Biberde (Capsicum annuum L.) Farklı Yöntemlerin Mikrospor Embriyogenesis Üzerine Etkisi

Year 2022, Volume: 51 Issue: (Özel Sayı 1) 13. Sebze Tarımı Sempozyumu, 85 - 94, 19.12.2022

Buse Özdemir Çelik , Ahmet Naci Onus

Abstract

Bu çalışmada ‘Erciyes F₁’ ve ‘Benino F₁’ biber çeşitleri kullanılarak ıslah amaçlarına uygun özelliklere sahip double haploid (DH)biber hatları elde edebilmek için etkili androjenik yöntemin belirlenmesi amacıyla üç farklı androjenik yöntem (anter, shed-mikrospor, mikrospor) karşılaştırılmıştır. Anter kültüründe anterler 4 mg l⁻¹ NAA 0.5 mgl⁻¹ BAP, %0.25 aktif kömür, 30 g l⁻¹ sakkaroz ve 15 mg l⁻¹ AgNO₃ içeren MS ortamında kültüre alınmıştır. Shed-mikrospor kültürü protokolünde ise %2 maltoz, %1 aktif kömür, %0.6 agar eklenmiş katı NN ortamı ve %2 maltoz, 2.5 μM Zeatin ve 5 μM IAA eklenmiş sıvı NN ortamından oluşan çift katmanlı kültür ortamı kullanılmıştır. Shed-mikrospor kültüründe petri kapları ilk bir hafta boyunca (1) 9℃ (karanlık) ve (2) 32℃ (karanlık) olmak üzere iki farklı ön sıcaklık uygulamasına tabi tutulmuştur. Mikrospor kültüründe ise izole edilen mikrosporlar sıvı NLN ortamı içerisinde kültüre alınmıştır. Çalışma sonucunda her iki çeşitten de anter kültürü protokolünden herhangi bir embriyo oluşumu meydana gelmemiştir. Mikrospor kültüründe ise sporofit bölünmeler gözlemlenmiş ancak bunlardan herhangi bir embriyo oluşumu meydana gelmemiştir. Shed-mikrospor kültüründen ise her iki çeşitte de embriyo ve bitki elde edilmiştir. Yapılan bu çalışma ile bu çeşitlerde haploid embriyo ve bitki gelişimi için shed-mikrospor kültürü yönteminin daha başarılı olduğu belirlenmiştir.

Keywords

Biber , mikrospor kültürü , anter kültürü , shed-mikrospor kültürü , haploid

Effects of Different Methods on Microspore Embryogenesis in Pepper (Capsicum annuum L.)

Abstract

This study aimed to establish an effective technique by comparing three different androgenic methods (anther, shed-microspore and isolated microspore culture) using ‘Erciyes F₁’ and ‘Benino F₁’ pepper varieties. In anther culture, anthers were cultured in MS medium containing 4 mg l⁻¹ NAA, 0.5 mg l⁻¹ BAP, 0.25% activated charcoal, 30 g l⁻¹ sucrose and 15 mg l⁻¹ AgNO₃. In the shed-microspore culture protocol, a double-layer culture medium consisting of 2% maltose, 1% activated charcoal, 0.6% agar added solid NN medium and 2%maltose, 2.5 μM Zeatin and 5 μM IAA added liquid NN medium was used. In shed-microspore culture, anthers in petri dishes were subjected to two different pre-temperature treatments as (1) 9℃ (dark) and (2) 32℃ (dark) during the first week. In microspore culture, isolated microspores were cultured in liquid NLN medium. In the anther culture studies embryo development and plant regeneration could not be obtained. In the microspore culture, some microspores were effectively induced to divide by the stress treatment and microspores with three or more cells were determined but these multicellular structures did not show any growth. According to the shed-microspore culture results were evaluated, although there were differences according to the genotypes embryos and plantlets were obtained from all genotypes. As a result, it was determined that the shed-microspore culture method was more successful in embryo and plant development from microspores in these varieties.

