c cho vo mi trng cy. Tuy
nhin, vai tr sinh l ca cc nguyn t ny th cha tht s hiu r. Ngi ta thy Nickel
l thnh phn ca enzyme urease bin i urea sang ammonium.
Hai loi ng mono v disaccharide c th nghim. ng sucrose thng
c s dng trong khong 2-5%. Khi s dng ng sucrose trong mi trng nui cy,
n c thy phn hon ton hoc mt phn thnh monosaccharide: glucose v fructose.
2.4.4 Vitamin
Vitamin thng c s dng nht l:
Nicotinic acid
Pyridoxine (vit B6)
Thiamine (vit B1)
Myo inositol
Vit C thng c s dng nh l cht chng oxy ha
2.4.5 Cht to gel
Agar: sn phm t nhin c trch t to
28
Gelrite: mt gellan gum (hetero-polysaccharide) c to ra bi vi khun Pseudomonas
elodea
Agar v c tnh gel tu thuc vo thng hiu ca nh sn xut trong cn lu n
tinh khit ca agar
Hm lng m cha trong agar
S sinh trng ca mu cy trong mi trng c agar theo tng loi thng hiu
Nng Agar c s dng s nh hng n
Th nng nc trong mi trng nui cy
cng ca mi trng
S sinh trng ca mu cy
Cc vn sinh l ca mu cy nh s tha nc (hyperhydricity)
S hot ng ca cytokinin trong mi trng c agar
Auxin t nhin
Auxin t nhin c tm thy thc vt l indole-3-acetic acid (IAA).N hin din
nng cao vng phn sinh m ca thc vt v c lin quan n nhiu p ng
sinh l. Trong lnh vc nui cy m, hiu qu chnh ca n l trn s phn chia t bo v
qu trnh gin phn. Mt trong nhng p dng chnh ca auxin l chng kch thch qu
trnh to r. Do , n c p dng trong vi nhn ging v to r ca cnh gim.
Auxin tng hp
Auxin tng hp c s dng rng ri nht l indole-3-butyric acid (IBA), hin
nay c chng minh l xut hin trong t nhin trong cy, alpha-naphthaleneacetic acid
(NAA) v 2,4-dichlorophenoxyacetic acid (2,4-D). Cc auxin ny thng c kt hp
lin kt vi cytokinin. Cc auxin ny n nh vi nhit v nh sng hn IAA. Do ,
chng c th c hp kh trng.
Cht ngn cn auxin
2,3,5-Tri-iodobenzoic acid (TIBA) th c s dng nh cht ngn cn auxin.
Cytokinin
Cytokinin l adenine c thay th N-6 kt hp vi auxin gy ra s phn chia t
bo thc vt. Zeatin l cytokinin t nhin c trch ra t ht bp ny mm. Kinetin v
benzyladenine c tng hp u tin, nhng gn y ngi ta chng minh l to ra t
nhin vi loi cy. Ngy nay, c hai c s dng rng ri trong nui cy m.
Cytokinins c tng hp r, t vn chuyn ln chi. Chng ng vai tr chnh
trong s thnh lp chi v c quan trong nui cy m. Nhiu cytokinin tng hp c
pht trin theo s khm ph, diphenylurea (DPU) c hot tnh ging nh cytokinin.
29
Thidiazuron (TDZ) c s dng rng ri trong nui cy m ngy nay. TDZ ngn cn
cytokinin oxidase. .
DPU c trch t nc da c s dng nh l thnh phn thm vo trong vi
nhn ging. Cht ny c chng minh cht l cytokinin chnh trong nc da. S khm
ph tnh c dn n s pht trin mt nhm cht iu ho sinh trng mi ging
cytokinin l thidiazuron.
Cytokinin thng c s dng kch thch s tng trng chi trong nui cy
m nh c chng minh bi s hnh thnh chi/hnh trn t l c ca Bowiea volubilis.
Benzyladenine c s dng kch thch s tng trng chi.
Gibberellin
Gibberellin mn cm vi nhit v thng khng s dng trong nui cy m. Khi
n s dng thng c lc v trng sau khi mi trng hp kh trng. Chc nng
chnh ca n trong cy l kch thch s sinh trng ko di phn sinh m chi. Do ,
chng c s dng kch thch ko di ca cy la ln, nh c chng minh
sinh trc nghim cy la ln. Trong s cc Gibberellin th Gibberellin 3 (GA
3
) thng
c s dng trong nui cy m.
Bng cch s dng cc cht ngn cn sinh tng hp Gibberellin, s sinh trng
chi c th c vn dng.
Abscisic acid (ABA)
Abscisic acid (ABA) l mt cht ngn cn s sinh trng ca cy mnh m. N
tng tc vi cht kch thch sinh trng khc. Trong nui cy m, n c s dng rng
ri trong trong s to phi v tnh (somatic embryogenesis) trong thao tc phi v bin
i phi thnh cy con. ABA c tng hp t mevalonic acid v ng vai tr chnh lin
h vi nc trong cy, s lo ha v s pht trin phi thc vt.
Ethylene
Ethylene l mt cht kh, l mt hormone bay hi. Hnh dng, kch thc v cch
y kn bnh nui cy b nh hng ca hormone ny. Sinh tng hp ca ethylene c th
c iu ha bi cc cht ngn cn nh aminoethoxyvinyl glycine (AVG) v amino
oxyacetic acid (AOA). Cc cht ny ngn cn s sn xut ra ethylene. Hat tnh ca
ethylene c th b c nh bng cch s dng ion bc (Ag
+
) nhiu dng khc nhau.
Ngi ta cho rng ion bc ngn cn chp nhn ethylene.
Tng tc auxin/cytokinin
Theo cc nghin cu ca nhiu tc gi cho thy t l auxin v cytokinin trong
mi trng nui cy nh hng n s thnh lp to chi v r. Mt t l cao cytokinin v
auxin thp thch hp cho s to chi. Trong khi t l cao auxin v cytokinin thp thch hp
cho s to r mc trung gian gia hai t l ny thch hp to callus. Hnh bn di l
th nghim cc mc BA v NAA khc nhau trn s thnh lp chi v r trn cy
Bowiea volubilis
30
Hnh 2.17 Hiu qu ca s tng tc NAA v BA trn cy Bowiea volubilis
(Debergh,2003)
BNG TM TT TC DNG CHNH CA CC CHT IU HO SINH
TRNG TRONG CY M (De Kler, 2001)
Auxin Tc dng chnh
trong h thng m
Cc cht iu tit bin dng, tc
ng hoc vn chuyn
Indol acetic acid (IAA)
Indol butyric acid (IBA)
1-Napthalene acetic acid
(NAA)
Phenylacetic acid (PAA)
2,4-Diclorophenoxy
acetic acid (2,4-D)
2,4,5-
Trichlorophenoxyacetic
acid (2,4,5-T)
Picloram
Dicamba
P-chloro phenoxy acetic
(CPA)
1. To r bt nh
( nng cao)
2. To chi bt nh
( nng thp)
3. Kch thch phi
sinh dng
4. Phn chia t bo
5. Thnh lp m so
v tng trng
6. c ch s pht
trin chi nch
7. c ch s pht
trin r
1. 2,3,4-triiodobenzoic acid (TIBA)
1-N naphthyl phthalamic acid (NPA)
c ch s vn chuyn auxin hu cc.
2. p-chlorophenoxy isobutyric
(PCIB) c ch hot ng ca auxin.
3. Hp cht phenolic (vd: ferulic
acid, phloroglucinol) c ch oxy ho
auxin
4. Riboflavin kch thch mnh s
quang oxy ha ca IBA v IAA
31
Cytokinin Tc dng chnh
trong h thng m
ng hoc vn chuyn
Zeatin (Z)
Zeatin riboside (ZR)
isopentenyladenine (iP)
isopentenyladenosine
(iPA)
6-benzylaminopurine
(BAP)
Kinetin
Thidiazuron (TDZ)
N-(2 chloro-4-pyridyl)-
N-phenylurea (CPPU)
1. To chi ph
( nng cao)
2. c ch to r
3. Phn chia t bo
4. Thnh lp m so
v tng trng
5. Kch thch mc
chi nch
6. c ch s vn
di chi
7. c ch s lo ha
l.
Cc hp cht c m t m c ch
tng hp cytokinin (lovastatin),
S phn gii v tc ng. Tuy nhin,
cc tc dng khc th cha c
nghin cu hay cn nghi vn
GIBBERELINS
Tc dng chnh
trong h thng m
ng hoc vn chuyn
Gibberelic acid (GA3)
Gibberelin 1 (GA1)
Gibberelin 4 (GA4)
Gibberelin 7 (GA7)
1. Ko di chi
2. Kh min trng
ht, phi sinh dng,
chi ngn v thn c
3. c ch vic thnh
lp r bt nh
4. Cc cht ch s
tng hp vic thnh
lp r
5. Cc cht c ch s
tng hp kch thch
s to thn c, r c,
gc c
6. Cht c ch tng
hp thc y s thch
nghi
Paclobutrazol, flurimidol v
ancymidol
c ch s tng hp gibberelin v vy
lm chi ngn li
ABSCISIC ACID
Tc dng chnh
trong h thng m
Cc cht iu tit, bin dng, tc
ng hoc vn chuyn
1. Lm trng thnh
phi sinh dng
2. Thc y s thch
nghi
3. To thn c v r
c
4. Kch thch s pht
trin min trng
Fluridone c ch tng hp ABA
thng qua s c ch tng hp
carotenoid, cy khng th tng hp
chlorophyll.
Tuy nhin, Fluridone khng c
Paclobutrazol cng c ch tng hp
ABA
32
ETHYLENE
Tc dng chnh
trong h thng m
Cc cht iu tit, bin dng hoc
tc ng
1. Lo ha l
2. Lm chn tri
3. Kch thch hoc c
ch s ti sinh ph
(ty vo thi im x
l hoc kiu gen)
1. 1-aminocyclopropane-1-
carboxylic acid (ACC) l tin cht
ca ethylene v c chuyn ha
thnh ethylene bi m
2. Aminothoxyvinylglycine (AVG)
c ch tng hp ethylene
3. Ag c ch hot ng ca ethylene.
Ag c p dng tt hn l Ag-
thiosulphate (STS)
POLYAMINES
Tc dng chnh
trong h thng m
tc ng
Putrescine
Spermidine
Spermine
1. Kch thch to r
bt nh
2. Kch thch to chi
3. Kch thch to
mm phi sinh
dng
1. DL-adifloromethylarginine
(DFMA) v adifloromethylornithine
(DFMO) ngn chn s tng hp
Putrescine.
2. Methylglyoxal bis
guanylthyrazone (MGBG) v
cyclohexylamine (CHA) ngn chn
s tng hp Spermidine v Spermine
3. Amino guanidine (AG) ngn chn
s phn gii Putrescine
JASMONIC ACID (JA)
Tc dng chnh
trong h thng m
tc ng
Methyljasmonate
(MeJa)
1. Kch thch to thn
c v r c
Gia tng s thnh lp
m phn sinh
2.4.7 Chelate
Chelate l cht c kh nng thnh lp phc cht vi cc cation kim loi (ch yu st) v
gip gi chng trong dung dch. Cc chelate.nh EDTA: (Ethylenediaminetetraacetic
acid), EDDHA: (Ethylenediamine-di (o-hydroxyphenyl) acetic acid) thng rt ph bin
trong nui cy m
2.4.8 Tc nhn thm thu (Osmotic agent)
Th nng thm thu ca mi trng nui cy m nh hng s hin din ca nc
v khong
Hiu qu ca ng 2% thng c quy cho l hiu qu thm thu v khng cho
chc nng ca n nh l ngun nng lng
33
Trong hu ht cc trng hp mannitol l mt alcohol ng khng bin dng v
ch c hiu qu thm thu.
2.4.9 Than hot tnh (activated charcoal):
Than hot tnh cho vo mi trng nhm mc ch lm ti mi trng nui cy
(mi trng to r), hp thu cc phn t, cc phn t thch hp c cu trc vng (v d
cht iu ha sinh trng). C th cha cc thnh phn kch thch nh polyamines. Mi
loi than rt quan trng v c kh nng ngoi hp khc nhau.ty vo cng ty sn xut
2.4.10 pH
pH thng c chnh gia 5.5 v 6 trc khi hp kh trng. pH quyt nh s ha
tan ca khong, nh hng s hp thu khong trong mi trng, nh hng trn s to
gel ca agar khi hp kh trng.
2.4.11 Acid hu c
Acid hu c cng nh acid citric, maliccng c thm vo mi trng. Hiu
qu ca chng kh r rt nhng chc nng tht s cha c bit.
- Dch chit nm men (Yeast extract), dch trch Khoai ty, chui nghin, nc Da,
dch trch tri cy nh C chua, Khm
2.5 CC KIU BNH CHA V NP Y S DNG TRONG NUI CY M
V T BO
Hnh 2.18 Cc kiu bnh c s dng trong nui cy
34
V mt nguyn tc, tt c cc bnh chai l no cng c th c s dng cho nui
cy m v t bo. Tuy nhin, cc bnh chai l c s dng phi tho mt s iu kin
sau:
- Phi chu ng nhit hp kh trng
- Phi cho nh sng xuyn qua
- Hu nh khng cn vi sinh vt sng st sau khi kh trng
- Phi c gi thnh r (nu s dng cho mc ch sn xut)
- D tm
Hin nay, ngi ta c th s dng cc chai l ng thc phm. Trong cng nghip
sn xut cc bnh nui cy bng nha s dng mt ln cng c p dng cc nc
cng nghip pht trin.
Cc ho cht tinh khit c trong cc t c lit k thnh danh mc d tm.
Ngi ta phn thnh cc cm ho cht nh cc khong a lng, cc khong vi lng,
ng, agar. Cc ho cht ny thng trn cc k c k hiu ni mt. Cc vitamine,
cc cht iu ho sinh trng th trong t lnh c nhit thp.
Cng vic chun b gm cc bc sau:
- Chun b ho cht pha mi trng
- Nu mi trng thng s dng ni nu bng thp khng g, hoc trong cc
cha bng plastic chu nhit s dng l vi sng (micro wave).
- o pH.
- Rt vo ng nghim hoc keo lo.
- Cho vo ni hp kh trng iu kin nhit v p sut mong mun
- Ly mi trng ra ngui
Hnh 2.19 T ng ha cht
35
2.6.2 Chun b dung dch gc (stock solution)
Dung dch gc thng c pha m c t 10 n 1000 ln. Khi pha thnh mi
trng nui cy th gim i 10 n 1000 ln.
Mt v d v thnh phn ca mt mi trng nui cy thng dng
Mi trng MS (1962). Pha cc dung dch gc (stock solutions) ca mi trng MS
(1962)
a lng MS Ha cht (g/l) Stock solution x10
(g/l)
1 MS1 NH
4
NO
3
1,65 16,5
KNO
3
1,9 19
MgSO
4
7H
2
O 0,37 3,7
2MS2 CaCl
2
0,33 3,3
3MS3 KH
2
PO
4
0,17 1,7
FeEDTA FeSO
4
7H
2
O
EDTA
27,8 mg/l
37,3 mg/l
m c 100 ln
2,78g FeSO
4
7H
2
O
3,73g EDTA 10ml/l mi
trng
Vi lng MS (mg/l) Stock x 1000 (mg/l)
H
3
BO
3
6,2 6200
MnSO
4
22,3 22300
ZnSO
4
7H
2
O 11,5 11500
Na
2
MoO
4
2H
2
O 0,25 250
CuSO
4
5H
2
O 0,025 25
KI 0,83 830
CoCl
2
0,025 25
Macro MS 100ml/l mi trng
Micro MS 1ml/ l mi trng
Cc Vitamin v cht iu ha sinh trng thng c pha nng
1mg/1ml.
36
Cng thc ca mt s mi trng nui cy m (mg/l)(Pauls, 2003)
Ha cht Mi trng
B5 White Heller
(NH4)
2
SO
4
134
(NH4)NO
3
NaNO
3
600
a lng KNO
3
2.500 80
Ca(NO
3
)
2
CaCl
2
2H
2
O
MgSO
4
7H
2
O
NaSO
4
KH
2
PO
4
NaH
2
PO H
2
O
KCl
300
150 75
250 720 250
200
125
150 16,5
65 750
FeSO
4
7H
2
O 27,8
Na
2
EDTA 37,3
FeCl
3
6H
2
O 1,0
Fe
2
(SO4)
3
2,5
Vi lng MnSO
4
4H
2
O 7 0,01
MnSO
4
H
2
O 10
ZnSO
4
7H
2
O 2 3 1
H
3
BO
3
3 1,5 1
KI 0,75 0,75 0,01
Na
2
MoO
4
2H
2
O 0,25
CuSO
4
5H
2
O 0,025 0,03
CoCl
2
6H
2
O 0,025 0,03
NiCl
2
6H
2
O
AlCl
3
0,03
vitamins
myo-inositol
nicotinic acid
pyridoxine HCl
thiamineCHCl
glycine
Ca D-pantothenic acid
100
1,0
1,0
10,0
0,5
0,1
0,1
3,0
1,0
0,03
1,0
carbohydrate sucrose 20.000 20.000 20.000
hormones kinetin
2,4-D
IAA
pH
0,1
0,1-1,0
5,5
6,0
5,5
2.6.3 Nu mi trng: i vi mi trng c, agar l thnh phn to gel c thm vo
v vy cn phi gia nhit. Gia nhit cho mi trng c th nu trn bp du, gas, in hoc
trn l vi sng (micro wave).

a b c
Hnh 2.20 Nu mi trng trn bp gas (a) hoc trn l vi sng (b,c)
37
2.6.4 o pH: khi agar c nu chy hon ton mi trng c o v chnh pH trong
khong 5.5-6 trc khi cho vo keo, ng nghim hoc bnh tam gic.
Hnh 2.21 o pH ca mi trng
Rt vo
ng
nghim
Rt vo keo l
Hp kh trng Mi trng sn sng
Hnh 2.22 Rt mi trng vo bnh cha, hp kh trng
2.7 IU KIN NUI CY
2.7.1 Nhit
- V mt thc hnh c th ti u ha nhit cho mi loi v mi giai on sinh
trng. Nhit chung cho tt c cc phng thng c iu chnh 22 2
o
C

- Ban m nhit thng thp hn
- Nhit trong cc keo l nui cy thng cao hn nhit phng vi do hiu
ng nh knh
- Mt trong cc vn ch yu l gi nhit phng n nh trong sut qu trnh
nui cy.
- Cn ci t mt dng c n nh nhit (thermostat), khi nhit phng qu cao
c th kim sot bng cch tt bt n.
2.7.2 nh sng:
- Thng s dng n hunh quang (n non) t song song vi k. Cc bin in
ca n nn t bn ngoi phng trnh s gia tng nhit trong phng
- Cc thng s nh sng sau y rt quan trng
- Quang k
- Cng nh sng
- PAR = s moles photon gia 400 v 700 nm mi m
2
mi giy (s)
- Lux khng l n v thch hp v n da trn c s s mn cm ca mt con ngi
- Quang ph
38
- Quang ph nh hng n hnh thi
- T l quan trng l xanh/o /cui (blue/red; red/far-red)
- Quang ph ty thuc vo kiu n
2.7.3 Quang k (photoperiod)
Trong hu ht trng hp quang k khng quan trng. Thng s dng ngy di l
16 gi.
Hnh 2.23 Phng tng trng
2.7.4 Cng nh sng (light intensity)
- Cng nh sng thch hp l khong 30 mol m
-2
s
-1
. Di iu kin ny, s
nui cy cn trong giai on tp dng (mixotrophic).
- i vi m t dng (autotrophic tissue culture) cng nh sng ln n 250
mol m
-2
s
-1
.
- Ty thuc vo np y ca keo nui cy m lm gim cng nh sng
- Bc x quang hp hu hiu (PAR = Photosynthetic active radiation, mol.m
2
.s
-1
)
- Ph nh sng (light spectrum)
- Ph nh hng p ng cho hiu qu hnh thi (chiu cao, s pht trin l)
- Cc t l quan trng l: blue/red v red/far red
- Ph ty thuc vo kiu n
2.7.5 m (Humidity)
- m tng i thng khng c kim sot trong phng nui cy
- Bnh ng cy tiu chun, khong trng bn trn thng c bo ha hi nc.
- m tng i trong bnh cha c th c kim sot bi cc phng tin khc
nhau nh s thng kh, lm lnh di y, nng agar.
39
Phng th nghim nui cy m hoc ni sn xut cy con t nui cy m nn phn
thnh ba khu vc.
- Khu vc sch: phng cy, phng sinh trng v ni ct gi mi trng
- Khu vc tng i sch: ni chun b mi trng nui cy hoc tit trng mi
trng nui cy
- Khu vc thng xuyn tip xc : vn phng, ni tip tn
KHUYN CO
o Hnh lang rng
di chuyn bn trong
o ni sch cn phi
tht s k lng
o Lun v sinh phng
th nghim:
Lau sch hng ngy,
bao giy bng nylon
khi tham quan, khu
trang, o
Khu vc sch: ni
ct mi trng
nui cy, phng
truyn, phng
sinh trng
Khu vc tng
i sch:
phng chun
b, v phng
tit trng
Khu vc tip
xc thng
xuyn: vn
phng, ni
tip tn
S tng qut ca cc khu vc khc nhau trong phng th
nghim
3.2C S H TNG
3.2.1 H thng in
H thng in cho phng nui cy nn thc hin theo ng yu cu v an ton v in.
Ngun in vo cc cng tc v cu dao t ng hoc chng git. C ni vi dy t
.
3.2.2 H thng chiu sng
H thng chiu sng nn s dng n hunh quang v nn t cc cc bin in
(transformer) ca h thng n ngoi phng lnh
H thng lnh phi gi c nhit phng 25
o
C
3.2.3 H thng phn hy
Nn c thm h thng phn hy cc cht thi ra t qu trnh nui cy nh bn lc vi
sinh
3.3 TRANG THIT B V HA CHT PHNG TH NGHIM NUI CY M
Tu theo mc ch nghin cu hoc sn xut m cc thit b nn c mua cho
thch hp sm. Di y l danh sch cc dng c ca phng nui cy. Ty theo qui m
m chn la thit b cho thch hp.
- T cy v trng
- T lnh ng ha cht
40
- T ng bng
- T sy cao lm kh dng c thy tinh
- T sy cao kh trng nhit
- Ni hp kh trng
- Xe y ng chai l
- Cn vi phn tch ( sai s mg)
- Mng lc vi cc ph kin i km
- Knh hin vi v knh lp c gn my nh
- My lc trn
- My ct vi mu
- My iu ha khng kh
- pH k
- T ht kh c
- My khuy t
- Nhit k
- n kh trng UV
- n ng hunh quang
- B m t bo
- B kh ion
- My ly tm
- Water bath ( 30
o
C)
- Gng tay dng mt ln
- Li ny lon
- ng ly tm
Dng c thy tinh
- Cc bnh tam gic v chai l
- ng chch nha hoc thy tinh
- Pipette thy tinh (ml)
- Pipette t ng (l)
- a thy tinh
Linh tinh
- Dng c cy: kp cy, dao m , kim cy, dao lam
- Giy nhm, nylon film, giy Parafilm, dy thun, vit du, s ghi chp
Ha cht v thnh phn c bn
Khong a vi lng
- N, P, K, Ca, Mg, S
Khong vi lng
- B, Mn, Zn, Cu, Mo, Cl, Ni, Al, Co, I, Fe, Na
- EDTA
Cc cht hu c
- Agar
- ng: sucrose, fructose, galactose, lactose
- Vitamine: A, D, E, C, biotin (vit H), Folic acid (vit M), nhm B (Thiamine HCl,
nicotinic (B
3
), pantothenic acid (B
5
), Pyridoxine (B
6
), Riboflavin (B
2
)
- Amino acid
- Cht iu ha sinh trng: auxin, cytokinin, gibberellin, abscisic acid, Ethylene
41
- Than hot tnh
Cc cht trch hu c:
- Nc Da
- Malt extract
- Yeast extract
- Nc trch C chua
- Chui
- Dch trch Khoai ty
- Casein hydrolysate
- Nc Cam
Nc ct:
- Nc kh khong
- Nc ct hai ln
Ha cht kh trng:
- Calcium hypocloride (Ca(OCl)
2
- Sodium hypocloride (NaOCl)
- Clorua thy ngn (HgCl
2
)
- H
2
O
2
- Cn
- Khng sinh
My lc xoay Mt my lc xoay trn vi gi ng nghim
42
My lc xoay bnh tam gic My lc xoay keo

