Efficiency of Microspore Culture for Doubled Haploid Production in the Breeding Project "Czech Winter Rape"

Abstract

Winter rape (Brassica napus L. var. napus) is the most widespread oil plant and the second most frequently cultivated crop after cereals in the Czech Republic. Brassica napus L. is an allotetraploid or amphidiploid species with n = 19 chromosomes, derived from 2 diploid species B. oleracea (n = 9) and B. rapa (n = 10). The microspore doubled haploid (DH) methodology is now employed in many B. napus breeding programmes around the world as an alternative/supplement to conventional methods of homozygous line production (KUČERA et al. 2002FERRIE & KELLER 2004;FRIEDT & ZARHLOUL 2005;KOPRNA et al. 2005 Abstract: A selected set of twenty inter-line F 1 hybrids of Brassica napus L. was evaluated for their capacity to produce microspore-derived doubled haploid (DH) plants subsequently utilized in the breeding project "Czech Winter Rape". All tested genotypes were able to produce microspore-derived embryos based on the standardized protocol previously adopted for the Czech winter rape varieties/breeding lines; nevertheless, significant differences were found in the frequency of microspore-derived embryo production as related to particular genotypes. However, the fertile DH regenerants were obtained only in 12 hybrid lines of this set. The other 8 lines failed to produce fertile DH plants for several reasons, mainly due to altered morphology of microspore embryos connected with limited germination/conversion potential and due to unsuccessful colchicine treatment resulting in haploid or mixoploid (chimeric) regenerants exhibiting partial or full sterility. In general, colchicine treatment induced diploid (doubled haploid), polyploid (triploid, tetraploid) and mixoploid (containing both euploid and aneuploid nuclei) plants. The fully aneuploid plants were recorded only sporadically. Some modifications of culture protocol (removal of embryo cotyledons before transfer to a germination medium; production of more plantlets from a single embryo by rooting multiple shoots; repeated colchicine treatment of unsuccessfully doubled plants in a later phase of growth) could enhance the final yield of DH fertile regenerants in our experiments. In total, 590 microspore-derived regenerants were produced in a series of experiments, 236 (40%) of them represented fully fertile DH plants. The R2 seeds (progeny of DH R1-generation) of the most promising hybrid-lines were included in the tests of agronomic performance in field conditions.

Microspore culture protocol for Indonesian Brassica oleracea

“…In other Brassica spp. it was reported that 33°C for 10 min, then 30°C for 40 min, and finally returned to 25°C continuously was optimal for B. napus (Anandarajah et al 1991), or 32°C for 8 days and 25°C continuously (Tian et al 2004), 30°C for 14 days continuously (Smýkalová et al 2006), 32°C for 3 days and 25°C continuously (Takahira et al 2010) for the same species; 32.5°C for 1 day and 25°C continuously for B. oleracea var. costata DC (Dias and Correia 2002); 32°C for 3 days and 25°C continuously (Chanana et al 2005), 35°C for 2 days then at 25°C continuously (Agarwal et al 2006;Ali et al 2008), or 4 ± 1°C for 10 days followed by 25°C continuously (Prem et al 2008) for B. juncea.…”

“…bud length) and developmental stage of immature pollen grains: most microspores at the late-uninucleate to early binucleate stage can undergo embryogenesis (Pechan and Keller 1988;Hansen and Svinnset 1993;Custers 2003;Prem et al 2005;Abraha et al 2008). The high response of microspore Zaki and Dickinson (1995) B. campestris Canadian Cv-2 5390 embryos/100 buds nr nr Guo and Pulli (1996) B. juncea Couve Algarvia 9 embryos/2 buds nr nr Dias and Correia (2002) B. juncea Rajat 3500 embryos/100 buds 52 nr Chanana et al (2005) Interspecific hybrid of B. napus L 365 embryos/3 buds nr nr Smýkalová et al (2006) HSH nr not reported, a under optimal conditions embryogenesis in the same developmental stage was also recorded in B. campestris (Guo and Pulli 1996), B. carinata (Abraha et al 2008), B. juncea (Chanana et al 2005;Agarwal et al 2006;Ali et al 2008;Prem et al 2008), B. napus cv. 'Topas' (Anandarajah et al 1991;Takahata and Keller 1991;Supena et al 2006), and B. oleracea var.…”

“…costata DC (Dias and Correia 2002), or 5 9 10 4 cells/ml in B. campestris (Guo and Pulli 1996). Separately, a higher microspore density (7 9 10 4 to 10 9 10 4 cells/ml) was needed to produce a high level of embryogenesis for B. napus (Smýkalová et al 2006), 124 9 10 3 cells/ml for B. carinata (Abraha et al 2008), and 15 9 10 4 cells/ml for Indonesian Brassica cultivars. A higher density was necessary because of the low vitality of microspores, i.e.…”

Doubled haploids as a material for biotechnological manipulation and as a modern tool for breeding oilseed rape ( Brassica napus L.)

“…Alternatively, in 1982, Lichter developed an isolated microspore culture system for B. napus. These techniques have been gradually developed and constantly improved (Cegielska-Taras et al, 2002;Forster and Thomas, 2005;Palmer et al, 2005;Smýkalová et al, 2006;Ferri and Keller, 2007;rev. Pratap et al, 2009;rev.…”

FEDERAL RESEARCH CENTER FOR VEGETABLE GROWING — 100-YEAR HISTORY AS A BASIS FOR FUTURE DEVELOPMENTS (review)

“…Базовый протокол технологии получения удвоенных гаплоидов с помощью культуры изолированных микроспор, разработанный в начале 1980-х годов (67) для рапса, адаптирован для представителей рода Brassica (68,69). Он успешно используется в разных странах, много сообщений по указанной тематике публикуют ученые из Индии (70), Канады (71), Чехии (72). В ФГБНУ ФНЦО разработан базовый протокол культуры микроспор in vitro для капустных культур 73 Исследования по получению удвоенных гаплоидов моркови в мире ведутся давно, но первые успехи достигнуты в последнее время.…”