Keywords

Pepper , microspore culture , anther culture , shed-microspore culture , haploid

References

• Ahmadian, P., Testillano, P.S., Gonzalez, P., Fadon, B., Prestamo, G., Jimenez-Duran, G., Risueno, M.C., 1998. Cell biology of the pollen developmental program and the induction of microspore embryogenesis in Capsicum annuum L. In:X Eucarpia meeting on genetics and breeding of Capsicum and eggplant, Avignon, France, pp:183-186.
• Alremi, F., Taşkın, H., Sönmez, K., Büyükalaca, S., Ellialtıoğlu, Ş., 2014. Effect of genotype and nutrient medium on anther culture of pepper (Capsicum annuum L.). Turk J. Agric. Nat. Sci. 1:108-116.
• Anonim, 2022. http://www.fao.com.
• Ari, E., Yildirim, T., Mutlu, N., Büyükalaca, S., Gökmen, Ü., Akman, E., 2016. Comparison of different androgenesis protocols for doubled haploid plant production in ornamental pepper (Capsicum annuum L.). Turkish Journal of Biology 40(4):944-954.
• Barany, I., Gonzalez-Melendi, P., Fadon, B., Mityko, J., Risueno, M.C., Testillano, P.S., 2005. Microspore-derived embryogenesis in pepper (Capsicum annuum L.):subcellular rearrangements through development. Biol Cell 9:709-722.
• Barroso, P.A., Rêgo, M.M., Rêgo, E.R., Soares, W.S., 2015. Embryogenesis in the anthers of different ornamental pepper (Capsicum annuum L.) genotypes. Genet Mol Res 14:13349-13363.
• Büyükalaca, S., Çömlekçioğlu, N., Abak, K., Ekbiç, E., Kılıç, N., 2004. Effect of silver nitrate and donor plant growing conditions on production of pepper (Capsicum annuum L.) haploid embryos via anther culture. Europ J. Hort. Sci. 69:206-209.
• Cheng, Y., Ma, R.L., Jiao, Y.S., Qiao, N., Li, T.T., 2013. Impact of genotype, plant growth regulators and activated charcoal on embryogenesis induction in microspore culture of pepper (Capsicum annuum L). S. Afr. J. Bot. 88:306-309.
• Comlekcioglu, N., Buyukalaka, S., Abak, K., 2001. Effect of silver nitrate on haploid embryo induction by anther culture in pepper (Capsicum annuum L.). In:11. Eucarpia Meeting on Genetics and Breeding of Capsicum and Eggplant, Antalya, Turkey, pp:133-136.
• Custers, J.B.M., 2003. Microspore culture in rapeseed (Brassica napus L.). In:M. Maluszynski, K.J. Kasha, B.P. Forster, I. Szarejko (Eds.):Doubled haploid production in crop plants:a manual. Kluwer Academic Publishers, Dordrecht, Boston, London.pp:185-194.
• Dolcet-Sanjuan, R., Claveira, E., Huerta, A., 1997. Androgenesis in Capsicum annuum L. effects of carbohydrate and carbon dioxide enrichment. J Am. Soc. Hort. Sci. 122:468-475.
• Dumas de Vaulx, R., Chambonnet, D., Pochard, E., 1981. In vitro anther culture in red pepper (Capsicum annuum L.):improvement of the rate of plant production in different genotypes by treatments at 35 C. Agronomie 1:859-864.
• Dumas de Vaulx, R., Chambonnet, D., Sibi, M., 1982. Stimulation of in vitro androgenesis in pepper (Capsicum annuum) by elevate temperature treatments. In:Earle E, Demarly Y (eds) Variability in plants regenerated from tissue culture. Praeger Publication, New York, pp:92-98.
• Ercan, N., Sensoy, F.A., Sensoy, A.S., 2006. Influence of growing season and donor plant age on anther culture response of some pepper cultivars (Capsicum annuum L.). Sci. Hort. Amsterdam 110:16-20.
