DETERMINING THE SEX OF PAPAYA FOR

IMPROVED PRODUCTION1)
Pabl i to M. Magdal i ta* and Charl es P. Mercado
*Frui t and Ornamental Crops Di vi si on
I nsti tute of Pl ant Breedi ng
Col l ege of Agri cul ture
Uni versi ty of the Phi l i ppi nes at Los Banos
Col l ege, Laguna, Phi l i ppi nes
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This Bulletin discusses the use of PCR to determine the sex of papaya plants. It discusses the three sex
types of papaya plants (male, female and hermaphrodite), and why predicting the sex of the plant is so im-
portant in papaya production. It outlines the PCR technique used to determine the sex of plants from three
genotypes of papaya: Cariflora, Cavite and Sinta hybrid. The expected frequency based on PCR testing was
a perfect fit with the observed frequency when the plants eventually flowered.
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The papaya, Carica papaya L., is a native of Central and South America. It is a member of the family Caricaceae. This
family consists of 55 species (Dallwitz 1980). Badillo (1971) placed these species into four genera, namely: Carica,
Cyclimorpha, Jacaratia and Jarilla. Carica is the largest genus, with 22 species (Purseglove 1968). Of these 22 species,
Carica papaya is the most economically valuable, and is widely cultivated for its edible fruit. The family is characterized
by trees or shrubs that are erect, small, soft-wooded and fast-growing. Often dioecious or monoecious, they grow to a height
of 3-6 meters. The trunk is usually unbranched, and crowned with large spirally arranged leaves which give the tree a palm-
like appearance. The stem and the leaves produce a milky sap when wounded, a feature found in all members of this family
(Purseglove 1968).
In general, there are three sex types in papaya: male, female and hermaphrodite. However, they cannot be
distinguished at the seedling and vegetative stages of growth. In an open-pollinated species such as papaya, the selection
of the appropriate sex type of the progeny for commercial planting would be beneficial, since only the female and
hermaphrodite plants are grown for fruit. The identification of sex types prior to propagation, especially in polygamous
plant species with a long juvenile cycle such as papaya, would result in higher fruit production and increased profitability.
In sub-tropical areas, dioecious varieties such as Honey Gold (Africa) and Richter Gold (Australia) are preferred,
because of their high yields (Allan 1981). Conversely, in the Philippines, hermaphrodite lines are preferred for commercial
production because of their high yield potential, inbreeding ability and crop uniformity over several generations. In both
cases, being unable to identify the sex type of the seedling prior to planting is a limiting factor.
The sex type of a papaya plant can only be identified 3-6 months after transplanting, when the flowers develop.
Several morphological characteristics such as seed coat color and root morphology have been associated with the sex type
of papaya. Females have been described as having a seed coat which is lighter in color and branched root morphology, while
males are believed to have darker seed coats and straight root morphology. However, these claims have not been proven
scientifically. A loose linkage between flower morphology and sex type has been identified (Storey 1953), but sex
determination based on flower morphology is not possible until four months after planting.
Cytological studies have also been conducted to determine sex types in papaya, in the hope of identifying the possible
existence of a heteromorphic pair, an unpaired chromosome or any chromatin body that may indicate a sex difference (Datta
1971). However, none have been recognized.
A colorometric test for total phenol content could distinguish females (86%) from males (77%), but could not detect
the hermaphrodites (Jindal and Singh 1976). Paper chromatography also indicated that transcinamic acid is dominantly
expressed in the leaves of hermaphroditic seedlings, but females and males could not be differentiated (Paller 1988). In
addition, isozymes (gene products that are phenotypically multiple forms of an enzyme) have been exploited to identify
markers that could co-inherit with sex type in papaya. Using the banding pattern of cationic peroxidase, males could be
differentiated from females, but females could not be distinguished from hermaphrodites (Sriprasertsak et al. 1988).
