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Cross compatibility of elite papaya inbred lines to an intergeneric hybrid of Carica papaya L. x Vasconcella quercifolia (Saint-Hil.) Hieron

Andres Godwin C. Sajise1, Simeona V. Siar1 and Juanito B. Sangalang2

1Institute of Plant Breeding and 2Department of Horticulture, College of Agriculture, University of the Philippines Los Baos (UPLB) College Laguna 4130,www.ipb-uplb.org.ph Emailandysajise@yahoo.com , moninasiar@yahoo.com and jay_bees2003@yahoo.com

Abstract

Cross compatibility of four papaya inbred lines to an F1 intergeneric hybrid, Carica papaya x Vasconcella quercifolia line 410 was studied. Parental lines used were characterized morphologically. Resistance to papaya ringspot virus (PRSV-P) and pollen viability of line 410 was evaluated under local conditions. The genotypic compatibility among four crosses between the F1 intergeneric hybrid and papaya inbreds (4108, 4172, 5648, and 5893) was evident. Crosses to inbreds 5648 and 5893 produced fruits filled with many non-viable seeds while 4108 and 4172 produced few seeds but had embryos that grew in vitro. Remarkable differences were observed in morphological characters among papaya inbreds as well as between papaya and F1 intergeneric hybrid line 410. Cluster analysis divided parent materials into three. Papaya inbreds derived from local selections (4108 and 4172) and inbreds that were introduced from other countries (5648 and 5893) were on separate clusters. Line 410 has a separate cluster of its own.

Symptomatology and serological test by indirect ELISA (Enzyme Linked Immunosorbent Assay) confirmed that intergeneric hybrid line 410 plants have resistance against PRSV-P. A pollen viability assay using tetrazolium showed some viable pollen under local conditions.

Media summary

Line 410 (C. papaya x Vasconcella quercifolia hybrid) had PRSV-P resistance and some pollen fertility in Philippines and showed cross compatibility with IPB inbreds 4108 and 4172.

Keywords

Backcross, symptomless, culturable embryos, plantlets and parthenocarpy

Introduction

Genetic variation for resistance to virus diseases is absent in domesticated papayas and this has motivated breeders to extend the search into a secondary gene pool. Many of the wild Vasconcella species, which until 2000 were members of genus Carica (Badillo, 2000) are intercompatible and can be cross-pollinated to produce hybrids with various degrees of fertility (Aradhya et al., 1999). Hybrids between C. papaya and V. quercifolia produced in Australia and Hawaii have demonstrated PRSV-P resistance and some fertility (Drew et al, 1998).

Litz and Conover (1983) reported in a study of C. papaya x V. cauliflora crosses that success varied considerably depending on the genotype of the papaya parent used. This conclusion was also confirmed by Manshardt and Wenslaff (1989) in Hawaii and Magdalita et al. (1998) in Australia.

The development of PRSV resistant papaya varieties with gene/genes from V. quercifolia through conventional means by introgressing resistant genotypes to elite inbred lines of IPB (Institute of Plant Breeding) is possible. However, the first and preliminary step to achieve this goal is to assess the compatibility of inbred lines to the fertile intergeneric hybrid that was available. Hence, cross compatibility of four elite papaya inbred lines to the F1 intergeneric hybrid, Carica papaya x V. quercifolia line 410 was studied. Parental lines used were characterized morphologically. Resistance to papaya ringspot virus (PRSV-P) and pollen viability of line 410 were also evaluated under local conditions.

Materials and methods

Four papaya inbred lines (4108, 4172, 5648, 5893) and an intergeneric hybrid of C. papaya x V. quercifolia line 410 were used as parents in the crosses. Line 410 was obtained from the hybrid selections of Dr. Rod Drew of Griffith University, Australia. All plant materials were initially grown in a greenhouse and the transplanted to the field. Plants were regularly fertilized with 14-14-14 (N-P-K) and 46-0-0 (N-P-K). Insect pests and weeds were also controlled. And irrigation was provided.

Morphological characters of the intergeneric hybrid line 410 and all inbred lines were scored using the descriptors list for papaya provided by the International Board for Plant Genetic Resources (IBPGR, 1988). Data were gathered from 10 representative plants. The characters documented were plant height to first flower, plant height and trunk diameter eight months after planting in the field. Other characters that were recorded were petiole color and length, tree habit, sex type, number of main veins, leaf length and width, flower size and color.

