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Transformation of plants can be successfully accomplished by employing two basic strategies. One is co-cultivation of protoplast with Agrobacterium and the leaf disc procedure. The first transgene was obtained by co-cultivation. Basic methodology involves Agrobacterium transformation of regenerating protoplast, plus screening and regeneration.
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Leaf-disc transformation is used extensively in most of the transformation work. Surface- sterilized leaf disc or pieces are incubated with Agrobacterium solution or co-cultured on regeneration medium for 2-3 days with Agrobacterium. Ideal explants for tomato and cotyledon transformation is the cotyledons. In Brassica napus, stem segments yield better result.
During co-cultivation bacteria attach to plant cells around wounded edge of the explant. Following co- culture, explants are then transferred to selection media containing carbencillin to kill the Agrobacterium. Inclusion of appropriate antibiotic in the medium could facilitate selection of transformed plant cells. Once regeneration is accomplished, shoots are then excised and rooted on rooting media.
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Hardenings of plants were followed before transferring them to soil. The leaf disc procedure exhibit definite advantage over protoplast involved co-cultivation method. One of the main problems lies with co-cultivation is the handling and regeneration of protoplast. Transgenic plants obtained through leaf disc culture require feasible regeneration process and the entire transformation process takes place within 4-6 weeks.
Pre-wounding of the apical meristem seems to be essential for Agrobacterium infection. Application of bacterial suspension at the tip of wounded apices was found to be more effective as compared to the explants co-cultivated in bacterial suspension in cow pea.
Transformation Efficiency:
Although Agrobacterium mediated gene transfer is the method of choice for plant transformation, its low transformation frequency is however, causing a major concern among plant bictechnologists.
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Agrobacterium mediated transfer is largely influenced by presence of inducer such as acetosyringone and actively dividing plant cell. Exogenous supply of inducers like syringone and acetosyringone during co-culture can enhance vir induction and better transformation efficiency.
Transformation of recalcitrant species such as rice and maize plants has been successfully achieved by introducing additional copies of the virulence regions.
Wenck (1999) constructed super binary vector by adding additional copies of constitutively active virG gene from octopine plasmid increased transformation efficiency. Implication of several other parameters has significantly increased transformation efficiency.
In sunflower, for example, presence of tiny wounds (microwounds) by particle bombardment and also shaking plant tissue with glass beads have been shown to improvise transformation process.
However, inflicting wounds on the plant material may incite considerable disruption of the surrounding tissue, which in turn hampers regeneration potential of plants. Therefore, exploitation of macerating enzymes such as pectinase, cellulase and macroenzymes, which are regularly used in protoplast isolation, may be feasible and wise to enhance transformation efficiency.
The utility of these enzymes could represent a less disruptive method. Following digestion with enzyme, area where Agrobacterium can attach to plant cells might increase. Further, digested cell wall may release inducers triggering the expression of bacterial vir genes. The frequency of explants with regenerated shoots expressing β-glucoronidase (GUS) or green fluorescent protein (GFP) increased following treatment with enzymes.
Sonification of explants by applying suitable frequency has also been found to enhance transformation efficiency. Comparison between sonification and macerozyme treatment emphasized that enzymatic treatment alone was superior to combined treatment of sonification and enzymes with respect to stable transformation.
