Indian Journal of Biotechnology

Vol 10, July 2011, pp 362-368

In vitro multiplication of Merton I. 793An apple rootstock
suitable for replantation
Mohit Soni, Manisha Thakur and Manju Modgil*
Department of Biotechnology, Dr Y S Parmar University of Horticulture and Forestry,
Nauni 173 230 , Solan, India
Received 27 April 2010 ; revised 6 September 2010; accepted 10 November 2010
In vitro shoot multiplication of Merton I. 793, a clonal apple rootstock, was achieved through terminal/axillary bud
culture using Murashige and Skoog (MS) basal medium supplemented with 0.5-1.0 mg/L 6-benzyl amino purine (BA) and
0.1 mg/L indole-3-butyric acid (IBA). Time of collection of explants, duration of surface sterilents and growth regulators
added in the medium influenced the survival of explants and their bud break. Although, the shoots were multiplied on
different concentrations of cytokinins and auxins, improved multiplication was achieved with 0.5 mg/L BA and 0.01 mg/L
IBA. Of the three types of explants evaluated for shoot multiplication, shoot tips on elongation were found suitable for
rooting whereas, nodal cuttings and cluster of small shoots for further multiplication. Low concentrations of -naphthyl
acetic acid (NAA) was proved to be more effective for rooting as compared to IBA and indole-3-acetic acid (IAA). To
overcome profuse callus formation during rooting, activated charcoal was added. In another experiment, auxin was
discontinued from the rooting medium after few days, thus, resulting in better rooting frequency (72%). Thirty plantlets of
Merton I. 793 were successfully hardened after 7 wks, with around 80% survival rate.
Keywords: Apple rootstocks, in vitro multiplication, Merton I. 793, in vitro rooting, micropropagation
Introduction
In recent years, productivity of apple orchards in
Himachal Pradesh has been decreased thereby
causing a serious concern for the apple growers
1
.
Main causes for decline in production and
productivity are (i) non-availability of high quality
and healthy planting material, (ii) proper selection of
varieties of rootstocks of; and (iii) inadequate
adoption of advanced production technologies. Most
of the commercial plantations are now more than 35-
40-year-old and have surpassed the commercial
bearing life and need to be replaced
1
. Because of
land limitation, growers are compelled to plant new
apples on old apple sites. There has been increasing
concern about poor growth, delayed fruiting and the
short life of apple trees planted on sites where apple
trees grew before
2
. This situation resulting in the
poor growth and making plantation uneconomical is
generally known as the problem of replantation.
However, a clonal rootstock of Merton Immune
series, Merton I. 793, is found resistant to diseases
spread during replantation in orchards
2
.
The Merton Immune series M.I. 788 to 793 was
introduced in 1930 as a result of joint breeding
programme by the John Innes Horticultural Institution
and the East Malling Research Station, followed by
Malling Merton (MM) series. In India, first rootstock
trial was initiated in 1937 at Chaubattia (Uttarakhand)
with Red Delicious, Jonathan, Rymer on M2, M13,
Merton I. 779, Merton I.793. Merton I. 793 is
obtained from a cross between `Northern Spy' (woolly
aphid resistant variety) M.2. It was introduced in
Himachal Pradesh (HP) in the year 2000-2001, from
New Zealand. It is vigorous, resistant to collar rot and
woolly apple aphid. In H P, the plant material is being
multiplied using conventional methods but is still in
short supply. It has been reported earlier that M12
rootstock should only be used where root diseases are
the problem, otherwise MM115 and M.I.793 are
recommended for lighter soils and M.I.793 for heavier
soils with chloropicrin fumigation
3
.
In vitro multiplication of plants is a commercial
venture in several countries with the Netherlands,
France and Italy as the world leaders. Among the fruit
crops, temperate fruits share significant portion of the
plants produced through tissue culture. Propagation of
apple rootstocks using tissue culture methods has
been developed by many scientists
4-7
. To the best of

*Author for correspondence:
Tel: 91-1792-252639; Fax: 91-1792-252242
Mobile: 09459241331
E-mail: manju_modgil@yahoo.com
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363
our knowledge, there are very limited reports
available on M.I.793 micropropagation
8-9
. Keeping in
view the recommendations of H P Directorate of
Horticulture to multiply Merton I. 793 on a large-
scale the present study was initiated
10
.

