OVULO EMBRYO CULTURE IN GRAPES - A REVIEW

Transcription

1 Agric.Re~.22(2): ,2001 OVULO EMBRYO CULTURE IN GRAPES - A REVIEW Anil Kumar, Suneel Sharma and D.S. Gill Department of Horticulture, CCS Haryana Agricultural University, Hisar - 12!;> 004, India ABSTRACT Seedless grapes are preferred as table grapes by consumer around the world. Seedlessness of table grapes (Vitis spp.) is caused by either stenospermocarpy or parthenocarpy. Techniques for in ovulo embryo culture have been reported for stenospermocarpic grapes, early ripening seeded grapes and interspecific-crosses. This technique provides an alternative to conventional breeding for seedless grapes by allowing recovery of progeny from abortive ovules of seedless crosses and also facilitates the study of inheritance of seedlessness. The present review deals with the work done in ovulo embryo culture with respect to the effect of genotype, culture media, age of ovule etc. in grapes. Seedless grapes have been prized as fresh and dried fruit for centuries and are preferred by consumers around the world. In these grapes cvs., the berries develop either through parthenocarpy or embryo abortion at an early stage of development (Stout, 1936). Stenospermocarpic cvs. comprise 85 per cent of the fresh table and raisin grape market (Davis and Severson, 1981). As all over the world seedless table grapes are preferred by consumers, hence the breeders place much emphasis on developing new stenospermocarpic cvs. Conventional breeding of seedless grapes involves pollinating seededgrapeswith pollen of seedless cvs. However the breeding of seedless grapes is hampered by several problems such as seedless cvs. can be used only as male parents, the frequency of gettingseedless progenies from crosses between seeded and seedless cvs. is very low (10-15%) (Weinberger and Harmon, 1964; Loomis and Weinberger, 1979). It has been suggested that intervarietal crossing of seedless grapes to obtain seedless progeny would be more effective. In ovu!o embryo culture technique provides an ilttractive alternative to conventional method of breeding seedless table grapes by allowihg recovery of progeny from abortive ovules of seedless x seedless crosses. Further, it would facilitate genetic studies leading to clear understanding of the inheritance and nature of the seedless fruits. The purpose of this paper is to review the available literature relevant to ovulo embryo culture in grapes. The relevant literature related to present topic has been summerised under various heads: A. Genotypic effect: Researchers have shown genotypic responses to o~urduring ovulo embryo culture. Goldy and Amborn (1987) worked on the ovules of 10 seedless grape clones and cv. Concord. Ovules were cultured six weeks after full bloom on Cain's et aj. (1983) medium containing 10 mm L cysteine. The embryo survival ranged from 0 for Reliance to 110 for Concord.Thirty days old embryos of cv. Baishing Jiao cultu~ed on modified B 5 medium promoted plantlet formation (Chen and Huang, 1984). The ovules of Thompson Seedless collected 68 days after anthesis and cultured on Cain's medium + 10 mm L-cysteine enhanced growth of embryo (Emershad and Ramming, 1984). The ovules of Perlette, Flame Seedless and Sultanina were taken 52, 49 and 42 days post anthesis, respectively and cultured on Nitsch and Nitsch (1969) medium supplemented with NAA (10. 5 M) + GA 3 (10-6 M). The ovules of Flame Seedless cultured 49 days after anthesis gave high per cent germination and viable plants (Spiegel Roy eta!., 1985). Genotype, culture media and their interactions significantly affected embryo enlarge-

2 128 AGRICULTURAL REVIEWS ment (Emershad et al., 1989). Similarly Gribaudo etal. (1993) also observed that genotype was the main factor affecting ovulo culture in grapes. but the medium and the interaction of genotype x medium also influenced it. Emershad et al. (1989) studied embryo enlargement and plant formation via in ovulo embryo culture in stenospermic grapes and reported that the genotype P60-58 in comparison to cv Thompson Seedless had the greatest number of enlarged embryos after 3 months of culture. The embryos from the reciprocal crosses of Kishmish Moldavski x Seedless Hybrid - 4 showed higher viability and produced higher number of normal plants (Tsolova, 1990). Brar et al..