Phng tr mi trng nui cy Phng hp kh trng Phng cy

T cy Phng tng trng
43
Chng 4 CC NG DNG CA K THUT NUI CY M V
T BO TRONG NHN GING
4.1 NHN GING HU TNH
Nhn ging hu tnh p dng trong nui cy m gii quyt cc vn m trong
iu kin t nhin kh thc hin nh ht ch c phi khng c phi nh (v d ht lan)
hoc kch thc ht qu nh kh ny mm. .
Gieo ht trong ng nghim
K thut gieo ht trong ng nghim l k thut nhn ging to ra cc cy con c
th khng ging cy m. N l mt trong cc phng php c s dng nhn mt s
loi hoa Lan v cc loi hoa trong iu kin t nhin kh ny mm. Ngoi ra, nhn ging
bng ht cng c p dng cho cng tc lai to ging mi, c bit l cc ging hoa Lan.
Gieo ht trong in vitro cng ging nh gieo ht trong iu kin t nhin, nhng k thut
ny i hi mt s hiu bit v pha ch mi trng gieo ht cng nh mt s thao tc kh
trng.
Mt th d c th l gieo ht hoa Lan
Ht Lan sau khi thu tinh v pht trin thnh tri. Khi tri n tui trng thnh
(trc khi kh v bung ra), tri s c thu hoch sau ra sch chun b cho thao
tc gieo ht. Cng vic gieo ht Lan gm ba bc. Chun b mi trng gieo ht, kh
trng tri Lan v gieo ht Lan vo trong bnh.
4.1.1 Chun b mi trng gieo ht:
C nhiu loi mi trng gieo ht Lan c khuyn co. Mi trng ph bin
nht l mi trng Knudson C (1946). Cc bc chun b mi trng (xem chng 2) c
th tm tt nh sau:
Thnh phn ca mi trng Knudson C (1946)
Ho cht Lng (g/lit)
Ca(NO
3
)
2
4H
2
O 1,000
(NH
4
)
2
SO
4
0,500
MgSO
4
7H
2
O 0,250
KH
2
PO
4
0,250
FeSO
4
0,025
MnSO
4
0,0075
ng 20
Agar 9
pH 5.6
44
Hnh 4.1 Mi trng nui cy (chai) v tri Lan chun b cy
- Nu mi trng cho tan agar sau o pH
- Rt mi trng vo trong chai, l hoc keo.
- Kh trng mi trng trong ni hp kh trng (chng 2)
- Ly mi trng ra ngui
Tri Lan khi n tui trng thnh (thi gian thu hoch tri tu theo loi) s c
thu hoch, ra sch vi bt git v sau kh trng tri theo cc phng php c
cp chng 2. gia tng s kh trng b mt tri Lan c th dung la ca n cn t
bn ngoi v tri (hnh 4.2)
Hnh 4.2. Kh trng b mt tri Lan bng n cn
Tri Lan sau khi kh xong s c tch ra v gieo thng vo keo hoc chai cha
mi trng. Do ht Lan c kch thc rt nh, v vy phn tn ht Lan u trn mi
trng, ngi ta c th rc cc ht Lan vo mi trng Knudson C (1946) lng c
chun b cng lc vi mi trng c. Sau , dng ng ht kh trng, rt mt th tch
dung dch v cho vo mi trng c, ri lc nh keo hoc chai cha mi trng dung
dch cha ht Lan phn tn u trn b mt ca mi trng. Sau theo di s pht trin
ca ht Lan sau khi cy.
45
Hnh 4.3 Gieo ht Lan vo trong chai
Vi nhn ging l vic nhn ng kiu cy (true-to-type) ca mt kiu gen c
tuyn chn bng cch s dng k thut in vitro. Vi nhn ging thng thng l phng
php nhn nhanh v gim gi thnh. Hnh 4.4 trnh by mt s tng qut quy trnh vi
nhn ging.
Hnh 4.4 Cc bc c bn ca vi nhn ging (George,1993)
Cy m
46
Chi ngang mt Chimeras Chi bt nh Cordyline
Hnh 4.5 S to thnh chi ngang v chi bt nh
Khi cy truyn nhiu ln, mi trng pht trin cc chi ngang c th chuyn sang
to ra cc bt nh (adventitious shoots). Kt qu l c th to ra cc bin d t bo soma.
V vy cy con to ra khng ng nht.
i vi mc ch vi nhn ging, s to chi ngang l k thut thch hp ang c
s dng. Theo nguyn tc, cch ny to ra ng kiu cy.
Vi nhn ging c Debergh v Zimmerman (1991) chia thnh 4 giai on khc
nhau, mi giai on c mt chc nng ring. S thnh cng ca cng vic vi nhn ging
ty thuc vo tt c cc giai on.
In vivo Giai on 0: s chun b ca cy m
In vitro Giai on 1: bt u tit trng
Giai on 2: nhn
Giai on 3a: ko di
Giai on 3b: to r v tin thun dng
In vivo Giai on 4: Thun dng
47
Hnh 4.6 Ghp nhiu tng
Giai on 0
Giai on 0 c hiu l ci thin iu kin v sinh ca cy m. Phng php ti
nh git cy m cho php ci thin tnh trng sinh l ca cy m, mu cy tun th
theo qui trnh cy. Tnh trng sinh l ca cy m cng nh ngun dng lm mu cy c
th c ci thin bi mt s k thut nh ghp nhiu tng (hnh 4.6). Ghp nhiu tng l
hnh thc ci thin mu cy lm cho mu cy tr thnh non hn, c bit l mu cy ca
cc cy thn g.
Giai on 1
Mc ch l thc hin thao tc cy. N l mt s kt hp gia mt phng php
tit trng y v mt t l sng cao ca mu cy v khng b nhim. Thng thng kh
t thnh cng 100 % trong k thut v trng mu. Cc hnh sau y l mt v d ca
thao tc v trng mu cy cy hoa Hng.
48
1/ Cc cy
m c ti
nh git
2/ Chi non c
ct ra
3/ Loi b l, ct
thn thnh on
ngn
4/ Ra cn 80%,
kh trng vi 10%
NaOCl trong 15
pht
5/Ra 3 ln
vi nc kh
trng
6/ Ct b y v
nh
7/ Ct mt 8/ Mi mt cha
mt chi bn
9/ t mt trong
ng nghim
10/ Hon tt
Hnh 4.7 Cc thao tc cy hoa Hng giai an 1 (Debergh,2003)
Phng php
Cc mu cy thng c s dng l cc mm ngang hoc m phn sinh. Trong
mt s trng hp s nui cy c bt u t cc phn khc ca thc vt. V d
rt kh tch ly mu mm ngang m khng b nhim ca cy Ficus lyrata v
Anthurium spp. V vy, cc mu cy c chn trn cc mu cy t l v cc chi
nguyn thy. K n kch thch to nhnh ngang ca giai on 2
Giai on 2
Qu trnh kch thch cc trung tm m phn sinh nh nh sinh trng, chi chnh,
chi bn. C nhiu kiu tng tc nhn nh cy mt (hnh 4.8). Trong k thut ny c
hai kiu to chi mt kiu to chi t mt mu cy ch c mt mt hoc mu cy nhiu
49
mt. C hai kiu u to ra s chi nhiu v h s nhn tu thuc vo cc on chi mi
to thnh giai on k ca nhn chi l to r. Phng php nhn ging ny thng c
ng dng trn nhng cy khng c mm bnh.
Hnh 4.8 Cy mt hoc nhiu mt (George,1993)
Phng php khc gia tng s chi giai on 2 l cy nh sinh trng
(meristem culture) hoc cy nh chi (shoot tip culture). nh sinh trng hoc chi c
th c ly t chi chnh hoc chi ngang (hnh 4.9). Theo cch cy ny hai kiu dng
chi c th c to thnh l chi di v cm chi. chi di, cc chi pht trin lng di
v cc chi ngang cng di. V vy, cy con c to ra t chi ny c dng bnh thng.
Trong khi cm chi to ra nhiu chi nhng lng ngn nn cy con c to ra c th
l cy n hoc cm cy con.
Hnh 4.9 Cy chi hoc nh sinh trng (George,1993)
50
giai on 2, vic gia tng s chi ph thuc nhiu vo vic s dng cytokinin. S dng
hm lng cytokinin cao c th gy ra mt s vn trong cc cy c nhn ging nh
s tha nc, to thnh bi rm, bin d v tnh, cc vn v r v chm ra hoa..
Hnh 4.10 Cc bc ca k thut nhn chi cy hoa Hng giai on 2
(Debergh,2003)
Giai on 3
c chia thnh hai giai on l giai on ko di (3a), kch thch to r v tin thun
dng (3b).
Giai on 3a: Ko di
Trong nhiu trng hp s ko di l mt yu cu cho s to r y ;
Mi trng ko di thng khng cha cytokinin hoc mt cytokinin yu hn
cytokinin c s dng trong giai on 2;
C th cn thit thm than trung ha hiu qu cytokinin cn li trong giai on
2;
Ty thuc vo kiu cy, s ko di c th xy ra trn cc chi n hoc cm.
Giai on 3b: Kch thch r v tin thun dng
Auxin thng c s dng kch thch to r. To r tt nht thng t c
trn mi trng vi hm lng khong thp (v d mi trng Knop 1/2).
Cc r pht trin trong in vitro th lun khng thch nghi vi iu kin nh li v
cc r qu di s gp kh khn khi trng.
Kch thch r c th xy ra trn chi n v cm.
51
Ngoi vic kch thch r, giai on ny cng thch hp cho s thun dng. Cc
cch lm tng kh nng t dng l cung cp cy con vi carbohydrate hoc lm thp m
tng i trong bnh cha, s dng nm r, s dng cht tr ...
Giai on 4: S thun dng (acclimatization)
(Chi tit chng 7)
Mc ch ca giai on ny l lm gim ti thiu s cht cy con, khi chuyn t in
vitro sang nh li hoc iu kin ngoi ng. Trong giai on ny cc yu t cn c
quan tm l:
- Tnh trng cy con khi chuyn ra mi trng trng;
- Cc yu t v mi trng nh nhit , m , nh sng
Hnh 4.11 S thun dng cy hoa Hng
Trong vi nhn ging, cng nhiu giai on cy truyn, cng tn nhiu chi ph v
cng sc. V vy, gi thnh s nng ln. gim chi ph, hai k thut c p dng l:
K thut hai lp (Double layer technique)
Hoa Hng trong bnh
cha plastic
Lm y mi trng
hai lp
S pht trin v sau
Hnh 4.12 K thut hai lp
Bng cch thm mt lp mi trng lng pha trn mi trng c, c th trnh
cy truyn, iu ny c th lm thp chi ph. K thut ny thng c s dng giai
on 2, v giai on ny cc cy cn mt thi k cy truyn lu di. V du hoa Lan cn
cy truyn 6 thng .Mi trng mi c thm vo sau ba thng v giai on cui
kch thch s ko di v to r.
52
Thnh phn c in ca mi trng hai lp bao gm: Nng 1/2 nguyn t
khong a lng, than, ng, auxin
T ng ha (Robotization)
.
Hnh 4.13. Cc kiu thao tc t ng trong cy m
Ngi ta c lng 70% chi ph gi thnh ca cy cy m l cng lao ng. V
vy, nhiu phng th nghim vi nhn ging hin nay l lm t ng ha cc thao tc
gim gi thnh lao ng
4.3 NHN GING T PHI V TNH (Somatic embryo)
4.3.1 Phi v tnh
Trong k thut vi nhn ging, ngoi vic lm gia tng s chi giai on 2 cn c
mt cch khc l to ra mt s lng phi v tnh nhiu giai on ny.
Phi v tnh l g?.
Trong t nhin c c ht, thc vt phi hon tt qu trnh th tinh bao gm
mt qu trnh dung hp hai t bo sinh dc c (ht phn) v ci (non) hnh thnh
phi (phi hp t, zygotic embryo) v phi nh.
Phi v tnh l phi c hnh thnh t cc t bo dinh dng (somatic cells) m
khng c s dung hp hai t bo sinh dc v cng khng c phi nh. C hai dng t bo
phi kh ging nhau v hnh thi, nhng khc nhau v qu trnh hnh thnh.
Mt phi thc vt c hnh thnh do ngun gc, hnh thi v s tin ha ca n .
Ngun gc: phi hp t l kt qu ca s dung hp ca cc t bo sinh dc,
thnh lp hp t; i vi phi v tnh s dung hp khng cn thit.
53
Hnh thi: mt phi pht trin y l mt trc hai cc vi phn sinh m chi
mt cc v phn sinh m r mt cc; n cng biu th c tnh mt kiu c bit
ca l , t dip.
Tin ha: s pht trin phi c biu th c tnh bi mt s k tip cc giai on
hnh thi tiu biu: dng hnh cu (globular), dng tri tim (heart shaped) v dng
ng li ( torpedo shaped) (hnh 4.14 )
Hnh 4. 14 Cc giai on hnh thi tiu biu trong qu trnh pht trin phi v tnh:
(A) hnh cu, (B) hnh tri tim, (C) torpedo
S thnh lp phi v tnh mt cch gin tip c th bt ngun t mt t bo n, t
mt cm t bo phi hoc t a bo.
- Ngun gc t bo n
Cc kho st v qu trnh hnh thnh c quan nh chi v r trong nui cy m trn
cc loi thc vt khc nhau. Ngi ta thy rng, qu trnh thnh lp phi v tnh hon
ton khc vi qu trnh hnh thnh lp c quan nh chi hoc r. Trc ht, phi l nhm
t bo c hai cc cc cc ny s hnh thnh phn sinh m chi v r sau ny. Nh vy,
vn t ra l c phi n pht sinh t mt t bo n hay khng ?
Ngi ta chng minh c l phi v tnh pht sinh t mt t bo n t callus
hoc t t bo trn (protoplast). Mt s thay i v mt phng phn chia t bo l mt
trong cc ch tiu u tin ca s thnh lp phi (hnh 4.15)
54
Hnh 4.15 S thnh lp cc t bo tin phi xut pht t b mt bt thng ca cc
t bo phn chia trong qu trnh thnh lp phi t cc t bo phi n (Trigiano v
cng s, 1989)
- Ngun gc a phi (polyembryos)
Hnh 4.16 S to phi phn chia hp t Pinus (George,1993)
Ngoi ngun gc pht sinh phi v tnh t t bo n, ngi ta cng pht hin thy
qu trnh thnh lp phi v tnh xut pht t cc m phi hnh cu ca Picea c gi l
s to a phi (polyembryogenesis) (hnh 4.16)
55
Thng thy cc mu cy ca phi tm (nucellus) ca cy c mi (Citrus).
Thng thng, khi cy phi tm s to thnh phi v tnh.
Cc giai on pht trin phi v tnh c trnh by hnh 4.17. N bao gm cc
giai on sau:
- Tin phi: mt cm nh t bo phn sinh m t phi v tnh pht sinh
- Giai on hnh cu: mt nhm t bo ln hn cha c dng ging phi c hnh
thnh
- Giai on tri tim: s hnh thnh cc phn sinh m r
- Giai on Torpedo: mt s ko di v hai cc ca giai on tri tim
- Cy con: s hnh thnh chi v r
Cy mt l mm v hai l mm c phn hi khc nhau mt s giai on (hnh 4.17,
4.18)
Hnh 4.17 Cc giai on pht trin phi v tnh ca cy hai l mm
(George, 1993)
56

Hnh 4.18 Cc giai on pht trin phi v tnh ca cy mt l mm (George, 1993)
4.3.3.1 Cht iu ha sinh trng
- Auxin
Auxin nh hng n qu trnh hnh thnh phi v tnh. Mt gi thuyt c
Okkels (1988) ngh rng, auxin gy ra s mt gc methyl ca DNA trong qu trnh
phn chia t bo. y l iu kin tt yu cho s thnh lp tin phi. Tri li, s pht trin
phi i hi s methyl ha DNA.
S thnh lp phi v tnh c th i theo con ng trc tip hoc gin tip di s
hin din ca auxin (George,1996). S bn di ch ra cc con ng hnh thnh phi
v tnh.
- Cytokinin
Hu ht cc cy hai l mm khi thm mt lng nh cytokinin (vi d 0,1 M BAP
hoc kinetin) vo trong mi trng c cha auxin c khuynh hng gia tng vn tc sinh
trng ca cc callus hnh thnh phi v tnh.
- Gibberellic acid (GA)
Mc d c cc kt qu bo co cho thy rng trong mi trng nui cy c s hin
din ca GA ngoi sinh lm gim s to thnh phi v tnh (Chen v cng s, 1990). Tuy
nhin, cng c cc kt qu khc cho rng s cung cp thm GA ngoi sinh cng gia tng
s thnh lp phi v tnh. GA ngoi sinh (1mg/l) khi c thm vo mi trng khng c
auxin c th gip chuyn ha, sinh trng v ny mm ca phi v tnh
- Abscisic acid (ABA)
ABA c hiu qu trong vic thnh lp phi v tnh nhiu loi c bit l phi v tnh
pht sinh t callus.
57
H
n
h

4
.
1
9

C
n
g

t
h
n
h

l
p

p
h
i

v
n
h

t
r
c

t
i
p

v

g
i
n

t
i
u

h
a

b
i

a
u
x
i
n

(
G
e
o
r
g
e
,

1
9
9
3
)
58
- Ethylene
S hin din ca ethylene lm gim s thnh lp phi v tnh. Mt s th nghim thm
cht c ch s thnh lp ethylene nh CoCl
2
hoc AgNO
3
lm gia tng s thnh lp
phi v tnh.
- Nitrogen
T l hm lng m NO
3
-
/ NH
4
+
s nh hng nhiu n s thnh lp phi v tnh.
Vi t l NH
4
+
/NO
3
-
l 80/20 trong mi trng cha 35 mM m tng s cho thy hiu
qu nht trong s thnh lp phi v tnh gin tip ca cy Betula pendula (Nuutila v
cng s, 1991). Tuy nhin, i vi cy Bp (Zea mays), Suprasanna v cng s, 1991 bo
co rng s p ng tt nht cho s thnh lp phi v tnh l s gia tng nng c NO
3
-
v NH
4
+
trong mi trng MS t 39,4 v 20,61 mM n 54 v 36 mM theo th t. Nh
vy, lm hiu qu gim t l NO
3
-
v NH
4
+
t 66/34 n 60/40
Hnh 4.20 Ba quy trnh thng thng pht trin phi v tnh (a) to phi v tnh
trc tip t hp t cy Redbud (Cercia) (b) to phi v tnh cc t bo cy treo ca
cy Carrot, (c) to phi v tnh ca cc t bo phi cy Thng (Hartmann v cng
s, 1999).
59
- ng lactose, galactose
ng sucrose nng cao v cc loi ng lactose v galactose c hiu qu cao
trong s hnh thnh phi v tnh cy Cam ngt (Citrus sinensis). Kochba v cng s,
1982 s dng ng galactose, lactose bin i callus thnh phi v tnh.
- Than hot tnh
Thm vo mi trng 1g/l than hot tnh gia tng s thnh lp phi. cy u than
hot tnh gia tng s thnh lp phi (Litz v Conover, 1980)
4.3.4 ng dng ca phi v tnh trong s to ht tng hp (Synthetic seed):
Khi nim ht nhn to c pht trin trong nhiu thp k qua. Mc d phi v
tnh c to ra u tin nm 1958, nhng mi n thp nin 1970 tng v ht tng
hp mi c c cng b. Nhiu loi thc vt nh c Alfalfa, Cn ty, rau Dip t
c thnh cng. Redenbaugh v Reyes (1988) ngh phi c bao li bng mt cht
gel gi l sodium alginate to ra mt ht nhn to n phi. n nay phm cht v
tin cy ca ht phi v tnh l yu t gii hn cho s pht trin ca ht nhn to.
Hai phng php c s dng bao phi l phng php bao bng gel nh git v
phng php c
Phng php bao bng gel nh git
Phi c pha trn vi sodium arginate theo t l 2% (trng lng/th tch) c
nh qua mt plastic pipette vo mt mui kim loi c 2 hoc 3 ha tr nh calcium nitrate
(100 mM). Mt phn ng trao i ion calcium s thay th sodium ngay v cc git c
to gel hon ton trong 30 pht (hnh 4.21)

(a) (b)
Hnh 4.21 Sodium hoc calcium alginate c s dng bao bn ngoi phi (a);
Cu trc phn t ca Calcium alginate (b)
60
Kch thc ca ht nhn to c th c kim sot bng ng knh trong ca u
pipette. V vy, mt u pipette c kch thc ng knh trong 4 mm th to thnh ht
nhn to c kch thc 5 mm ng knh.
Phng php c khun
Phi c th c pha trn trong mt loi gel ph thuc nhit nh gelrite hoc agar.
Phi c t trong cc ging nh trong di tao gel khi nhit h thp. Sau , calcium
nitrate hoc calcium chloride c thm vo.

