Carbon sources and polyethylene glycol on soybean somatic embryo conversion

Abstract

-Somatic embryogenesis is an efficient method for the production of target cells for soybean genetic transformation. However, this method still offers low percentages of plant regeneration, and perhaps is related to the maturation process and high morphological abnormalities of the matured embryos. This study aimed to identify a maturation medium that could contribute to the outcome of more efficient plant regeneration results. Embryogenic clusters, derived from cotyledons of immature seeds of the soybean cultivars Bragg and IAS5, were used as starting material for embryos development. Different maturation media were tested by using 6% maltose, 3% sucrose or 6% sucrose, combined with or without 25 g L -1 of the osmotic regulator polyethylene glycol (PEG-8000). The histodifferentiated embryos were quantified and classified in morphological types. Percentages of converted embryos were analyzed. Cultivar Bragg resulted in higher matured embryo quantities, but lower percentages were obtained for the conversion in comparison to cultivar IAS5. While the addition of PEG did not affect the number of embryos converted into plants, 6% sucrose enhanced the conversion percent significantly.Index terms: Glycine max, PEG-8000, sucrose, osmotic regulation. Fontes de carbono e polietilenoglicol na conversão de embriões somáticos de sojaResumo -A embriogênese somática é um eficiente método na produção de células-alvo para a transformação genética de soja. Entretanto, este método ainda apresenta baixas porcentagens de regeneração de plantas, o que pode estar relacionado ao processo de maturação e às altas anormalidades morfológicas dos embriões maturos. Este estudo visou a identificar um meio de maturação que pudesse contribuir com resultados mais eficientes de regeneração de plantas. Foram utilizados conjuntos embriogênicos derivados de cotilédones imaturos de sementes de soja das cultivares Bragg e IAS5 para o desenvolvimento de embriões. Testaram-se diferentes meios de maturação, com a utilização de 6% de maltose, 3% de sacarose ou 6% de sacarose, combinadas ou não com 25 g L -1 do regulador osmótico polietilenoglicol (PEG-8000). Os embriões histodiferenciados foram quantificados e classificados em tipos morfológicos. A porcentagem de embriões convertidos em plantas foi analisada. A cultivar Bragg apresentou maior quantidade de embriões maturos, mas foram obtidas menores porcentagens de conversão em comparação à cultivar IAS5. A adição de PEG não afetou o número de embriões convertidos em plantas. Entretanto, a concentração de 6% de sacarose aumentou significativamente a porcentagem de conversão.Termos para indexação: Glycine max, PEG-8000, sacarose, regulação osmótica.

Strategies for Improvement of Soybean Regeneration via Somatic Embryogenesis and Genetic Transformation

“…Carbohydrates are commonly used as carbon sources for in vitro development of tissues, and maltose and sucrose have been usually added to soybean tissue culture media in an optimal concentration range of 3% to 6% (Samoylov et al, 1998b;Körbes & Droste, 2005;Schmidt et al, 2005). Carbon source type and concentration required for histodifferentiation/maturation appear to differ between solid and liquid media.…”

“…Körbes & Droste (2005) compared conversion frequencies when 6% maltose was replaced by 3% maltose (MSM3) or 6% sucrose (MSS6). Results showed that maturation in MSS6 medium leads to an increment in rates of histodifferentiated embryos with normal morphology, as well as in plant recovery.…”

“…The quality of somatic embryos can be positively influenced by a low osmotic potential in maturation medium Körbes & Droste, 2005). Carbohydrates can act as an osmotic agent (Li et al, 1998).…”

“…Nevertheless, differences observed in embryos matured in media supplemented with maltose or sucrose have been related to these compounds' osmotic potentials (Samoylov et al, 1998b;Körbes & Droste, 2005). Influence of other osmotic agents (sorbitol, mannitol and polyethylene glycol) has also been tested in soybean somatic embryogenesis Körbes & Droste, 2005;Schmidt et al, 2005), but positive effects were only reported for sorbitol ), as described above.…”

“…MSM6 -MS salts, B5 vitamins, 6% maltose and 0.25% Gelrite or 0.3% Phytagel (pH 5.8) (Finer & McMullen, 1991). MSS6 -MS salts, B5 vitamins, 6% sucrose, 0.3% Phytagel (pH 5.8) (Körbes & Droste, 2005). MSO -MS basal salts, B5 vitamins, 3% sucrose, and 0.2% Gelrite, at pH 5.8 role in the sequence of events leading to desiccation tolerance (Hoekstra et al, 2001).…”