\ 1990) studied the ovuio embryo culture in seedless grapes. Maximum survival of ovules was observed in cultivar Perlette (90.91 t Yil) when cultured 30 days after anthesis on MS medium with supplements in comparison with other cultivars. Singh and Brar (1992) examined the ovule development in three seedless cvs. viz. Perlette. Thompson Seedless. Beauty Seedless and the seeded cultivar Anab-e-Shahi to identify the proper developmental stage of the embryo for in ovuio embryo culture. Among seedless cultivars the growth of ovules was least in Thompson Seedless and Beauty Seedless however. ovule development was faster in Anab-e-Shahi than in seedless cultivars. Gribaudo et al. (1993) examined ovules of 14 seedless cultivars which were collected days after anthesis and cultured in vitro on 2 different media. Plant development occurred at variable rates, the best percentage was found in Flame Seedless, Perlon, Imperatrice, Coarina, Perlette and Ruby Seedless. The main factor affecting ovule response to culture proved to be genotype. Pommer etal. (1995) worked on eighteen seedless grape cvs. differing in ripening season (early, mid season and late). Seed traces of small, medium and large size were harvested at 6, 10, 14, 18 and 22 weeks after flowering and found that genotype had a significant influence on embryo culture traits. Late maturing cultivars had fewer rescued embryos, germinated embryos and regenerated plantlets than early and mid season cultivars. Selfed Perlette and Perlette x Flame Seedless ovules cultured 52 days after anthesis developed 11 per cent seedlings whereas, reciprocal crossing showed 16 per cent development of seedlings (Spiegel Roy et al., 1985). The cross combination has been found to be a major factor influencing growth response of ovules (Kanamadi et ai., 1996: Sharma et al ). Cancellier et al. (1990) reported the work on embryo and seed culture in grape to increase the percentage of plants produced by selfing of the cv. Regina and crossing it with various seedless cvs. Sultanina, Perlette, Perlona, Pasiga and Neroma. They observed the percentage of plants derived by crossing or selfing, plants of low germinability was increased from 3.5 to 9.3 per cent by in vitro culture and the percentage of plants obtained from crosses of the seedless type was raised to 21 per cent. Tsolova (1990) reported that the embryos from the crosses of Kishmish Moldavski x Seedless Hybrid VI-4 when cultured on Nitsch and Nitsch (1969) medium containing GA j (10' 6 M) 1M (1 O-i, M), activated charcoal (0.3 t!t(l) and Kin (10t, M) on 52 and 66 days after anthesis showed higher viability and produced higher number of normal plants. Crosses were made between M(J!lukka and Mukaku Shiro as seedless cvs. and Campbell Early as seeded one.with the transfer of pollen of Monukka and Mukaku Shiro on to the stigma of Campbell Early as many as 74 seeded berries were produced. However, in reciprocal pollination Monukka and Mukaku Shiro showed 21 per cent and 39 per cent

3 Vol. 22, No.2, berry set, respectively but their embryos were ovules. However, Spiegel Roy et a!. (1985) almost abortive (Wang eta!., 1993). recovered thirteen plants out of 27 embryos Marasali (1992) studied effect of pol- from'open pollinated ov~les ~f cv. Thom~son len parent on seed germination. He examined See~less cultured on soh~ Nltsc~ and NItsch cv. Cavus which had high frequency of empty medium supplemented with 10 M 1M and.. seeds and high percentage (>99l!lil) of abortive 10-6 M GA3 embryos along with Karasakizas a pollinator Gray et a!. (1987) compared in ovulo cv. of Cavus and six Cavus hybrids and found embryo culture using liquid vs. solid medium that germination rate of Cavus hybrid seeds culture methods and reported that an equivawas very low suggesting that germination abil- lent number of embryos per ovule were obity seems to be only little influenced by pollina- tained in Orlando Seedless x Arkansas 1105 tor cvs. The reciprocal crosses were made be- with the exception of selfed Orlando Seedless tween diploid Muscat of Alexandria and its for which the solid medium culture method was 4n mutant Cannon Hall Muscat with an objec- superior. However, moreembryosgerminated tive of creating a new seedless grape and re- from ovules cultured on solid medium than the covered per cent of ovules from the 4n liquid medium. Ovules of the genotype P 60 x 2n cross while per cent from the re- 58 ovules cultured on Cain's etal.