a b

c d
Hnh 4.22 Ht nhn to v s ny mm
a/ ht nhn to; b/ ht nhn to trong da petri c/ ht nhn to ny mm
d/ cy con t ht nhn to
(Bioreactor)
Bioreactor c s dng u tin bi cc nh vi sinh vt hc. Mc ch ca vic s
dng cc bioreactor l gia tng s trao i kh trong mi trng nui cy gia tng sinh
khi. C nhiu kiu bioreactor: kiu n gin l cho lung khng kh sch c qua
mng lc v i vo mi trng nui cy. Kiu khng kh c nng ln, kiu c hc, kiu
rung ng khuch tn kh. V th tch ca cc bioreactor c nhiu loi kch thc khc
nhau t vi lt n vi chc lt. Kch thc cng ln cng d nhim. Cc bioreactor khi s
dng phi:
- c hp kh trng tt c cc thnh phn
- bung sinh trng ca bioreactor l vt liu trong sut nh sng i xuyn qua
61
- vt liu lm bioreactor phi c bn chu nhit v t b v
- s dng trong nui cy m nn s dng loi kch thc nh trnh nhim
Hnh 4.23 Cc kiu bioreactor. (A) kiu n gin l cho lung khng kh sch
c qua mng lc v i vo mi trng nui cy,(B) kiu khng kh c nng ln
(air lift), (C) kiu c hc, (D) kiu rung ng khuch tn kh
Bioreactors kch thc ln khng thch hp cho vi nhn ging bi v chng qu t
v nhiu ri ro. Bioreactors nh th uyn chuyn hn, v d nh cc kiu sau y Nalgene
TM
filter units, RITA
TM
(Cirad, Montpellier) v LifeReactor
TM
t Osmotek
Nalgene
TM
filter units RITA
TM
Lifereactor
TM
Hnh 4.24 Cc kiu bioreactor c kch thc nh
Hu ht cc t bo v m c cy trong cc bioreactor th chm trong mi trng
lng. Cch ny ging nh phng php cy treo. Tt c cc kiu trn u thch hp cho
vic to phi v tnh. Tuy nhin, bt li chnh ca bioreactor l mi trng nui cy rt d
b nhim.
62
Chng 5 CC NG DNG CA K THUT NUI CY M V
T BO TRONG TO GING
Di iu kin t nhin, s thnh lp hoa bt u khi cy t c trng
thnh. Tui ca cy t c trng thnh ra hoa thay i theo loi thc vt. S ra
hoa trong ng nghim u tin c bo co trn cy Khoai M, khng c s dng cht
iu ha sinh trng trong mi trng. S pht sinh hoa t mm ngang ca cy Morus
alba v Hibiscus cannabinus thng qua cy pht hoa. Mc d, c nhiu kt qu bo co
v s ra hoa trong ng nghim ca nhiu loi thc vt khc nhau, nhng n nay c ch
ca hin tng ny vn cha c bit r.
S ra hoa trong ng
nghim ca cy ngy
ngn
Plumbago indica
S ra hoa trong ng nghim ca cy
ngy ngn Streptocarpus
S ra hoa trong
ng nghim ca
cyTre
Hnh 5.1 Cc dng ra hoa trong ng nghim
Gen quyt nh cc tnh trng ca thc vt. Tnh trng tng ng vi mt gen gi
l tnh trng n gen v cc tnh trng c lin quan n nhiu gen gi l tnh trng a gen.
Hai gen nm trn vng c cng mt v tr nht nh (mt locus) trn hai nhim sc
th tng ng gi l alen (allele, gen ng ng). Tuy cng tham gia quyt nh mt tnh
trng, v d mu hoa, mt alen c th c th mang cc thng tin di truyn c mu , alen
kia mang thng tin di truyn to nn mu trng. Trong trng hp ny, ta c cc c th d
hp v tnh trng mu hoa. Nu c hai alen u mang thng tin di truyn cho mu
(hoc mu trng), ta c cc c th ng hp v tnh trng mu hoa. i vi trng hp d
hp, mt trong hai alen c th l alen tri (dominance), alen cn li l alen ln (recessive).
Mu hoa ca cy do alen tri quyt nh. Trong v d trn, nu trn thc t ta thu c
cc hoa mu hng th l trng hp cc alen tri khng hon ton (incomplete
dominance).
63
Cc t bo, m hay c th n bi gi l bn hp (hemizygous), v mi tnh trng
ch c mt alen. cc c th n bi kp (double haploid) cc gen u l ng hp hon
ton.
Th bi l danh t dng ch s b nhim sc th (chromosome set) c trong t
bo m, c th thc vt, vi quy nh chung l cc t bo sinh sn c b nhim sc th
l n, c gi l th n bi (haploids). Hp t, sn phm dung hp ca hai giao t n
bi, c th bi l nh bi (diploids) vi s nhim sc th 2n. Tt c cc t bo soma hnh
thnh do s phn hp t u l nh bi th khc nhau, cc m khc nhau ca c th thc
vt (4n, 8n...). l hin tng a bi ha do ni gin phn (endomitosis). Trong cng
mt loi c th gp nhiu mc bi. Cc th bi ln hn nh bi c gi l a bi.
Khong mt na thc vt bc cao mc a bi.
d dng hn cho s tm hiu t bo hc ca cc loi mc a bi ny, ngi ta
s dng khi nim s nhim sc th c bn ca loi x (l s n bi nh nht trong dy a
bi). Cc c th c x nhim sc th c gi l th nht bi (monoploids) phn bit vi
th n bi (haploid).
V d: Cy La M c hp t 2n = 42. Trn thc t, La M l mt th lc bi
(hexaploids) 6x, trong s th nhim sc c bn ca loi l x = 7. Th n bi ca cy
La M lc bi c n = 3x = 21 th nhim sc. Cy Thuc L (Nicotiana tabacum) l mt
th t bi 4x c 48 th nhim sc: s nhim sc th c bn x bng 12. Th n bi ca
cy Thuc L ny c n=2x=24 th nhim sc. Mt loi Thuc L khc Nicotiana
sylvestris c cng s x=12, nhng v l mt cy nh bi trong t nhin nn cc t bo hp
t cha 2n=2x=24 th nhim sc v cc t bo sinh sn cha n=x=12 th nhim sc.
Trng hp ny t n bi ng ngha vi nht bi.
Cy t bo n bi c thc hin cho cc mc ch to ging. S quan tm ch
yu cy n bi l trn c s tao thnh cc cy ng hp t, c to ra bng cch nhn
i nhim sc th. y l con ng to ging ngn nht. Cy t bo n bi cn c
nghin cu di truyn nh lng (quantitative genetic). Cc nghin cu nh th bao gm
pht hin s tng tc gen, c lng s bin d gen, pht hin s lin kt, c lng s
gen nh hng mt c tnh nh lng v v tr ca a gen. Ngoi ra, cy t bo n bi
cn c nghin cu cho s chuyn ha t bo.
K thut cy ti phn
iu kin sinh trng ca cy m
cy ti phn, tnh trng sinh l ca cy m rt quan trng (v d: quang k, nhit
v s dng nng dc). iu kin sinh trng ca cy m c s dng cung cp
ti phn cho nui cy c nh hng ng k trn s thnh cng ca vic nui cy. Mi
loi cn mt ch mi trng khc nhau.Cc yu t mi trng bn ngoi nh nhit ,
quang k v cng nh sng v d v nui cy ti phn c thc hin hon ho
nht trn cy Thuc L (Nicotiana tabacum) trong iu kin quang k ngn (8 gi) v
cng nh sng cao (16.000 lux) l rt c li cho nui cy ti phn. Trong khi , cy
La Mch (Hordeum vulgare) cn gim nhit (12
o
C) v cng nh sng cao
(20.000 lux) c khuyn co.
64
Tui cy m v giai on pht trin hoa
Ngi ta khuyn co l ti phn nn c ly giai on cy ra hoa, nhng hoa
cha gi. Mi loi ra hoa theo chu k sng khc nhau, c cy sau khi ra hoa th cht,
nhng c cy ra hoa sau sinh trng ri li ra hoa tip. Sc sng ca ht phn ty
thuc nhiu vo sc sng ca cy m. Cy m cn non cung cp hoa mang ht phn c
sc sng mnh hn cy m gi. Qu trnh hnh thnh v pht trin hoa thay i theo tng
loi. i vi cy Thuc L, hoa c ly giai on 2 (hnh 5.2) th cy d thnh cng
Hnh 5.2 Cc giai on khc nhau ca hoa c s dng trong cy ti phn. Giai
on 2 l giai on tt nht ca s pht trin s dng trong cy ti phn cy
Thuc L (Debergh, 2003)
Giai on c bit ca s pht trin ti phn thi gian cy l yu t quan trng
nht t c thnh cng trong s thnh lp cy n bi. cy hin hoa b t (cy ht
kn), c nhiu ti phn kh xc nh cho mi mm hoa, c th chn vi ti phn cc
giai on khc nhau ca s pht trin ht phn. cc loi c nhiu ti phn trn mi hoa,
mt lot mm hoa phi c xc nh cho tt c cc giai on ca s pht trin ca ht
phn. Mt s c tnh c th quan st c v phn bit d dng c th s dng vo giai
on pht trin thch hp nht v d giai on 2, cnh hoa hi ln hn i hoa.
Giai on pht trin ht phn
Giai on ti u v s pht trin ca ht phn vo thi gian thu hoch mm hoa rt
quan trng. cy Thuc L, giai on phn bo ht phn (Pollen grain mitosis = PGM)
cho kt qu tt nht, trong khi cy ng cc v cy Ci (Brassica) giai on ti u l thi
k n nhn (uninucleate).
Cc bo co cho thy rng ti phn ri vo mt trong ba loi: trc phn bo
(premitotic), phn bo (mitotic) hoc sau phn bo (postmitotic).
S p ng tt nht t cy ti phn m cc vi bo t hon tt giai on
gin phn (meiosis), nhng cha bt u phn chia ht phn ln u v d cy
Hyoscyamus. p ng tt nht t cy ht phn vo thi gian phn chia ht phn u tin
v d cy thuc l (Nicotiana tabacum). p ng tt nht t ht phn giai on hai t
bo u ca s pht trin ht phn (Atropa belladonna).
65
Hnh 5.3 Vng i ca cy ht kn. Cy nh bi trng thnh to ra th giao t l
ht phn pht tn ra bn ngoi. Trng cn gi li trong bu non. Khi c s thu
tinh xy ra ht c thnh lp v pht trin thnh cy mi (Pauls, 2003).
Hnh 5.4 Cc kiu pht trin ht phn Nicotiana tabacum v Hordeum vulgaris
(Dixon, 1985)
Cc phng php kh trng
S kh trng c thc hin vi cc ha cht kh trng nu chng 2. Thng
thng kh trng ton b pht hoa trc khi kh trng tng hoa.
66
Tin x l mm hoa hoc ti phn
X l hoa hoc pht hoa cha ht phn giai on thch hp c cho thy l c
s ci thin sn lng phi hnh thnh mt s cy;
X l nhit trc trong 2-30 ngy nhit 3-10
o
C, khi cy Nicotiana, Datura
hoc 35
o
C cy Capsicum c th kch thch s to phi;
Ngm hoa c ct vo trong nc trong vi ngy;
Ly tm ti phn 3-5 gs
-1
;
p dng chn khng.
Phng php tch ra tng phn nh
Mt s loi c ti phn d tch ra t hoa. Tuy nhin, mt s loi cy ly ht vng
nhit i nh Panicum, Penisetum v Setaria vic tch cc thnh phn ti phn nh rt
kh khn v tn nhiu thi gian, nn ngi ta khuyn co l nn cy ton b pht hoa v
c th t trong mi trng lng, lc chm.
S phng thch ht phn ngu nhin
Ht phn c ly t ti phn bng c hc hay bng s nt t nhin ca ti phn.
Ht phn c tch ra, cy vo mi trng agar hoc mi trng lng v s pht trin ca
ht phn s xy ra bn trong ti phn.
K thut tch
K thut ny bao gm ct vch ti phn phng thch bo t tt hn l nt ra
t nhin. K thut ny thnh cng trn cy Thuc L v cng thnh cng trn cc cy
khc nh Pennisetum typhoides.
Ti phn ly t mm hoa non c lm ng nht vi my ng nht mu. Theo
cch ny, mt dung dch ht phn th c lm sch. Dung dch c lc, ly tm v c
gn ly phn bn di v chng bng pipette trn mi trng da petri (Debergh, 1974
trch t Pierik, 1987) (Hnh 5.5)
Hnh 5.5 Ti phn (B) c ly t mm hoa non (A) c lm ng nht vi my
lm ng nht mu (C). Theo cch ny mt dung dch ht phn th (D) c lm
sch. Dung dch c lc (E), ly tm (F), c gn ly (G) v c chng bng
pipette (H,J) trn mi trng da petri (K) (Debergh, 1974 trch t Pierik, 1987)
67
Mi trng cy
+ Mi trng rn hoc lng: Trong nhiu nm nghin cu, ngi ta nhn thy rng, hu
ht cc kiu agar c s dng lm cng mi trng cha cc hn hp gy hi cho sc
sng ca ht phn. Mt dng gel c ngh s dng l agarose hoc s dng mi
trng lng. Tuy nhin, bt li ca mi trng lng l ym kh, mc d to nhiu callus
nhng kh nng bin i thnh cy thp.
+ p sut thm thu: Mc d t c thng tin v vn ny, nhng hm lng ng cao
trong mi trng (8-12%) cng hn ch s pht trin ca mu cy. Cc kt qu cho thy,
hm lng ng trong mi trng thp (2-4%) cho kt qu tt hn.
+ Cht iu ha sinh trng: Mt s loi khng cn cht iu ha sinh trng, nhng
mt s loi khc cn cht iu ha sinh trng; trong 2,4-D, BAP v IAA th rt cn.
+ Cht hu c v nitrogen khc: Khoai Ty l thnh phn mi trng thch hp cho nui
cy ti phn ca cc cy ly ht.
+ Hn hp khc: Than hot tnh l thnh phn ch yu c khuyn co nn s dng cho
cy ti phn, bi v cht ny c th hp thu cc cht nh vitamin, phenol, st .. c
phng thch ra t m gi. Mt kh nng khc l s ngoi hp bi than ca
5(hydroxymethyl)-2-furfural, mt sn phm phn hy ca ng b hp kh trng.
Cc cy con ti phn v
ht phn trn mi
trng rn cha than
hot tnh
Cy con ti phn v ht
phn trn mi trng
lng cha than hot tnh
Cy con ti phn v ht phn trn
mi trng hai lp. Mi trng
rn nm bn di, trong khi mi
trng lng nm bn trn cha
cht iu ha sinh trng
Hnh 5.6 Cc cy con ca cy ti phn v ht phn trn mi trng rn cha than
hot tnh, mi trng lng v mi trng hai lp
iu kin
+ Nhit : Nhit l mt yu t quan trng trong qu trnh nui cy. Ti phn ca
hu ht cc loi nui cy thng thch hp nhit 25
o
C. Tuy nhin, mt vi loi c
bit cn nhit 35
o
C trong mt thi gian ngn nh loi Brassica.
+ nh sng: t c bng chng hiu qu ca nh sng trong qu trnh nui cy. Tuy nhin
kinh nghim cho thy, cy ti phn nn trong ti cho n khi thnh phi hay callus.
+ Mt s ti phn: Ngi ta khuyn co l nu cy trong mi trng lng t nht 60 ti
phn c t trong mi ml mi trng.
68
Hnh 5.7 Phng php c bn to cy n bi t cy ti phn (Dodds v Robert,
1988)
Cc t bo ca cy n bi thc s cha mt b nhim sc th n. Kiu hnh ca
n l s biu hin mt bn n thng tin di truyn.
Mt lnh vc thng c cp trong cy ti phn l to t bo c
(androgenesis). Tuy nhin, chng thng c s dng theo mt ngha hn ch ch t
cy n bi hoc n bi nhn i t cc ti phn ny .
Khi bt u nui cy t cy t bi (tetraploid), kt qu to n bi c mt b i
thng tin di truyn c gi l nh bi n (dihaploid).
Trc khi s phn bo hnh thnh ht phn u tin xut hin, ngi ta gi n l vi
bo t (micropore) v sau ny n c gi l ht phn.
Li ch ca cy vi bo t (microspore) trong cy ti phn:
Trong qu trnh pht trin vi bo t thng khng tin trin qua phn chia u do
cht ngn cn ti phn.
S sinh si nhanh ca m vch ti phn c th xy ra kt qu nh bi (diploid)
khng ng nht.
Bt u vi vi bo t c tch ri s lun to ra cc cy ng nht, ngay c nu
chng l diploid (trng hp ca cc ht cc khc) hoc ngay c triploids (Petunia).
Cy vi bo t cho mt qun th ng nht ca cy, kt qu t s pht trin vi bo
ts hoc t ht phn, trong khi ly i m vch ti phn
69
Hnh 5.8 Vi bo t
Hnh 5.9 Hai quy trnh cy ht phn v ti phn (Pauls, 2003)
- Sn phm ca nui cy
Sn phm tao ra phi l cy n bi.
a bi ha
Mt s k thut p dng nhn i nhim sc th ca cy n bi.
70
S ti sinh bng phng php cy m: cc l gi hn c kh nng ti sinh c cy
n bi (haploid) ln cy nh bi (diploid). Nh bi dn n nhn i ni sinh thng xy
ra trong cy cy m.
Nhn i bng ha cht colchicines. S nhim sc th c th c nhn i bng
cch p dng ha cht colchicines cho phi hoc cy bi. Ngm ti phn cha cy con
mi c thnh lp trong dung dch lng colchicines (0,5%) trong 24-48 gi) hoc p
dng colchicines trong lanolin dn vo (0,4%) mm ngang.
S dng cc lai ha cht khc v d nh thuc dit c oryzaline hoc amiprophos-
methyl.
Li Bt li
Kiu hnh bng kiu gen
S hin din ca mt b nhim sc th lm
d dng tch t bin ngu nhin hoc dn d
Nh bi c th t c bng cch nh bi
ha nhim sc th
Bng cch lai hoc hi giao c th t c
cc dng tinh khit nhng tn nhiu thi
gian. p dng cy ti phn v ht phn c
th t c vt liu trong vng vi tun v
nhn i nhim sc th lm ng nht cy
nh bi.
Cy n bi c th lm vt liu hu ch cho
nghin cu di truyn t bo soma
Nh bi c th t c s ng nht v gen
trong cc trng hp, thng kh khn
t c (alen khng tng thch).
n bi c th c s dng chn lc tr li,
c nh cc c tnh nh lng v xc nh
cc cch lai cao cp.
Ht phn hoc vi bo t cha gen gy
c s khng bao gi pht trin v do ,
c th p ng cho s tiu hao gen.
Thng khng c s sinh trng v pht
trin trong in vitro hoc s sinh trng
ca phi b cht non.
Nh bi v t bi thng to ra cng
thi gian nh n bi.
i vi cc cy ng cc, s ti sinh
thng gp hin tng cy bch tng.
Nhn i nhim sc th khng lun lun
cho kt qu sn phm ng hp t. Cc
cy nh bi i khi cho thy c s phn
ly cc th h sau.
S phn chia t bo chn lc phi xy ra
cc ht n bi v khng phi cc m
nh bi khc. Nhng thng c s xut
hin cc m nh bi.
5.3 S CU PHI (rescue of embryos)
K thut cu phi thng c s dng trong chng trnh lai to vt qua tr
ngi do h phi v phc hi cy con m c th b mt nu hin din ngoi ng. K thut
ny c chng minh l rt hu ch trong gii quyt vn lai to nhiu loi cy trng.
V mt k thut, cu phi cng ging nh k thut cy phi v to phi v tnh.
Cng vic ca cc nh chn ging c th gp kh khn do nhng vn sau: ht
phn khng ny mm trn nm, s sinh trng ca ng phn trong vi nhy cc b hoc
b nh tr, khng c s th tinh xy ra, t bo trng th tinh nhng khng pht trin, s
rng bu non xy ra trc khi hon chnh. Nu th tinh khng xy ra sau khi t th phn
hoc th phn cho, trng hp ny gi l s khng tng thch trong t th hoc th
phn cho. Trong mt s trng hp, cc nh lai to phi c phng cch c bit gy
71
ra s th tinh bng cch no gip cho s th tinh t c kt qu. Cng vic ny
c thc hin trong ng nghim, bng cch mang ht phn t ngoi vo th tinh vi
non trong ng nghim.
Th tinh trong ng nghim c th xy ra ba con ng khc nhau:
S th tinh nm (stigma fertilization)
Phng php ny tng t nh phng php th tinh thng thng, nhng khc
vi phng php th tinh thng thng l ti phn v bu nhy c kh trng v th
tinh trong in vitro (hnh 5.10). S dng phng php th tinh nm thnh cng trn
Thuc L (Nicotiana tabacum), u (Pisum sativum), Bp (Zea mays)...
Hnh 5.10 S th tinh trong ng nghim
S th tinh bu nhu (placental fertilization)
Mt hoa nguyn c kh trng v mu cy l bu nhy (placental) vi non cha
th tinh c ct ra di iu kin knh hin vi v c cy trn mi trng dinh dng.
Ti phn c m ra di iu kin v trng, ht phn c t gn non. Sau giai on
ny, ht phn ny mm v pht trin trong s kt hp vi non. S th tinh ni sinh sn
c thc hin trn Caryophyllaceae, Gossypium v cy Bp (Zea mays).
S th tinh non tch ri khng c vch bu nhu (placenta)
S th tinh ca mt non c tch ri khng c vch bu nhu (placenta).
Phng php ny ging nh phng php trn v thi gian non c tch trong in vitro.
Phng php ny t t thnh cng, v n cc kh trong vic kch thch thnh lp phi
trong in vitro ca non th tinh.
S th tinh trong ng nghim c th c s dng cho cc trng hp sau:
S th phn theo bu nhy (placental) c th c thc hin khi cy khng tng
thch hon ton trong iu kin bn ngoi (in vivo).
Th phn cho c th c thc hin trong in vitro hoc ngay c trong in vivo. Cc
nghin cu cho thy c th to ra cc cy lai sau khi th tinh trong ng nghim v d non
cy Nicotiana alata vi ht phn Nicotiana tabacum. Lai lin gen (intergenic) cng c th
t c trong in vitro vi cc thnh vin khc trong h Caryophyllaceae, vi h ny
72
dng nh ht phn ny mm tt theo kiu th tinh placental hn th tinh nm nhy
(stigma fertilization).
S rng hoa hoc bu nhy (ovary) khng th trnh khi, trong trng hp nh
th, s th tinh nm c th hiu qu.
Nghin cu sinh l ca s th tinh
Ni chung, s hiu bit cc iu kin th tinh trong in vitro vn cn t. Tuy nhin,
mt s iu cn lu l:
- Ht phn v non phi ng tnh trng sinh l.
- Chn mi trng dinh dng cc k quan trng
- Khi kh trng hoa s dng cn nhu rt mn cm vi cc ha cht kh
trng.
- Th tinh theo phng php nm th tinh (stigma fertilization) khng ly i l
i trn hoa, v n cn thit cho s pht trin bu non.
- Nhit c th l yu t quyt nh. mt s loi, nhit thp khng thch
hp.
KHNG
Phng php chn lc
- Chn lc trn mi trng lng
Chn lc cc t bo thc vt c nhng c tnh khng vi iu kin mi trng v
khng vi ho cht hoc sinh hc. Cc th nghim chn lc t c mt s lng ln
t bo t bin. y l phng php tng i r tin v d p dng, c bit cho cc yu
t khng vi mi trng nh mn, acid, nhit cao, nhit thp v cc ha cht nh
thuc dit c hoc sinh vt nh bnh hi cy trng.
Hu ht phng php p dng thnh cng cho n nay u thc hin trn mi
trng lng bng cy treo (suspension culture). Mt th tch nh ca mi trng lng c
th cha hng triu t bo.
t c cc kch thc ng nht trong qu trnh chon lc, cc khi callus s
c lc qua li lc c nhng kch thc khc nhau. Li lc c kch thc ln (1000
m) cho cc cm t bo c kch thc ln i qua, trong khi li lc c kch thc nh s
gi li cc cm t bo c kch thc ln. Kch thc cng nh, cc t bo cng c c hi
tip xc vi tc nhn gy c v p lc chn lc s tng.
a b c
Hnh 5.11 Cc loi kiu lc ly i cc cm t bo c kch thc ln
a: phu lc bng li thp khng g hay bng plastic
b: phu lc lm bng ng chch nha c li lc
c: phu lc bng li thp khng g
73
Li lc c th t ch bng cc vt liu r tin nh ng chch nha. Kch thc
li lc c th thay i tu theo mc ch ca th nghim. Vt liu lm c th l vi, li
thp hay plastic. Tuy nhin, vt liu lm phu hoc li phi l vt liu chu nhit v c
th hp kh trng c.
a b c
Hnh 5.12 Cch lm li lc bng ng chch nha
a: ct ng chch nha
b: h nng trn n
c: p phn h nng ln mt li
Nng ngn cn (nng khng)
Nng ngn cn c s dng trong th nghim chn lc s tu thuc mn
cm ca t bo thc vt.
- Chn lc trn mi trng rn
Chn lc trong mi trng rn thng thc hin sau giai on nui cy callus
trong mi trng lng. Cc callus c nui cy trn mi trng lng c ly ra mt th
tch 1-2ml v tri trn b mt ca mi trng chn lc rn, sau dng que thy tinh
kh trng tri u. Nim da petri li bng giy parafilm. Sau 8 tun chuyn cc callus
no vn tip tc tng trng trn mi trng chn lc sang mi trng chn lc tip tc.
Hnh 5.13 Phng php chn lc trn mi trng rn. Ly th tch dung dch c
cha callus cho vo mi trng rn. Callus c tri ln trn b mt mi trng
74
S n nh ca cc callus c chn lc v nhn v tnh cc callus ny
S n nh ca tnh khng
To tc nhn gy t bin
Mc ch: to bin d bng x l ha cht hoc phng x
X l t bin bng vt l: X-rays, tia Gamma
X l t bin bng ha hc: EMS (Ethylmethylsulfonate), Sodiumazide
Hnh 5.14 Chn lc s khng hydroproline ca cy thuc l trn mi trng rn c
agar v nng hydroproline 10 mM sau 8 tun. Hnh bn tri khng c
hydroproline. Hnh bn phi c hydroproline 10 mM. Ch c vi callus khng (Dix,
1990)
Hnh 5.15 Chn lc s khng nhm ca cy c mi (Citrus) trn mi trng lng
nng 1000 M Al sau 8 tun. Hnh bn tri callus khng nhm (mi tn) trn mi
trng c nhm. Hnh bn phi ch c vi callus khng (mi tn), phn cn li khng
khng (Toan v ctv, 2004)
75
Ngun vt liu ban u (du hiu khng/khng khng)
Bin i thnh Callus
(Mi trng nui cy c 2,4-D)
Chn cc callus ri T bo trn
Nui cy trong mi trng lng
nh lng s t bo/ cm callus (thng 5-7 t bo)
S t bo/ml dung dch( 5.10
5
tb/ml)
Chuyn vo mi trng chn lc (rn / lng)
Chn lc cc callus/t bo trn sng
Bin i thnh cy cc callus c chn lc
To phi v tnh v bin i thnh cy con
Th nghim cc cy vi mi trng chn lc v ngoi t nhin
Hnh 5.16 Quy trnh thc hin s chn lc tnh khng
C th x l cc tc nhn gy t
bin
(2)Tia X,, hoc UV, Tc nhn
ha hc: MMS,EMS,nPMS
C th x l cc tc nhn gy t bin
(1) Tia X,, hoc UV hoc tc nhn ha
hc: MMS,EMS,nPMS
76
Cy quyn (Azalea) (Rhododendron simsii)
Chn hng ngn chi chiu x t c cy t bin
Hnh 5.17 Cc dng hoa ca cy quyn (Azalea) t bin tia X
X l t bin bng vt l: tia X, tia Gamma v d chn ging Chui t bin bng
tia Gamma
Phn sinh m chui Chiu x bng tia
gamma
Cy c ti sinh t phn sinh m chiu
x
Cy truyn trong ng nghim Cy c ti sinh Trng ngoi ng v tip tc chn
Ging Chui c chn t t
bin
Hnh 5.18 Quy trnh to t bin vi bc x tia gamma trn cy Chui
(Debergh,2003)
77
To t bin trn cy hoa hng
Nui cy hoa Hng trong
ng nghim
Chi c chiu x
Chi c chiu x c nhn
v cy truyn 5 ln
Ging nguyn thy mu
hng
t bin thnh hoa mu
t bin thnh hoa