Materials and Methods

Collection of Plant Material
The shoots of apple rootstock Merton I. 793 were
collected in plastic bags throughout the year from the
nursery of H P Horticulture Department, Kwagdhar,
District Sirmour (HP), India. The procedure for explant
collection, excision and approaches to overcome
phenol exudation were similar to as described
previously
5
. The terminal and axillary buds (0.5-2.0
cm) were washed under running tap water for about 1 h
and treated with 70% alcohol followed by surface
sterilization using sterilents 0.1% HgCl
2
for 3-4 min
and 2% NaOCl for 20-25 min separately. The buds
were rinsed with sterile distilled water to remove
traces of the sterilent. The explants were placed on to
petridish lined with filter paper, and cultured in 100
mL flasks containing basal liquid Murashige and
Skoog medium (MS)
11
. The flasks were rotated on a
shaker for one day to rinse the phenols, which exuded
from the cut surface of explants.

Culturing of Explants For Establishment
After rinsing of phenols in liquid medium, the
explants were inoculated on MS medium having
adsorbent polyvinyl pyrollidone (PVP) (10 g/L) and
supplemented with different combinations of benzyl
adenine (BA) (0.5-2.0 mg/L) and indole-3-butyric
acid (IBA)/ napthyl acetic acid (NAA) (0.1 mg/L).
The medium was solidified with 0.8% agar. The
explants which showed vigorous bud break were
selected for transfer to fresh medium for further
growth and multiple shoot production.

Shoot Multiplication
The shoot cultures were maintained and multiplied
by the method of enhanced release of axillary buds.
MS medium consisted of different combinations and
concentrations of plant growth regulators like BA,
kinetin (Kn), 2-isopentyl adenine (2ip), IBA,
gibberellic acid (GA
3
). Best cytokinin and auxin
concentration for multiple shoot formation was
determined by recording the number of shoots
produced per explant and length of shoots (placed
vertically or horizontally) after 5 wks of culture.
Further, the effect of explant type on shoot
multiplication was observed by comparing single shoot
tips with other explant types such as nodal cuttings and
cluster of 2-3 shoots. 6-10 cultures were examined per
treatment and experiment was repeated twice.

In vitro Induction of Rooting
Vigorously growing shoots from proliferating
cultures were used to make cuttings of more than
1 cm long (microshoots) and cultured on 1/2 strength
MS medium consisting of 25 g/L sucrose, 100 mg/L
inositol, 0.5 mg/L thiamine and 6 g/L agar, which was
further supplemented with different concentrations
and combinations of auxins viz. IBA (0.3-0.7 mg/L),
IAA (Indole-3-acetic acid) (0.5-1 mg/L) and NAA
(0.1-0.5mg/L) in one-step method. Activated charcoal
(0.2%) was supplemented in some of the above
combinations. Two-step rooting procedure was
used where microshoots were first cultured in NAA
(0.1-0.5 mg/L) enriched liquid rooting medium. After
one wk, these were transferred to same medium
devoid of NAA but solidified with agar. Each
experiment consisted of three replications with 30
cuttings. Rooting response was noted after 4 wks.
All the cultures were incubated at 252C. Light
consisted of white fluorescent tubes and an
incandescent lamp. The intensity of light at the level
of cultures was 4000 lux with photoperiod of 16 h
light and 8 h dark.

Hardening
Plantlets with adequate roots were removed from
the culture vessels and cleaned under running tap
water. After washing, plantlets were treated with
0.25% bavistin for 30 min and transferred to pots
filled with coco peat which was already drenched
with 5 g/L bicontrol agent (Trichoderma viridae with
commercial name Defence or Ecoderma). They
were maintained in shade in glass house at 202C
with the help of misting, cooling pads and fans. Plants
were irrigated with Knops nutrient solution and
fungicide after every 15 d.