( 1983) basal ciprocal cross. Ten to twenty per cent of the medium + L-serine or L-glutamine and of cv. embryos from the 2n x 4n cross developed in Thompson Seedless ovules cultured on Cain's to rooted plantlets and proved to be triploids et.a!. (1983) basal medium + L-cysteine or L (Okamoto et al. 1993). asparagine produced greatest number of en- B. Culture Media effect: Work done larged embi'yos (Emershad et aj ). to date by several workers on this aspect The ovules of 14 seedless cultivars colshowed that it is possible to recover embryos lected OPA were excised and cultured and to obtain plants of newly created in vitro on 2 different media: Nitsch and Nitsch stenospermocarpic cultivars belonging to (1969} media + 1 ~l M GA ~IM IAA and Sultanina group using the solid medium (Cain Nitsch and Nitsch (1969) media + 1 ~l M GA 3 et al., 1983; Emarshad and Ramming, 1984; + 20 ~IM IAA + 2g11 activated charcoal. Plant Speigel Roy et a!., 1985; Goldy and Ambron, development was affected by medium used and 1987; Gray et al., 1987). also by genotype. The addition of 2g/1 acti- Emershad and Ramming (1984) inves- vated charcoal to the medium as teste? on the tigated the in ovulo embryo culture of cv. Th- cv, Perletteand the types ofadded auxins (lba, ompson Seedless as a method to enable the IAA, NM) tested on cvs. Perlette and Sultanina hybridization of sternospermocarpic seedless ex~ibited enhanced plantlet development grapes. Fertlized ovules taken 68 OPA were (Grtbaudo et al., 1993). cultured on two basal liquid media (i) Cain's Yamashita et al. (1995) reported that et al. ( 1983 ) medium (ii) Stewart and Hsu's the germination of both immature and abnor (1977) medium both tested with five seperate mal embryos improved when they were culaddenda (IBA, Ergostim, tartaric acid, L- tured on a modified MS medium without amglutamine and L-cysteine). Addition of 10 mm moniumsalts butsupplemented with 250,500 L-cysteine to Cain's medium promoted the or 1000mgLI Casein hydro-lysate. Although growth of more embryos. They were able to most of the germinatedembryos stopped growrecover one plant from thirty embryos derived ing onthese germination media, theyproduced from selfed Thompson Seedless (Sultanina) leaves ormultiple leaves budsafterbeing trans-

4 130 AGRICULTURAL REVIEWS ferred to MS medium supplemented with 0.2, 2 or 5 mgll BA and then formed shoots. Roots were initiated when shoots were transferred to MS medium without phytohormones. C. Effect of age of ovules.: Cain et al. (1983) reported that the culturing of ovules 0-31 days after anthesis, produced callus whereas culturing 38 days after anthesis showed visible embryos. Plantlets formed from ovules cultured days after anthesis. The embryos of grapecv: Baishan Jjiao taken 15 days after flowering, failed to produce plantlets when cultured on modified B 5 medium, whereas embryos taken at 30 OPA produced green plantlets Chen and Huang (1984). However, Emershad et al. (1989) reported no effect of culturing dates on embryo enlargement. Excision andculturing of HameSeedlessovules 49 days after anthesis gave higher germination percentage and more viable plants than excision and culturing at anthesis plus 28. Selfed Perlette and Perlette x Flame Seedless ovules cultured at 52 days gave 11 per cent and the redprocal cross over 16 per cent well developed seedlings (Spiegel Roy etai., 1985). Tsolova (1990) reported that the early date of ovule cultivation by the 50 th day after anthesis prove to be more suitable in Kishmish Moldevski x Seedless hybrid \ii-4. Cancellier et al. (1990) working with RegIna, Sultanina, Perlette, Pasiga and Nerona cvs. of grapes found that the best stage for isolation of embryo was 60 day after selfing, while the ripening stage was best for isolation of seeds. Gray etal. (1990) fertilized ovules of seedless bunch grapes (Vitis spp.) by using pollen from seedless parentsand cultured them on NN 69 medium with GA 3, laa, sucrose, agar and charcoal. They noticed that more ovules cultured at 10 days or at days after pollination became brown compared to those cultured at days and cultured ovules developed with or without endosperm. More embryos and plants were recovered at 40 or 60 days than at 10 or 20 days after pollination. Ovules and seeds of cv. Barbera were collected 40 and 55 days after anthesis and at fruit maturity. Samples of the first two dates were cultured on Nitsch and Nitsch medium with 10-6 M GA M IAA. Ovules collected 40 days after anthesis developed only. callus. The ovules collected after 55 days were similar to those seeds obtained from fully