A Preliminary Research on Microspore Culture in Eggplant (Solanum melongena L.)

Öz

 Microspore embryogenesis is a process in which immature male gametophytes are induced to divert them from their gametophytic pathway toward embryo development during in vitro culture. In this study, therefore, it was aimed to determine the response of two eggplant cultivars to microspore culture. For this purpose, the microspores were firstly isolated from the anthers at the appropriate stage of microspore development (containing mostly vacuolate microspores and young bicellular pollen) and subjected to pre-treatment at 35°C  for 3 days in dark conditions. After pre-treatment, the microspores were cultured in liquid NLN culture medium supplemented with 2% sucrose, 0.5 mg/l naphthaleneacetic acid (NAA), and 0.5 mg/l, 6-benzylaminopurine (BAP), pH 5.9, and kept in the dark at 25°C for one month. During this culture process, the microspore embryogenesis induction was microscopically analyzed and focused on the initial stages of this developmental process. Immadiately after induction, before the microspores develop into callus, they were induced to divide symmetrically and form multinucleated structures, and then microspores did not form direct embryos and formed callus. At the end of one month, only callus formation occurred from microspores and total callus number per petri was  analyzed. The average calli for G07-1 cultivars was 288 calli/petri, while it was 64 calli/petri dish for G07-2 cultivars. It is thought that this research will guide both practical and basic researches on the development of microspore culture technique.

Anahtar Kelimeler

• Eggplant,Microspore culture,Haploid,DH,Microspore embryogenesis

Patlıcanda (Solanum melongena L.) Mikrospor Kültürü Üzerine Bir Ön Araştırma

Öz

Mikrospor embriyogenesis olgunlaşmamış erkek gametofitlerin in vitro kültür süresince gametofitik gelişimden embriyo oluşturmak üzere uyarıldığı bir sistemdir. Bu araştırmada, iki adet patlıcan (Solanum melongena L.) çeşidinin mikrospor kültür tekniğine tepkisinin belirlenmesi amaçlanmıştır. Bu amaçla, ilk olarak uygun mikrospor gelişme dönemindeki mikrosporlar (çoğunluğu vakuol mikrospor ve genç çift çekirdekli polen) anterlerden izole edilerek 35°C‘de 3 gün karanlık koşullarda ön uygulamaya maruz bırakılmıştır. Ön uygulama işleminden sonra mikrosporlar %2 sakkaroz, 0.5 mg/l naphthaleneacetic acid (NAA) ve 0.5 mg/l 6-benzylaminopurine (BAP), pH 5.9, içeren NLN ortamında kültüre alınmış ve bir ay boyunca 25°C‘de karanlıkta bekletilmiştir. Kültür süreci boyunca mikrospor embriyogenesis indüksiyon süreci mikroskobik olarak analiz edilerek bu gelişimsel sapmanın ilk evrelerine odaklanılmıştır. Mikrosporların indüksiyondan hemen sonra kallus haline gelmeden önce simetrik bölünme ve çok çekirdekli yapılar meydana getirdiği daha sonra ise mikrosporların direkt embriyo oluşturmadıkları ve kallus oluşturduğu tespit edilmiştir. Araştırmada bir ay sonunda mikrosporlardan yalnızca kallus oluşumu meydana gelmiştir ve petri başına toplam kallus sayısı belirlenmiştir. G07-1 çeşidinde ortalama 288 kallus/petri elde edilirken G07-2 çeşidinde 64 kallus/petri meydana gelmiştir. Bu araştırmanın mikrospor kültürü tekniğinin geliştirilebilmesi üzerine hem uygulamalı, hem de temel araştırmalar için yol gösterici olacağı düşünülmektedir.

Anahtar Kelimeler

• Patlıcan,Mikrospor kültürü,Haploid,DH,mikrospor embriyogenesis

Kaynakça

1. [1] Forster, B.P., Herberle-Bors, E., Kasha, K.J., Touraev, A. 2007. The resurgence of haploids in higher plants. Trends in Plant Science, 12(8), 368–375.
2. [2] Dunwell, J.M. 2010. Haploids in flowering plants: origins and exploitation. Plant Biotechnol J., 8: 377-424.
3. [3] Germanà, M. 2011. Gametic embryogenesis and haploid technology as valuable support to plant breeding. Plant Cell Rep., 30: 839-857.
4. [4] Segui-Simarro, J.M. 2010. Androgenesis revisited. The Botanical Review., 76: 377-404.
5. [5] Maluszynski, M., Kasha, K.J., Szarejko, I. 2003. Published doubled haploid protocols in plant species. pp 309-335. Maluszynski, M., Kasha, K.J., Forster, B.P., Szarejko, I. eds. 2003. Doubled haploid production in crop plants A manual. Kluwer Academic Publishers, Dordrecht, Netherlands, 428.
6. [6] Maraschin, S.F., De-Priester, W., Spaink, H.P., Wang, M. 2005a. Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. J Exp Bot., 56: 1711–1726.
7. [7] Segui-Simarro, J.M., Corral-Martinez, P., Parra-Vega, V., Gonzalez-Garcia, B. 2011. Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep., 30: 765-778.
8. [8] Raina, S.K., Iyer, R.D. 1973. Differentiation of diploid plants from pollen callus in anther cultures of Solanum melongena L. Z Pflanzenzucht, 70: 275-280.