Results
It has been observed that only 6-12% explants died
due to phenol exudation and contamination in the buds
collected during spring or summer season (Table 1).
Further, the explants collected in summer survived
maximum (85%) in cultures after 4 wks whereas
those collected in spring showed maximum bud break
(43.90%). Effect of sterilizing agents on explants
revealed that treatment with 0.1% HgCl
2
for 4 min
resulted in 90% uncontaminated buds but only 18%
INDIAN J BIOTECHNOL, JULY 2011


364
explants survived after 4 wks, while 3 min duration
resulted in 85% explants survival. On the other hand,
bud explants survived after 4 wks were more (71%)
by using 2% NaOCl for 25 min, as compared to
explants treated for 20 min duration.
Terminal/apical buds elongated into small shoots
after 4-6 wks on MS medium containing 1 mg/L BA
and 0.1 mg/L IBA (Fig. 1a) which increased in length
upon subculturing on fresh medium (Fig. 1b) while
stimulation of axillary buds led to rosette type of
leaves. It is evident from Table 2 that the growth
regulator composition significantly affected the
establishment of cultures. Out of 14 combinations
attempted, maximum bud break (87.50%) was achieved
on medium containing 1 mg/L BA and 0.1 mg/L IBA
followed by 0.5 mg/L BA and 0.1 mg/L IBA (70.83%).
Medium supplemented with BA (0.5-2.0 mg/L) alone
resulted in reduced number (12-54%) of bud breaks.
On the other hand, moderate callus formation was
observed in bud cultures with BA (0.5-2.0 mg/L) and
NAA (0.1 mg/L) which were discarded.

In vitro Shoot Multiplication
When shoots, excised from established multiple
shoot cultures, were placed vertically, multiplication
was seen in all the combinations tested (Table 3).
Highest multiplication rate (8-fold) with 2-3.5 cm long
shoots was obtained on medium having 0.5 mg /L BA
and 0.01 mg/L IBA (Fig. 1c). From each explant 2-5
shoots were obtained on 1.0 mg/L BA, 0.05 mg/L
IBA and 0.5 mg/L GA
3
where some of the shoots
were vitrified. However, this rate of shoot
multiplication was not observed in subsequent
subcultures. Five-fold multiplication and longer
shoots were achieved when 1.0 mg/L 2ip was
substituted with BA in above combination. However,
five shoots per explant were obtained in very few
cultures. A 4-fold shoot multiplication was achieved
when BA alone at low concentration (0.5 mg/L) was
used, while at higher concentrations (1.0 & 1.5 mg/L),
shoot production and length reduced. Addition of
Kn (0.5-1.0 mg/L) alone or in combination with BA
(0.5 mg/L) and IBA (0.05 mg/L) did not show any
increase in multiplication of shoots.
When the explants were placed horizontally on the
medium (Table 3), 2-3 small shoots were observed to
arise from nodes. While investigating the type of
explant on medium supplemented with 0.5 mg/L BA
Table 1Effect of time of year on browning, contamination and survival of explants in M.I. 793, on MS medium
supplemented with 0.5 mg/L BA and 0.1 mg/L IBA
S.No. Time of year Explants browning due to
phenol exudation (%)
Explants
contaminated (%)
Explants survived after
4 wks (%)
Explants showing bud break
(%)
1. Spring 10.37
(18.79)
12.26
(20.50)
77.35
(61.61)
43.90 (42.48)

2. Summer 6.60
(14.74 )
8.49
(16.95 )
84.90
(67.17)
35.55 (35.41)

3. Autumn 37.73
(37.91)
32.07
(34.51)
30.18
(33.34)
28.12 (32.04)

4. Winter 29.24
(32.75)
53.77
(47.19)
16.98
(24.35)
22.22 (28.04)

S.E. 0.78 0.81 0.36 1.15
C.D.0.05 1.81 1.88 0.83 2.65
Values in parenthesis are arc sine transformed values.


Fig. 1a-iMicropropagation of clonal apple rootstock M.I. 793:
a, Bud break in terminal/axillary buds on MS medium with 1.0
mg/L BA and 0.1 mg/L IBA; b, Elongation of shoot after 2
months; c, Shoot production on MS medium with 0.5 mg/L BA &
0.05 mg/L IBA; d-f, shoots with roots and callus development on
0.1 mg/L NAA, 0.7 mg/L IBA. and 0.8 mg/L IAA + 0.2 mg/l
IBA, respectively; g, Rooted shoots in two-step procedure; h,
Rooted shoots on activated charcoal supplemented medium; i,
Hardened plants grown in paper cups.
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365
& 0.01 mg/L IBA, it was found that the shoot tips
grew longer (3-4 cm), with few multiple shoots (2-5)
while nodal cuttings and clusters of 2-3 shoots
produced smaller (2.0-2.5 cm) but comparatively
more number of multiple shoots (3-8).

