Induction of symmetrical nucleus division and multi-nucleate structures in microspores of eggplant (Solanum melongena L.) cultured in vitro

Abstract

A modification of a protocol used to induce tobacco microspore embryogenesis was tested in eggplant (Solanum melongena L.). In tobacco, uninucleate microspores are subjected to stress treatment by culturing in mannitol containing "B" medium at 33°C for six days. The microspores are then transferred to maltose containing AT3 medium for further development. In the experiment presented here late uninucleate and bi-nucleate microspores of the eggplant cultivar Bambino were pre-cultured in B medium and then incubated at +4°C, 25°C and 33°C, respectively, for two days. After the pre-treatments, microspore cultures were transferred to AT3 medium containing 0.25 M maltose and maintained at 25°C in the dark. Presence of symmetrical division and multinucleate structures was checked with DAPI staining of the nucleus after one and two weeks. Symmetrical division of the nucleus and multinucleate structures were observed only in uni-nucleate microspores pre-treated at 33°C for two days. The frequency of multinucleate structures was 19.4% under these conditions. We demonstrated that eggplant is responsive to the modified tobacco protocol in the production of symmetrically division and multinucleate structures. These results may be used as a basis for adaptation fully of the tobacco system in eggplant. Key words: haploid, microspore culture, symmetrical division, mannitol starvation INDUÇÃO DE DIVISÃO NUCLEAR SIMÉTRICA E ESTRUTURAS MULTINUCLEADAS EM MICRÓSPOROS DE BERINJELA (Solanum melongena L.) CULTIVADOS IN VITRORESUMO: Uma modificação de um protocolo utilizado para induzir embriogênese a partir de micrósporos de tabaco foi testada em berinjela (Solanum melongena L.). Em tabaco, micrósporos uninucleados são submetidos a um tratamento de estresse em meio "B" contendo manitol a 33°C por seis dias. Os micrósporos são então transferidos para um meio AT3 contendo maltose para as fases posteriores de desenvolvimento. Neste estudo, micrósporos uninucleados e binucleados tardios do cultivar de berinjela Bambino foram pré-cultivados em meio "B" e, posteriormente, incubados a 4°C, 25°C e 33°C por dois dias. Após os pré-tratamentos, as culturas de micrósporos foram transferidas para o meio AT3 contendo maltose 0,25 M e mantidas a 25°C no escuro. A presença de divisão simétrica e estruturas multinucleadas foram verificadas com coloração DAPI do núcleo após uma e duas semanas. Foram observadas divisões simétricas dos núcleos e estruturas multinucleadas somente em micrósporos uninucleados, pré-tratados a 33ºC por dois dias. A frequência de estruturas multinucleadas foi de 19,4% sob essas condições. Nós demonstramos que a berinjela responde ao protocolo modificado de tabaco para produção de divisões simétricas e estruturas multinucleadas. Esses resultados podem ser utilizados como base para uma completa adaptação do sistema de tabaco em berinjela. Palavras chave: haplóide, cultura de micrósporos, divisão simétrica, estresse com manitol

Androgenesis in recalcitrant solanaceous crops

“…As in tomato, the development of a method for androgenesis induction from isolated eggplant microspores would be highly desirable, and would avoid the problems mentioned above of tapetum effect, appearance of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 somatic regenerants, and low efficiency. In this regard, it is striking that very few studies have been published on the successful production of DH plants from isolated microspores (Miyoshi 1996;Corral-Martínez et al 2008;Bal et al 2009). In the studies These results are extending the still scarce knowledge on eggplant microspore cultures.…”

Development of Androgenesis Studies on Eggplant (Solanum melongenaL.) in Turkey from Past to Present

“…The first haploidization study in eggplant began in 1991 in Turkey with Karakullukçu's [19] anther culture study. This was followed by other anther culture [20,[35][36][37][38][39][40][41][42][43][44][45][46], isolated microspore culture [32,34,47] and shedmicrospore culture [19] studies. Although the number of studies on this subject is small at the beginning, the efforts to obtain DH plants through anther culture have been accelerated in recent years thanks to the increase in the state-supported projects and the engagement of private sector.…”

“…Differently, Özzambak and Atasayar [71] used MS [72] and NN [73] media, while Alpsoy [38] and Doksöz [20] investigated the effect of MS medium as well as DDV-C and DDV-R media. In the microspore culture studies [32,34,47], NLN [74] medium was used.…”

“…However, incubation in the dark at +35°C for 8 days is generally used successfully in eggplant anther cultures. To date, a number of factors affecting microspore embryogenesis have been investigated, and their effects have been demonstrated in several eggplant haploidy studies [11,[17][18][19][28][29][30][31][32][33][34] conducted in the world and Turkey; however, this article will mainly focus on studies in Turkey.…”

Pseudo-embryogenic structures in anther and isolated microspore cultures in vitro: a cautionary guide

“…True microspore-derived multicellular structures (Figures 1c, e) are compared with pseudo-structures (Figures 1d, f ). In the isolated microspore cultures of eggplant, sporophytically developing microspores are swelled and compartmentalized, containing several nuclei (Bal et al 2009) similar to trichome head as shown in Figure1f.…”