versão On-line ISSN 1678-4596
Cienc. Rural v.38 n.1 Santa Maria jan./fev. 2008
Diferenças genotípicas no desenvolvimento de pró-embrióides e regeneração de plântulas verdes via androgênese em genótipo de cevada
Paula WiethölterI, 1; Maria Irene Baggio de Moraes FernandesII; Sandra Patussi BrammerIII; Euclydes MinellaIII; Edson Jair IorkzeskiIII
IDepartamento de Plantas de Lavoura, Universidade Federal do Rio Grande do Sul (UFRGS). Av. Bento Gonçalves, 7712, 91540-000, Porto Alegre, RS, Brasil. E-mail: firstname.lastname@example.org
IIUniversidade de Passo Fundo (UPF), Passo Fundo, RS, Brasil
IIIEmpresa Brasileira de Pesquisa Agropecuária-Trigo-Passo Fundo (EMBRAPA), Passo Fundo, RS, Brasil
The development of in vitro haploid plants followed by spontaneous or induced genome duplication allows to achieve, in one generation, the recovery of total homozygosis. The efficiency of the haplodiploidization process through in vitro anther culture of barley is variable among genotypes. This study was aimed at determining the androgenetic response of nine barley genotypes from the breeding program of Embrapa Trigo, analysing proembryoid development and green plantlets regeneration in anthers cultivated in vitro. Cultivar 'BR2' presented the highest average of proembryoids (104/anther) and 'MN698' presented the highest average of green plantlets (0,41/anther). There was a significant variation among the average values of barley genotypes for embryo formation and green plantlets regeneration, making possible the selection to combine androgenetic capacity and good agronomic traits.
Key words: haploid, in vitro androgenesis, androgenetic capacity, Hordeum vulgare L.
A obtenção, na cevada, de plantas haplóides in vitro e a posterior duplicação natural ou artificial do genoma permitem alcançar a homozigose completa, em uma geração. A eficiência da haplodiploidização pela cultura de anteras é variável entre os genótipos. Foi avaliada a resposta androgenética através da formação de pró-embrióides e da regeneração de plântulas verdes em nove cultivares do programa de melhoramento de cevada da Embrapa Trigo, em anteras cultivadas in vitro. A cultivar BR2 apresentou maior média de pró-embrióides (104/antera), enquanto MN698 mostrou a maior média de plântulas verdes (0,41/antera). Houve variação significativa entre os valores médios dos genótipos em relação à formação de pró-embrióides e à regeneração de plântulas verdes, indicando a possibilidade de seleção para combinar a capacidade androgenética com boas características agronômicas.
Palavras-chave: haplóides, androgênese in vitro, capacidade androgenética, Hordeum vulgare L.
After GUHA & MAHESHAWARI (1966) discovered in vitro androgenesis in cultured anthers of Datura innoxia, it become possible, in responsive crops, to obtain, in one step, fully homozygous lines, saving time and resources in breeding programs. Androgenetic capacity is defined as the ability of the male gametic cell to reverse its development, giving rise to haploid green plantlets that become completely homozygous and fully fertile after spontaneous or colchicine induced genome duplication (PICARD et al., 1990; PETERS et al., 1999). Microspores of androgenetic genotypes are able to develop haploid proembryoids which may be regenerated in green haploid plantlets. In barley, as well as other responsive species, inherited factors have been reported as involved in successful haploid plant development (FAROUGHI-WEHR et al., 1982; LARSEN et al., 1991; HENRY et al., 1994). But androgenetic response is possible only when androgenetic genotypes have appropriate environmental conditions that include physiological state of the donor plant, culturing anthers at the right stage of development of microspore and the right culture medium composition (OLSEN, 1987; MORAES-FERNANDES, 1990; HOEKSTRA, 1996; GRANDO & MORAES-FERNANDES, 1997). If some of these factors are inadequate, the anther response may be absent even in androgenetic genotypes. The androgenetic ability may be introduced by crossing in genotypes with good agronomic traits speeding up breeders selection that can be made directly in uniform progenies (FAROUGHI-WEHR & FRIEDT, 1984; QUÉDRAOGO et al., 1998; MORAES-FERNANDES et al., 1999; SMYKAL, 2000). The purpose of this research was the analysis of in vitro proembryoid development and green plantlets regeneration, in in vitro cultivated anthers, in order to investigate the androgenetic capacity of barley cultivars belonging to crossing block of Embrapa Trigo Barley Breeding Program that had good agronomic traits.
