RESUMO
With the accelerating advances in genetics and genomics research in Arabidopsis and Brassica, transformation technologies are now routinely being exploited to elucidate gene function as well as contributing to the development of novel enhanced crops. When a researcher's desired goal is simply to modify or introduce candidate genes into a Brassica, the availability of easy-to-follow protocols and knowledge of readily transformable genotypes becomes a valuable resource. In this chapter we outline a basic A. tumefaciens-mediated transformation method, using 4-day-old cotyledonary explants, that has been successfully applied to a range of different B. oleracea and B. napus genotypes. For demonstration purposes, we focus primarily on the diploid species B. oleracea using a model doubled haploid genotype, AG DH1012. After only 3-4 weeks on kanamycin selection the first transgenic shoots can be isolated. Transformation efficiencies are typically in the region of 15-25 % (based on 15-25 PCR-positive independent shoots from 100 inoculated explants). Most explants will produce multiple shoots (1-3+ per explant) and so the total number of transgenic shoots produced will exceed 15-25 per 100 explant experiment. The protocol is also applicable to B. napus and modifications specific to this species are highlighted accordingly. For researchers wishing to use their own plant genotype, tissue culture phenotypes that are conducive to efficient transformation are also highlighted within this chapter.
Assuntos
Brassica/genética , Técnicas Genéticas , Plantas Geneticamente Modificadas , Agricultura/métodos , Agrobacterium tumefaciens/genética , Brassica/crescimento & desenvolvimento , Brassica napus/genética , Brassica napus/crescimento & desenvolvimento , Técnicas de Cocultura , Cotilédone/genética , Germinação , Haploidia , Brotos de Planta/genética , Sementes/genética , Seleção Genética , Esterilização , Transformação BacterianaRESUMO
Within this chapter we outline an A. tumefaciens-mediated transformation method for B. rapa using 4-day-old cotyledonary explants and the genotype R-o-18. Transformation efficiencies are typically achieved in the region of 1% (based on 2 PCR-positive independent shoots from 200 inoculated explants). This system has been developed to work with gentamicin selection.
Assuntos
Brassica/crescimento & desenvolvimento , Brassica/genética , Engenharia Genética/métodos , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/crescimento & desenvolvimento , Técnicas de Cocultura , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Ambiente Controlado , Germinação , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Esterilização , Transformação GenéticaRESUMO
GM risk assessments play an important role in the decision-making process surrounding the regulation, notification and permission to handle Genetically Modified Organisms (GMOs). Ultimately the role of a GM risk assessment will be to ensure the safe handling and containment of the GMO; and to assess any potential impacts on the environment and human health. A risk assessment should answer all 'what if' scenarios, based on scientific evidence. This chapter sets out to provide researchers with helpful guidance notes on producing their own GM risk assessment. While reference will be made to UK and EU regulations, the underlying principles and points to consider are generic to most countries.
Assuntos
Técnicas de Transferência de Genes/efeitos adversos , Organismos Geneticamente Modificados/genética , Animais , Conservação dos Recursos Naturais , União Europeia , Saúde , Humanos , Funções Verossimilhança , Marketing , Plantas Geneticamente Modificadas , Medição de Risco , Segurança , Controle Social FormalRESUMO
A better understanding of the genetic basis underlying the genotype dependence of Brassica oleracea transformation is enabling researchers to distinguish between recalcitrant and successful candidate genotypes for routine transformation. In this chapter we outline an A. tumefaciens-mediated transformation method for B. oleracea using 4-d-old cotyledonary explants and a model B. oleracea doubled haploid genotype, AG DH1012. After only 3 wk on kanamycin selection, the first transgenic shoots can be isolated. Transformation efficiencies in the region of 10 to 25% (based on 50-125 PCR-positive independent shoots from 500 infected explants) are typically achieved. For researchers wishing to use their own plant genotype, we highlight the tissue culture phenotypes that are conducive to efficient transformation.