• Erim, F.B. 2019. Farklı biber (Capsicum annuum L.) genotipleri üzerinde shed-mikrospor kültürü tekniğinin uygulanması ve mikrospor gelişim aşamalarının belirlenmesi (Yüksek Lisans Tezi). Akdeniz Üniversitesi Fen Bilimleri Enstitüsü Bahçe Bitkileri Anabilim Dalı, Antalya, 53s.
• Fan, Z., Armstrong, K., Keller, W., 1988. Development of microspores in vivo and in vitro in Brassica napus L. Protoplasma, 147:191-199.
• Ferrie, A.M.R., Caswell, K.L., 2011. Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tiss Organ Cult. 104:301-309.
• George, L., Narayanaswamy, S., 1973. Haploid Capsicum through experimental androgenesis. Protoplasma 78:467-470.
• Gonzalez, J., Jouve, N., 2005. Microspore development during in vitro androgenesis in triticale. Biol Plant, 49:23-28.
• Grozeva, S., Todorova, V., Cholakov, T., Rodeva, V., 2013. Effect of temperature and growth period of donor plants on pepper anther culture. In:3. International Conference Research People and Actual Tasks on Multidisciplinary Sciences. Lozenec, Bulgaria, pp:60-64.
• 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.
• Irikova, T., Grozeva, S., Rodeva, V. 2011-a. Anther culture in pepper (Capsicum annuum L.) in vitro. Acta Physiol Plant 33:1559-1570.
• Irikova, T., Grozeva, S., Popov, P., Rodeva, V., Todorovska, E., 2011-b. In vitro response of pepper anther culture (Capsicum annuum L.) depending on genotype, culture medium and duration of cultivation. Biotechnol Biotech Eq 25:2604-2609.
• Jacquard, C., Mazeyrat-Gourbeyre, F., Devaux, P., Baillieul, F., Clement, C., 2006. Plant defense mechanisms are triggered in the anther during the pre-treatment process. In:The international conference haploids in higher plants III, Vienna, Austria, 12-15 February 2006, 29p.
• Kaltchuk-Santos, E., Mariath, J.E., Mundstock, E., Hu, C.Y., Bodanese-Zanettini, M.H., 1997. Cytological analysis of early microspore divisions and embryo formation in cultured soybean anthers. Plant Cell Tiss Org. Cult., 49:107-115.
• Kim, M., Kim, J., Yoon, M., Choi, D.I., Lee, K.M., 2004. Origin of multicellular pollen and pollen embryos in cultured anthers of pepper (Capsicum annuum). Plant Cell Tiss Org. Cult., 77:63-72.
• Kim, M., Jang, I.C., Kim, J.A., Park, E.J., Yoon, M., Lee, Y., 2008. Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture. Plant Cell Rep 27:425-434.
• Kim, M., Park, E.J., Lee, Y., 2013. High-quality embryo production and plant regeneration using a two-step culture system in isolated microspore cultures of hot pepper (Capsicum annuum L.). Plant Cell Tiss Org. Cult. 112:191-201.
• Koleva-Gudeva, L. 2003. The effect of incubation treatment on the pepper (Capsicum annuum L.) androgenesis. Institute of Southern Crops, Strumica, Yearbook, pp:87-94.
• Koleva-Gudeva, L., Spasenoski, M., Trajkova, F., 2007. Somatic embryogenesis in pepper anther culture:the effect of incubation treatments and different media. Sci. Hort. 111:114-119.
• Koleva-Gudeva, L., Trajkova, F., Dimeska, G., Spasenoski, M., 2009. Androgenesis efficiency in anther culture of pepper (Capsicum annuum L.). Acta Hort. 830:183-190.
• Koleva-Gudeva, L., Trajkova, F., 2012. Anther culture of pepper:morphological characteristics of fruits of androgenetic pepper lines (Capsicum annuum L.). J. Res. Agric. 1:136-145.