9. [9] Isouard, G., Raquin, C., Demarly, Y. 1979. Obtention de plantes haploides et diploides par culture in vitro d'anthères dáubergine (Solanum melongena L.). C R Acad Sci, Paris, 288: 987-989.
10. [10] Dumas de Vaulx, R., Chambonnet, D. 1982. Culture in vitro d'anthères d'aubergine (Solanum melongena L.): stimulation de la production de plantes au moyen de traitements à 35ºC associés à de faibles teneurs en substances de croissance. Agronomie, 2: 983-988.
11. [11] Misra, N.R., Varghese, T.M., Maherchandani, N., Jain, R.K. 1983. Studies on induction and differentiation of androgenic callus of Solanum melongena L. pp 465-468. Sen, S.K., Giles, K.L. eds. 1983. Plant cell culture in crop improvement. Plenum Press, New York, 502p.
12. [12] Borgel, A., Arnaud, M. 1986. Progress in eggplant breeding, use of haplomethod. Capsicum Newsl, 5:65–66.
13. [13] Rotino, G.L., Falavigna, A., Restaino, F. 1987. Production of anther-derived plantlets of eggplant. Capsicum Newsl, 6:89–90.
14. [14] Tuberosa, R., Sanghineti, M.C., Conti, S. 1987. Anther culture of eggplant Solanum melongena L. lines and hybrids. Gene´tica Agra´ria, 41:267–274.
15. [15] Rotino, G.L. 1996. Haploidy in eggplant. pp 115-141. Jain, S.M., Sopory, S.K., Veilleux, R.E. eds. 1996. In vitro haploid production in higher plants, Kluwer Academic Publishers, Dordrecht, The Netherlands, 412p.
16. [16] Rotino, G.L., Sihachakr, D., Rizza, F., Vale, G., Tacconi, M.G., Alberti, P., Mennella, G., Sabatini, E., Toppino, L., D'Alessandro, A., Acciarri, N. 2005. Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives. Acta Physiol Plant, 27: 723-733.
17. [17] Alpsoy, H.C., Seniz, V. 2007. Researches on the in vitro androgenesis and obtaining haploid plants in some eggplant genotypes. Acta Hortic, 729:137–141.
18. [18] Salas, P., Prohens, J., Seguı´-Simarro, J.M. 2011. Evaluation of androgenic competence through anther culture in common eggplant and related species. Euphytica, 182:261–274.
19. [19] Salas, P., Rivas-Sendra, A., Prohens, J., Segui-Simarro, J.M. 2012. Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica, 184:235–250.
20. [20] Başay, S., Ellialtıoğlu, Ş. 2012. Effect of genotypical factors on the effectiveness of anther culture in eggplant (Solanum melongena L.). Turk J Biol, 37: 499-505.
21. [21] Gu, S.R. 1979. Plantlets from isolated pollen cultures of eggplant (Solanum melongena L.). Acta Bot Sin, 21: 30–36.
22. [22] Miyoshi, K. 1996. Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L.). Plant Cell Rep, 15:391–395.
23. [23] Corral-Martinez, P., Segui-Simarro, J.M. 2012. Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica, 187:47–61.
24. [24] Corral-Martinez, P., Segui-Simarro, J.M. 2014. Refining the method for eggplant microspore culture: effect of abscisic acid, epibrassinolide, polyethylene glycol, naphthaleneacetic acid, 6-benzylaminopurine and arabinogalactan proteins. Euphytica, 195: 369–382.
25. [25] Bal, U., Abak, K. 2005. Induction of Symmetrical Nucleus Division and Multicellular Structures from the Isolated Microspores of Lycopersicon Esculentum Mill. Biotechnology & Biotechnological Equipment, 19(1): 35-42.
26. [26] Bal, U., Ellialtioglu, S., Abak, K. 2009. Induction of symmetrical nucleus division and multi-nucleate structures in microspores of eggplant (Solanum melongena L.) cultured in vitro. Sci Agric. 66: 535–539.
27. [27] Tuncer, B., Yanmaz, R. 2011a. Effects of colchicine and high temperature treatments on isolated microspore culture in various cabbage (Brassica oleraceae) types. International Journal of Agriculture and Biology, 13(5): 819-822.
28. [28] Tuncer, B., Yanmaz, R. 2011b. Induction of microspore embryogenesis in ornamental kale by gamma irradiation and high temperature stress. Asian Journal of Biotechnology, 3: 415-421.
29. [29] Tuncer, B., Cig, A., Yanmaz, R., Yasar, F. 2016. Effect of heat shock treatment on microspore embryogenesis in Brassica oleracea species. Journal of Agricultural Scieces, 22(4): 548-554.
30. [30] Tuncer, B. 2017. Callus formation from isolated microspore culture in radish (Raphanus sativus L.). The Journal of Animal & Plant Sciences, 27(1): 277-282.
31. [31] Custers, J.B.M. 2003. Microspore culture in rapeseed (Brassica napus L.). pp 185-194. Maluszynski, M., Kasha, K.J., Forster, B.P., Szarejko, I. Eds. 2003, Doubled haploid production in crop plants: a manual. Kluwer Academic Publishers, Dordrecht, Netherlands, 428.
32. [32] Touraev, A., Pfosser, M., Heberle-Bors, E. 2001. The microspore: a haploid multipurpose cell. Adv Bot Res, 35:53–109.
33. [33] Segui-Simarro, J.M., Nuez, F. 2008. How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiologia Plantarum, 134: 1-12.
34. [34] Indrianto, A., Barinova, I., Touraev, A., Heberle-Bors, E. 2001. Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo. Planta, 212: 163–174.
35. [35] Maraschin, S.F., Vennik, M., Lamers, G.E.M., Spaink, H.P., Wang, M. 2005. Time-lapse tracking of barley androgenesis reveals position-determined cell death within pro-embryos. Planta, 220: 531–540.
36. [36] Zaki, M.A.M., Dickinson, H.G. 1991. Microspore-derived embryos in Brassica: the significance of division symmetry in pollen mitosis I to embryogenic development. Sexual Plant Reproduction, 4:48–55.