The failure of morphological tags, cytological evidences and isozyme markers to determine the sex type of papaya
at the seedling stage has led to the analysis of the DNA to determine sex differences. Markers for sex type in papaya that
could be generated through DNA analysis using PCR technology is seen as a reliable strategy. This is because direct analysis
of the genomic DNA allows an accurate determination of genetic variations and assessment of similarities between
individuals and populations, as well as between species and cultivars, thereby eliminating the components of phenotypic
variation and interaction between different alleles of a locus or different loci (epistasis). For example, through PCR, markers
were generated by DNA amplification fingerprinting (DAF) for male, female and hermaphrodite progenies of a cross
between Khaeg Dum (Thai cultivar) and Richter Gold (Australian cultivar) (Somsri et al. 1997). In addition, randomly
amplified polymorphic DNA (RAPD) markers were also developed for interspecific Carica hybrids (Magdalita et al. 1998).
However, these techniques are more complicated than ordinary PCR, so that their use is very limited. Therefore, the
development of simple PCR markers linked with the sex determining locus, and those that co-segregate with sex, is needed
as a reliable genetic marker for predicting the sex of papaya at the early seedling stage.
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Propagation of papaya by seed is still the most practical method of raising the crop, because it is efficient and
economical. Vegetative methods of propagation, such as the use of cuttings, grafting and tissue-cultured materials, are
available but they are laborious and expensive. Commercial papaya planters and most backyard growers depend solely on
seed as planting materials. There are several reasons why a desirable sex type of papaya plant be known to the grower before
planting.
The papaya usually flowers 3-6 months after transplanting, and produces ripe fruits at the age of 9-14 months. The
waiting time from planting to harvesting is long, so growers need to be sure that a seedling is a female or a productive
hermaphroditic plant, either of which will give a good harvest. The determination of the sex type of papaya seedlings prior
to the flowering stage would avoid the need for removing undesired sex types (e.g. males) from the field, thus saving labor,
time and other resources.
Under Philippine conditions, since papaya ringspot virus (PRSV) is often present, the grower should be assured that
the seedling he is planting will bear fruit a year after planting. Papaya is presently being grown as an annual crop because
of the virus problem. The hermaphroditic papaya exhibits sex reversibility depending on prevailing environmental
conditions. Selection of the productive hermaphroditic type (ie. elongata) with desirable fruit form is necessary to assure
the grower of a productive crop.
A knowledge of the sex type of papaya is important in selecting parents for use in hybridization work. Crosses
between females and hermaphrodites will give all fruit-bearing progenies. In addition, for micropropagation, the early
detection or identification of the sex type of a particular papaya seedling would be advantageous, since the desired sex type
can be selected prior to micropropagation. This will ensure that the resulting micropropagated plants are 100% either
females or hermaphrodites.
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There are three types of flowers in papaya: male, female and hermaphrodite or bisexual (Fig. 1). The inflorescence
is a cyme (Storey 1969). Male inflorescences are long, pendulous and freely branching. They are in panicles, 25-100 cm
long, pendent, sessile with a small five-toothed cup-shaped calyx, trumpet-shaped corolla with light yellow lobes and 10
stamens in two whorls alternating with the petal lobes. The female flowers are solitary or in few-flowered cymes, 3-5.5
cm long with a yellowish green cup-shaped calyx. The corolla has five petals that are twisted, whereas the ovary has
numerous ovules and a fan-shaped stigma. There are two types of hermaphrodite flowers: the elongata type with flowers
with short peduncled petals, 10 stamens in two series and an elongated ovary, and the pentandria type, with flowers similar
to female flowers but with five stamens. Intermediate forms occur in which some or all of the stamens become carpelloid
and produce ridged or irregularly shaped or cat-faced fruits. The proportion and type of hermaphrodite flowers produced
may vary, depending on the variety, the age of the tree, the season, the soil moisture, the nitrogen content of the soil, the
temperature, daylength, defoliation, defloration, etc. (Awada 1958).
Papaya flowers are pollinated by natural agents. They may be wind-pollinated, since the pollen is light and abundant.