• Koleva-Gudeva, L., Gulaboski, R., Janevik-Ivanovska, E., Trajkova, F., Maksimova, V., 2013. Capsaicin inhibitory factor for somatic embryogenesis in pepper anther culture. Electron J. Biol. 9:29-36.
• Kuo, J.S., Wang, Y.Y., Chien, N.F., Ku, S.J., Kung, M.L., Hsu, H.C., 1973. Investigations on the anther culture in vitro of Nicotiana tabacum L. and Capsicum annuum L. Acta Bot. Sin. 15:47-52.
• Lichter, R., 1982. Induction of haploid plants from isolated pollen of Brassica napus. Zeitschrift für Pflanzenphysiologie 105:427-434.
• Liu, F., Zhao, H., Chen, B., Zhang, Y.Y., 2007. Embryogenesis of microspore derived multicells in Capsicum annuum L. Fen Zi Xi Bao Cheng Wu Xue Bao 40:371-379.
• Lantos, C., Juhász, A.G., Somogyi, G., Ötvös, K., Vági, P., Mihály, R., Kristóf, Z., Somogyi, N., Pauk, J., 2009. Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via coculture with ovary of pepper or wheat. Plant Cell Tiss Org. Cult. 97:285-293.
• Lantos, C., Juhasz, A.G., Vagi, P., Mihaly, R., Kristof, Z., Pauk, J., 2012. Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.). Plant Biotechnol Rep. 6:123-132.
• Maraschin, S.F., Priester, W., Spaink, H.P., Wang, M., 2005. Androgenic switch:an example of plant embryogenesis from the male gametophyte perspective. J. Exp. Bot. 417:1711-1726.
• Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
• Mityko, J., Andrasfalvy, A., Csillery, G., Fari, M., 1995. Anther culture response in different genotypes and F₁ hybrids of pepper (Capsicum annuum L.). Plant Breed 114:78-80.
• Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen grains. Science,163:85-87.
• Niklas-Nowak, A., Olszewska, D., Kisiała, A., Nowaczyk, P., 2012. Study of individual plant responsiveness in anther cultures of selected pepper (Capsicum spp.) genotypes. Folia Hort. 24:141-146.
• Novak, F., 1974. Induction of a haploid callus in anther cultures of Capsicum sp. Z Pflanzenzücht 72:46-54.
• Nowaczyk, L., Nowaczyk, P., Olszewska, D., Niklas-Nowak, A., 2015. Effect of 2,4-dichlorophenoxyacetic acid pretreatment of Capsicum spp. donor plants on the anther culture efficiency of lines selected by capsaicinoid content. BTa JBCBB 96:179-183.
• Nowaczyk, P., Kisiała, A., 2006. Effect of selected factors on the effectiveness of Capsicum annuum L. anther culture. J. Appl. Genet. 47:113-117.
• Ochoa-Alejo, N., Ramirez-Malagon, R., 2001. In vitro chili pepper biotechnology. In Vitro Cell Dev Biol. Plant 37:701-729.
• Olszewska, D., Kisiała, A., Niklas-Nowak, A., Nowaczyk, P., 2014. Study of in vitro anther culture in selected genotypes of genus Capsicum. Turk J. Biol. 38:118-124.
• Özkum, D., Tıpırdamaz, R., 2007. Effects of silver nitrate, activated charcoal and cold treatment on the in vitro androgenesis of pepper (Capsicum annuum L.). Acta Hort. 729:133-136.
• Özsoy, B. 2019. Genotip, besin ortamı ve stres uygulamalarının biberde androgenesis üzerine etkileri (Yüksek Lisans Tezi). Tokat Gaziosmanpaşa Üniversitesi Fen Bilimleri Enstitüsü Bahçe Bitkileri Anabilim Dalı, Tokat, 66s.
