ABSTRACT
O uso da biobalística na transformação genética de plantas requer a otimização de diversos parâmetros, entre eles, o desenvolvimento de protocolos para regeneração de plantas a partir de células transformadas. Este trabalho teve como objetivo avaliar a eficiência de regeneração de plantas de milho a partir de calos embriogênicos e de explantes sementes divididas, submetidos à transformação por biobalística, utilizando o gene bar como marcador de seleção. Foram submetidos 2000 agrupamentos de calos embriogênicos de milho e 2000 explantes sementes divididas à transformação por biobalística. A seleção dos eventos de transformação foi realizada em meios de cultivo suplementados com glifosinato de amônio. As plântulas selecionadas foram aclimatadas e transplantadas para vasos em casa de vegetação. Com a utilização de embriogênese somática, a eficiência de regeneração foi de 0,2%, enquanto que, com a utilização de sementes divididas, a eficiência de regeneração foi de 1,3%. Embora ambos os protocolos possibilitem a regeneração de plantas de milho submetidas à transformação por biobalística, há a necessidade de aumentar sua eficiência.
The use of biolistic on genetic transformation of plants requires an optimization of several parameters, including the protocol development for plant regeneration from transformed cells. The objective of this research was to evaluate the regeneration efficiency of maize plants from embryogenic callus and explants from split seeds submitted to transformation by biolistic using the gene bar as a selectable marker. It was submitted 2000 clusters of embryogenic callus and 2000 divided maize seeds to transformation by biolistic. The selection of transformation events was in culture medium supplemented with ammonium glifosinate. Selected seedlings were acclimatized and transplanted to pots in a greenhouse. Using somatic embryogenesis, the regeneration efficiency was 0.2%, while using split seeds explants the regeneration efficiency was 1.3%. Although both protocols allow the maize plant regeneration after transformation by biolistic, there is a need to increase its efficiency.
ABSTRACT
Insects pests and weeds are the main factors that reduce the yield of sugar beet. Genetic engineering breeding is an effective method to breed insect-resisitant and herbicide-resisitant sugar beet. A transformation system for foreign genes in sugar beet chloroplast was established. The expression of the foreign genes can confers resistance in transgenic sugar beet plants to insects pests and weeds. The chloroplast transformation vector pSKARBt/bar, which carries Bt cry1Ac gene and bar gene expression cassettes, was constructed by using molecular method. The Bt gene expression cassette contained the 3.5 kb Bt cry1Ac gene under the control of psbA promoter and terminator cloned from sugar beet chloroplast genome. The bar gene expression cassette contained the bar gene, 16 S promoter and terminator cloned from sugar beet chloroplast genome, The atpB and rbcL gene cloned from sugar beet chloroplast genome were used as homologous fragment, the bar gene was the selective marker. Plasmid pSKARBt/bar were transformed into the petioles of sugar beet with particle bombardment method. The petioles were planced onto the shoot-inducing selection medium which contained spectinomycin (20 mg/L), 6-BA (1.5 mg/L) and NAA (0.2 mg/L) at first. And when the green shoots regenerated, the green shoots were transfered into the shoot-propagation medium for optimal shoot development which contained spectinomycin (20 mg/L) and 6-BA (0.5 mg/L) and NAA (1.0 mg/L) one subculture at 20-day intervals, and then the shoots were transfered into the shoot-propagation medium for optimal shoot development with herbicide (PPT 10 mg/L) several subcultures. The shoots were transfered into the root-induction medium with herbicide (PPT 10 mg/L) and the transgenic plants were obtained at last. The transgenic sugar beet plants were testsed by PCR and Southern blot. The results showed that the Bt gene and bar gene had been transferred into the chloroplast genome of sugar beet. The transgenic plants had tolerance to both PPT and bioassays testsed. The insecticidal activity (the mortality of larvaes was 33%～80%) and herbicide resistance of the transgenic plants indicated that the relevant protein had been expressed already in sugar beet. The study showed that the bar gene can also be used as an efficient selective marker gene besides antibiotic resistant markers in plant transformation. Efficient transformation system in sugar beet chloroplast had been established.