mu trng
Hnh 5.17 Quy trnh to hoa Hng t bin (Debergh, 2003)
Phng iu khin
My to tia gamma
Chiu x mu thc vt
Hnh 5.19 Thit b x l bng phng tin ht nhn (Debergh, 2003)
5.6 NUI CY T BO TRN (PROTOPLAST CULTURE)
T bo trn thc vt l mt loi nguyn vt liu khi u cho nhiu k thut nhm
ti s thay i di truyn ca cc t bo thc vt v ton cy. T bo trn ca mt loi ny
c th c s dng dung hp vi t bo trn ca mt loi khc. V vy, k thut ny
to c hi cho vic to thnh cc loi lai mi (lai t bo soma). T bo trn cng l mt
vt liu s dng trong cng vic chuyn gen to ra mt loi mang mt gen mi.
T bo trn c hiu nh th no?
l t bo m cc nguyn sinh cht c tch ri do vch t bo b ly i bng
c hc hay bng enzyme.
Mt trong nhng s khc nhau gia t bo ng vt v thc vt l s cu to ca
vch t bo thc vt. Vch t bo thc vt to nn s cng chc cho t bo, c chc nng
78
bo v cho t bo v nh l mt hng ro chn ca t bo. Cc nh gii phu thc vt
cng nhn rng vch t bo thc vt bao gm vch s lp, vch hu lp v lp chung.
Hnh 5.20 Thnh phn cu to ca vi si cellulose (Taiz v Zeiger, 1998)
Vch s lp c thnh lp trong qu trnh pht trin trong khi vch hu lp c
tch ly sau khi t bo ngng sinh trng. Cu to ca vch s lp gm hai pha: mt pha
gm cc vi si cellulose v mt pha khc gm cc vt liu nn (matrix materials). Pha vi
si gn cht vo mt khi v nh hnh. Cellulose bao gm mt chui thng ca cc phn
t glucose c ni ha tr thnh lp mt cu trc si di t 0,25 - 5,0m t 40 - 70
chui c kt cht bi ni hydrogen gia nhm OH ca phn t ng hnh thnh
cu trc tinh th gi l vi si vi ng knh 3 nm. Cellulose rt n nh v hu nh
khng ha tan. Cc vi si ny c xp song song hay theo chiu ngang vi n to cho
vch t bo c cu trc v chc nng trong qu trnh sinh trng v pht trin. Ngoi
cellulose vt liu nn ca vch s lp c th chia thnh hai phn chnh: pectin v
hemicellulose.
Vch hu lp khc vch s lp v thnh phn v c tnh c hc. Vch hu lp
thng dy hn vch s lp. cy song t dip vch s lp bao gm 25 - 30% cellulose,
15 - 25% hemicellulose, 35% pectin v 5 - 10 % protein trn c s trng lng kh. Vch
t bo hu lp cha cellulose vi t l cao hn vch t bo s lp. Vch t bo hu lp
thng c t pectin hn do n kt hp t nc v m c hn vch s lp. Ngoi ra,
79
nu tt c polysaccharide ca vch t bo bao gm c cellulose bi tiu hy ht, phn cn
li l glycoprotein cc amino acid him nh hydroproline nhng thnh phn
glycoprotein ny c gi l extensine.
Lp chung l lp nm gia kt dnh cc t bo k cn li vi nhau. Thnh phn ch
yu ca lp chung l pectin. Pectin cng c th b ph v do cc enzyme.

(a) (b)
Hnh 5.21 Vch t bo thc t vt c quan st di knh hin vi in t (a) v t
bo trn (b)
T bo trn c th c s dng dung hp to ra mt s lai t bo m khng th
to ra bng lai to c in do khng tng thch.
T bo trn c tch ra c th lm ngun vt liu cho s du nhp nhn, lc lp, ty
th, plasmid, vi khun v virus.
T bo trn c th c s dng nghin cu s tng hp v phn hy vch.
T bo trn c th c nghin cu nh l h thng t bo n.
Dung dch ng trng : Chc nng ch yu ca vch t bo l s dng p sut
vch trn t bo trn ngn cn s hp thu nc d tha. Trc khi vch t bo c ly
i, t bo c ra trong mt dung dch ng trng nh mannitol hoc sorbitol 13 %
(cc ng alcohols ny t b bin dng trong t bo thc vt).
Tch c hc t bo trn
Ct c hc cc m trong iu kin co nguyn sinh v sau lm mt co nguyn
sinh dn n s ni rng v phng thch t bo trn t phn cui ca t bo b ct.
Trong thc hnh k thut ny kh thc hin v to ra cc t bo trn sng v c
kch thc dp. Tuy nhin, mt li ch ca phng php c hc l khng s dng enzyme
phn hy vch t bo v cc hiu qu c hi ca cc enzyme phn hy vch trong s bin
dng ca t bo trn b loi b.
80
Hnh 5.22 Phng php c hc tch t bo trn (Debergh, 2003)
Phng php ha hc l s dng enzyme phn hy vch
S dng cc enzyme dn n to nhiu t bo trn ng nht sau khi ly i cc mnh t
bo. T bo trn c ngun gc t nhiu ngun khc nhau: nh knh hoc vt liu ngoi
ng, cy c vi nhn ging, cc callus .
..
Hnh 5.23 Cc t bo trn c tch cn nguyn
81
Hnh 5.24 Cc bc ch yu trong vic tch t bo trn bng enzyme
(Debergh, 2003)
Cc yu cu v k thut c bn:
- Vt liu v trng
- Ra trong mt dung dch thm thu (osmoticum) thch hp
- Lm d dng s thm enzyme
- X l enzyme tun t hoc pha trn
- Lm tinh khit t bo trn c tch, (ly i cc enzyme v cc mnh t bo)
- Chuyn vo mi trng thch hp
Cc enzyme dng tch t bo trn
Tch t bo trn thnh cng bng cch s dng cellulase kt hp pectinase v
hemicellulase.
S chun b cc enzyme thng mi c ly t vi sinh vt v c th cha
ribonucleases, proteases v mt s enzyme c khc.
C hai phng php s dng cc enzyme phn hy vch t bo
Phng php pha trn: pectinase v cellulase hoc hemicellulase c p dng
ng thi.
Phng php tng bc: bao gm x l vi pectinase (lm ni lng t bo), theo
sau bi cellulase hoc hemicellulase.
Chun b cc enzyme thng mi
Onozuka R10, cellulase
Cellulysin, cellulase
Driselase, cellulase
Macerozyme R10, pectinase
Pectinase
Rhozyme HP150, hemicellulase
82
PATE (pectic acid acetyl transferase)
Phng php tiu biu tch t bo trn t t bo tht l:
- Ly l khe mnh v ra nc, kh trng v ra li nc v trng.
- Cc bc k tip c thc hin di iu kin v trng.
- Khi l c ra ln cui, biu b di c lt ra khi l hoc lm try vi ln.
- Ct l thnh cc mnh nh v chuyn vo trong dung dch enzyme c kh trng bng
lc.
- Bao li bng giy nhm ( qua m)
- Khuy nh nh vi ci kp phng thch t bo trn
Lm tinh khit t bo trn (lc, ly tm v ra)
Hnh 5.25 ng tim c s dng lc dung dch enzyme
Hnh 5.26 Lm tinh khit cc t bo trn c tch (Debergh, 2003)
Vic lm tinh khit cc t bo trn bng cch tch chit (ly cc enzyme v cc
mnh t bo) v truyn vo mi trng thch hp:
- T bo trn c lc xuyn qua mt li nylon (64 micrometer) ly i cc m cha
b tiu hy v cc mnh vch t bo.
83
- Chuyn cc phn lc c vo trong ng ly tm v quay + 75 g/5 pht.
Cc mnh (ni trn mt) c ly cn thn ra (t bo trn thnh lp mt vin trn
y ng).
- T bo trn c cn thn cy treo li trong mi trng cy (cng 13% mannitol), v
quy trnh c lp li ba ln.
- T bo trn c xc nh mt s v sc sng.
Trc khi cy t bo trn, iu cn thit phi th sc sng ca n bng
- Fluorescein Diacetate
Fluorescein Diacetate c th tch ly ch bn trong mng t bo ca t bo trn sng, c
th c pht hin vi knh hin vi hynh quang hoc UV
- Evans blue: t bo trn sng nguyn, loi nhum Evans blue. S khng
thm ca t bo Evans blue ch mt t bo sng.
- S vn ng vng hoc dng nguyn sinh cht c th l mt ch tiu o
lng sc sng.
Bng 1: Mt s mi trng (MT) c s dng trong nui cy t bo trn
Thnh phn b sung
mg/l
MT
VN
MT
EO
MT
2N
MT
BA1
MT
CC2
Mi trng c bn MS MS MS MS MS
Casein 200
Hydrolysate
Glutamine 800
L-serine 100
Glutathione 30
IAA 0,1
2,4-D 1
NAA 0,5 0,5 0,5
BA 0.5 0,625 0,625 5 0,25
2iP 0,625 0,625 0,25
Kinetin 0,625 0,625 0,25
Zeatin 0,625 0,625 0,25
Mannitol 100.000 50.000
Sucrose 30.000 15.000 15.000 15.000
Agarose 6.000 4.000 4.000 4.000
PH 5,7 5,7 5,7 5,7 5,7
Phng php kh
trng
lc
ni hp kh
trng
ni hp kh
trng
ni hp kh
trng
ni hp kh
trng
Ch thch MS l mi trng c bn Murashige v Skoog (1962).
Ngun (Pauls,2003)
84
Mi trng nui cy t bo trn cng l cc mi trng nui cy m thng dng.
Tuy nhin c cn lu mt s im sau
- Gim mc ammonium
- Tng mc Ca
2+
- Thm nhiu cht hu c nh vitamin, acid hu c, casein hydrolysate
- Cht n dnh thm thu nh mannitol hoc sorbitol)
- Auxin v cytokinin
Hiu qu to da ti ho (t bo trn thuc l) 5 x 10
4
t bo trn/cm
3
. Nng ca
t bo trn c th c xc nh bng Fuchs-Rosenthal c b sung lam m t bo
(haemocytometer) vi su 02 mm. C th chnh nng ca t bo trn bng cch pha
long nng sau khi ly tm.
Cch nui cy t bo trn: t bo trn c th c cy theo mt s cch bn di
Hnh 5.27 Cc hnh thc nui cy t bo trn pht trin (Pauls, 2003)
T bo trn ca t bo tht l bt u ti sinh mt vch t bo mi trong vi gi sau
khi tch, mc d n c th cn vi ngy hon tt s tng hp vch t bo. iu ny c
85
th quan st di knh hin vi bng cch s dng Calcofluor White. Thuc nhum trng
kt hp vi vch v hunh quang di nh sng xanh dng.
Mt khi t bo trn ti sinh mt vch mi, chng chu s phn chia v thnh lp
mt callus. Callus ny c th c cy truyn. Callus c th pht trin s to phi
(embryogenesis) hoc to c quan (organogenesis) sau 3-4 tun, trong cc iu kin cy
thch hp. Phi (embryoids) hoc c quan (organs) c th pht trin nh hu ht cc cy
con c nui cy.
Hnh 5.28 Cc bc nui cy t bo trn (Pauls, 2003)
86
t bo trn c
tch t trc h
dip la
S phn chia ca
mt t bo trn 4
ngy sau khi tch
T bo trn xut
pht t cm t bo
la trong agar
T bo trn xut
pht t cm t bo
la trong khi
agar ni trn mi
trng
T bo trn bin
i thnh cy con
trong ng nghim
Hnh 5.29 Nui cy t bo trn cy la (Debergh, 2003)
Thao tc di truyn l phng php di truyn phn t ca vic truyn vt liu di
truyn t t bo ny sang t bo khc trong in vitro, m khng xy ra s giao phi. C
nhiu kiu khc nhau trong thao tc di truyn.
- Lai t bo soma (somatic hydridization)
- Lai t bo cht (cytoplasmic hydridzation)
C hai c thc hin bng phng php dung hp t bo trn. Cc bc dung hp c
thc hin nh sau
- Tch mt lng ln t bo trn
- Kch thch s dung hp gia cc t bo trn cn sng
- Chn lc t bo dung hp
- Kch thch s phn chia t bo dung hp
- Bin i thnh cy t cc callus dung hp
- Xc nh c tnh cy dung hp lai
Mt s tc nhn kch thch t bo dung hp
- lipid:lysophosphatidyl choline, oleoyl glycerol, alcohol, benzyl alcohol
virus
- ion: sodium nitrate, Ca
2+
- Cc hn hp tan trong nc polyethylene glycol (PEG) HOCH
2
-(CH
2
-O-CH
2
)n-
CH
2
OH; polyvinyl alcohol
- Gy sc in
87
Hnh 5.30 S dung hp t bo trn (Pauls, 2003)
5.7 NUI CY PHI NH
5.7.1 Phi nh
Ngoi cc thnh vin ca h Orchidaceae, Podostemaceae v Trapaceae hu nh
khng c phi nh. Tt c cc cy b t u c phi nh. M phi nh nh v trong ht
non di iu kin bnh thng n hot ng nh ngun cung cp dinh dng cho phi
pht trin. N l phi tam bi do s dung hp mt nhn ca tinh trng ca ht phn v hai
nhn nh v trong dch phi (Frost v Soost, 1968). M phi nh th ng nht m, thiu
s chuyn ha c quan v mch.
5.7.2 Nui cy phi nh
cy phi nh trng thnh, tri c kh trng b mt phi nh v phi c
tch ra di iu kin v trng.
cy phi nh non, ton ht c kh trng v m phi nh cn thn tch di
iu kin v trng s dng knh lp ni. iu cn thit l phi tch phi nh khi phi.
Nu tch phi nh khng thnh cng s c s hin din ca m phi (m nh bi).
88
S dng cy phi nh
T nm 1965, cc chi tam bi v cy con t c t phi nh ca nhiu loi.
To cy khng ht t t bo tm bi l mt trong nhng phng cch ci thin ging cy
trng.
Tnh ton nng ca cc t bo tam bi ca phi nh to ra mt phng php
mi sn xut ra nhiu cy tam bi
M phi nh l ni d tr thc n v vn chuyn trong qu trnh pht trin ht v
ny mm ht. Do , n cung cp mt h thng th nghim hon ho trong nghin cu
sinh tng hp v bin dng ca sn phm t nhin ny.
Pauline Mooney v ctv, 1985 Trung tm nghin cu Kerikeri, New Zaeland
thnh cng trong vic nui cy phi nh ca cy Cam Dweet tangor. Phng php c
tm lc nh sau: Tch ly phi t ht non v t trong iu kin cy dn d hnh
thnh phi v tnh. Qu trnh hnh thnh phi v tnh, ni m thnh lp phi trong cy m
c hai bc ring bit. Trc ht, kch thch t bo m phi nh bin i thnh callus, ri
thnh tin phi. Giai on k l pht trin tin phi thnh phi qua cc giai on hnh cu,
tri tim v torpedo. Cui cng thnh cy con. Callus to phi xut hin 6 thng sau khi
cy ca ging a phi Dweet tangor. Hu ht phi nh c cy l tri non c thu
hoch 10-12 tun sau khi ra hoa, nhng mc d thnh cng ca phng php ny tng
i thp.
Hnh 5.31 Cy con pht sinh t cy phi nh
5.8 CNG NGH DI TRUYN (genetic engineering)
Thao tc di truyn thc vt c thc hnh cch y hng trm nm vi thnh
cng rt ln, cc nh to ging thc hin bng cch s dng cc qu trnh chn lc t
nhin. Phng php lai hu tnh ca cy trng c s dng du nhp v duy tr cc c
tnh mong mun v gia tng nng sut. Bt li ca s ti t hp hu tnh l c tnh ch
c th c du nhp thng qua cc loi tng thch, v khng th du nhp cc c tnh
mong mun v loi i mt s c tnh khng mong mun.
K thut ti t hp DNA c kh nng loi b cc tr ngi lin quan n di truyn
c in, cc nh di truyn xc nh v nhn cc gen c bit cho cc c tnh mong mun
v a cc c tnh ny vo trong cc ging cy trng. Cc gen c du nhp vo thc
vt cng c th c ngun gc t vi sinh vt hoc ng vt, trong khi di truyn c in
khng th thc hin c. S chuyn mt gen mi (gen ngoi) vo t bo thc vt c
gi l s chuyn np gen. S chuyn np gen c th gin tip hoc trc tip.
89
Agrobacterium tumefaciens
Hin tng khi u v vi khun Agrobacterium tumefaciens
Khi u (crown gall tumour) l mt loi bnh thc vt xut hin gn gc cy hoc
r. Bnh ny do vi khun Agrobacterium tumefaciens gy ra. Vi khun Agrobacterium
l mt loi vi khun sng trong t gram m, di chuyn, dng hnh que. Khi u gy ra do
nhim Agrobacterium tumefaciens hu ht cy song t dip, rt t cy n t dip.
Hnh 5.32 Khi u, mt loi bnh thc vt xut hin do vi khun
Agrobacterium tumefaciens gy ra.
Hnh 5.33 Mt khi u ln hnh thnh y cy hoa Hng (A). Mt lot khi u hnh
thnh nhnh cy Nho (B)
Hnh 5.34 Vi khun Agrobacterium tumefaciens nhn di
knh hin vi in t
S nhim vi khun Agrobacterium tumefaciens
Agrobacterium tumefaciens nhim vo cy t cc v tr vt thng. Ngi ta c th
chng minh d dng s nhim trong iu kin phng th nghim bng cch chng vi
90
khun ln gc cy C chua. Sau 5 tun, v tr chng hnh thnh khi u (hnh 5.35C). Trn
vng m phn sinh chung quanh h thng mch trung tm ca c Carrot cng pht trin
khi u sau hai tun chng (hnh 5. D).
Hnh 5.35 S nhim Agrobacterium tumefaciens trong phng th nghim
c tnh ca Agrobacterium
A. tumefaciens l vi khun c quan h gn vi vi khun Rhizobium c nh m
cy h u. Hin nay, ngi ta ghi nhn c phm vi cy ch ca A. tumefaciens bao
gm 331 ging v 643 loi.
c tnh ca Ti-Plasmid
Plasmid l mt dng DNA vng, nm ngoi nhim sc th ca vi khun c kh
nng t sao chp c lp trong t bo. Mi vi khun c th c hng trm plasmid. Plasmid
c th chuyn np t vi khun ny sang vi khun khc.
Hnh 5.36 Vi khun A. tumefaciens cha Ti Plasmid v nhim sc th
Vi khun Agrobacterium tumefaciens cha plasmid v nhim sc th. Khi u do vi
khun gy ra c th tch ri v nui cy trong iu kin in vitro. Cc khi u ny tip tc
sinh trng trong in vitro sau khi git cht vi khun, m khng thm bt c hormone thc
vt no. Ti sao c hin tng ny? Ngi ta cho rng, t bo thc vt nhn v c
gn n nh cc tc nhn gy khi u (tumor inducing principle = TIP) vo trong nhim sc
th ca n. Cc nghin cu chng minh rng TIP l mt phn plasmid ln (gi l Ti-
plasmid) ca vi khun. T-DNA m ha cc enzyme to ra hormone thc vt v cc
91
enzyme tng hp opine (opine l cc cht bin dng, amino acid hoc dn xut t
ng, do thc vt tng hp v c vi khun s dng).
Nghin cu cc kiu gen T-DNA ca vi khun, cc nh nghin cu chng minh
c l T-DNA cha hai kiu gen: gen gy ung th (oncogenic gen), m ho cc enzyme
cho s tng hp auxin v cytokinin, p ng cho s thnh lp khi u v cc gen m ha
cho s tng hp cc opine. Bn ngoi vng T-DNA l v tr cc gen lm thoi ha opine
v cc gen trong qu trnh chyn np T-DNA t vi khun sang t bo thc vt (Hooykaas
v Schilperoort, 1992; Zupan v Zambrysky, 1995). Mi dng vi khun A. tumefaciens c
th s dng mt trong ba loi opine khc nhau: octopine, nopaline v agropine. V d mt
trong cc kiu thng thng ca T
i
plasmid (c tm thy dng vi khun A.
tumefaciens C58; m ho s sn xut nopaline v agrocinopine A.
Hnh 5.37 Trnh by n gin ha cc vng chnh ca Ti plasmid ca A.tumefaciens.
T-DNA khi chn vo trong nhim sc th (DNA) ca cy ch, iu hnh s tng hp
Nopaline ri c s dng bi vi khun cho mc ch ring. T-DNA cng iu hnh
t bo thc vt phn chia theo cch khng kim sot c hnh thnh khi u.
C ch ca s nhim
Agrobacterium tumefaciens v cc loi gn gi chng A. rhizogenes, v A.vitris l
cc loi gy bnh thc vt bng cch chuyn DNA vo trong cy.
S b sung di truyn ca thc vt xy ra l kt qu ca s kt hp vo genome
thc vt ca mt on DNA c bit, gi l T-DNA, hoc DNA chuyn np, c ngun
gc t Ti plasmid ca vi khun. Biu hin ca nhng gene ny dn n s thnh lp
Opines; mt loi oligosaccharide c bit c vi khun A. tumefactiens s dng nh l
mt ngun carbon. Ton b qu trnh c iu ho v kim sot bi mt b gen c
bit nh l Vir gen, c hot ho bi s pht hin cc hn hp phenol khi thc vt b tn
thng.
Tn hiu thc vt cm ng
A. tumefactiens thc hin mt h thng hai thnh phn m thng dng nhiu
loi thc vt bao gm cc protein VirA v VirG (Tzfira v Citovsky, 2000). VirA hot
ng nh mt protein mn cm mng, trong khi VirG iu ho s p ng t bo cht
cho cc hn hp phenol t bo thc vt b thng v kch thch s hot ho ca tt c Vir
gen. VirG tng tc c bit vi hp vir (vir box); mt chui 12 cp base bo th nh v
trong chui kch thch ca tt c vir gen. VirG cng c th kch thch s biu hin ring
92
ca n, khi n c to ra t mRNA c trong hin din ln trong vng mt ca cc hn
hp phenol (Stachel v Zambryski, 1986). Cc hn hp c bit kch thch biu hin
Vir gen bao gm lignin, tin cht flavanoid v acetosyringone (Stachel v cng s, 1985).
Con ng to tn hiu c bt u khi phenol thc vt c tit ra t vt thng tng
tc vi VirA trc tip hoc gin tip, tu thuc vo tnh cht ca tn hiu b ngn chn
(Tzfira v Citovsky, 2000).
Cc Vir Gen v chc nng ca n
Vir Gen Chc nng
VirA,
Vir G
Cc hn hp cm nhn phenol t t bo thc vt b thng v kch thch s
biu cc virulence gen khc
VirD2
Enzyme Endonuclease; ct T-DNA ba phi bt u s tng hp si T (T-
strand)
Vir D1 Topiosomerase; gip Vir D2 nhn ra v ct trong chui ba 25bp
Vir D2
Gn ho tr vo v tr cui 5 ca si T (T-strand), v vy thnh lp phc cht
T-DNA (T-DNA Complex). Hng dn phc cht T-DNA ngang qua l nhn
Vir C Kt dnh vo vng tng tc kch thch s tng hp si T hiu qu cao
Vir E2
Kt dnh vo T-strand, bo v n khi s tn cng ca enzyme nuclease v
xen vo gia vi lipid thnh lp cc knh trong mng t bo thc vt m
phc cht T i qua
Vir E1 Hot ng nh mt chaperone lm n nh Vir E2 trong Agrobacterium
Vir B v
Vir D4
Lp t vo trong mt h thng tit m m rng mng t bo vi khun trong
v ngoi. Cn cho s i ra ca T-complex v Vir E2 vo trong t bo thc vt
S kt dnh v s xm nhp
A. tumefaciens di chuyn qua trung gian ca mt ui (flagella). C ch chnh xc
kim sot s hng ho ny cha c bit, nhng ngi ta cho rng s di chuyn hng
n ng amino acid tch lu chung quanh r cy vng r. Mt s dng vi khun cng
c thu ht vo cc hn hp c bit c phng thch t cy b thng nh
acetosyringone.
S kt dnh vo thc vt l mt qu trnh hai giai on, trc ht bao gm s bm
vo t bo thc vt, ri vi khun tng hp cc si cellulose gn cht chng vo trong b
mt t bo thc vt b thng. Mt s gen vi khun quy nh cho qu trnh ny c
xc nh, c tn l chvA, chvB, pscA v att. Khi lm t bin bt c mt trong cc gen
ny, vi khun c gen t bin khng th bm vo t bo thc vt.
Qu trnh chuyn np c th chia lm hai bc; bc t bo vi khun v bc t
bo thc vt (Tinland, 1996).
93
Hnh 5.38 Qu trnh xm nhp ca vi khun (Agrobacterium tumefaciens) gy khi u
trn cy
Bc t bo vi khun
Bc t bo vi khun bao gm tt c qu trnh dn n s sn xut v i ra ca
phc cht T-DNA (T-DNA complex), cha thng tin cn thit gy ra s nhim. Phc cht
T c to ra t nucleoprotein; mt si n T-DNA c bao ph bi VirE2 protein, vi
VirD2 protein gn v tr cui 5 (Howard v Citovsky, 1990). VirD2 hot ng nh mt
enzyme endonuclease c bit nhn ra, ct ba bn phi v bn tri ca T-DNA vi
VirD1. VirE2 hot ng nh mt protein v bao T-DNA, v th bo v n khi s tn
cng ca enzyme nuclease khi n i vo trong t bo thc vt (Tinland, 1996). Cc VirD2
v VirE2 protein c xem nh l c vai tr trong vic gn T-DNA vo nhn t bo, khi
n vo trong t bo thc vt. Mt khi T-DNA c bao ph n s c bo v v
khng b phn hu khi bn ngoi t bo vi khun, n c cp nh T phc cht (T-
complex).
94
Hnh 5.39 S xm nhp ca phc cht T (T-complex) vo t bo thc vt
Phc cht T qui nh mt h thng ng i ra ring a n xuyn qua mng t
bo vi khun, mng t bo thc vt v vo trong t bo cht thc vt. S vn chuyn ca
T phc cht ra khi vi khun xy ra thng qua mt c ch iu tit kiu IV (hnh 5.39),
bao gm mt pilus si v mt phc cht vn chuyn chuyn ch cc cht xuyn qua mng
t bo (Salmond, 1994).
A. tumefaciens, h thng tit kiu IV c tp hp t protein m ho bi VirD4
gen v VirB operon (Tzfira v Citovsky, 2000). Mi mt VirB protein c m ho bi
VirB operon, tt c ng vai tr trong s vn chuyn ca phc cht T i xuyn qua mng.
VirB1 bt u kt hp v VirB2 l protein cu trc chnh trong pilus. Pilus c ngh
bi Zupan v cng s (1998) cm ng lin h vi mt t bo thc vt.
95
5
.
4
0

C

c
h
a

s

n
h
i
m
96
Bc t bo thc vt
Ngi ta tin rng, phc cht T-DNA i xuyn qua vo trong nhn t bo thc vt
bng hp thu nhn hot ng, khi kch thc ca phc cht T-DNA (12,6 nm ng knh)
(Citovsky v cng s, 1997) vt qu kch thc ca ng knh ca l nhn (9nm)
(Forbes, 1992), mc d kch thc ca l nhn c th gia tng n 23nm (Forbes, 1992).
Khng nh cc yu t di truyn khc nh retroviruse, T-DNA khng m ho cc chc
nng cho s vn chuyn v kt hp, do chui DNA khng c bit. Chnh c tnh ny
ca T-DNA lm cho n rt hu ch; bt c chui DNA chn gia cc ranh gii T-DNA s
c chuyn vo genom thc vt, cho php sn sinh hiu qu cy chuyn gen.
S kt hp vo trong genom t bo thc vt xy ra c bn ngu nhin: Mt qu
trnh c tin l c kim sot bi yu t cy ch. Nh kt qu nghin cu cy
Arabidopsis t bin, mt yu t sao chp c bn VIP1 c xc nh xut hin lm
d dng s xm nhp ca phc cht T vo trong nhn. Mt protein th hai VIP2 hot
ng nhm ti phc cht T sang DNA hot tnh sao chp. Mt khi c kt hp vo trong
genom thc vt, gen sinh tng hp auxin v cytokinin s c biu hin, dn n s pht
trin khng kim sot v sinh trng ca khi u. Gen sinh tng hp opine cng c biu
hin v cc opine ny c s dng bi khi u nh l ngun carbon duy nht, lm cho n
hu nh c lp trong cy.
Cc vector T-DNA da trn mt s nguyn tc quan trng:
- Ranh gii 25 cp base l yu cu duy nht nhn bit T-DNA gia. iu ny
c vai tr quan trng cho s pht trin ca vector chuyn gen. Qu tht tt c DNA gia
ranh gii c th b trit tiu (bao gm gen gy khi u) v c thay th vi DNA c
chn (m ta mun du nhp vo cy). Tri vi cc phng php chuyn gen khc, iu tiu
biu cho h thng ny l DNA a vo c nh ngha r. i khi c T-DNA ngn
hn c du nhp hoc lp li T-DNA. Thng c s thng nht ch mt locus, nhng
s thng nht 2 hay nhiu locus cng c th xy ra.
- Protein virulenee c th nhn ra T-DNA Cis (trn cng plasmid c s dng
cc vector cy kt hp) hoc Trans ( vector nh phn). Protein nhn bit T-DNA v
chuyn n vo trong nhn t bo thc vt c m ha bi gen Virulence (vir). Cc gen
ny tr nn kch thch do cc phn t phng thch t vt thng t bo (v d
acetosyringone).
VirA l protein mng t bo nhn tn hiu ny truyn n sang VirG, mt protein
hot ha s sao chp tt c gene Vir. Ranh gii c cng nhn bi mt phc cht ca
VirD1, VirD2 v protein VirD2 gn vo v tr cui 5
'
(ranh gii bn phi) ca DNA b ct.
Si T-DNA y sau phng thch. Si T-strand c bo v bi VirE2 protein, c
chuyn sang t bo thc vt v nhn t bo, c hng dn bi VirD2 protein. , T-
DNA ha nhp vo trong nhim sc th thc vt. S chen vo n nh ny l ngu nhin,
ngoi tr dng nh l ni thch hp cho cc vng hot ng sao chp. Ngi ta
chng minh l s chen vo T-DNA ny n nh v c di truyn nh gen bnh thng
theo lut Mendel.
- Gen th khm (Chimeric gene) trn c s cc gen chng li khng sinh c th
c s dng chn lc cc t bo thc vt bin i
- Mt cht khng sinh khc c th c s dng git Agrobacterium v sau
cc t bo thc vt bin i c th ti sinh thnh cy chuyn gen.
97
Trong thc t, vi m cy c chung vi vi khun Agrobacterium tumefaciens
trong 48 gi (cng nui cy). Sau , vi khun c ra sch v cc t bo thc vt c
trn mi trng chn lc c cha khng sinh git vi khun v cc t bo khng c
bin i. Cng nui cy c th c thc hin bi protoplast ca cy, nhng thng s
dng cc nhnh l v r.
Phng php u tin c s dng bin i cy s dng Agrobacterium c
gi l cng kt hp (co-integration).
Gen c chn vo trong plasmid cha mt on T-DNA. Plasmid ny c gi
l vector con thoi ri bin i sang E. Coli. Thnh phn bin i c chn trn c s s
khng vi cht khng sinh c m ha bi gen cha trong plasmid. Plasmid sau
c chuyn t E. Coli sang Agrobacterium bi qu trnh c gi l kt hp. Mt khi i
vo trong Agrobacterium, vector con thoi ha hp chung vo trong Ti plasmid bng
phng tin ti t hp tng ng ca T-DNA trn c hai plasmid. Agrobacterium cha
thnh phn ti t hp Ti plasmid c chn lc v s dng nhim vo t bo thc vt.
Cc gen nh du cha Ti plasmid m ha cho s khng li cht khng sinh v cc c
tnh sinh l khc c s dng chn cc cy c bin i.

Hnh 5.41 Phng php cng kt hp
98
H thng nh phn (Binary system)
Hin nay, phng php chun s dng Agrobacterium chuyn gen vo thc vt
l h thng nh phn. Vng T-DNA mt vect nh phn b loi v mt gen khng
Kanamicine c chn vo cho php chn lc cy. Mt Ti plasmid sa i gi l Ti
plasmid helper b thay i n khng cha T-DNA nhng vn mang gen cn thit
cho s nhim. Ti plasmid helper lm d dng s bin i ca DNA t vect nh phn vo
trong t bo thc vt. Thnh phn bin i c chn lc bi s khng cht khng sinh
Cc k thut mi h tr cho A.tumefaciens
Cc k thut mi cho s bin i cy ang pht trin n nh. Mt k thut xm
nhp chn khng pht trin gn y c s dng gia tng hiu qu bin i. Thc vt
v Agrobacterium c t p sut khng kh thp, v vy gip s xm nhp
Agrobacterium vo trong m cy. C ch xm nhp t nhin ca virus thc vt v
transposon (yu t bin i DNA = transposable DNA elements) cng c khai thc
to ra GMO (genetically modified organisms=sinh vt b bin i di truyn)
DNA c th c a vo t bo bng phng tin vt l hn l sinh hc. Cc k
thut ny bao gm xung in (electroporation), sng bn gen (gene guns), vi v a tim
(macro- and micro-injection). Cc k thut mi pht trin mt cch n nh lm cho s
bin i cy c hiu qu hn.
Xung in (Electroporation)
K thut xung in i hi t bo thc vt c nui cy di dng t bo trn
(protoplast). Plasmid DNA cha gen c chn c thm vo trong dung dch nui cy
t bo trn v s trn ln c cho mt dao ng xung in 200 n 600 Volt/cm
2
. Dao
ng xung in dn n thnh lp cc l trong mng protoplast. DNA c th di chuyn
xuyn qua cc l ny vo trong t bo, xy ra ti t hp vi DNA. T bo trn c
sinh trng trong iu kin nui cy m trong mt hoc hai tun cho chng n nh trc
khi chn thnh phn bin i.
c tnh ca xung in mng t bo
Mng t bo thc vt phc v nh chc nng sng ca qu trnh hp thu cc cht
vo v ra khi mng. Mng t bo bao gm mt lng ln cc lipid k nc. Tuy nhin,
cc phn t lipid c th b ph v bi mt in trng mnh lm gia tng dn in v
s khuch tn cc cht ngang qua mng. Hiu qu ny dn n s thnh lp cc l nc
trong mng m thay i th nng in xuyn qua mng. Xung in ca mng t bo thc
vt c s dng nh mt dng c a DNA vo trong t bo thng qua cc l mng
(Tsong, 1991). Xung in cng l c ch c bn ca s tn thng m khi b chm n
in th cao (Lee, 1994).
Xung din xy ra lm thay i cc phn t lipid ca mng i thnh lp cc l
a nc trong mng. S phn b ca cc l (c kch thc ln s lng) xc nh c tnh
in ca mng t bo. Cc thay i v ng knh l c th b nh hng ca lc cng b
mt trn vch ca l, s khuch tn cc phn t nc vo v ra khi l mt lc in
trng.
99
Dng c to xung in
.
Hnh 5.42 M hnh nguyn tc n gin ca xung in.
Mt cc cha dung dch c t bo trn, sau thm vo mt dung dch cha cc
on DNA cha gen quan tm. DNA c th bao gm mt reporter gen nh DNA
chloramphenicol acetyltransferase (CAT). Hai in cc c gn vo h thng in bao
in tr t in v ngun cung cp in. T in c tch in bng cch ng cng tc
phi. Khi t in tch in, xung in c to ra trong dung dch nui cy bng
cch ng cng tc tri.
Xung in lm h tm thi mng v lm di chuyn DNA vo trong t bo. DNA
a vo s kt hp vi nhim sc th ca cy ch
Sng bn gen (Gene gun)
y l phng php bao gm vic bn cc vin n cc nh c o bn ngoi cc
DNA cha gen cn chuyn np vo t bo thc vt. Ht kim loi bao gm hai loi: ht
bng vng v ht bng tungsten. Cc ht tungsten c kch thc 1m ng knh v c
ph DNA cn chuyn. Cc ht c bn ra t sng c vn tc 430 m/giy. Cc t bo trn
ng bn trc tip bi tiu dit nhng cc t bo ngoi vi th c th sng st.
.
a b c
Hnh 5.43 Mt kiu sng bn gen (a) v cc vin n tungsten (b); DNA c bao
bn ngoi vin n (c)
100
Hnh 5.44 Cu to v cch vn hnh sng bn gen
Tt c cc thao tc thc hin chuyn np gen bng sng c thc hin trong iu
kin v trng.
Hnh 5.45 Sng bn gen c t trong t cy
Mt s t bo thc vt b cht, nhng mt s t bo khc vn cn nguyn DNA bao
bn ngoi vin n xm nhp vo trong. DNA sau s kt hp vi DNA ca t bo ch.
Cui cng, cc t bo c chuyn np gen s c bin i thnh cy con.
101
So snh Agrobacterium vi sng bn gen
Agrobacterium ch hiu qu trn cy hai l mm v mt s cy mt l mm. Sng
bn gen hiu qu trn cy hai l mm v mt l mm.
Agrobacterium cn 3-5 thng hon tt; sng bn gen cn thi gian lu hn
hon tt khong 5-7 thng.
Agrobacterium chuyn DNA ch vo trong nhn ca t bo thc vt; sng bn gen
chuyn DNA vo bt c phn no ca t bo thc vt nh lc lp, ty th, nhn.
Phng php a, vi tim (Macro- and micro-injection):
a tim (Macroinjection)
y l mt phng php tim DNA tng t mt ging cho vo trong phi ca mt
ging nhn. Mt dung dch ca gen khng kanamycin (trong plasmid) c chn vo
trong khong trng bao hoa ang pht trin
Vi tim (Microinjection)
Vi tim l mt trong cc k thut chnh xc nht a cc i phn t vo trong
khong trng gian bo ca nhng t bo sng. Cc vi pipette (micropipette) v knh hin
vi c s dng a DNA vo trong t bo trn. Dung dch DNA c a vo trong
nhn bi ng pipette cc nh.
Hnh 5.46 Phng php vi tim
102
H
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5
.
4
7

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p
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n
g

p
h
p

c
h
u
y
n

g
e
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t
r
o
n
g

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u
i

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y

m
103
Trc ht, s chuyn gen thnh cng ca DNA ngoi vo mt t bo thc vt c
thy bi kiu to khi u.
T-DNA ca Agrobacterium tumefaciens thng mang cc gen to ra auxin v
cytokinin, khi t bo thc vt nhn cc DNA ny s sinh trng thnh cc callus khng
chuyn ha. Sau khi nhim vi A.tumefaciens, cc t bo thc vt c chuyn gen nhim
c xc nh bng s sinh trng khng kim sot nh l mt khi u. Khi u ny d
dng c s dng ti to cy con, nhng n khng thch hp, khi cn ti sinh cy
bnh thng. Do , cc marker chn lc khc phi c xc nh.
T bo thc vt mn cm vi nhiu khng sinh ngn cn ribo th ca vi khun.
Bng cch cung cp cc gen khng hat ha khng sinh tng ng, cc marker s dng
theo nguyn tc ny trn c s l gen phosphostranferase neomycin ca vi khun m lm
gim c tnh ca nhiu khng sinh amino glycoside bng cch thm nhm phosphate.
Gen ny lm khng li kanamycin, neomycin, gentamycin v G418. biu hin gen,
gen ny c dung hp vo promoter nopaline synthase.
Mt Marker chn lc thng c s dng khc l gen phosphinothricin
acetyltransferase t Streptomyces hygroscopius. iu ny lm mt hot tnh
phosphinothricine hoc gluphosinate, cc cht ny c s dng nh thuc dit c. Ngoi
ra, trong chn lc in vitro i vi cc cy m biu hin enzyme ny, s chn lc cng c
th c thc hin trn t trng cy bng cch phun chng vi thuc dit c.
Cc v d khc ca cc marker chn lc l: hygromycine phophotransferase, khng
streptomycin v glyphosate
Gen Reporter
Gen reporter l cc chui nucleic acid m ha cc protein. Chng c s dng
xc nh s thnh cng ca chuyn gen thc vt. Danh sch di y l cc gen reporter
thng dng
Protein Hot ng v s o lng
CAT (chloramphenicol cetyltransferase) chuyn np nhm acetyl phng x n
chloramphenicol, pht hin bng
phng php sc k v phng x t ghi
GAL (-galactosidase) Thu phn galactosides khng mu
thnh sn phm c mu
GUS (-glucuronidase) Thy phn glucuronides khng mu
thnh sn phm c mu
LUC (luciferase) Oxy ha luciferin, pht x photon
GFP (green fluorescent protein) Fluoresces trn s pht x vi tia t
ngoi (UV)
Gen reporter c th gn vo cc chui khc protein reporter c thc hin hoc
protein reporter c dung hp vi protein khc.
Vng iu ha DNA c th c kho st bng cch t cc chui ny vo trong
phn pha trn ca cu trc plasmid hoc pha trc ca mt reporter gen. Mc ca
biu hin gen sau c o lng nh chc nng ca chui DNA. Gen E. Coli cho
enzyme B-glucuronidase (GUS) c bit hu ch nh l reporter gen. Enzyme ny s
104
dng glucuronide nh l cht nn. Li ch ca n nh reporter gen i vi thc vt m
thc vt c cc mc khng th pht hin thc s ca GUS. Khi t bo thc vt biu
hin B-glucuronidase c vi X-glucuronide (X-gluc) mt mu xanh c to ra c
th c pht hin ha cht nhum m (histochemically).
S chn lc cho cc cy bin i
Cc t bo thc vt nhn thnh cng T-DNA t Agrobacterium hoc t chuyn
gen t sng bn gen cn c xc nh li. C hai cch c bn:
a/. Sc sng ca cc cy bin i trn mi trng chn lc. Mi trng sinh trng cha
mt cht khng sinh nh hygromycin hoc mt cht dit c nh RoundUp (glyphosate)
tng ng vi gen marker chn lc t trong gen c chuyn. Ch nhng t bo c
bin i di truyn vi gen c chuyn c th to ra protein (thng l mt enzyme) m
cho s khng t bo i vi thuc khng sinh hoc thuc c. Tt c t bo khc (hoc cy
con) cht mt cch n gin.
Cc hnh nh di y cho thy cy cht hon ton trng do khng sinh git cht lc lp
ca chng, lc lp pht trin t vi khun v v th rt mn cm vi khng sinh.

Hnh 5.48 Cy c chuyn gen da petri trn cng khng vi thuc dit c
RoundUp (glyphosate). Cy khng chuyn gen da petri bn di th cht hon
ton
b/ S biu hin ca gen reporter. Trong chin lc ny gen marker lm cy c mu sc
di cc iu kin c bit, thng thng nht l mu xanh l cy v mu xanh dng:
105

Hnh 5.49 Mu xanh l cy t c bi mt gen reporter c gi l GFP (Green
Fluorescent Protein).