The experiment was carried out at the Biotechnology Laboratory of Embrapa Trigo. The cultivars used were 'BR2', 'BRS180', 'BRS195', 'EMBRAPA127', 'FM404', 'GIMPEL', 'MN684', 'MN698', and 'SCARLETT'. To minimize environmental effects, donor plants were cultivated in a growth chamber with controlled temperature, humidity and photoperiod. A complete randomized experimental design was used. Three plants of each genotype were grown per pot, with three replications (nine plants per genotype). When the main spikes reached the size of development in which uninucleated microspores could be cytologically identified in the anthers of central flowers, these spikes were asseptically cleaned with a 70% ethanol and a 10 days pretreatment at 4°C, in dark, was made; 36 anthers from 12 central flowers of each spike (three anthers/flower, 324 anthers/genotype), were cultured on modified Hunter medium (HUNTER, 1985). The Petry dishes were wrapped in aluminum foil and placed in a growth chamber at 25oC. After 18 days of in vitro culture, six main anthers of six central flowers were fixed in an acetic acid - ethanol solution (3:1). For optical microscope cytological studies, the anthers content was stained with acetic-carmine and cultured microspores were analyzed. After that, the remaining 24 anthers from each Petry dish were left 12 days more, covered with aluminum foil (total of 30 days in dark) and then left in light for monitoring green plantlets emergence. As soon as green plantlets were formed they were transferred to Potato II regeneration media (MORAES FERNANDES & PICARD, 1983). Therefore, from a total of 2916 anthers cultured in vitro proembryoids were counted in 486 anthers and green plantlets in 1944 anthers that remained in the medium. Proembryoids identification was made when three or more identical nuclei were observed in microspores with non disrupted membrane or clear embryo like structures. Data were submitted to statistical analysis and means were compared by using the Duncan test at 1% probability, using the Statistical Analysis System (SAS).
A very large variation in proembryoid occurrence was observed among cultured anthers of the same spike and among spikes of the same genotype (Table 1). Genotypes BR2 and BRS195 presented the highest value of proembryoids/anther and FM404, BRS180 and SCARLETT presented the lowest value. The statistical analysis of the regenerated green plantlets data showed that the barley cultivar 'MN698' was the best one for this trait, presenting a significantly higher number of regenerated plantlets: 41 green plantlets per each 100 cultured anthers (Table 1). There was no significant correlation between frequencies of proembryoids and frequencies of regenerated plantlets. But, BRS195, GIMPEL and MN684 had statistically similar means of proembryoids and green plantlets regeneration. However, MN684, EMBRAPA127, MN698 and SCARLETT, although having statistically similar means as far as proembryoid development, were different in relation to green plantlet regeneration.
QUÉDRAOGO et al. (1998), evaluating the androgenetic capacity of agronomic lines of barley and their hybrids, observed that Igri was less responsive than Léger in relation to proembryoid development, but similar in green plantlet regeneration. However, Cadette was similar to Igri in proembryoid development, but produced fewer green plantlets. STIVAL et al. (1997) studied heterozygous populations resulting from crosses between barley lines and found two groups of independent genotypes, one presenting high proembryoid induction and another group with high green plantlets regeneration. All genotypes presenting high regeneration capacity were descendent from line PFC9104, which is descendent from Igri which is referred as an androgenetic genotype, confirming the transmission of androgenetic ability. Therefore, the literature reports as well as the results observed here among cultivars of barley crossing block makes possible the recommendation for use as breeding strategy the simultaneous selection for androgenetic capacity and agronomic traits in order to increase genetic progress.