In vitro Root Induction
When IBA, IAA or NAA were tested, rooting was
observed in a few cases only (Table 4). Basal portion
of the shoots formed callus and later root initials
developed after about 15 d. Among the three
concentrations of NAA used, highest rooting
percentage (66.78) was observed with low
concentration (0.1 mg/L) of NAA (Fig. 1d). Around
50% of shoots were rooted on medium supplemented
with 0.7 mg/L IBA (Fig. 1e) whereas 27% rooted in
case of 1 mg/L IAA. Lower levels of IBA and IAA did
not initiate roots. In the presence of activated charcoal
(AC), thin roots were initiated after 2 wks and 55%
rooting was observed with 0.1 mg/L NAA. It is clear
from Table 4 that AC did not enhance rooting in any
treatment but suppressed callus formation (Fig. 1f).
In two-step rooting procedure, root initials were seen
one week after transferring the shoots to basal solid
medium. At the end of 4 wks, 72% rooting (Fig. 1g,
Table 4) was observed in shoots dipped in 0.1 mg/L
NAA followed by 57 and 35.9% rooting of shoots in
0.3 and 0.5 mg/L NAA, respectively. Combined effect
of IBA and IAA showed that (Table 5), 0.2 mg/L IBA
and 0.8 mg/L IAA resulted in 36.36% rooting with
emergence of callus (Fig. 1h), whereas low
concentrations of IAA (0.2, 0.4 & 0.6 mg/L) along with
0.2 mg/L IBA gave reduced rate of rooting (22-28%)
without callus. There is not much difference in rooting
percentage (30-33%) on medium containing 1 mg/L
IAA with 0.1 and 0.3 mg/L IBA.

Acclimatization in Potting Mixture
Rooted plantlets (30) without callus were
successfully acclimatized with an average of
80-85% survival rate while plantlets with callus
survived less. Knops nutrient solution seemed to
enhance the growth of the plantlets (Fig. 1i).

Discussion
Although axillary bud cultures of Merton I. 793
could be initiated at any time of the year, the explants
from actively growing shoots at the beginning of spring
season generally gave best results which showed that
the success in the establishment of explants was
dependent on the time of year of collection and
physiological state of the parent plant at the time of
explant excision. In addition, surface sterilization was
found to be the most critical factor for successful
establishment of bud cultures. Both the sterilants
(HgCl
2
and NaOCl) were effective but HgCl
2
proved
better. Webster and Jones supported our findings
12
. In
the present study, though HgCl
2
treatment for 4 min
provided highest number of uncontaminated buds, the
survival of explants in later stages was more in
3 min duration. It explains that finding the exact
duration of sterilant treatment is necessary for
determining the survival of explants, and higher
durations may be toxic to the explants itself.
Out of all the combinations, BA (0.5-1.0 mg/L) and
IBA (0.1 mg/L) were found the most suitable for
establishment of explants. When GA
3
was used with
BA and IBA, the results were not satisfactory.
In other apple rootstocks, many workers found
maximum bud break on media supplemented with
GA
3
along with BA and IBA
13-15
. This difference may
be due to the different genotypes used in our studies.
There is an earlier report on in vitro shoot
proliferation of Merton I. 793 where the explants were
established successfully

on MS medium supplemented
with BA (0.5 mg/L) alone
8
.
During shoot multiplication, BA proved the most
effective cytokinin, and a relationship between BA
concentration, shoot number and shoot size was
Table 2Effect of different combinations of growth regulators
in MS for explant initiation in M.I. 793
Concentrations of growth
regulators (mg L
-1
)
BA IBA NAA GA
3

Explants
showing bud
break after 6
wks (%)
Survival of
explants after 1
st

sub-culture (%)
0.5 - - -
0.5 0.1 - -
0.5 - 0.1 -
0.5 0.1 - 0.5
1.0 - - -
1.0 0.1 - -
1.0 - 0.1 -
1.0 0.1 - 1.0
1.5 - - -
1.5 0.1 - -
1.5 - 0.1 -
2.0 - - -
2.0 0.1 - -
2.0 - 0.1 -
S.E.
C.D.
0.05