Hnh 5.50 Mu xanh dng t c khi s dng mt reporter gi l GUS.
Phc hi cy chuyn gen
Mt khi to c cy chuyn gen, mt s trc nghim cn c thc hin lm
r gen mi c kt hp v hot ng ng.
Xc nh cy cha marker: trng n trn mi trng chn lc
Xc nh tan b gen c chuyn c v kt hp ng:
Phn tch PCR s dng primer ring cho tng b gen c chuyn
Chc chn cy c sc sng
- Quan st cy c cc c tnh mong mun: Cy c biu hin kiu hnh mi hay khng?
- Trng cy n khi trng thnh.
- Xc nh kh nng sinh sn.
- Xc nh chc chn cy con cng cha gen c chuyn
Cc k thut pht hin
Mt khi mt gen c nhn v bin i vo trong t bo ch s hp nht gen phi c
xc nhn trong cy ch. Gen c nhn c th c s dng phn tch biu hin gen
v t chc gen trong b gen DNA. iu ny c th thc hin bng cch s dng Southern
blotting v Northern blotting
Southern blotting l mt phng php mnh c s dng nghin cu cu trc
gen v xc nhn s hp nht ca gen trong cy chuyn gen. DNA ca cn xc nh c
ct bng mt hoc vi enzyme hn ch. Cc on DNA sau c phn tch in di
agaro. DNA trong gel c lm bin tnh ha hc thnh mt si n (a single stranded
form), ri chuyn vo mng nitrocellulose hoc s dng hot tnh mao dn.
Phn t DNA c gn vo mng thnh lp mt bn sao ca DNA trn gel. Mng
c th c thm d bi mt gen c nhn hoc oligonucleotide (DNA c tng hp).
Mu d (probe) c hon li thnh mt dng n thng qua s bin tnh nhit hoc ha
cht. ssDNA mu d s lai vi cc phn t ring cha mt chui b sung. Mu d khng
106
kt dnh s c ra khi mng v mu d kt dnh sau c pht hin bng phng
php mu phng xa hoc khng phng x.
Northern blotting ging phng php Southern blotting, nhng RNA c s
dng. mRNA c trch t thc vt v cphn tch theo kch thc in di
agarose. mRNA l si n c chuyn vo trong mt mng cng cch nh
DNA.. Northern blot sau c thm d bng gen c nhn bin tnh (the denatured
cloned gene) hoc cDNA (K thut ny hu ch trong vic pht hin hoc l kch thc
ca cDNA c nhn di).
Cc v d ca cy c thc hin cng ngh di truyn c s dng thng mi
S dng k thut ti t hp DNA c nhiu li ch trong con ng ci thin i
sng c bn nu n c p dng mt cch hp l. S mong i u tin ca cc k
thut mi ny khng thc t do s hiu bit qu t v gen. Khi chng ta nghin cu nhiu
v chc nng iu ha ca gen v vy s p dng trong nng nghip tr nn r rng hn.
Mt trong cc v d u tin mt cy p dng k ngh di truyn c thng mi
ha l cy C chua FLAVRSAVR c pht trin bi Calgene Inc., ca Davis
California. Ngi ta yu cu cy C chua FLAVRSAVR cn thn di, khi chn vi mi
thm y trc khi thu hoch n th trng m khng b dp; thu hoch chng vn
cn xanh v khi chn c kch thch kh ethylene. C chua FLAVRSAVR vn cn cng
chc sau khi thu hoch bi v thng qua k thut di truyn, sn lng ca enzyme
polygalacturonase (PG) b ngn cn. PG ph v pectin c tm thy vch t bo gy
ra s chn c chua v lm mm tri. PG b ngn cn bng cch a vo mt bn sao
ngc ca gen PG a vo trong cy gi l mt antisense gen.. V th, cy C chua
FLAVRSAVR c mc thp PG cho php chng lm mm chm hn nhiu.
Mt phng php khc ci thin ging cy trng l p dng ha cht trong qun
l cy trng. Ha cht dit c c s dng loi s cnh tranh c di, lm ti u nng
sut cy trng. Hin nay, cc nng dn trng u nnh p dng thuc dit c chm phn
hy. Tuy nhin thuc dit c cng lm tn hi n cc cy khng phi l i tng
dit. Thng qua k thut di truyn, mt ging u nnh khng vi thuc dit c Roundup
c pht trin. Roundup th khng bn, khng th di chuyn t do trong t v phn
hy thnh cc hn hp v hi nhanh chng.
Mt s cy chuyn gen thng phm (Birch, 1997)
Cy trng v ngy phng
thch
Tn Cng Ty c tnh mi
C chua (1994) Flavr Savr Calgene Chn c mi thm
C chua (1995) Zeneca Bn khi di dng bt nho
Bng vi,
Khoai ty, Bp
(1996-97)
Bollgard
NewLeaf
YieldGuard
Monsanto
C cha c cht t
Bacillus thuringiensis
khng su
u nnh, Nho, Bng vi
(1995-96)
Roundup
Ready
Monsanto
Khng thuc c
Glyphosate
107
NUI CY M V T BO
Cy b nhim virus, mycoplasma, vi khun v nm c th rt kh tr. Khng th
loi b cc cy nhim bnh bng cch s dng ha cht. C th dit virus bng cch
s dng cc ch phm t tin nh Vira zole (ribavirine) v vidarabine (khng bin
dng) trong mi trng dinh dng. mt s trng hp s dng, Virazole s to ra
cc cy sch bnh. S dng cht khng sinh cho vo mi trng khng c kt qu,
virus cng c th truyn qua nhiu th h khi nhn ging .
6.1.1.1 X l nhit:
X l nhit l cch hiu qu lm bt ng mt s virus v mycoplasma. Mt
s trng hp x l nhit i khi khng hiu qu, c th do bn thn thc vt qu mn
cm vi nhit. Nhit v thi gian x l thch hp c chn cy (cnh hoc
nhnh) sng trong khi virus b bt ng. X l nhit tht s hiu qu trn cy n tri,
Ma, Khoai M. Cc cy Nho sch bnh c th t c bng cch x l chi trong
ng nghim trong 21 ngy 35
o
C (George, 1996)
nh sinh trng l ni c vn tc phn chia t bo nhanh hn vn tc xm
nhim ca virus. V vy, t bo v tr ny hu nh sch virus. Holmes (1948,1955)
thnh cng trong cy nh sinh trng to ra cc cy hoa Thc Dc sch virus.
Morel v Martin (1955) thnh cng trong vic loi b virus ca cy Khoai Ty b
nhim. Do kch thc ca nh sinh trng rt nh, vic phn lp phi c thc hin
di knh phng i. Hnh 6.1 trnh by cu trc ca nh sinh trng.
a b
Hnh 6.1 Cu trc nh sinh trng: a: cu trc gii phu; b: cu trc
thc t
108
Hnh 6.2 Quy trnh sn xut cy sch virus bng nui cy nh sinh trng
(Walkey,1980)
Cy nh sinh trng c th hoc khng th kt hp vi tr liu do nhit (>35
oC
)
ca cy m v bao gm cc tc nhn khng virus ha hc (v d: virasol) trong mi
trng. nh sinh trng cng nh cng c c hi loi virus hoc vi khun. Quy trnh
to cy sch bnh c trnh by hnh 6.2
109
Hnh 6.3 Quy trnh phc hi cy nhim virus bng cch cy phn sinh m chi
(George, 1996)
Hnh 6.4 Quy trnh test vi khun v virus ca ging Khoai ty, sau l vi nhn
ging (Kristensen, 1984)
110
6.1.1.3 Vi ghp
Vi ghp l ghp mt chi hoc phn sinh m chi rt nh ln trn gc ghp cy
con. Vi ghp c th thc hin trong in vitro hay in vivo
Navarro (1988) ch ra 4 li ch ca k thut vi ghp
t c cc cy sch virus v cc bnh nh virus
Mt phng php loi virus v cc sinh vt nh virus nhim vo cy
Gip nghin cu s khng tng thch gia chi (mm) ghp v gc ghp, cng
nh nghin cu kha cnh m hc v sinh l hc ca qu trnh ghp
Mt phng php c s ri ro thp nht trong vic nhp khu vt liu thng qua
kim dch
Hnh 6.5 Cc v tr thch hp thc hin vi ghp trn cy c mi (Citrus)
( Navarro v cng s, 1975)
Hnh 6.6 Hai phng php vi ghp vi mm (chi) ghp c kch thc ln
(Pliego-Alfaro v Murashige, 1987; Navarro v Juarez, 1977)
111
CC HNH BN DI L CC BC THC HNH VI GHP TRN CY
C MI
1) Gieo ht cy c mi
lm gc
ghp vo ng nghim
ny
mm thnh cy con
2) Ly cy con ra khi ng
nghim
trong iu kin v trng
3) Chun b ct b t
dip
4) Ct b t dip
5) Ct b r ca cy con 6) Ct b phn trc thng
dip
v rch ngang phn thn
7) Rch dc phn thn
to vt
ct ch T ngc
8) Chn chi non phn
lp nh
sinh trng
112
9) Ct ly chi non
10) Ct b l bao quanh
nh sinh
trng
11) Phn lp nh sinh
trng
12) nh sinh trng c
phn lp
c kch thc 0,1-0,3 mm
13) t nh sinh trng
vo v
tr ghp
14) nh sinh trng
c t
vo v tr ghp (mi tn)
6.2 CY T BO, CALLUS
Nui cy t bo thc vt thng di dng cm t bo. Vic nui cy t bo
n thc vt ch thc hin c di hnh thc cc t bo giao t (ht phn, trng)
hoc t bo trn. Nui cy callus l s kch thch t bo pht trin v trt t di dng
callus. Cc callus c th cha rt nhiu t bo, nhng cng c th cha 6-7 t bo.
Nui cy to callus c s dng cho nhiu mc ch khc nhau nh: nhn ging,
chn to ging, to cht bin dng th cp
6.2.1 To Callus
nh ngha callus: Callus bao gm mt khi v nh hnh ca cc t bo nhu m c
vch mng c sp xp lng lo
S to callus
Trong t nhin, vt thng c th dn n s thnh lp ca callus ni b ct
hoc phn thn hay r b tn hi. S thnh lp callus c quan st hu ht cc nhm
thc vt sng.
113
Hnh 6.7 Callus vt thng trn thn cy Erythrina
Callus pht
sinh t m l
Callus pht sinh
t m mch
Callus pht
sinh t m r
Callus pht sinh
t m phi
Hnh 6.8 Cc ngun gc hnh thnh callus t mu cy
S hnh thnh callus vt thng l s p ng bo v ca thc vt; s phn
chia t bo c hot ha do cc thay i trong s cn bng ni sinh ca hormone thc
vt. S tn thng c hc, s xm nhp ca vi khun v s tn cng ca cn trng c
th dn n vic to callus.
Ngun gc ca callus:
Callus pht sinh t cc t bo sinh si ca m cha m. Trong nui cy callus
c hnh thnh t mt mnh nh ca mu cy trn mi trng c cung cp cht
sinh trng di iu kin v trng. Hormone thay i bin dng ca t bo t th
ng sang hot ng.
S hnh thnh callus
S hnh thnh callus trong in vitro c th chia lm ba giai on: kch thch, phn
chia v chuyn ha
114
S kch thch
Hu ht cc loi thc vt, callus c th c thit lp
tng i d dng; song t dip (cy hai l mm), n
t dip (cy mt l mm), kha t, dng x, ru. M ca
nhiu c quan c th c tim nng phn chia v sinh si
trn mi trng nui cy thch hp; Tuy nhin, mt s m
th chu s phn chia t bo nhanh hn cc m khc
S phn chia t bo
S phn chia t bo hot ng, cc t bo bin i sang
tnh trng m phn sinh.
S chuyn ha
S xut hin v chuyn ha t bo v s biu hin ca cc
con ng bin dng th cp.
Hnh 6.9 S hnh thnh callus trong in vitro ba giai on: dn d, phn chia v
chuyn ha t bo
115
Cc kiu callus:
Mt s callus c th b lignin ha nng n v lm cng cu trc trong khi cc
callus khc c th b v thnh cc mnh nh gi l callus ri rc. Callus c th c s
xut hin mu sc khc nhau: vng, trng,
Callus ri rc Callus cng Callus tit nhiu cht
ng
Callus to r
Callus cha nhiu
anthocyanin(Oxalis)
Callus trng
(Oxalis)
Callus xanh l cy
(Gloriosa)
Callus vng
(Gloriosa)
Hnh 6.10 Cc kiu callus
S chuyn ha callus
S chuyn ha t bo xut hin thc vt han chnh v m nui cy tr thnh
b mt chuyn ha khi cc t bo chuyn ha pht sinh cc t bo mi cha chuyn
ha vi d callus. Trong s mt chuyn ha sn phm d tr bin mt v m phn sinh
mi pht sinh to ra cc t bo nhu m khng chuyn ha, khng c bt c trt t cu
trc no. Thiu t chc bn khi callus sinh si, mc d khi s sinh trng tin trin
mt s loi t bo chuyn ha c th mt ln na c thnh lp ngu nhin. Callus
xut hin khng c kiu khun mu t chc, c nh v cc trung tm hot ng
phn sinh m thng hin din v vng tng tng s ng c th xut hin vi cc
din tch cho thy s chuyn ha mch.
116
M g S hot ng ca
phn sinh m
M khng t chc
(unorganised tissue)
Hnh 6.11 S chuyn ha callus ca Lampranthus v Erytrina
S chuyn ha v t chc callus
C s bin i ng k trong s pht trin v kiu chuyn ha t bo cc
callus nui cy. Mt callus ng nht bao gm ton b cc t bo nhu m him khi
thy c.
Hnh 6.12 Cc kiu t bo khc nhau trong cy t bo
S chuyn ha t bo xy ra hnh thc mch, ng sng, cc t bo sube ha, t
bo tit v t bo c lng.
Mt din tch nh ca cc t bo phn chia thnh lp nhu m hoc khi mch c
th tr thnh trung tm cho s thnh lp chi, mm r v phi.
Hnh 6.13 S thnh lp chi (caulogenesis) (a) v pht trin r (b v c) trn callus
cy Hypoxis
Callus khng n nh v di truyn
Nui cy callus thng gp phi vn khng n nh di truyn. iu ny c
th dn n cc bin d v kiu hnh trong cng lc nui cy. S bin d ny c mt c
s di truyn v biu sinh (epigenetic).
Cy truyn callus (callus subculture)
- Sau mt thi gian cn thit cy truyn callus vo trong mi trng mi, do ht dinh
dng trong mi trng v gel agar tr nn kh i.
- Cc cht thi do callus tit ra cng c th tch ly n mc c trong mi trng.
117
- Callus c cy truyn phi c kch thc bo m s sinh trng v lun
c cy li trn mi trng mi.
Cch cy callus mt cch c hiu qu l cy treo. Cc loi song t dip c
khuynh hng d to cy treo hn cc loi n t dip.

Dn d Callus Bt u cy treo
Hnh 6.14 Kch thch callus trn mi trng c v lng
Hu ht phng php cy treo i hi callus trc ht xut pht t in vitro. C
ba loi chnh dn d:
S dng callus ri rc (friable callus): S ri rc ca callus th rt quan trng, v n
lm tch ri tng cm callus mt cch d dng trong mi trng lng, to thnh cm
callus nh. Trong s cy truyn lp li v lc, s cy treo c th gim cc cm t bo
rt nh ri rc cc t bo n. ly mt callus ri rc thch hp, cn thay i t l
auxin v cytokinin trong mi trng bng cch nng nng auxin.
S dng callus khng ri rc: Chuyn callus vo mi trng lng. Nui cy c lc
v cy truyn u n n khi callus t ri rc thch hp..
S dng enzyme
Callus c thm vo mi trng lng cha nng thp ca enzyme phn hy t bo
(cellulase v pectinase) n khi s cy treo t c .
Khi thit lp cy treo, cn thit cy truyn lp li lin kt vi lc gim kch thc
ca cm.
Cc kiu t bo
118
Cy treo him khi bao gm t bo n, ch bao gm hu nh
cm t bo cng nh cc t bo tri ni t do. Cc t bo trong
cm t bo th trong mt vi mi trng khc vi cc cm tri
ni t do. iu ny quy cho s thiu s ng nht ca mt
t bo v kch thc, hnh dng v s bin dng c quan
tm. y l mt vn trm trng. T bo khng n nh dn
n khng ng u nhim sc th v bin ng kiu gen.
Hnh 6.15 Mt kiu cm t bo c hnh thnh trong qu trnh cy treo callus
cy Erythrina
Bioreactor cy treo lin tc bung kim sat My lc cho cy treo
Cc kiu my lc cho cy treo Bnh cu cho my lc Bnh tam gic c vi cho my
lc
119
Bnh tam gic cho my lc My lc xoay gn ng
nghim
Bnh cu s dng my lc xoay
Hp nui cy Giy lc lm cu
Hnh 6.16 Cc thit b nui cy callus
Bung m t bo (Haemocytometer)
Dng c lc o trng lng ti v kh ca
t bo
Hnh 6.17 Cc phng tin xc nh mt s v trng lng t bo
6.3 SN XUT CC CHT BIN DNG TH CP BNG PHNG PHP
NUI CY T BO
6.3.1 Cht bin dng th cp
T xa xa, thc vt khng nhng l ngun cung cp thc phm cho con ngi,
m cn l ngun cung cp dc liu, cc cht to mi thm, cc cht to mu v cc
cht dit cn trng. Ngy nay, mc d cng nghip ha hc pht trin rt cao,
nhng vn cha tng hp c mt s ho cht c trong thc vt hoc c th tng hp
cc cht ny nhng c gi thnh qu cao.
Cht bin dng th cp l g? Trong qu trnh sng ca thc vt, cc sn phm
bin ng ch yu l cc sn phm trung gian ca qu trnh quang hp v h hp. a
s cc sn phm ny c gi l sn phm bin ng s cp (primary metabolites).
Cht bin dng s cp l cc hn hp cn trong cc qu trnh c bn, m gi cho thc
vt sng nh carbohydrate, protein v lipid. Cc sn phm bin dng s cp, mt s
s l tin cht cho cc sn phm bin dng th cp. Cht bin dng th cp l cc
hn hp c to ra trong cy, khng cn thit cho cc chc nng c bn ca cy.
Cht bin dng th cp bao gm terpen, cardiac glycoside, hn hp steroid, alkaloid
v nhiu cht khc na nh antibiotic, resin, tannin v saponin. Mt s cht bin
dng th cp c to ra nh cht bo v ha hc chng li vi khun v ng vt.
Cht bin dng th cp c s dng trong k ngh dc, cht to mi phm nhum
v trong nc hoa.
120
6.3.2 Sn phm ca cht bin dng th cp
Sn phm dc
Khong 25% n thuc s dng th gii phng Ty c ngun gc thc vt.
Khai thc thc vt bng cch gieo trng theo cch c truyn cc cy thuc th tng
i c gi thnh cao. Ngi ta hy vng l sn lng dc cht trong nui cy t bo
di iu kin kim sot s l mt s la chn ng. Tuy nhin, cc cht bin dng
th cp th thng khng c to ra trong cy t bo v khng mc lm
thnh sn phm thng mi, ngoi tr vi trng hp. Hin nay, cc thnh phn thuc
to ra trong nui cy t bo v c quan bao gm: diosgenin, codeine, morphine,
atropine, hyoscyamine, scopolamine, digoxin, digitoxin, quinine, reserpine,
artemisinin v taxol.
Mt v d cy t bo c hu ch l sn xut Paclitaxel (taxol). Paclitaxel c
s dng tr ung th t cung v v ph n. Hn hp ny c trch t v ca cy
Taxus baccata. Paclitaxel rt kh tng hp v n c 10 trung tm lp th
(stereocenters) v qun th t nhin ca T. baccata khng th cung cp lm thuc. .
Bng 6.1 ch vn tc sn xut Paclitaxel t cy, t bo v cy nm (mt loi
nm sng trn cy v cng to ra paclitaxel). Ngi ta thy l cy m t bo c th to
ra mc ca paclitaxel cao hn v c th thc hin c trong sn xut
Bng 6.1 Sn lng ca Paclitaxel to ra
.
Vt liu thc vt
Vn tc sinh tng
hp
Hm lng paclitaxel trung bnh
(% trng lng kh)
V ca cy gi
(100 tui)
4,70 X 10
-6
mg/g/day 0,017
Taxus n in
(4 tui)
0,34 X 10
-6
mg/g/day 0,005
Taxus nui cy
m
0,64 mg/l/day 0,200
Taxomyces
andreanae
400 x 10
-6
mg/l/day -
S chuyn ha
nhiu h thng cy treo callus, ngi ta tm thy l cc t bo khng sn sinh
ra cc cht bin dng th cp c tm thy cy m. Tuy nhin, khi chuyn ha xy
121
ra s thnh lp c quan chi/r hoc to phi v tnh, cc m c chuyn ha bt u
to ra cht bin dng th cp nh cy m.
a b c
Hnh 6.18 a: cy m (Thapsiagarganica); b: callus khng to ra Thapsigargin; c:
cy con c hnh thnh to ra Thapsigargin
Cc l do dn n thiu sn lng cht bin dng th cp trong t bo c th
l:
- S lm lch cc tin cht thng thng trong bin dng s cp
- Loi b hoc lm gim biu hin ca enzyme ch yu trong bin dng th cp
- Thiu v tr tr thch hp, nh cc t bo tit, c cc l nh hoc dch bo, nha (latex
tissue) hoc tuyn lng (glandular hairs) xy ra trong m c chuyn ha.
Sn xut sc t
Mt s nui cy t bo tch ly sc t mc cao. Mt v d nui cy
Lithospermum erythrorhizon to ra sc t c s dng trong m phm.
Nui cy cc t bo khc to ra anthocyanins, hoc vng xanh, c th s dng
to mu t nhin trong thc phm.
Hnh 6.19 Cy callus cy Oxalis reclinata c mu vng trong nh sng, nhng khi
c chuyn vo trong ti n bt u to ra sc t anthocyanin .
S kch hot (Elicitation)
Vi cht bin dng th cp nh cht alexin thc vt (phytoalexins) c tch
ly trong cy sau khi nhim do vi sinh vt. Nhng hn hp ny c hot ng khng vi
khun v v th c chc nng nh l cht ha hc bo v cy.
Cht kch hot (Elicitor) S nhn ra cht kch hot s tng hp Enzyme s
tng hp Phytoalexin
122
Cc hn hp phng thch ra do bnh hoc cc phn ca vch t bo bnh c th
dn n phn ng tng t. Cc hn hp ny c t tn l cht kch hat v qu
trnh dn d sn xut ra alexin thc vt (phytoalexin).
Ngoi ra, cc hn hp c ngun gc t bnh thc vt hoc c cc tc nhn
stress, dn n s tch ly sn phm. Tc nhn stress bao gm: bc x UV, nhit
thp, ethylene, thuc tr nm, khng sinh, mui kim loi nng v nng mui cao.
S bin i sinh hc (bioconversion)
Nui cy t bo cha nhiu enzyme c th to nhiu phn ng sinh hc. Trong
s cc enzyme ca thc vt s sinh tng hp ra cc cht bin dng th cp mt cch
bnh thng v cc enzyme ny ch c tm thy cc thc vt c bit . Nui cy
t bo to cc cht bin dng th cp c nhiu li ch quan trng hn l c tng
hp bng ha hc, v cy t bo to ra cc sn phm tinh khit.
Bng cch cung cp mt tin cht, thng r v c sn, nng sut cc cht bin
dng th cp c th c gia tng. Tin cht c bin i s dng cng con ng
sinh tng hp nh thc vt nguyn. Tuy nhin, i khi cy t bo bin i tin cht
thnh cc hn hp mi khng tm thy trong cy.
Mt kh nng khc l cung cp mt hn hp ngoi lai vo t bo. Cy t bo
lc c th s dng b my enzyme bin i hn hp ny thnh hn hp khc.
Hnh 6.20 Th d ca s bin i sinh hc trong cy t bo. Coniferin c thm
vo trong dung dch cy Podophyllum hexandrum v podophyllotoxin c to
ra. Podophyllotoxin c s dng to ra thuc chng ung th.
Khng c mt phng php chung cho s nui cy t bo to cht bin
dng th cp. Tuy nhin, nhng yu t sau y cn c lu :
- Mi trng h tr cho s tng hp cc cht bin dng th cp
- Cht iu ho sinh trng
- Thnh phn khang
- H thng nui cy bao gm: chng, thu thp t bo chng, chuyn t bo sang
mi trng sn xut
- Theo di s thnh lp sn phm
- S khng n nh ca nng sut
- Phn tch sn phm
- Trch mu
6.4 TR LNH THEO CNG NGH SINH HC
6.4.1 Mc ch:
123
- Tr mu vt thc vt trong mt thi k ngn (lm chm s sinh trng ca
cc mu nui cy)
- Tr mu vt thc vt trong mt thi k lu di
- Duy tr sc sng v s n nh di truyn
- Duy tr s pht trin y tim nng v chc nng
- Tit kim cng lao ng
- Thc hnh s dng: ngn hng gen
6.4.2 K thut tr c bn:
C hai k thut c bn c p dng hin nay l tr mu vt thc vt trong mt
thi k ngn v tr mu vt thc vt trong mt thi k lu di
6.4.2.1 Tr mu vt thc vt trong mt thi k ngn (lm chm s sinh trng n
mc ti thiu)
Lm chm s sinh trng n mc ti thiu l mt trong cc cch duy tr
tnh trng mu trong iu kin in vitro. Thng thng hai yu t c lu l iu
kin mi trng (nhit , nh sng) v thnh phn mi trng nui cy. C th lm
gim s sinh trng bng cch lm gim nhit v nh sng. Mt cch khc l
thng c kt hp s gim nhit l p dng cht c ch sinh trng hoc thnh
phn dinh dng.
Phng php lm chm s sinh trng thch hp cho bo qun chi v cy con,
nhng hn ch cho cy callus (Withers, 1987).
Kim sot nhit v nh sng
Lm gim nhit ca phng nui cy mc thp hn nhit ti ho cng
lm gim s sinh trng. Bn cnh , s gim cng nh sng cng gim i s
sinh trng ca mu vt ang nui cy.
Tr trong iu kin nhit thp: Chi v cy con ca nhiu loi thc vt c bo
co tr c thnh cng nhit thp gn nhit ng c. Phi v tnh c
tr thnh cng trong iu kin nhit thp trn 0
o
C. Phi cy Carrot c tr 10
o
C
trong 120 ngy, sau cho phc hi v phi pht trin bnh thng.
Tr trong ti
Tr trong ti ch thch hp cho mt s loi. Chi cy Prunus c th tr trong ti
nhit -3
o
C sau 10 thng vn cn sc sng. Cy Kalmia v cy Populus c th gi
trong ti 3-4
o
C trong 1-2 nm (Chun v Hall, 1986; McCulloch, 1988)
Thay i thnh phn ca mi trng nui cy
Gim carbohydrate v dinh dng s lm gim s sinh trng ca mu vt nui cy.
Kathar v cng s, 1981 bo co rng chi cy C ph c th tr mt cch c hiu qu
trong iu kin nhit 26
o
C, quang k 16 gi, trong vng 2 nm, vi mi trng
nui cy l MS nng v khng c ng.
Gim khong v c: gim lng khong v c cng lm gim s sinh trng ca mu
cy
Thay i th nng thm thu
- Nng ng cao cng l yu t hn ch s sinh trng ca mu nui cy. Schenk
v Hildebrandt, 1972 cho rng nng ng cao (4-5%) l yu t duy tr mu cy
min trng trong mt thi k lu.
- S dng cht iu ha sinh trng
Mt s cht iu ha sinh trng c tc ng lm chm s sinh trng. Cc cht ny
a s l cc cht c ch sinh trng. Chng gm CCC (Cycocel) 2g/l; B-nine (n-
dimethyl-succinamic acid) 50mg/l hoc ABA (Abscisic acid) 10mg/l
- X l kt hp
124
S kt hp nhit thp vi mi trng c hm lng khong thp hoc l s hin
din ca cc cht c ch sinh trng hoc s dng nng ng cao.
6.4.2.2 Tr mu vt thc vt trong mt thi k lu di (tr ng lnh)
y l phng php lm ngng sinh trng ca t bo. Vic lm ngng sinh
trng ca t bo thng nhit rt thp ca s ng lnh. thc hin c quy
trnh ny cc giai on sau y cn lu :
Phng php tr ng lnh
Ba phng php tr ng lnh c cp l lnh rt nhanh, lnh nhanh v
lnh chm (hnh 6.21).
s tr lnh thnh cng, thc vt phi c lm lnh dn dn t -30 n -
60
o
C, trc khi chuyn vo nitrogen lng. S lm lnh t t, lm kh nc trong t
bo tp trung lin tc vo thnh phn t bo v lm suy gim im ng bng ca n.
Mt khi dung dch t bo c tp trung lm lnh chm, s gi nc c th b bin
thnh thy tinh do ng bng vt liu thc vt ht sc nhanh. mt s trng hp,
mu vt thc vt nh c th chuyn ngay n -196
o
C khng tn hi m cy.
Hnh 6.21 Cc s bin i ha l trong ba phng php tr lnh. thnh cng
vt liu phi c tr lnh 30
o
C n 60
o
C trc khi chuyn vo nitrogen lng,
hoc c lm lnh rt nhanh trong mt th tch nh ca cht lng
(George,1996).
125
Quy trnh thc hin
+ Trc s sinh trng
Trc khi bt u tr lnh, hu ht cng on cy i hi thao tc v iu kin
cy c bit gip cho mu chu ng ng bng ti a. Yu cu s thay i t h thng
nui cy ny sang h thng nui cy khc v thay i theo loi.
+ Cht bo v s ng lnh
X l mt s ha cht cho mu cy trong khong 1 gi trc khi ng bng s
gy ra nhng thay i v tnh thm ca t bo, im ng bng v cc p ng i vi
cc stress v ng lnh v lm tan bng. Hn hp thng dng nht l
dimethylsulphoxide (DMSO) v glycerol. Cht u i khi c s dng n, trong
khi glycerol t khi hiu qu tr phi kt hp vi DMSO hoc cc hn hp khc nh
sorbitol, manitol, ng, glucose, proline hoc polyethylen glycol.
Cht bo v thng c chun b trong mi trng nui cy nng gp
i, c pha long lc s dng, chnh pH ca mi trng chun v lc kh trng
trnh to ra caramen ha trong qu trnh hp kh trng.
+ ng bng
Gia nhit 0
o
C v 100
o
C s thay i c ngha xy ra khi thnh lp nc
sinh ra t bn trong v bn ngoi t bo. Tnh cht ca nhng s thay i tu thuc
mt phn vo mu vt v x l cht ng bng, nhng tu thuc ln vo vn tc lm
lnh. phng php lm lnh chm, s ng lnh bn ngoi t bo gy ra s mt
nc bn trong t bo n im ng lnh t bo cht. phng php lm lnh nhanh
gy ra s ng bng bn trong t bo xy ra sm v tng i t mt nc.tr phi lm
lnh qu nhanh, tinh th nc thnh lp trong t bo s gy hi.
+ Tr
trnh s tn thng gy ra do s kt tinh nc li, iu cn thit nn tr
mu vt iu kin lnh 100
o
C. Trong iu kin thc t, cho tin li ngi ta c
th tr trong nitrogen lng 196
c
C hoc nitrogen lng bay hi 156
o
C.
Hnh 6.22 a: ng tr mu c t trong mt bnh chn khng v trong t lnh
70 n 80
o
C; b: ng tr mu c t trong khi polysterene c l v c
t trong t ng ging nh a; c: ng tr mu c b li v treo vi que thy
tinh t trong bnh cha nitrogen lng bay hi, np c bao li bng ht
polysterene; d: ng tr mu c gi trong polysterene v ni bn trong bnh
cha cn lnh
126
Hnh 6.23 ng tr mu c t trong cc ngn ko xp chng c ngm trong
nitrogen lng

