RESUMO
Production of transgenic plants with desired agronomic and horticultural traits has gained great importance to fulfill demands of the growing population. Genetic transformation is also a fundamental step to study basics of plant sciences. Different transformation protocols have been developed and used which are reliable and efficient. These protocols used antibiotic or herbicide resistance genes incorporated along with gene of interest to identify transformed plants from non-transformed ones. These marker genes may pose a threat to human and environment. Use of visual markers enables direct and easier observation of transformed plants with more precision. In current study a gene cassette with 'pigment production hydroxylase (PPH) gene under fiber specific promoter (GhSCFP) and downstream Nos-terminator was designed. After checking the structural and functional efficiency of codon optimized gene using bioinformatics tools, the cassette was sent for chemical synthesis from commercial source. The pigment gene cassette (PPH_CEMB), cloned in pCAMBIA-1301, was transformed into Agrobacterium through electroporation. Agrobacterium-mediated floral dip method was used to transform Camelina sativa inflorescence. After seed setting a total of 600 seed were observed for change in color and out of these, 19 seeds developed a reddish-brown coloration, while the remaining 581 seeds remained yellow. The transformation efficiency calculated on basis of color change was 1.0%. PCR analysis of leaves obtained after sowing reddish seeds confirmed the transformation of pigment production gene, while no PCR amplification was observed in leaves of plants from wild type seeds. From the results it is evident that Agrobacterium-mediated transformation of C. sativa inflorescence is very efficient and environment friendly technique not only for detection of transformed plants but also to study basic cellular processes.
A produção de plantas transgênicas com características agronômicas e hortícolas desejadas vem ganhando grande importância por atender às demandas da crescente população. A transformação genética também é um passo fundamental para estudar os fundamentos das ciências das plantas. Foram desenvolvidos e utilizados diferentes protocolos de transformação, ââque são confiáveis ââe eficientes. Esses protocolos usaram genes de resistência a antibióticos ou herbicidas incorporados ao gene de interesse para identificar plantas transformadas e não transformadas. Esses genes marcadores podem representar uma ameaça ao ser humano e ao meio ambiente. O uso de marcadores visuais permite a observação direta e fácil de plantas transformadas com mais precisão. No estudo atual, foi projetado um cassete de genes com o gene da hidroxilase de produção de pigmentos (PPH) sob promotor específico de fibra (GhSCFP) e terminador NOS a jusante. Após verificar a eficiência estrutural e funcional do gene otimizado por códons usando ferramentas de bioinformática, o cassete foi enviado para síntese química a partir de fonte comercial. O cassete do gene do pigmento (PPH_CEMB), clonado em pCAMBIA-1301, foi transformado em Agrobacterium por eletroporação. O método de imersão floral mediado por Agrobacterium foi usado para transformar a inflorescência de Camelina sativa. Após a formação de sementes, foi observado um total de 600 sementes com mudança de cor, das quais 19 desenvolveram uma coloração marrom-avermelhada, enquanto as 581 restantes permaneceram amarelas. A eficiência de transformação calculada com base na mudança de cor foi de 1%. A análise de PCR das folhas obtidas após a semeadura de sementes avermelhadas confirmou a transformação do gene produtor de pigmentos, enquanto não foi observada amplificação por PCR em folhas de plantas de sementes do tipo selvagem. A partir dos resultados, é evidente que a transformação da inflorescência de C. sativa mediada por Agrobacterium é uma técnica muito eficiente e favorável ao ambiente não só para a detecção de plantas transformadas, mas também para estudar processos celulares básicos.
Assuntos
Rhodococcus , Biomarcadores , Plantas Geneticamente ModificadasRESUMO
Production of transgenic plants with desired agronomic and horticultural traits has gained great importance to fulfill demands of the growing population. Genetic transformation is also a fundamental step to study basics of plant sciences. Different transformation protocols have been developed and used which are reliable and efficient. These protocols used antibiotic or herbicide resistance genes incorporated along with gene of interest to identify transformed plants from non-transformed ones. These marker genes may pose a threat to human and environment. Use of visual markers enables direct and easier observation of transformed plants with more precision. In current study a gene cassette with 'pigment production hydroxylase (PPH) gene under fiber specific promoter (GhSCFP) and downstream Nos-terminator was designed. After checking the structural and functional efficiency of codon optimized gene using bioinformatics tools, the cassette was sent for chemical synthesis from commercial source. The pigment gene cassette (PPH_CEMB), cloned in pCAMBIA-1301, was transformed into Agrobacterium through electroporation. Agrobacterium-mediated floral dip method was used to transform Camelina sativa inflorescence. After seed setting a total of 600 seed were observed for change in color and out of these, 19 seeds developed a reddish-brown coloration, while the remaining 581 seeds remained yellow. The transformation efficiency calculated on basis of color change was 1.0%. PCR analysis of leaves obtained after sowing reddish seeds confirmed the transformation of pigment production gene, while no PCR amplification was observed in leaves of plants from wild type seeds. From the results it is evident that Agrobacterium-mediated transformation of C. sativa inflorescence is very efficient and environment friendly technique not only for detection of transformed plants but also to study basic cellular processes.