A few insects, including thrips and moths, can assist in pollinating the sweet-smelling flowers (Purseglove 1968). The
stigma is receptive prior to anthesis, and remains receptive for several days or until the stigmatic lobes turn brown. Self-
pollination of hermaphrodite flowers can easily be achieved by bagging each flower in a glassine bag before anthesis, and
then keeping it covered for at least five days until the fruit begins to develop. Cross-pollination is done by dusting the pollen
onto the stigma of a female flower that is at the balloon stage, and then covering the flower with a glassine bag immediately
to prevent contamination. Fresh pollen is preferred for hybridization. However, pollen stored for about six months at 10oC
and 10% relative humidity can be also used (Cohen et al. 1989). The pollinated fruit is ready for harvest 4-5 months after
pollination. Fruits that develop from hermaphrodite flowers are generally elongated, cylindrical, obovoid or pyriform,
depending on the variety, while those from female flowers are rounded or spherical to oval in shape (Fig. 2). In Australian
varieties, fruits that develop from sexually ambivalent males (SAM) are elongated in shape (Aquilizan 1986).
GENETICS OF SEX EXPRESSION
Sex expression in papaya is controlled by a single gene, with three alleles which have a pleiotropic effect (Hofmeyr
1941, Storey 1953). The sex homologues were designated as: M for male, MH for hermaphrodite and m for female. All
combinations of dominant alleles, such as MM, MHMH and MHH, are lethal to the zygote. This makes all males and
hermaphrodite into enforced sex heteroxygotes. Twenty-five percent of the seeds in their fruits are non-viable. The
genotypes for sex are Mm for female, MHm for hermaphrodite and mm for female. Using these sex genotypes, there are
eight possible cross combinations that could be made with various segregation ratios, as indicated in Table 1.
Self-pollination in males, cross-pollination between males, and cross-pollination between males and hermaphrodites,
can all be done using the sexually ambivalent males (SAMs) that produce perfect flowers during certain periods of the year.
Male and hermaphrodite trees undergo various degrees of sex reversal, depending on seasonal changes and climate (Awada
1958). The female tree is the most stable form.
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Our understanding of the complex genomes of a eukaryotic organism has been enhanced by the discovery of the in
vitro synthesis of a specific stretch in the DNA. This technique of gene amplification was originally conceived by Kary
B. Mullis in 1985, while he was pondering a proposed experiment on DNA sequencing (Mullis 1990). The technique is
now popularly known as polymerase chain reaction (PCR). It is an in vitro and sterile method for the enzymatic synthesis
of specific DNA sequences, using two oligonucleotide primers that hybridize into opposite strands and flank the region of
interest in the target DNA. It replicates a specified nucleic acid sequence in a complex DNA template, and consequently
produces millions and even billions of copies of DNA fragments with defined length and sequence. This in vitro synthesis
requires deoxynucleotide triphosphates (dNTPs), two oligonucleotide primers, a relatively pure DNA template and a DNA
polymerase that remains stable and active at high temperatures.
The cycles involved in PCR are a reflection of in vivo DNA synthesis. PCR is made up of three basic cycles: 1)
denaturation of the heteroduplex DNA template, 2) annealing of the primers at the homologous region sequences of the
template and, 3) binding of the polymerase enzyme and subsequent extension of the primer to synthesize the specified DNA
segment. After the first cycle, two double-stranded DNA fragments are produced which serve as templates for the following
reaction. The repetition of the cycles to about 40 therefore leads to an exponential amplification of the defined DNA
segment.
In predicting the sex type of papaya, the four 20-Mcr oligonucleotide primers shown in Table 2 are used in the PCR
amplification. Primers T1-F and T1-R (primer sequence provided by the University of Hawaii) produced a 1.3 kb PCR
product in both females and hermaphrodites, while W11-F and W11-R produced a 0.8 kb PCR product in hermaphrodites
only. Thus, the hermaphrodites are distinguished by having two distinct bands (1.3 and 0.8 kb). Females have a single band
(0.8 kb), while males have no band.
The standardized reaction mixture being used for PCR has a volume of 25 ?L. The components of the reaction are
indicated in Table 3. The genomic DNA should be loaded individually into each reaction tube. The other components are
mixed together in one tube and suspended over ice. This mixture is called the master mix. From the master mix, 21 ?L is
transferred by aliquot into each reaction tube. The reaction tubes are placed into the PCR block, which has already been
brought to 95oC. In addition to this initial denaturation at 95oC, the denaturation, annealing and polymerization steps are
run for 25 cycles. After these are completed, the reaction remains in the reaction block at 4oC until ready for electrophoresis.
Conditions used for PCR using a MJ PCR machine are shown in Table 4.
To test the reliability of the PCR technique in predicting the three sex types in papaya, three genotypes producing
progenies with known segregation ratios were used. These genotypes included the following.
Cariflora. This is a dioecious inbred line producing male and female progenies only. It is used as a female parent
of the Sinta F1 hybrid. It was released originally by Conover, Litz and Malo (1986) in Florida as a variety tolerant to papaya
ringspot virus.
Cavite. This is a gynodioecious inbred line producing hermaphrodite and female progenies. It is the male parent of
the Sinta F1 hybrid. The original tree, which has moderate tolerance to papaya ringspot virus, was selected in Silang, Cavite
in 1983. Inbreeding, selection and screening for PRSV were conducted on the progeny until a tolerant line was derived.
Sinta hybrid. This is a gynodioecious hybrid from a cross between Cariflora x Cavite (Villegas 1995). It produces
hermaphrodite and female progeny only. This hybrid is moderately tolerant to papaya ringspot virus. The sex types and
ratios of the progeny of these genotypes are shown in Table 5.
Forty-five Sinta hybrids, and 25 Cariflora and 25 Cavite Special plants, were used. All plants were at the vegetative
stage (before flowering commenced). The genomic DNA of these samples was extracted using the Doyle and Doyle (1987)
method, and used for PCR. After the gel was stained with ethidium bromide and visualized under UV light, it was found
that there were 23 and 13 hermaphrodite plants of the Sinta hybrid and Cavite Special, respectively. Three hermaphrodite
plants had two distinct bands: a 1.3 and 0.8 kb fragment. On the other hand, there were 22 female plants of the Sinta hybrid,
while there were 12 Cavite Special and 14 Cariflora hermaphrodite plants. The female plants were found to have only one
distinct band, a 1.3 kb fragment. In addition, it was found that there were 11 male plants of Cariflora, none of which had
a band. In the male plants, there is no available binding site for any of the primers used, hence the failure to flank any stretch
in the DNA template, resulting in the absence of PCR products. In this way, the male genotype can be differentiated from
the female and the hermaphrodite. The negative control did not show any PCR product.
A limiting factor in the use of this marker is the possible occurrence of false-negative results. Thus, during the PCR
run, the male should be run under an optimal genotype and the negative control should always be present. In addition, the
PCR conditions and the PCR reaction mixture should be accurately prepared, to ensure the accuracy and consistency of PCR
amplification reactions.
The 45 Sinta hybrid plants and the 25 plants each of Cavite Speical and Cariflora were inspected during the flowering
stage to determine their sex type, in order to verify the results of the prediction obtained using the PCR technique. The results
of the prediction by PCR was consistent with the observed sex type of the plants tested, as shown in Table 6. This result
shows a 100% accuracy in the prediction of the three sex types in Sinta hybrid, Cavite Special and Cariflora varieties. For
instance, in the Sinta hybrid, 22 females and 23 hermaphrodites were identified using PCR. This prediction corresponded
with the number of females and hermaphrodites actually observed during the flowering stage in the field (Table 6). A sample
of the gel representing the female and hermaphrodite genotypes of the Sinta hybrid is shown in Fig. 3 and Fig. 4.
For the Cavite Special, 12 females and 13 hermaphrodites were identified using the PCR technique. The same number
of females and hermaphrodites were observed at the flowering stage in the field (Table 6). The gel representing the females
and hermaphrodites of the Cavite Special variety is shown in Fig. 5. For the Cariflora variety, 14 females and 11 males were
predicted by PCR. A sample of the gel representing the females and males is shown in Fig. 6. The same number of females
and males were actually observed in this variety when the same plants grew to the flowering stage in the field (Table 6).