a b c d
Hnh 6.24 a: ng ng mu; b: cc kim loi ng ng ng mu; c: t mu vo
bnh nitrogen lng; d: bnh cha nitrogen lng
+ Lm tan
S h hi do s kt tinh li nc s xy ra trong t bo c lm tan qu
chm. V vy, s lm tan nhanh cn c lu . iu ny d thc hin v c th b
mu lm tan trong nc m
+ Phc hi
Sau giai on lm tan, mu c th c cy truyn sang mi trng phc hi
m khng cn ra mu loi cc cht bo v lnh.

127
Chng 7 S THUN DNG
Cc cy con sau giai on cy m, khi mang trng trc tip ra nh li hoc mi
trng t nhin thng c t l cht rt cao. C nhiu nguyn nhn gy ra hin tng ny.
Trc ht, cy con t trong phng nui cy cha thch nghi vi iu kin mi trng bn
ngoi. Hu ht, cy cy m c nui trong iu kin mi trng c nh sng thp, m
cao v dinh dng c kim sot. Trong khi iu kin t nhin c nh sng cao, m
thp, ch dinh dng kh kim sot.
S thun dng (acclimatization) l cng vic lm cho cc cy con cy m thch
nghi trong iu kin mi trng mi. Trong chng ny chng ta s xc nh cc yu t
gii phu, hnh thi v sinh l gii thch cho s thch nghi km ca cc cy con cy m
khi a ra mi trng t nhin.
Mt vn thng gp ca cc cy con cy m chuyn t ni nui dng ra mi
trng t nhin l s khng hong nc (water stress). S stress nc lin quan n s
thot hi nc qu nhiu b mt l hoc s hp thu nc qu yu ca r. Nguyn nhn
ca stress nc lin quan nhiu n cu trc hnh thi v gii phu ca l.
7.1 .1. Lp cutin
Kho st lp cutin ca cy cy m v cy trng t nhin trong nh li cho thy
rng, cu trc tinh th lp cutin ca cy cy m khc bit rt r vi cy trng trong nh
li. Cu trc c quan st di kinh hin vi in t qut (scanning electron
microscope) cho thy lp cutin trn b mt l ca cy cy m c dng ht v que nhm
(Preece v Sutter, 1991). Nguyn nhn gy ra s thiu lp cutin ny c gii thch l do
m trong bnh nui cy qu cao (Sutter v Langhans, 1982). S thiu lp sp dy trn
b mt l l mt trong nhng nguyn nhn lm cho cy cy m d mt nc v d cht
khi trng trong iu kin t nhin.

Hnh 7.1:S pht trin bt thng lp sp biu b ca cy cy m
(hnh phi) c so snh vi cc cy trng trong nh knh (hnh tri)
7.1 .2. Kh Khu (stomata)
Cu trc kh khu ca cy cy m khc vi cc cy trng trong nh li. cy cy
m, kh khu c dng trn so vi cy bnh thng trng trong nh li c cu trc hnh
elip. m kh khu ca cy cy m ln hn m kh khu ca cy trng trong nh
li. V s kh khu trn din tch l, kt qu nghin cu ca nhiu tc gi cng cho thy,
cy cy m c s kh khu trn n v din tch l nhiu hn cy trng trong nh li
128
(Blanke v Belcher 1989; Capellades v cng s, 1990). Nhiu nghin cu cng cho thy,
kh khu ca cy cy m cha c chc nng ng m khi chuyn t bnh nui cy ra mi
trng ngoi, hu ht khu u m. V vy, trong nhng ngy u khi chuyn cy cy m
ra mi trng ngoi, s mt nc ca cy rt cao. Mt s k thut cng c ngh
ci thin tnh trng ny nh l lm ni lng np bnh cy giai on cui tng s trao
i kh, tng cu trc khu hnh elip, gim s khu trn n v din tch v kh khu hot
ng ng m tt hn.

a b
Hnh 7.2: Cu trc kh khu ca cy cy trng trong nh li (a) v cy cy m (b)
7.1.3. L
Cu trc gii phu l ca cc cy cy m cho thy rng, l ca cc cy cy m
mng hn l ca cc cy trng trong nh li. Cc t bo m giu pht trin ngho nn v
t cc khong trng bn trong. mt s loi, l ca cy cy m ch pht trin mt lp m
giu, trong khi l ca cc cy trong trong nh li pht trin hai n ba lp m giu.
Cung l pht trin thiu lp m gia (collenchyma) v gim cc si libe v h
mch so vi cc cy trng trong nh li (Smith v cng s, 1986)
7.1.4.Thn
Thn ca cy cy m thiu m gia (collenchyma) v cng m (sclerenchyma) l
nhng m h tr cho s cng chc ca thn. V vy, cy cy m thng yu hn cy
trng trong nh li, h mch km pht trin
7.1.5 R
R ca cy cy m thng cha c chc nng. Mt s loi, r cha pht trin lng
ht, cha hon chnh s lin kt gia h mch r v thn.
129
Hnh 7.3: S thnh lp r ca cy con Cercis canadensis trong iu kin cy m (a);
cy con c to r mi trng t nhin (b); gii phu phn r ca cy con cy m
cho thy s pht trin cu trc bn trong cha hon thin (c); v cu trc gii phu
ca cy con c to r trong mi trng t nhin cho thy cu trc r hu nh
hon thin (d) (Hartmann v ctv., 1997)
7.2. QUANG HP
Cy cy m thay i chc nng sinh l cc nhanh khi chuyn t mi trng nui
cy ra iu kin t nhin. Trong mi trng nui cy, ngun cung cp carbon ch yu l
ng. Trong khi , hm lng CO
2
rt gii hn, cy con phi thay i chc nng sinh
l ca cy trc y d dng sang chc nng sinh l l t dng. V vy, quang hp c
vai tr quan trng cho s sng. Di iu kin t nhin, thc vt l cy t dng v s
dng ngun carbon ca mi trng l CO
2
. Trong cy m, ngun carbon chnh l ng
(carbohydrate) c trong mi trng nui cy v CO
2
t khong trng bn trn bnh cy.
Khi so snh quang hp ca cy trng trong nh li v cy cy m, kt qu cho thy hu
ht trng hp mc quang hp c ghi nhn, cc cy cy m.thp hn cy trng
trong nh li. Grout v Aston, 1978 kho st s hp thu CO
2
phng x, h tm thy rng
s hp thu CO
2
khng ng k trong qu trnh nui cy. Sau khi chuyn trc tip sang nh
knh, cy cy m phi pht trin mt cng c quang hp. Mt s nh nghin cu chia cc
cy c cy thnh hai nhm ring bit: Mt nhm cc l in vitro khng bao gi pht
trin kh nng quang hp khi trng trn mi trng vi ng v mt nhm l c th
thch nghi cc iu kin t dng.
Khi hoa Hng c trng trn mi trng c hm lng ng thp (1 v 3%),
mt gi tr quang hp ti a cao hn v im b nh sng thp hn c tm thy so vi
cc cy con c x l vi ng 5% sucrose. ng ngoi sinh trong mi trng c
khuynh hng lm gim quang hp thun ca cy c cy m. Cy Spathiphyllum c
130
cy trn mi trng c cha 3% ng sucrose, quang hp thun c o cui giai
on in vitro. Khi nng ng c tng gp i, cy tr nn tp dng. S thun ha
cn khong 2 tun trc khi s t dng tr nn tp dng hon ton v vn tc quang
hp 3% ng.
Hiu qu ca nng ng trong mi trng trn s d tr carbohydrate:
Khi nng ng trong mi trng nui cy ca Spathiphyllum c gia tng t
3 n 6%, cc nng ng cao hn v tinh bt c tm thy trong l cui giai on
in vitro. Trong tun u sau khi chuyn vo nh li, s d tr ng c s dng
vt qua thi k stress ban u (Debergh, 2003).
hoa Hng, mt lng ln tinh bt (tng t vi cc cy trng trong nh knh)
c tm thy cc lc lp ca giai on 3a cc chi con pht trin trong mi trng
cy vi 5% ng. ng knh ht tinh bt gia tng vi s gia tng nng ng ca
mi trng cy. Ngi ta gi nh l tinh bt ny c s dng nh l d tr dinh dng
cc chi con in vitro v cng thch hp cho s to r v sinh trng trong s thun ha
trong nh knh (Debergh, 2003)
c tnh gii phu v sinh l ca cy con cy m cho thy, chng cn c thi gian
hon thin cu trc v chc nng sinh l thch nghi vi mi trng t nhin. Cc k
thut thun dng c mc ch lm cho cy cy m dn dn hon thin cu trc. Cc yu
mi trng cn t n nh lm thp m , tng dn cng nh sng. Qu trnh thun
dng c th bt u giai on cui ca cc cy cy m, cn trong bnh nui cy v giai
on sau khi chuyn cy con ra ngoi mi trng t nhin.
131
H
n
h

7
.
4

Q
u
y

t
r
n
h

t
h
u
n

d
n
g

c
y

c
o
n

t
h
e
o

G
e
o
r
g
e

(
1
9
9
6
)
132
7.3.1 Trong bnh nui cy c th lm tng qu trnh thun dng bng cch:
- Lm gim m ca mi trng chung quanh nh mang cc bnh nui cy ra
ngoi phng tng trng, ni m m khng kh tng i thp
- Ni lng np bnh nui cy tng s trao kh vi mi trng xung quanh.
Tuy nhin, theo cch ny bnh nui cy d b nhim. Nhng vn nhim trong
giai on ny khng quan trng, v vi sinh vt gy nhim cha hn l sinh vt gy
bnh.
- C th gim m ca khong trng bn trn np bnh bng cch lm lnh y
bnh (Maene v Debergh, 1987).
- m
Cy cy m sinh trng trong mi trng c m cao, v vy khi chuyn sang
mi trng t nhin d b mt nc do m thp. Nhiu k thut to m cng c
nghi bi cc tc gi nghin cu. Trong cc khuyn co, ngi ta ngh l khng nn
s dng phng php to m bng cch phun sng, v lm nh th cy con rt d b
mt khong do ra tri. Phng php c nhiu tc gi ngh l trm kn plastic trong
to m cao v t trong iu kin nh sng thp.
- nh sng
Cy cy m c cu trc hnh thi, gii phu l mng v t t bo tht l (t bo
giu) nn d thch nghi trong mi trng c nh sng thp. Cy cy m t di nh sng
cao d b chy l. Khi t trong mi trng mi, thng thng cng nh sng bng
hoc ln hn mt t. Sau , tng dn cng nh sng ln. Mt s tc gi ngh, thi
gian thch nghi ny cn khong 4 tun (Miller, 1983; Pocock, 1983; Griffis v cng s,
1983).
- t
Mi trng cy cy con rt quan trng, trong pH t, gi m, thong
kh v dinh dng cn ch . Mc d nhiu nghin cu cho thy rng, r ca cc cy cy
m c to ra trong qu trnh nui cy cha c chc nng hon ton, m cc r ny cn
c thi gian mi hon thin cc cu trc bn trong; V vy, trong thi gian u cn trnh
lm cho cy con b mt nc.
- Bnh v cn trng
Cy cy m khi chuyn sang mi trng t nhin thng gp phi cc vn v
bnh v cn trng tn cng. Do mi trng thun dng c m cao l iu kin tt
cho nm bnh pht trin. Ngoi ra, cy cy m cng l thc n a thch ca cn trng,
nn phng php thng c p dng cho cc nh li hoc vn dng cy l lun v
sinh sch s mi trng ra cy. Trong mt s trng hp, cy con trc khi cy vo t
c ngm trong thuc dit nm bnh, cng nh t m cy cn c s dng thm
thuc tr su.
133
- Nhit
Nhit khng kh l yu t quan trong trong qu trnh thun dng cy con.
Nhit khng kh lin quan nhiu n nh sng chiu vo mi trng. nh sng trc x
chiu vo cc vm ph plastic gy ra hiu ng nh knh, lm gia tng nhit bn trong,
dn n gia tng h hp cy con.
- Phn bn
Phn bn cung cp cho cy con trong giai on u cha quan trng lm, v giai
on u r cy cn thi gian hon thin cu trc. Cung cp phn bn cho cy con l
cn thit.
Cc vm nh thun ha cy: kim sot kh hu ti ho l yu t quan trng
to s thnh cng ca cy cy m. cc lu hoc cc vm c to, m tng i
cao.
Hnh7.5 Trm kn plastic trong theo kiu nh vm
Hnh 7.6 Nh li m cy con cy m
Trong mt s trng hp, cc ni thun ha l phng tng trng ng kn hon
ton. Cc cy con lc c trng t trn cc k tit kim khng gian.
134

Hnh 7.7 Phng tng trng
7.3.4.T ng ha
Cy v chuyn ch cy con t in vitro vo ni thun dng ch yu l lao ng tay
chn. Cc cng vic ny c th c t ng ha gim gi thnh.
Hnh 7.8. S vn chuyn t ng ha
TRNH THUN DNG
m : Kim sot m nn trnh cht nn qu t, nn s dng
cc thit b to sng m.
Nhit : Nhit cao nn trnh. Nhit bn di nng c th thch
hp cho to r.
CO
2
: Mt s trng hp nng CO
2
cao (900 ppm) ci thin
qu trnh thun dng
nh sng: Cng nh sng qu cao giai on chuyn vo trong
nh li s c hi.
Quang k Chn ng quang k
Phn bn: giai on u khng cn phn bn.
Thuc BVTV: S dng ti thiu thuc BVTV
135
Trong qu trnh nui cy m, c nhiu s c gp phi. Cc s c ny lm cn tr
cho cng vic nui cy cng nh gy ra nhng kiu hnh bt thng. C nhiu nguyn
nhn gy ra hin tng ny. S hiu bit v cc nguyn nhn cng nh cch cha tr l
iu cn thit ci thin cc k thut nui cy m.
Trong t nhin, thc vt c th tip xc hoc nhim nhiu loi vi sinh vt trn b
mt hoc vi sinh vt xm nhim vo bn trong m cy hay t bo. Tuy nhin, trong nui
cy m th khng th chp nhn s tn ti ca vi sinh vt trn b mt mu cy hoc bn
trong m hay t bo.
C nhiu loi vi sinh vt c th nhim trong qu trnh nui cy m.
- Virus
- Bacteria
- Fungi
- Mycoplasma
8.1.1 Nhim do virus
Cc bnh do virus thc vt gy ra s tn tht to ln trong nng nghip. Trong lnh
vc cy m, iu quan trng c bit l cc cy c to ra vi s lng rt ln v phi
hon ton sch bnh. Mt mu cy b nhim virus c th to ra v s cy b nhim virus,
v d nh trong vi nhn ging t cc mu vt b nhim c th dn n hng triu cy con
c to ra nhim bnh. T sch bnh i khi b lm dng; sch bnh cha hn sch
virus, ch khi no cc cy c test virus mi bit chc chn cy sch bnh, bao gm c
sch virus (George, 1996).
Nui cy nh sinh trng kt hp nhit tr hoc ha tr cng khng m bo cc
phn ca cy cy m hon ton sch virus. Cc phng php trn i khi loi virus thc
s, nhng i khi cng lm gim s hin din ca virus. V vy, thc s kh xc nh cy
cy m c hon ton sch virus hay cha. Mt s st nh khng xt nghim trc khi
chuyn ra ngoi ng c th gy thit hi ln sau ny. Phng php xt nghim s hin
din ca virus c bit nh l phng php ch th virus (virus indexing). Cy qua
phng php ny c xem nh cy c xt nghim virus.
loi tr vic nhim virus, ta cn bit thc vt thng b nhim virus no. Cc
virus c chia thnh 35 h hoc nhm trn 700 virus c bit (George, 1996).
Nhiu k thut xc nh dng virus c thit lp:
Dng cy ch th
- Chng virus trn cy ch xt nghim virus gy bnh phi c bit trc
Phng php ny c thc hin bng cch chng vo trong cy (cy ch th), cy
phn ng vi s nhim virus bng cch sn xut ra cc vt thng biu hin trn l.
Cc cy ny l nhng cy d mn cm vi phm vi rng ca cc loi virus, v d cy
136
Gomphena globosa v Nicotiana spp. Cy ch th c s dng rng ri cho vic gim
nh virus.
- Ghp cy
S nhim virus khng phi lc no cng c pht hin bng cch chng trn cc
cy ch th. Phng php ghp cy cng l phng php xt nghim s nhim virus.
Damiano (1980) s dng ghp l xt nghim virus trn cy Du Ty trc khi sn
xut hng lot.
Knh hin vi in t cho kch thc hnh thi ca virus
Xc nh dch trch hoc mu vt ct ngang di knh hin vi in t c th pht
hin s hin din ca virus. Dng hnh que v hnh si d phn bit hn dng hnh cu. S
kt hp gia quan st di knh hin vi v xt nghim bng huyt thanh s cho kt qu
chnh xc hn.
Xt nghim bng huyt thanh/ELISA
Protein virus hot ng nh mt khng nguyn, cht c kh nng kch thch s p
ng min dch ng vt bng cch kch thch sn xut khng th. Khng th c th c
s dng theo nhiu cch pht hin virus trong vt liu thc vt.
Sn xut khng th
Khng huyt thanh cha cc protein khng th l globulin c c t mu ng vt
(thng l th) c tim trc mt lng nh virus c trng. Mt khi c c huyt
thanh c th tr trong mt thi gian lu v c s dng cho xt nghim lp li. Xt
nghim bng huyt thanh cung cp kt qu nhanh v c li ch l khng nguyn virus
trong cc phn trch t thc vt c tr thch hp v d pht hin sau vi ngy, trong khi
s nhim c xt nghim vi cy ch th th biu hin mt i rt nhanh.
- Phng php ELISA (Enzyme-linked immunosorbent assays) s dng khng th
chng li virus. C nhiu quy trnh, nhng nguyn tc chung c trnh by trong cc
hnh bn di.
Trc ht, virus c gn vo b mt ging ca mt da ELISA, ri khng th
c xy dng chng li virus c thm vo (hnh 8.1). Khng th c thm vo d
tha, v vy tt c virus s c mt khng th gn vo n. Phn thng d ca khng th
c ra sch. Lng khng th cn li sau cn bng vi s virus. Khng th c
enzyme lin kt vi n. Mt cht nn khng mu c thm vo ging xt nghim.
Enzyme bin i cht nn ny thnh mt hn hp c mu, sau c th c o quang
ph. Mu cng m cng nhiu virus.
137
Hnh 8.1 Phng php xt nghim ELISA sandwich khng th i i vi virus
138
Hnh 8.2 Phng php xt nghim ELISA gin tip
Hnh 8.3 Ging xt nghim virus
139