mature berries. Ovules collected after 55 days from vines pollinated by 2n cvs. gave only per cent plants (Vallania etal., 1990). According to Gray and Hanger (1993) in eleven cvs. investigated the percentage of ovules producing zygotic embryos increased significantly with maturityat harvest. Ovules sampled at 20,30, or 40 OPA ( days post anthesis ) produced significantly more embryogenic cultures than those sampled at 10OPA. Braretal. (1990) observed that Perlette and Thompson Seedless ovules cultured 10,15 and 20 days after anthesis on White"s medium supplemented with L-asparagine and L-cysteine, the ovule survival was not affected much but the growth of surviving ovules improved. The.embryo shrivelling occurred within 20 days after anthesis in these seedless cultivars. The number of shrivelled ovules started increasing 20 days after anthesis and at the same time, the number of viable ovules also started declining in all the seedless cvs. viz. Perlette, Thompson Seedless and BeautySeedless. The embryos in Perlette, Thompson Seedless and Beauty Seedless cultivars may be rescued in vitro prior to 20 days post anthesis to obtain plantlets from these abortive ovules (Singh and Brar, 1992). Yu et al. (1992) suggested that the culturing of embryos at mature stage of fruits show significantly better results than culturing 45 days after anthesis. The ovules removed 50 days after crossing (Muscat of Alexandria x Cannon Hall Muscat) developed embt'9os on a liquid medium (Okamoto

5 et al., 1993).The number of growing plantlets derived from ovules harvested at 10 weeks after anthesis and at maturity was significantly higher than those collected 8 weeks after anthesis in seedless cultivars (Aguero etal., 1995). Consumer demand for seedless grapes is apparent from the increases in newly planted areas of stenospermic cvs. Breeding of seedless grapes have become more efficient by us- Vol. 22~ No.2, ing in ovuloembryo culture. Thetechniquefor in ovulo embryo culture have been reported for stenospermocarpic grape, early ripening seeded grapes and interspecific crosses with the current breeding emphasis, it appears that in future we can expect many new types of seedless grapes that are better adapted to the environment, easier to produce and hopefully better tasting than before. REFERENCES Aguero, C. et al. (1995). Vitis. 34 : Brar, S.J.S. et al. (1990).!n: Horticulture - new technologies and applications. Proc. Int. Sem. New Frontiers in Horticulture, Indo-American Hybrid Seeds, Banglore, 1990 pp. : Cain, D.W. etal. (1983). Vitis. 22: Cancellier, S. etal.(1990). Rivista-di-viticaltura-e-di-Enologia.43 : Chen, Z.G. and Huang, D.L. (1984). PI. Physiol. Commun. 6 : Davis, A.R. and Severson, S. (1981). California crop and livestock reporting service. USDA Statistical Reporting Service. p. 4. Emershad, R.L. and Ramming, DW. (1984). Am. J. Bot. 71 : Emershad, RL et ai. (1989). Am. J. Bot. 76 : Goldy, RG and Amborn, U. (1987). Hort Sci. 22 : 952. Gray, D.J. et al. (1987). Hort. Sci. 22 : Gray, D.J. and Hanger, L.A. (1993). Hort. Sci. 28 : 227. Gray, D.J. et al. (1990). J. Am. Soc. Hort. Sci. 115 : Gribaudo, I. etal. (1993). Vitis. 32: Kan~madi, V.C. et al. (1996). In : Souvenir of National Symposium on Horticulture Biotechnology, Bangalore. Loomis, N.H. and Weinberger, J.H. (1979). J. Am. Soc. Hort. Sci. 104: Marasali, B. (1992). Thesis, Ankara Univ. Dept. Hort., Turkey. Nitsch, J.P. and Nitsch, C. (1969). Science, New York, 163: Okamoto, G. et al. (1993). Scientific Reports of the Faculty of Agriculture, Okayama University. 82 : Pommer, C.v. et ai. (1995). Bragantia. 54 : Sharma, S.. et al. (1996). In : Souvenir, National Symposium on Horticulture Biotechnology, Bangalore (India). Singh, Z. and Brar, S.J.S. (1992). Vitis. 31 : Spiegel-Roy, P. etal. (1985) J. Am. Soc. Hort. Sci. 110: Stewart, J.M and Hsu, c.l. (1977). P/~nta, 137 : Stout, A.B. (1936). Seedlessness in grapes. NY State Agric. Exp. Stat. Tech. Bull. 238 : Tsolova, V. (1990). Vitis. 29 : 1-4. Vallania, R etal. (1990). Quaderni della scuola di specializzazione in.viti coltura ed Enologia, Univ. Torino. 14: Wang, J. et al. (1993). J. Japanese Soc. Hort. Sci. 62 : 1-7. Weinberger, J.H. and Harmon, F.N. (1964). Proc. Am. Soc. Hort. Sci. 85 : Yu, D. etal. (1992). J. Fruit Sci.9: 1-7. Yamashita, H. etal. (1995). In vitro culture of embryos obtained by crossing.tetraploid grape cultivar "Kyoho" with deploid cultivars. J. Japan. Soc. Hort. Sci. 63: