ANO

AOVANCES //////////

IN GENETICS /////// ANO BREEOING //// OF CAPSICUM ////// ANO EGGPLANT ///

Editors Jaime Prohens Adrián

Rodríguez-Burruezo

EDITORIAL UNIVERSITAT POUTECNICA DE VALENCIA

The publication of this book has been funded by Ministerio de Ciencia e Innovación (grant reference: AGL2009-07831-E/AGR) and by Conselleria d'Educació de la Generalitat Valenciana (grant reference: AORG/2010/014).

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Sub-title Proceedings of the XIVth EUCARPIAMeeting on Genetics and Breeding of Capsicum ti Eggplant, 30 August - 1 September 2010, Valencia, Spain Publisher Editorial de la Universitat Politecnica de Valencia Camino de Vera s/n, 46022 Valencia, Spain Tel. 963877012. Fax 963877912 Ref. 2010.2354 © Jaime Prohens and Adrián Rodríguez-Burruezo

Printed by LAIMPRENTA CG ISBN: 978-84-693-4139-1 Depósito Legal: V-2687-2010

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Eds. J. Prohens 8: A. Rodríguez-Burruezo Advances in Genetics and Breeding of Capsicum and Eggplant, (2010) Editorial de la Universitat Politecnica de Valencia, Valencia, Spain

Improvement in doubled haploids production through culture of isolated eggplant microspores P. Corral-Martínez,

in vitro

J.M. Seguí-Si marro y Mejora de la Agrodiversidad

Instituto

para la Conservación

Valencia.

Ciudad Poli técnica de la Innovación

(CPI), Edificio

Valenciana

(COMAV). Universidad

Politécnico

de

8E"Escalera l. Camino de Vera sIn, 46022 Valencia.

SPAIN. Contact: [email protected]

Abstract Production of androgenic doubled haploids by means of isolated microspore cultures is a promising alternative to classic breeding techniques to obtain pure lines with fewer resources. But unfortunately, this technique is not optimized in eggplant and there is only a previous published work, where doubled haploids are obtained from calli, not embryos. In this work we have improved this existing procedure, increasing significantly the number of calli by two ways: the co-culture with Brassica napus induced microspores and the disruption of calli through different mechanical methods. We have also analyzed the ploidy of the calli and regenerants obtained by flow cytometry. The calli were fundamentally mixoploids, although haploid and doubled haploid callí were also observed. Nevertheless, the majority of the regenerants studied were doubled haploids, with only few haploid and mixoploid individuals.These results open new ways to improve the efficiency of isolated microspore cultures in eggplant. Keywords: androgenesis, Solanum melongena.

Brassica napus, co-culture,

haploid,

microspore

embryogenesis,

Introduction The production of doubled haploids by means of androgenesis allows for the shortening of breeding programs, obtaining pure lines in much less time and with fewer resources, both human and material (Forster et al., 2007; Seguí-Simarro and Nuez, 2008). This is the reason why these methods are the alternative of choice in those crops where the technique is optimized. Eggplant (Solanum melongena L.) is a crop of great interest for the Spanish agriculture. In spite of it, in eggplant only one of the two techniques for induction of androgenesis, the culture of anthers, is well set up. The second one, the culture of isolated microspores, although technically more complex, presents a number of practical advantages that makes it worth to investigate on its optimization. Up to date, there is only a previous study published on regeneration of plants from cultures of isolated microspores (Miyoshi, 1996).

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Advances in Genetics and Breeding 01 Capsicum and Eggplant

We have previously designed a method for the culture of eggplant isolated microspores which overperforms the previous report in terms of number of calli and doubled haploids obtained (Corral-Martínez et al., 2008). In addition, Miyoshi (1996) described only formation of callus from isolated microspores, whereas we have observed that globular embryos are formed prior to their transformation into microcalli. In this work, we have studied the variation in ploidy level of the calli and the regenerants obtained from microspore cultures using flow cytometry. Rapeseed (Brassica napus) is a model system for microspore embryogenesis where doubled haploids embryo can be easily obtained. We have also evaluated in this work the possible effect of co-culturing eggplant microspores with those of rapeseed, in order to evaluate their possible effect in the improvement of the efficiency of our system. Finally, we have assessed different methods to multiply the number of calli in the cultures, in order to have more material to further optimize the protocol in a genotypeindependent manner. Our results may represent an important advance in the development of a highly efficient microspore culture system in eggplant for the production of androgenic doubled haploids.

Material

and methods

Plant material and growing conditions We have used individuals of a commercial hybrid of eggplant, Bandera, as donor plants. They were obtained from the CONlAVgermplasm collection. Plants were grown in the CONlAV glasshouses at the Universidad Politécnica de Valencia, under 18°C and naturallight. In vitro isolated microspores culture, co-culture with Brassica napus and plant regeneration Flower buds at the appropriate stage (anther lengh about 6 mm with microspores at the vacuolated stage) were excised "and surface sterilized. With this size the microspores were mainly at vacuolated stage: Microspores were isolated, placed in a 6 cm petri dish with sterile distilled water and pretreated according to Miyoshi, 1996. Later on, plates were transferred in NLN medium at 25°C (Miyoshi, 1996) and incubated into a growth chamber at 25°C and 16/8 h photoperiod. Induced calli were transferred to solid MS medium a month later, where they regenerated shoots and then full plantlets. For the co-culture with rapeseed microspores, after the application of the inductive treatment separately for both microspore types, they were mixed and equally distributed in plates either with eggplant or rapeseed culture medium. The culture medium for rapeseed microspores was prepared according to Custers, (1994). Flow cytometry Small pieces of green calli and young leaves from regenerated plants were analyzed using the CyStain UV Precise P Kit (Partec). The plant material was crushed on ice for 1 minute with 500 ~l of NEB (nuclei extraction buffer). Then, 2 ml of DAPI were added and incubated for 5 minutes. The obtained extract was filtered away with 30 ~m cell Tricks · 370

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After isolating the calli, they were subjected to several mechanical procedures to disgregate their cells and increase the number of microcalli dones. These procedures allowed for the disgregation of cells or groups of cells (Figure 2A). When those cells were transferred to fresh culture medium for calli regeneration, a week later the cells presented a normal, undisturbed appearance (Figure 2B). Each method yielded different efficiencies in terms of number of calli (Figure 2C). The highest number of calli was obtained by crushing the original calli with a syringe pisto n in sterile conditions. But in this case, the viability of calli was lower than 10% (Figure 20). The second highest number of calli was obtained incubating them for seven days under constant agitation in fresh liquid medium. In this case, the viability was very low too (Figure 20). In the other two cases the number of calli obtained were very similar (Figure 2C). The viability of calli obtained after two days in constant agitation in liquid medium was approximately 60%, while after 3 minute vortexing, the viability was higher, nearly 80% (Figure 20). Our results indicated that the best procedure for disgregation is 3 minutes in vortex. Increase in number of calli through co-culture of eggplant with rapesefd microspores. In order to improve the efficiency of the culture of eggplant isolated microspores, we have co-cultured them with isolated and induced rapeseed microspores (Figure 3). After two weeks of co-culture, some initial divisions of eggplant were observed when the co-culture was carried out in rapeseed medium (Figure 3A). In these conditions, rapeseed embryos were not induced. In the eggplant medium, calli of eggplant could be identified but rapeseed embryos or dividing microspores were not observed (Figure 3B). After a month of culture, a significant observed in the culture with eggplant alone (Figure 3C). An increase in the experiments was also observed (data were observed, either for rapeseed or 372

increase in the number of total eggplant calli was medium compared to the eggplant microspores mean size of the calli compared to the control not shown). However, neither calli nor embryos for eggplant in the rapeseed medium (Figure 30).