The observed frequency of females, hermaphrodites and males, identified as such both by PCR and field observation,
in the three varieties (Sinta hybrid, Cavite Special and Cariflora) were tested for goodness of fit (using Chi-square) with
the expected sex segregation ratios given by Storey (1953). The derivation of the female, hermaphrodite and male progenies
of Sinta hybrid, Cavite Special and Cariflora, including the result obtained by Chi-square tests, are as follows. Female and
hermaphrodite progenies of Sinta hybrid were obtained from a cross between female Cariflora and hermaphrodite Cavite
Special. The expected sex types are female and hermaphrodite at a ratio of 1:1. Chi-square goodness of fit at 1% level of
significance indicates that the observed frequencies match the predicted segregation with a X2 value of 0.36 (Table 7). The
sibbed (cross of female x male) female Cariflora that is expected to segregate at 1 female: 1 male also matched with the
expected sex segregation ratio, with a X2 value of 0.36.
The primers used in this prediction study are highly specific, because of the large number of bases (20-mer
nucleotides). Hence, the chance that the primers would find a homologous sequence in any DNA other than the target site
is low. The technique of sex prediction by PCR is much more precise than the colorometric test (Jindal and Singh 1976),
chromatographic analysis (Paller 1988) or isozyme markers (Sriprasertsak et al. 1988).
The PCR-based markers for sex prediction in papaya have the advantage that they are not affected by environmental
conditions in which the plants are grown or by epistasis (gene interaction). Thus, sex prediction can be done at any
developmental stage of plant growth. However, the PCR technique for sex prediction in papaya at this point in time is costly.
It also requires technical expertise and sophisticated equipment (PCR machine, electrophoresis). At present it would seem
that the technique is useful only for experimental purposes, particularly in developing countries. However, if this technique
could be converted into a dip-stick type method (eg. color reaction type) that is fast, reliable, practical and cheap, it would
become affordable and of great value to plant biologists and papaya growers.
kL|LkLN0LS
Allan, P. 1981. Clonal Honey Gold papaws. Citrus and Sub-tropical Fruit Journal. October 1981.
Aquilizan, F.A. 1986. Breeding systems for fixing stable papaya inbred lines with breeding potential for hy-
brid variety production. In: The Breeding of Horticultural Crops, Proceedings of the International Sympo-
sium held at the National Chung Hsing University, Taiwan, December 1986. FFTC Book Series No. 35.
Taipei, Taiwan, pp. 101-106.
Awada, M. 1958. Relationships of minimum temperature and growth rate with sex expression of papaya
plants (Carica papaya L.). Hawaii Agric. Expt. Stn. Bull. 38: 16 pp.
Badillo, V.M. 1971. Monografia de la Familia Caricaceae. Maracay, Venezuela. 222 pp.
Cohen, E., U. Lavi and P. Spiegel-Roy. 1989. Payaya pollen viability and storage. Scientia Horticulturae
40: 317-324.
Conover, R.A., R.E. Litz, and S.E. Malo. 1986. Cariflora a papaya ringspot virus tolerant papaya for
South Florida and the Caribbean. HortScience 21: 1072.
Jindal, K.K. and R.N. Singh. 1976. Sex determination in vegetative seedlings of Carica papaya by phenolic
tests. Scientia Horticulturae 4: 33-39.
Dallwitz, M.J. 1980. A general system for coding taxonomic descriptions. Taxon 29: 41-46.
Datta, P.C. 1971. Chromosomal biotypes of Carica papaya Linn. Cytologia 36: 555-562.
Doyle, J.J. and Doyle, J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue.
Phytochemistry Bulletin 19: 11-15.
Hofmeyr, J.D.J. 1941. Genetics of Carica papaya L. Chronica Botanica 6: 245-247.
Magdalita, P.M., R.A. Drew, S.W. Adkins and I.D. Godwin. 1997. Morphological, molecular and cytological
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229.
Mullis, K.B. 1990. The unusual origin of the Polymerase Chain Reaction. Scientific American 262: 36-43.
Paller, E. 1988. Difference in phenol content of the male, female and hermaphrodite trees of rambutan
(Nephelium lappaceum L.), pili (Canarium ovatum Engl.) and papaya (Carica papaya L.) through paper
chromatographic analysis. University of the Philippines at Los Banos, Philippines. Unpublished B. Sc.
Thesis.
Purseglove, J.W. 1968. Tropical Crops: Dicotyledons. Essex: Longman Group and Co. Ltd. BurntMill,
United Kingdom.
Somsri, S., M. Jobin, R.A. Drew, W. Lawson and M.W. Graham. 1998. Developing molecular markers for
sex prediction in papaya (Carica papaya L.). Acta Horticulturae 461: 141-148.
Sriprasertsak, P., S. Burikam, S. Attathom and S. Piriyasurawong. 1988. Determination of cultivar and sex
of papaya tissues derived from tissue culture. Kasetsart Journal (Natural Science Supplement) 22: 24-29.
Storey, W.B. 1953. Genetics of papaya. The Journal of Heredity 44: 70-78.
Storey, W.B. 1969. Papaya. In: Outlines of Perennial Crop Breeding in the Tropics, H. B.V. Veenman
Zonen, F.P. Ferwerda and F. Wit (Eds.). Wageningen, Netherlands, pp. 389-407.
Villegas, V.N. 1995. Sinta Hybrid Papaya. Leaflet, IPB, UPLB-CA, College, Laguna, Philippines.
Fig. 1. DiIIerent se types oI papaya I|owers (A) Iema|e (pisti||ate) (B) hermaphrodite (biseua|/
perIect); and (C) ma|e (staminate)
Fig. 2. The shapes oI papaya Iruit are re|ated to the se oI the I|ower types oI papaya p|ants.
Hermaphrodites produce e|ongated Iruits (A), whi|e Iema|es produce rounded Iruits (B).
Fig. 3. PCP banding pattern oI 'Sinta' papaya p|ants grown in the Iie|d generated by primers T1-F,
T1-P, W11-F and W11-P. Lanes 1 and 13. mo|ecu|ar size markers (???? X174 DNA, digested
with Hae III, |ane 2. negative contro| (N, without DNA temp|ate), and |ane 3-12. 'Sinta'
papaya hybrids
H. Hermaphrodite
F. Fema|e
Fig. 4. PCP proIi|e oI 'Sinta' papaya hybrids cu|tured in vitro. Lanes 1 and 17. mo|ecu|ar size marker
(?, ??X 174 DNA digested with Hae III), |ane 2. negative contro| (N, without DNA temp|ate),
and |ane 3-4. 'CariI|ora' Iema|e, |ane 5-6. 'Cavite Specia|' hermaphrodite, and |ane 7-16. their
progenies ('Sinta' papaya hybrids)
H. Hermaphrodite
F. Fema|e
Fig. 5. PCP proIi|e oI the 'Cavite Specia|' generated by primers T1-F, T1-P, W11-F and W11-P. Lane
1 and 13. mo|ecu|ar weight markers (???? X 174 DNA digested with Hae III), |ane 2. negative
contro| (N, without DNA temp|ate), and |anes 3-12. predicted se types (Iema|es and
hermaphrodites) oI the representative p|ants oI 'Cavite Specia|'.
H. Hermaphrodite
F. Fema|e
Fig. 6. PCP proIi|e oI the 'CariI|ora' papaya generated by primers T1-F, T1-P, W11-F and W11-P
Lanes | and 13. mo|ecu|ar weight markers (???? X174 DNA digested with Hae III), |ane 2.
negative contro| (N, without DNA temp|ate, and |ane 3-12. predicted se types (ma|es and
Iema|e) oI 'CariI|ora'.
F. Fema|e
M. Ma|e
Tab|e 1. Cross combinations invo|ving the three se genotypes oI papaya, and the resu|ting se
segregation ratios oI the progenies
Tab|e 2. Primers used Ior PCP and their nuc|eotide sequences. The primer sequence was provided by
the University oI Hawaii.
Tab|e 3. Components oI the PCP miture and the vo|ume needed Ior one standard reaction used Ior se
determination in papaya
Tab|e 4. The PCP cyc|ing conditions used Ior se determination in papaya
Tab|e 5. Genotypes, and the ratios oI the se types oI the progeny
Irom each genotype
Tab|e 6. Comparison oI the predicted se type shown by PCP, and the actua| se type oI the p|ants as
observed in the Iie|d at the I|owering stage Ior the three papaya genotypes
Tab|e 7. Segregation ratio oI various crosses oI the three types oI sees invo|ving Sinta hybrid, Cavite
Specia| and CariI|ora