Indirect ELISA test were performed for two plants of line 410 that were vigorous and symptomless after one year in heavily PRSV infected field. Young leaves of line 410 and PRSV-P infected Kapoho were collected from the pollination block. Pollen viability of line 410 plants were determined by the stainability of pollen grains in tetrazolium chloride under a microscope. Flowers were harvested between 9:00am and 10:00am.

Papaya inbreds as female parents were hand pollinated with pollen from the intergeneric hybrid line 410. Ten male flowers were used to cross each female flower. Pollinated flowers were bagged and labeled. Data were taken from 20 successful pollinations per cross. Ten bagged flowers that were not pollinated were used as control. The parameters measured were weight and size of mature pollinated fruit, number of seeds and number of embryos. Fruits were harvested 90 to 120 days after pollination for embryo rescue. Recovered embryos were grown in vitro under laboratory conditions.

ANOVA was used to determine the differences of all parameters in the crosses. All mean comparisons were tested using Least Square Difference (LSD) in SAS software.

Results and discussion

Morphological characters

Morphological differences among the papaya inbred lines and the hybrid were remarkable. Highly significant differences were observed on all quantitative vegetative characters. Variations between C. papaya and intergeneric hybrid 410 and within C. papaya in leaf characters were highly significant. Differences in color of leaf petiole were also observed. Flower size of papaya was generally intermediate to large with cream color or with red purple shades whereas the intergeneric hybrid had small and greenish flowers. Flowering habit of the intergeneric hybrid was comparable to a male papaya.

Cluster analysis of the parent materials

Morphological data collected were categorized into qualitative and quantitative (Fig.1) types. Both qualitative and quantitative traits showed similar trend in clustering. In both traits, clustering of inbreds were related to geographical origins. Inbred lines 4108 and 4172 were local accessions while 5648 and 5893 were lines developed from foreign accessions. The results also show that the inbred papaya lines are quite distant genetically from line 410.

In qualitative data, genetic distances were generated using simple matching coefficient using the NTSYS-pc software. Likewise, Euclidian distances for the quantitative morphological data were measured using the same software. Based on the genetic distance matrices, cluster analysis was done using Unweighted Pair-Group Method, Arithmetic Average (UPGMA).

Traits of V. quercifolia like greenish petiole color, branching tree habit and leaf color were retained in the hybrid. The trend in clustering obtained was in accordance with the study of Lasalita (2001) using isozyme markers where inbred lines 5648 and 5893 were contained in one cluster while inbred line4108 belonged in another cluster.

PRSV-P infection in the field and ELISA

All papaya trees in the field were infected by PRSV-P and developed a range of symptoms: mosaic and chlorosis on leaves, water-soaked oily streaks on the petiole and upper part of the trunk, and distortion of young leaves. Concentric ringspots appeared on the skin of the fruit.

A year after, line 410 plants remained symptomless in the field. No virus was detected from the ELISA test. Mean absorbance (450nm) single wavelength reading of 0.161 and 0.195 for plants 1 and 2 was noted respectively. Threshold value obtained was 0.242. The values signify that PRSV reactions were negative on the two line 410 plants used in the crosses.

Pollen viability

The percentage of functional pollen grains was higher in papaya (66.8%) than in F1 intergeneric hybrid 410 (1.6%). The difference is highly significant (Pr >F) between papaya and the intergeneric hybrid 410. Viable pollen grains were oblate subspheroidal in shape.

Variable chromosome count, presence of univalents, chromosome elimination and marked meiotic aberrations characterized the meiotic behavior of intergeneric hybrids (Siar et al., 1998). Meiotic irregularities lead to formation of non-functional pollen. The very low count of fertile pollen of line 410 would be a reason for inadequate fertilization in the crosses that were made.

Hybridization with F1 intergeneric hybrid line 410

Seed development in the hybridization involving different genotypes of female parent with diverse genetic background to one common male parent was assessed. Significant differences in average fruit size, fruit length, and number of seeds were observed among four crosses. Inbred line 5893 had the highest seed count. However, these seeds were similar with inbred 5648 and had no fertile embryos (Table 1). Both crosses had seeds that totally filled the fruit cavity. Though few seeds were obtained in crosses from lines 4108 and 4172, well-developed and viable embryos were produced. The majority of pollinations failed to produce embryos. A few plump seeds typically contained embryos in inbred lines 4108 and 4172. All bagged flowers that were not pollinated did not produce seeds. Results suggested the presence of parthenocarpy in all papaya inbred lines.