We would like to thank Embrapa Trigo for making possible the development of this research and also, thanks to Gilney Müller, Adrimara Radin, and Regina Fontaneli for their help.
FAROUGHI-WEHR B. et al. On the genetic improvement of androgenetic haploid formation in Hordeum vulgare L. Theoretical and Applied Genetics, v.62, p.233-239, 1982. [ Links ]
FAROUGHI-WEHR, B.; FRIEDT, W. Rapid production of recombinant barley mosaic virus resistant Hordeum vulgare lines by anther culture. Theoretical and Applied Genetics, v.67, p.377-382, 1984. [ Links ]
GRANDO, M.F.; MORAES-FERNANDES, M.I.B. Two points deterministic model for acquisition of in vitro pollen grain androgenetic capacity based on wheat studies. Brazilian Journal of Genetics, v.20, p.467-476, 1997. [ Links ]
GUHA, S.; MAHESHWARI, S.C. Cell division and differentiation of embryos in the pollen grains of Datura in vitro. Nature, v.212, p.97-98, 1966. [ Links ]
HENRY, Y. et al. Genetic analysis of in vitro plant tissue culture responses and regeneration capacities. Euphytica, v.79, p.45-58, 1994. [ Links ]
HOEKSTRA, S. Androgenesis in Hordeum vulgare L.: Physiological and molecular aspects. Amsterdam: TNO Nutrition and Food Research Institute, 1996. 121p. [ Links ]
HUNTER, C.P. The effect of anther orientation on the production of microspore-derived embryoids and plants of Hordeum vulgare cv. Sabarlis. Plant Cell Reports, v.4, p.267-268, 1985. [ Links ]
LARSEN, E.T. et al. Nuclear genes affecting percentage of green plants in barley (Hordeum vulgare L.) anther culture. Theoretical and Applied Genetics, v.82, p.417-420, 1991. [ Links ]
MORAES-FERNANDES, M.I.B.; PICARD, E. Viability of haploid production by anther culture using brazilian wheat genotypes. Revista Brasileira de Genética, v.6, n.2, p.261-277, 1983. [ Links ]
MORAES-FERNANDES, M.I.B. Obtenção de plantas haplóides através da cultura de anteras. In: TORRES, A.C.; CALDAS, L.S. Técnicas e aplicações da cultura de tecidos de plantas. Brasília: ABCTP/EMBRAPA-CNPH, 1990. Cap.4, p.311-332. [ Links ]
MORAES-FERNANDES, M.I.B. et al. Haplodiploidização: genética e melhoramento. In: TORRES, A.C. et al. Cultura de tecidos e transformação genética de plantas. Brasília: Embrapa, 1999. V.2, cap.3, p.613-650. [ Links ]
OLSEN, F.L. Induction of microspore embryogenesis in cultured anthers of Hordeum vulgare: the effects of ammonium nitrate, glutamine and asparagine as nitrogen sources. Carlsberg Research Communication, v.52, p.393-404, 1987. [ Links ]
PETERS, J.A. et al. Produção de haplóides e duplo haplóides. In: TORRES, A.C. et al. Cultura de tecidos e transformação genética de plantas. Brasília: Embrapa, 1999. V.2, cap.3, p.569-611. [ Links ]
PICARD, E. et al. Wheat (Triticum aestivum): In vitro production and utilization of double haploids. Biotechnology in Agriculture and Forestry, v.12, p.101-124, 1990. [ Links ]
QUÉDRAOGO, J.T. et al. Effect of amino acids, growth regulators and genotype and androgenesis in barley. Plant Cell, Tissue and Organ Culture, v.53, p.59-66, 1998. [ Links ]
SMIKAL, P. Pollen embryogenesis the stress mediated switch from gametophytic to sporophytic development. Current status and future prospects. Biologia Plantarum, v.43, p.481-489, 2000. [ Links ]
STIVAL, A. L. et al. Production of androgenetic barley doubled haploid lines. Pesquisa Agropecuária Brasileira, v.3, p.1159-1166, 1997. [ Links ]
1 Autor para correspondência.