Hnh 8.4 Da xt nghim virus c mu v khng mu (bn tri), my c mu (bn
phi)
Phn tch DNA, RNA ca virus.
Hin nay, ngi ta cng s dng phng php PCR xc nh s hin din ca virus
trong cy ch.
Cch cha tr
- Cy nh sinh trng
- Vi ghp
- Nhit tr (thermotherapy)
8.1.2 Vi khun:
Nhim do vi khun thng gp trong nui cy in vitro. Mt s vi khun gy nhim
c th l vi khun gy bnh cho cy ch, nhng mt s khc khng phi l vi khun gy
bnh. Cc vi khun ny c th l nhng vi khun sng t do trong khng kh. S hin
din ca cc vi khun trong mi trng nui cy gy nhiu bt li. N c th gy cht cy
hoc lm chm sinh trng ca mu nui cy.
Nhim do vi khun c th c nhng bt li sau:
Rt kh chng minh mt s nui cy sch vi khun.
i khi cc triu chng ca nhim bnh c thy r rng trn thc vt hoc trong
mi trng cy m.
Cc triu chng thy c xut hin chm trong mi trng cy hoc sau vi ln
cy truyn.
Chng c th khng xut hin hon ton, nhng to vn ca giai on u giai
on thun dng.
Khng phi tt c vi khun u gy bnh, nhng i khi chng c th l vi khun
c li.
Cc vi khun gy nhim thng gp trong qu trnh nui cy:
140
Agrobacterium
Bacillus
Enterobacter
Pseudomonas
Lactobacillus
Staphylococcus
3 %
13 %
12 %
19 %
11 %
26 %
Hu ht cc loi vi khun c quan st thng xuyn trong cy m (Leifert v
Waites, 1990)
Hnh 8.5 Cc triu chng ca nhim vi khun. Hnh bn tri: khng c vi khun;
hnh bn phi: nhim. (Ngun Debergh, 2003)
Cch cha tr: Cy nh sinh trng, dng khng sinh
- S dng khng sinh:
Khng c khng sinh ring s dng cho thc vt. Tt c khng sinh hin ti c
s dng cho ngi v ng vt.
Cc khng sinh c in c hiu qu km hm, nhng khng hiu qu git vi khun
cc nng chu ng ca cy. Chng c th ngn cn tm thi s xm nhp ca vi
khun.
- n nay khng c dng c chn on sn c khng nh mt cy no sch
vi khun.
S xt nghim bng mt ca mi trng phn y cy c th cung cp bng
chng ca s nhim do vi khun.
- pht hin trng hp nhim khng thy, cn thit l to cc iu kin thch
hp cho nm v vi khun pht trin. Khng phi tt c nhim do vi khun c th pht hin
c. Ngi ta thng chn lc trn hai, ba mi trng xc nh vi khun. Xt nghim vi
141
khun bng cc lt ct thn v c chng trong mi trng lng v mi trng c vi
agar trong ba tun nhit 30
o
C.
Hnh 8.6 Chai bn tri nhim vi khun, chai bn phi khng nhim (Debergh,2003)
Nu s sinh trng ca mu th c quan st l nhim th c th lm sch bng
cch s dng phng php chun i vi vi khun v s dng cc th nghim sinh hc
nh nhum Gram, quan st s vn ng, gelatinase, oxidase v s oxid ha /s ln men.
8.1.3 Nm
Nm l vi sinh vt kh ph bin v rt d nhim trong qu trnh nui cy. C
nhiu loi nm khng gy bnh cho thc vt nhng hin din nhiu trong khng kh hoc
cc mu cy. Nhiu loi nm thng gp trong nui cy m l Aspergillus, Candida,
Microsprium, Phialophora.
Hnh 8.7 S nhim mt s loi nm thng gp trong da petri nui cy
(Debergh,2003)
S nhim nm rt d pht hin sau 1-2 tun cy. S hin din ca nm trong mi
trng nui cy c th lm cht mu cy, cy con, i khi c callus. Tc pht trin ca
nm c th quan st bng mt thng t khi hnh thnh cc cm, khi khun ty n khi
pht trin ra tan b bnh nui cy trong thi gian rt ngn (vi ngy n tun).
Nguyn nhn nhim nm l:
Kh trng b mt cha sch hoc ho cht kh trng cha thch hp
Nhim trong thao tc cy truyn.
S du nhp cc bo t nm bi nhng cn trng nh.
S nhim nm d dng pht hin bng mt.
142
Cch cha tr: Tit trng thch hp trnh s hin din ca cc cn trng nh.
8.1.4 Mycoplasma
Pht hin Mycoplasma rt kh bi v chng kh nui v kh nh danh.
8.1.5 Cc con nhn v b tr (mite v thrips)
Hu ht phng th nghim nui cy m lu nm rt d nhim cc con nhn v b
tr. Cc con nhn v b tr th v hi, nhng gy ra vn du nhp bo t nm v vi
khun vo mi trng.
Hinh 8.8 Giai on u trng ca con b tr (Debergh, 2003)
Hnh 8.9 Nhim do nhn (Debergh, 2003)
Hnh 8.10 Nhim do b tr (thrip)
143
Hnh 8.11 Nhim nm do nhn (Debergh, 2003)
Vng i
Cc con nhn ci bm vo cy ch v ht thc n. T lc , trn c th ca con
nhn bt u pht trin v trng. Mui ngy sau , trng ca con nhn n. Nhn n ra
u tin lun lun l con ci, theo sau khong 150 con c. iu ny gii thch s sinh
trng tim tng ca tp on nhn (Debrgh, 2003).
Cch cha tr: Ph lp nng dc hoc t cc bnh cha trn b mt c keo.
8.2. SINH L SINH THI (ECOPHYSIOLOGY)
8.2.1 Kh nng gi nc:
Hnh 8.12 S mt nc ca cy cy m v cy trong nh knh (Debergh, 2003)
o lng s mt nc ca l theo thi gian l mt tng v kh nng gi nc
ca cy. Cc biu hin cho thy, cy cy m c kh nng gi nc rt km. Bng cch
144
gim m tng i trong bnh nui cy xung 75%, s biu hin ng cong mt nc
gn vi kiu cy trong nh knh. S kim sot nc trong cy c ci thin do lm tt
chc nng ca khu.
8.2.2 S vn chuyn nc (water transport)
Hnh 8.13 (A): Thit lp th nghim: trong mt bnh nui cy mt ly nh c gn
gia; 90ml ca mi trng giai on 2 c rt vo chung quanh ly, 7ml ca mi
trng giai on 2 c chun b vi nc c lm thng vi ly v s thot hi
nc t ly b ngn cn (B): S di chuyn nc theo chu k 48 gi trong bnh nui
cy giai on 2 ca cy Rosa multiflora. Cc gi tr (%) i din cho s ti phn
phi ca hot ng nc c thm vo u tin trong ly nh. D kin l cc gi tr
trung bnh ca cc th nghim khc nhau s dng cc agar v nng ng khc
nhau v cy tui khc nhau trong mi trng nui cy (Debergh, 2003).
S di chuyn nc lin tc hin din trong mt bnh cha. Nng ng m cc
chi c duy tr khng cn hiu qu ch yu trn s di chuyn nc, trong khi nng
agar c. Nng agar cng cao cng nhiu nc c chuyn ch thng qua chi n
vng xung quanh. Cc chi ny c kh nng gi nc tt hn.
- Loi thc vt
Thnh phn kh bn trn khong trng ca bnh nui cy c to ra tu thuc
loi, ging v giai on pht trin. giai on nhn hoc to r kt qu to ra mt thnh
phn kh trn khong trng bnh nui cy khc nhau. C hai yu t c dn chng trong
hnh bn di l s phng thch CO
2
v ethylene.
145
Hnh 8.14 S phng thch CO
2
khong trng bnh cy m trong giai on nhn cy
ca mt s loi (Debergh, 2003)
Hnh 8.15 S phng thch CO
2
khong trng bnh cy m trong giai on to r
ca mt s loi (Debergh, 2003)
146
Hinh 8.16. S phng thch ethylene khong trng bn trn bnh cy trong giai
on nhn chi mt s loi (Debergh, 2003)
Hnh 8.17. S phng thch ethylene khong trng bnh cy trong giai on to r
mt s loi (Debergh, 2003)
147
Ngy/m
Chu trnh v s phng thch CO
2
ngy/ m trong thi k cy.
CO
2
thay i trong 24 gi v c o cc thi gian khc nhau trong thi k cy
ca cy hoa Hng. Trong thi gian ti, nng CO
2
nng ln do h hp kh cao. Trong
thi gian sng, nng gim nhiu theo ly tha. Khi thi k nui cy ko di nng
cao CO
2
t c. Cng nn lu l trong cc bnh nui cy khc nhau c s bin ng
cao nng CO
2
c o.
Kh nng quang hp
Phc ha dng carbon cho mt cy in vitro.
Trong h thng nui cy m, thc vt c hai ngun carbon chnh. Sucrose, c
hp thu t mi trng c th c s dng thnh lp mch carbon ca sinh khi hoc
duy tr h hp. Khi ng c s dng cho nhu cu nng lng, tt c carbon s c
bin i v phng thch CO
2
. Ngoi ra, s kt hp ng ty thuc vo tp dng ca
qu trnh nui cy, s c nh CO
2
c th cung cp mt ng gp ng k cho s sinh
trng. hoa Hng 15 n 25% ca s gia tng sinh khi do s c nh CO
2
(Hnh 8.18).
Hnh 8.18 S i din dng carbon khc nhau trong mt cy cy m (Debergh,
2003)
148
Hnh 8.19 Thit lp th nghim o CO
2
t khong trng trn bnh cy (Debergh,
2003)
- Cng thc mi trng
Tnh trng: lng hoc cng
S phn chia v h s khuch tn (s ha tan)
Ngun carbon bn ngoi, cn bng hormone, cung cp dinh dng
S dng cht ngn cn sinh tng hp v hot ng
- Bnh nui cy
Cc c tnh ca kiu np y
Vt liu, cu trc, dy, dng...
S trao i kh:
S trao i khng kh mi gi
Thnh phn ca khong trng bnh cy
Th tch khong trng bn trn v mt cy
149
Hnh 8.20: S trao i khng kh t mi trng bn ngoi v bn trong bnh nui
cy (Debergh, 2003)
Hnh 8.21 Thit b thc hin th nghim (Debergh, 2003)
- Cc iu kin mi trng bao quanh
Nhit , p sut, nh sng, m tng i, thnh phn kh
- Bn ngoi v bn trong bnh cha
- Chu k ngy m
- nh hng ca lm lnh y, s thng gi,
150
Bnh nui cy m c ng tht cht ngn cn s nhim. Tuy nhin, mt bnh
cha c ng cht c th tch ly kh m dn n s sinh trng bt bnh thng ca
cy trong bnh cy nh gim s sinh trng (hnh 8.22) v bch tng (hnh 8.23).
Hnh 8.22. Gim s sinh trng (Debergh, 2003)
Hnh 8.23. Bch tng
8.3. S HA NU (BROWNING)
8.3.1 Triu chng
- Mu cy v mi trng thng tr nn nu hoc en sau khi cy. S sinh trng b
ngn cn v m thng cht.
- S sinh trng b nh hng trm trng nht cc loi trong t nhin c cha mc
cao ca tannin hoc hydroxyphenol khc.
- S ha nu c th b nh hng theo thi gian ca nm m m c ly mu, s hin
din ca cht gy nhim, phng php kh trng....
151
8.24. Ha nu mu cy (Debergh,2003)
8.3. 2 Nguyn nhn
M ha en l do hot ng ca enzyme oxidase cha ng (v d polyphenoloxidases
v tyrosinases) c phng thch hoc tng hp v hin din trong iu kin oxy ha
khi m b thng.
Hnh 8.25: Mu cy b ha nu sau khi cy (Debergh, 2003)
Cht nn cho cc enzyme ny thay i cc m khc nhau.
Phenol c mt chc nng t nhin quan trng trong s iu ha s oxy ha IAA
(cht bo v auxin = auxin protector).
S ngn cn sinh trng khng th phc hi li khi phenol b oxy ha sang cc hn
hp quinone hot ha cao, lc cyclise, polymerise hoc oxy ha protein thnh lp
hn hp melanin.
152
Hnh 8.26 S tch ly polyphenol (P) cha trong:
1 Cc t bo cy np bnh (Nepenthes)
2 T bo thn cy np bnh
3 T bo l cy np bnh
4 T bo c l kn (Sarracenia)
8.3. 3 Cc bin php cha tr
Ly hn hp phenolic c to ra
B sung th nng kh
Lm mt hot ng phenolase enzyme
Gim hot ng phenolase v cht nn hin din.
Lm en m ngn cn lm en
Hnh 8.27 Cch loi b s ha nu
Tin x l: ngm trong nc my
trong nc kh trng 2-3 gi sau khi tch
Chuyn sang mi trng mi (cht tit tr nn b gi bi agar v tr nn m c)
[5-6 chuyn 2-3 ngy lun phin n khi s oxy ha phenol ngng]
153
Cy trc trn mi trng lng
S ngoi hp trn than hot tnh
Hnh 8.28. Cc hn hp ngn cn ngoi hp (1-5 g/l) t mi trng, nhng cng c
cc thnh phn khc (ch yu l phn t hu c)
Cc tc nhn lm gim hoc chng oxy ha
Cc cht chng oxy ha
o Ascorbic acid, citric acid, L-cysteine hydrochloride, dithiothreitol, glutathione,
mercaptoethanol [trc khi kh trng v hoc sau khi kh trng, impregnate filter
paper]
Cc hn hp ny c th c s dng n hoc kt hp.
Mt s cht chng oxy ha tr thnh cht oxy ha mnh sau khi chng b oxy ha,
v d: Ascorbic acid. V vy cc cht ny a vo mi trng cy m c th c hi cho
cc mu cy hoc cy con trong mt thi gian di.
Ngn cn hot ng ca enzyme phenol oxidase bi
Tc nhn l chelate (sequester ions): chelate ca ng
Diethyldithiocarbamate
Dimethyldithiocarbamate
Hot ng ca Phenolase
Hot ng Polyphenol oxidase ln nht pH 6,5 v gim pH thp hn
Cc mc gim ca cht iu ha sinh trng
Gim KNO
3
Glucose thay v sucrose
Lm ti hoc gim nh sng
154
8.4. S BIN D T BO SO MA (Somaclonal variation)
Hnh 8.29. S bin d v tnh cy Rhododendron xut pht t cc chi bt nh
Hin tng bin ng cao cc c th ca cy cy m hoc cc chi bt nh c
t tn l s bin d v tnh (somaclonal variation). S bin d v tnh khng b hn ch,
nhng c bit cc callus t nhng phn c ti sinh (regenerants). S bin d c th l
kiu hnh hoc kiu gen, sau ny c th hoc di truyn hoc c ch biu sinh (epigenetic)
theo ngun gc. S thay i di truyn tiu biu l s thay i s nhim sc th
(polyploidy v aneuploidy), cu trc chromosome (s chuyn dch (translocations), s
mt an (deletions), nhn i (duplications) v chui DNA (t bin base). Epigenetic
tiu biu lin quan n cc bin c l s khuch i gen (gene amplification) v s methyl
ha gen (gene methylation).
Nu s thay i hnh thi khng thy c xut hin, cc phng php chn lc
thc vt khc phi c p dng. C c li v bt li ca bin d v tnh:
Li ch
Li ch chnh ca bin d v tnh l ci thin cy. S bin d v tnh dn n to ra
thm bin d di truyn. Cc c tnh t bin v tnh c th lm giu trong cy m bao
gm s c cht do bnh (pathotoxins), thuc tr c v chu ng vi stress mi trng
v ha cht cng nh gia tng bin dng th cp.
Bt li
Mt bt li ca bin d v tnh xy ra trong cc hot ng m cn s ng nht
dng nh trong k ngh cy cnh hoc cy rng, cy m p dng cho nhn nhanh
cc kiu gen u t.
Cc cch gim s bin d v tnh
Cc bc khc nhau c th trnh c s bin d v tnh. Ngi ta bit rng gia
tng s ln cy truyn rt c th gia tng s bin d v tnh, v th s cy truyn nn gi
ti thiu. Bt u li vi cc mu cy mi c th gim s bin d theo thi gian. Mt cch
khc l gim s bin d l trnh s dng 2,4-D trong mi trng nui cy, v hormone ny
c bit l tng s bin d.
155
8.5 S THA NC (Hyperhydricity)
8.5.1 Triu chng (Symptoms)
Triu chng khng ging nhau tt c cy, c th chia thnh ba kiu: hnh thi,
gii phu v sinh ha.
Hnh 8.30.Triu chng ca s tha nc; hnh bn tri: bnh thng; bn phi tha
nc
Biu hin ca s tha nc
HNH THI GII PHU SINH HA
Lng ngn hn Khong trng gian bo ln Cha r
L thnh cm S pht trin h mch gim
Mng nc, dn Lp biu b h
Mu sc bt thng Lp sp thay i
Hot ng khu km
Hnh 8.31 S tha nc l mt qu trnh pht trin nhng cy nui cy m
(Debergh, 2003).
156
8.5.2 Nguyn nhn
Nhiu nguyn nhn gy nn s tha nc, nhng nhng yu t ny s hot ng khi cc
iu kin khc trong h thng nui cy khng t ti ho bao gm.
Mu cy: loi, ging, di thi gian t khi cy truyn cui cng, s ln cy
truyn; cch gim cnh, t mu trn mi trng
Mi trng: Nng v kiu gel agar; s hin din ca mt lp cht lng; mc
v loi cytokinin; nng mui.
Bnh cha: thnh phn v nng kh
Mi trng bao quanh: Nhit , cng nh sng v phm cht nh sng; m
tng i, s di chuyn khng kh
8.5.3 Cha tr
Mi trng: s dng mi trng c vi nng agar cao hoc s dng cytokinin
nng thp.
Bnh cha: c mng thm kh v plastic thm kh gia tng s trao i kh
Mi trng xung quanh: lm lnh y; gia tng cng nh sng; tr lnh trn
mi trng khng c BA.
S dng k thut hai phase (lng v c)
Gim nng ion ammonium trong mi trng nui cy
Thm cc acid hu c nh citric acid, succinic acid, malic acid gip ng ho
ion ammonium
Gim nng vi lng trong mi trng
Thay th ng sucrose bng frucose hoc galactose
157
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158
8.6 S CHT CHI (Shoot necrosis)
S cht chi thng gp kh cy cc cy thn g. C nhiu nguyn nhn dn n
cht chi: thiu calcium, thiu hormone ni sinh, khong thi gian cy truyn, s nhim
kh bn trong bnh cy do s dng cn kh trng dng c cy
Cha tr: cung cp thm calcium trong mi trng cy, gim s nhim kh, s
dng mi trng hai lp c v lng
8.7 CU TRC BT THNG (Abnormal structures)
Trong nui cy m t bo thc vt, hu ht cc c quan hnh thnh t cc mu cy
hoc t cc callus c mt cu trc d cng nhn, v d nh l chi, r hoc phi v tnh
(somatic embryo). Tuy nhin, nu trong qu trnh bin i thnh cc c quan ny c mt
s qu trnh c iu tit bn trong ca c th khng c hon tt hoc ngng li hn.
iu ny c th dn n kt qu hnh thnh nn cc c quan mi c cc cu trc bt
thng. Cc cu trc ny c nhiu kiu hnh nh:
- Cu trc ging l (Leaf like structure)
- R bt thng (abnormal roots)
- C quan ging nh hoa (floral organoids)
- Gi mm (Pseudobud)
- Cu trc ging c (Tuber like structure)
- Gi hnh (Pseudobulbils)
- Phi bt thng (abnormal somatic embryo)
Hnh 8.33 S bt thng v cu trc c th gp phi trong qu trnh nui cy in
vitro (George, 1993)
159
Hnh 8.34 Cu trc bt thng dng cm chi v bnh thng ca cy Delphinium
S hnh thnh phi v
tnh cy c mi
S pht trin cc cu trc bt thng ca phi v tnh
Hnh 8.35 Cc giai an thnh lp chi v r t phi bt thng ca cy c mi
(Toan, 2003)
8.8 CC DNG TH KHM(Chimeras)
Hnh 8.36 Dng th khm hoa Rhondodendron simsii Aquarel
T chc phn sinh m chi:
Phn sinh m chi thc vt bc cao l a bo v nhiu lp
Hu ht cy song t dip: 3 lp phn sinh m:
160
o 2 Lp tunica (L1 v L2)
o 1 Lp corpus (L3)
Cy n t dip: 2- hoc 3-lp phn sinh m chi
Thng thng, mt mt phng phn chia t bo ngn cn s pha trn ca cc lp t
bo ny.
Hnh 8.37 nh sinh trng nhiu lp
Mi lp t bo phn sinh m gp phn hnh thnh nn hnh thi thc vt:
L1: Biu b l
Biu b thn (bao gm gai)
Biu b hoa
L2: Nhu m dip nhc v ba l
Giao t (gametes)
Cnh hoa
Thn
L3: Nhu m xp ca l, mch
R
Phn trung tm ca than
Th khm (Chimeras):
nh ngha: Mt th khm (chimeras) ca cy l do t bo ca mt hoc hai lp phn sinh
m c kiu di truyn khc nhau.
Hnh 8.38 Dng th khm l cy Draceana
161
Cc kiu th khm
Th khm hnh qut
(Sectorial
chimera)
Mt ci nm ca cc lp t bo
khc nhau v di truyn hin din
xuyn qua tt c cc lp t bo
phn sinh m chi
Th khm vng cung
(Mericlinal
chimera)
Mt phn ca mt hoc hai lp t
bo phn sinh m di truyn ring
bit
Th khm theo chu vi
(Periclinal
chimera)
Mt hoc hai lp t bo phn sinh
m hon ton di truyn khc nhau
Nhn ca s t bin lc lp
Bt k s thay i di truyn t bo ca phn sinh m chi c th to chimeras
t bin sc t t bin lc lp
Hnh 8.39 t bin sc t v t bin lc lp
Cc yu t di truyn i ch
mt s loi thc vt, mt lot cc yu t di truyn c th c i ch thng
xuyn t mt v tr trn nhim sc th sang mt v tr khc, hoc gy ct t nhim sc
th, dn n mt th khm di truyn. Khi mt s ngn cn nhp vo vi chc nng ca
gen sc t, s to thnh cc m mu khc nhau xy ra. Hin tng ny c m t cy
Bp v cy D Yn Tho (petunia).
162
Hnh 8.40 t bin Gen Petunia
Th khm do ghp (Graft chimeras)
Th khm do ghp t pht (Spontaneous graft chimeras)
Anno 1674 'Bizzarria' orange: Mt nh lm vn Florentine ghp mc ghp
ca cy Cam Chua trn trn gc ghp ca cy Chanh Thanh Yn (citron). Mc
ghp khng thnh cng nhng mt mm pht sinh t callus pht trin trn gc
ghp pht trin thnh cy bt thng. Trn cng cy c l, hoa v tri ging vi
cam hoc ging vi citron v c cc tri hn hp vi hai loi xen k nhau hoc to
thnh hnh qut theo nhiu cch khc nhau.
Th khm ghp tng hp (Synthethic graft chimeras)
Trong h Solanaceae nhiu th khm ghp c tng hp. V d:
Lycopersicum pimpinellifolium + Solanum nigrum (Brabec, 1960)
Solanum tuberosum + Lycopersicum esculentum (Jorgensen 1927)
Nicotiana tabacum + Nicotiana glauca (Marcotriguano v Gouin, 1994)
S tng hp trong in vitro:
Callus pha trn (Mixing callus)
Cc chi th khm c th c ti sinh in vitro t callus bao gm cc t bo c cc
kiu gen khc nhau. Callus nh th c th c ngun gc do t bin c kch thch hoc
do s pha trn callus ca hai (hoc nhiu hn) cy c di truyn khc nhau. Cc chi c
kch thch phi c ngun gc a bo.
163
Hnh 8.41 Callus pha trn (Debergh, 2003)
Ghp trong in vitro
Trong in vivo cng nh in vitro, cc t bo ca gc ghp ln mt ghp c th ng
gp cho s to ra cc chi bt nh ni tip hp. Sau khi s ni ghp c lm lnh li,
tt c mt ghp b ly i tr mt lp mng. Tt c cc chi bt nh khng pht sinh t
ni ghp c ly i (hnh 8.42).
Hnh 8.42 Ghp trong in vitro
Nhn chimeras in vitro
Chimeras phi c nhn t chi nh hoc chi ngang. Khng bao gi s dng
chi bt nh hoc phi v tnh (somatic embryos) bi v trong hu ht trng hp chng
ti sinh t mt t bo ca lp L1, L2 hoc lp L3.
164
Hnh 8.43 Draceana th khm c nhn t chi ngang
S phn hy ca th khm (chimeras) in vitro
Chimeras c th b phn hy bng cch kch thch chi bt nh hoc phi v tnh
bi v hu ht chng c ngun gc t t bo n.
Hnh 8.44 Th khm Azalea ny c th b phn hy thnh ging (carmine) v
trng
165
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