Advances in Genetics and Breeding of Capsicum and Eggplant

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•... Figure 3. Co-culture with Brassicanapus. A: Mixed microspores of eggplant and rapeseed in rapeseed medium. Two weeks after the culture, the first dividing eggplant microspores were observed. B: Mixed microspores of eggplant and rapeseed in eggplant medium. After two weeks of culture, calli of eggplant could be identified. C: Comparisonof the efficiency between the CQ-culturewith rapeseed (left dish) and the culture of eggplant microspores alone (right dish).D: Number of eggplant calli obtained by the culture of eggplant microspores alone (control), and obtained by the co-culture of both microspore types. Discussion

- .•••.1

In this work, the analyses of the calli ploidy level revealed that not all of the cells undergo genome duplications. This heterogeneity is the responsible of the mixoploidy observed. This does not seem to be an important problem, since a high percentage of doubled haploids (60%), more genetically stable, are obtained. In tomato, it was described that mixoploidy is frequent in young calli and regeneration is favoured over the 2C regions (Seguí-Simarro and Nuez, 2007). This spontaneous genome doubling may be due to the effects of the in vitro culture conditions, and more specifically, to the effect of the growth regulators added to the medium. In this system, the disruption of microcalli supposes an effective method to increase considerably the material, obtaining clonic calli and regenerant lines useful to evaluate the effect of different factors regardless of genotype. Among the procedures used in this work to disgregate the cells, the most efficient in terms of number of calli obtained was crushing, but viability was the lowest. This might be due to the aggressive disruption of the calli in individual cells, killing or damaging many of them. By means of a soft and continued agitation, we obtained the highest number of microcalli. Two days of agitation is better because, although we obtained the lowest number of calli, the damage to the cells was minimized and the calli regenerated from them had higher size and viability.

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By vortexing, we obtained the lowest number of calli, but they are the biggest in size, and the highest in viability. In addition, it is the most rapid and simple method. Vortexing released a sufficient number of cells that proliferated and regenerated shoots, probably due to reduced damage they have suffered.

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Advances in Genetics and Breeding of Capsicum and Eggplant

We have shown that the co-culture of isolated eggplant and rapeseed microspores can help to improve the efficiency of the system. In the co-culture in eggplant medium, the number of calli obtained was almost twice than the control culture. This allows to increase number and size of the calli. Our results suggest that rapeseed secretes to the culture medium something that favours the proliferation of eggplant calli. On the contrary, when the co-culture is in rapeseed medium neither eggplant calli nor rapeseed embryos were observed. The absence of eggplant calli could to be due to the lack of hormones in the culture medium, while the absence of rapeseed embryos could be due to the secretion of some inhibitory substance by the eggplant microspore. Further experiments will help to elucidate this hypothesis.

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Ackowledgements We want to acknowledge the staff of the COMAV greenhouses for their valuable help. P.C-M is a FPI predoctoral fellow from spanish Generalitat Valenciana. This work was supported by grants AGL2006-06678 from the spanish Ministry of Education and science (MEC) and ACOMP12007/148 from spanish Generalitat Valenciana to JMss.

References Corral-Martínez, P.; Nuez, F.; segui-simarro, J.M 2008. Recent advances in eggplant microspoea cultures for production of androgenic double haploids, pp. 104-108. In: Prohens, J.; Badenes, M.L. (eds.) Modern variety breeding for present and future needs, Editorial de la Universidad Politécnica de Valencia, Valencia, spain. Custers, J.B.M.; Cordewener, J.H.G.; Nollen, Y.; Dons, H.J.M.;Van Lockeren Campagne M.M.1994. Temperature controls both gametophytic and sporophytic development in microspore cultures of Brassica napus. Plant Cell Reports. 13:267-271. Dumas de Vaulx, R.; Chambonnet, D. 1982. Culture in vitro d'antheres d'aubergine (Solanum melongena L.): stimulation de la production de plantes au moyen de a 35°C associés a de faibles teneurs en substances de croissance. traitements Agronomie 2:983-988. Forster, B.P.; Heberle-Bors, E.; Kasha, K.J.; Touraev, A. 2007. The resurgence of haploids in higher plants. Trends in Plant science 12:368-375. Miyoshi, K. 1996. Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L). Plant Cell Rep. 15: 391-395. Seguí-Si marro, J.M.; Nuez, F. 2007. Embryogenesis induction, callogenesis, and plant regeneration by in Vitro culture in tomato isolated microsporas and whole anthers. Journal of Experimental Botany 58: 1119-1132. seguí-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.

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