41.66 (40.22)
70.83 (57.34)
33.33 (35.28)
20.83 (27.17)
54.16 (47.51)
87.50 (69.33)
20.83 (27.17)
8.33 (16.78)
20.83 (27.17)
29.16 (32.70)
12.50 (20.71)
12.50 (20.71)
8.33 (16.78)
4.16 (11.77)
0.32
0.66
50.00 (51.69)
76.47 (61.01)
25.00 (30.23)
20.00 (26.57)
61.53 (45.02)
90.47 (72.08)
20.00 (26.57)
0.00 (0.00)
40.00 (39.25)
28.57 (32.33)
33.33 (35.28)
33.33 (35.28)
50.00 (45.02)
0.00 (0.00)
1.20
2.46
Values in parenthesis are arc sine transformed values.
S.E.-Standard error
C.D.-Critical difference
INDIAN J BIOTECHNOL, JULY 2011


366

also observed in the present investigation. Higher
concentration of BA alone may increase the shoot
number with decreased shoot length whereas low
concentrations led to longer shoots. BA (0.5 mg/L)
combined with IBA (0.01 mg/L), was found to
increase the rate of shoot production as well as length
of shoots. After supplementing GA
3
(0.5 mg/L) with
above combination, number of shoots produced were
not constant in each subculture. This combination had
also been found to be better for the micropropagation
of several other apple rootstocks
5,14,15
. Use of 2 ip in
place of BA in combination with 0.05 mg/L IBA was
not found effective and on adding GA
3,
the

rate of
shoot production increased in few cultures only.
However, in case of Java apple, shoot proliferation
was stimulated by the addition of BA (1 mg/L) to MS
basal medium
16
but shoot elongation and shoot
number was enhanced with 2 ip (1 mg/L) and NAA
(0.1 mg/L). This difference may be due to the
different genotype and type of auxin used with 2ip.
Kinetin with BA or IBA did not enhance rate of shoot
multiplication in M.I. 793. It has been suggested that
differential sensitivity to a particular cytokinin and
auxin, to the concentration of each, and to the ratio
between them are important factors, especially in the
establishment and proliferation of cultures
17
.
Removal of tip and inoculating shoots horizontally
or inverted in the medium reduced the apical
dominance, thus encouraging the development of
Table 3Effect of different combinations of plant growth regulators on the multiplication of shoots (after 5 wks)
MS medium supplemented with (mg L
-1
)
BA IBA Kn 2 ip GA
3

Shoot placement No. of shoots obtained
from a single shoot
Length of shoots
(cm)
No. of cultures
examined
0.5 - - - - Vertical
Horizontal
1 : 1-4
1: 1-2
1.0-3.0 8
1.0 - - - - Vertical 1 : 2-3 1.0-1.5 10
1.0 0.1 - - - Vertical 1 : 2-4 1.5-4.0 24
1.5 - - - - Vertical 1 : 1-2 1.0-2.0 6
0.5 - 0.5 - - Vertical 1 : 2-3 1.0-3.5 12
- - 1.0 - - Vertical 1 : 1-2 1.0-2.0 6
0.5 0.05 0.5 - - Vertical 1 : 1-2 1.0-1.5 6
0.5 0.01 - - 0.5 Vertical
Horizontal
1 : 3-4
1 : 2-3
1.0-2.5 10
1.0 0.05 - - 0.5 Vertical 1 : 2-3 1.0-2.0 10
- 0.05 - 1.0 - Vertical
Horizontal
1 : 1-2
1 : 1-3
1.0-1.5 8
- 0.05 - 1.0 0.5 Vertical
Horizontal
1 : 2-5
1 : 1-2
1.0-2.5 8
1.0 0.05 - - - Vertical 1 : 1-3 1.0-2.0 12
1.0 0.05 - - 0.5 Vertical 1 : 2-5 0.5-1.5 7
1.0 - - - 0.5 Vertical 1 : 2-4 0.5-1.0 7
0.5 - - - 0.5 Vertical 1 : 2-4 1.0-3.0 6
0.5 0.01 - - - Vertical
Horizontal
1 : 3-8
1 : 1-2
2.0-3.5 32
Table 4Effect of different concentrations of auxins along with
the presence or absence of AC (0.2%), and two-step procedure on
rooting of microshoots
Growth regulators
(mg/L)
Rooting percentage
in one-step
Rooting
percentage
in two-step
IBA NAA IAA Without AC With AC
0.3 - - 0.00(0.00) - -
0.5 - - 0.00(0.00) - -
0.7 - - 50.00(4.041)+ 32.6(34.45) -
- 0.1 - 66.78(54.83)+ 55.2(48.04) 72 (57.90)
- 0.3 - 42.00(40.40)+ 44.0(41.55) 57 (48.79)
- 0.5 - 52.18(46.25)+ 33.0(34.86) 35.9(37.76)
- - 0.5 0.00(0.00) - -
- - 0.8 0.00(0.00) - -
- - 1.0 27.70(31.75) - -
CD
(0.05)
1.1362 1.449 1.510
Callus formation indicated by + or -
Figures in parentheses are arc sine transformed values.