• Parra-Vega, V., González-García, B., Seguí-Simarro, J.M., 2013-a. Morphological markers to correlate bud and anther development with microsporogenesis and microgametogenesis in pepper (Capsicum annuum L.). Acta Physiol Plant 35:627-633.
• Parra-Vega, V., Renau-Morata, B., Sifres, A., Seguí-Simarro, J.M., 2013-b. Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.). Plant Cell Tiss Organ Culture 112:353-360.
• Pulido, A., Bakos, F., Castillo, A., Valle´s, M., Barnabas, B., Olmedilla, A., 2005. Cytological and ultrastructural changes induced in anther and isolated-microspore cultures in barley:Fe deposits in isolated-microspore cultures. J. Struct. Biol. 149:170-181.
• Qin, X., Rotino, G.L., 1993. Anther culture of several sweet and hot pepper genotypes. Capsicum Eggplant Newsl. 12:59-62.
• Reynolds, T.L., 1993. A cytological analysis of microspores of Triticum aestivum (Poaceae) during normal ontogeny and induced embryogenic development. Am. J. Bot. 80:569-576.
• Rodeva, V., Irikova, T., Todorova, V., 2004. Anther culture of pepper (Capsicum annuum L.):comparative study on effect of the genotype. Biotechnol Biotechnol Equip 3:34-38.
• Roshany, G., Kalantarai, S., Naderi, R., Hassani, M.E., 2013. Callus formation via anther culture in Capsicum annuum L. with differences in genotypes, media and incubation temperature. Tech. J. Engin. & App. Sci. 3:3847-3853.
• Seguí-Simarro, J.M., P. Corral-Martínez, V. Parra-Vega, B. González-García, 2011. Androgenesis in recalcitrant solanaceous crops. Plant Cell Rep. 30:765-778.
• Sibi, M., Dumas de Vaulx, R., Chambonnet, D., 1980. Androgenese in vitro chez le Piment Capsicum annuum L.:Impact des pretraitements sur le taux de plantes regenerees. In:Reunion CR (ed) EUCARPIA application de la culture in vitro a l’Amelioration des Plantes Potageres, Versailles, 16-18 April, pp:143-149.
• Sunderland, N., Wicks, F.M., 1971. Embryoid formation in pollen grains of Nicotiana tabacum. J. Exp. Bot. 22:213-226.
• Sunderland, N. 1974. Anther culture as a means of haploid induction. In:K.J. Kasha (Eds):Haploids in higher plants:advances and potential. The University of Guelph, Guelph.
• Supena, E., Suharsono, S., Jacobsen, E., Custers, J., 2006. Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.). Plant Cell Rep. 25:1-10.
• Supena, E., Custers, J., 2011. Refinement of shed-microspore culture protocol to increase normal embryos production in hot pepper (Capsicum annuum L.). Sci. Hortic. 130:769-774.
• Szakacs, E., Barnabas, B., 1988. Cytological aspects of in vitro androgenesis in wheat (Triticum aestivum L.) using fluorescent microscopy. Sex Plant Reprod, 1:217-222.
• Taşkin, H., Büyükalaca, S., Keleş, D., Ekbiç, E., 2011. Induction of microspore-derived embryos by anther culture in selected pepper genotypes. Afr. J. Biotechnol. 10:17116-17121.
• Wang, Y.Y., Sun, C.S., Wang, C.C., Chien, N.J., 1973. The induction of pollen plantlets of Triticale and Capsicum annuum anther culture. Sci. Sin 16:147-151.
• Wang, L.H., Zhang, B.X., 2001. Advancement in the anther culture of Capsicum annuum L. China Veg. 3:52-53.
• 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.
• Zhao, J., Zhou, X., Zhang, Z., Yang, B., Zhou, S., 2010. Effects of culture media on anther culture of chili pepper (Capsicum annuum L.). J. Hunan Agric. Univ. (Nat Sci) 36:181-184.