The major barriers to intergeneric gene flow were postzygotic and include ovule and embryo abortion, as well as lack of endosperm development (Manshardt et al, 1998). Similar inference could be deduced in this study because of the presence of numerous seeds without endosperm and embryo from the fruits of four crosses.

Based on these results on wide hybridization, classifying parental materials by their place of origin may help breeders in identifying probable genotypes that may show similar compatibility.

Table 1. Mean number (no.) of seeds and embryos, no. of fruits harvested (20 fruits of each cross) with seeds and with viable embryos, no of plantlets developed and percent parthenocarpy of inbred lines used.

PARAMETERS

CROSSES

4108 x 410

4172 x 410

5648 x 410

5893 x 410

Mean no. of seeds**

5 b

1b

69 b

340 a

Mean no. of embryosns

2.3

0.5

0

0

No. of fruits that produced seeds

7

1

17

19

No. of fruits with viable embryos

5

1

0

0

No. of seeds produced (range)

0-64

0-15

0-264

0-1054

No. of plantlets developed

17

7

0

0

Parthenocarpic fruits (%)

100

100

100

100

ns not significant. Mean separation by LSD at 5 % level.

**significant at 1% level. Means with the same letter are not significantly different

The tissue culture protocol developed by Magdalita et al (1996) and Drew et al (1998) for embryo rescue and plantlet production for F1 intergeneric hybrids was proven suitable for the backcross hybrids. The freshly dissected embryos from 90 to 120 days after pollination have two broad flattened cotyledons and comparatively small hypocotyls. The first two leaves and a few root strands developed 10 days after.

Conclusion

Cluster analysis of morphological characters established the genetic distance among the inbred lines used in these experiments Papaya inbred lines were genetically distant from line 410. Symptomatology and serological test by ELISA confirmed the PRSV-P resistance of line 410 and a tetrazolium test proved low pollen viability of the line 410 plants under local conditions. Parthenocarpy is common to all papaya inbreds tested. The genotypic compatibility among four crosses between interspecific hybrid (C. papaya L. x V. quercifolia) and the papaya genotypes (4108, 4172, 5648 and 5893) was also evident. Ninety to 120 day-old embryos when cultured on medium for papaya grew and developed normally into plantlets.

References

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Badillo VM (2000). Carica L. vs.Vasconcella St.-Hil (Caricaceae) con la rehabilitacion de este ultimo. Ernstia;10(2):74-79

Drew RA, O’brien CM and Magdalita PM (1998). Development of interspecific Carica hybrids. Acta Horticulturae 461: 285-292.

IBPGR (1988). Descriptors for papaya. International Board for Plant Genetic Resources, Rome.

Lasalita FC (2001). .Morphological and Biochemical Characterization of selected papaya (C. papaya) inbreds and hybrids. M.S. Thesis. University of the Philippines Los Baos. 72 p.

Litz RE and Conover RA (1983). High-frequency somatic embryogenesis from Carica suspension cultures. Ann. Bot. 51: 683-686.). An improved embryo rescue protocol for a Carica interspecific hybrid. Australian Journal of Botany 44: 343-353.

Magdalita PM, Adkins SW, Godwin ID and Drew RA (1996).An improved embryo rescue protocol for a Carica interspecific hybrid. Australian Journal of Botany 44:343-353.

Magdalita PM, Drew RA , Godwin ID and Adkins SW (1998).An efficient interspecific hybridisation protocol for Carica papaya L. x C. cauliflora Jacq.. Australian Journal of Experimental Agriculture 38:523-530.

Manshardt RM and Drew (1998). Biotechnology of papaya. Acta Hort. Sci.461: pp 65-73.

Manshardt RM and Wenslaff H (1989). Zygotic polyembryony in interspecific hybrids of Carica papaya and Carica cauliflora. J. Amer. Soc. Hort. Sci. 114: 684-689.

Siar SV, Geronimo SB, Sierra Z and Villegas VN (1998). Cytology of Carica papaya, C. cauliflora and their F1 Interspecific Hybrids. Philipp J. Crop. Sci. 23(2):91-96.

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