Table 5Combined effect of different concentrations of
IBA and IAA on rooting in M.I. 793
IBA Conc. (mg/L) IAA Conc. (mg/L) Rooting percentage
0.1
0.2
0.2
0.2
0.2
0.1
0.3
0.1
0.2
0.4
0.6
0.8
1.0
1.0
0.00 (0.00)
28.38 (32.19) -
22.20 (28.11) -
27.20 (31.44) +
36.36 (37.09) +
30.33 (33.49) +
32.97 (35.04) +
CD(0.05) 0.438
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367
axillary buds
18
, which might have resulted in superior
utilization of BA. However, there was no advantage
seen in shoot production by horizontal placement of
shoots in M.I. 793. This is in contrast to earlier studies
on other apple rootstocks where higher multiplication
rate was reported by horizontal positioning of the
explants
19
. This factor may be dependent on genotype
and growth regulator combinations. Three types of
explants assessed for shoot multiplication in the present
study resulted in multiple shoot production but, the
main advantage of such type of evaluation was that
shoots grew from shoot tips were used for rooting
being longer whereas shoots produced by other
explants were spared for further shoot multiplication.
Among the three auxins, NAA was found superior
to IAA and IBA, though thick and callused roots were
obtained in one-step procedure. Low levels of NAA
resulted in the highest rooting whereas high
concentrations gave rise to more root initials, which
eventually developed into callus rather than rootlets.
In contrast to our studies, earlier reports on Merton I.
793 reported that IBA stimulated high rooting
abilities
8,20
. This may be due to the different age of
cultures, or growth regulators present in shoot
multiplication medium used by them differed from
ours, or these differences of in vitro rooting ability
may be due to clonal variation in the rootstock used.
In our experiments, after supplementing the medium
with activated charcoal (AC), callus formation was
stopped, but the rooting frequency decreased. These
results are supported by Magyar et al

that the presence
of AC in root elongation medium decreased the
rooting rate and number of roots depending upon the
cultivar and the concentration
21
. The favourable effect
of AC on rooting was mainly due to the adsorption of
NAA. In our previous studies
22
, efficacy of different
concentrations of IBA in combination with activated
charcoal was tested and found that the formation of
roots in apple cultivar shoots growing in vitro was
variable with considerable genotypic difference.
During the present study, combining IAA and IBA in
different concentrations did not support good rooting
efficiency. On the other hand, for rooting of shoots,
two-step procedure appeared to be more effective than
one-step because it had the major advantage of
leading to increase in rooting efficiency without callus
development. It seems that both auxin type and
concentration influence rooting in Merton I.793 and
continuous contact with auxins led to the callus
formation followed by the emergence of roots. Since
too long treatment of auxin leads to root inhibition it
has been suggested that the auxin treatment must be
appropriately interrupted by transplanting the root
induced shoots on hormone free medium
23
.
In vitro rooting stage is followed by hardening,
which involves the transfer of plants from in vitro
conditions to soil to impact some tolerance to
moisture stress, for conferring a degree of resistance
against certain pathogens and conversion of the plant
from heterotrophic to autotrophic stage. We have
observed that rooted plantlets without callus
successfully established in peat. Better root and shoot
development before hardening determines the survival
which is also supported by other workers
4
. Fertigation
during hardening of Merton I. 793 improved the
vigour of rooted plantlets, but the nutritional
requirement must be adjusted to plant size and age
24
.
Application of complete nutrient solution similar to
that used for raising bedding plants can be applied
as both liquid and foliar feed
26
.
In conclusion, our results suggest that a
micropropagation method has been accomplished in
Merton I. 793. For the commercial scale production of
plants, further efforts are in progress to increase the
rooting efficiency, and thereby hardening.

Acknowledgement
We acknowledge the Department of Biotechnology,
New Delhi, Govt. of India for financial assistance
in the form of a project, under Network Programme
on Apple.

References
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prospects, in The apple: Improvement, production and post
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