Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.

The efficiencies of different transformation methods of E. coli DH5α strain, induced by several cations like Mg2+, Mn2+, Rb+ and especially Ca2+, with or without polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were compared using the two commonly used plasmid vectors pCAMBIA1201 and pBI121. The widely used calcium chloride (CaCl2) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl2 method were found to further augment the transformation efficiency (TR)E for both the vectors like repeated alternate cycles of heat shock, followed by immediate cold, at least up to the third cycle; replacement of the heat shock step by a single microwave pulse and even more by double microwave treatment and administration of combined heat shock-microwave treatments. The pre-treatment of CaCl2-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)E, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl2 method suggested in the present study may thus find use in more efficient E. coli transformation.

In vitro response of encapsulated somatic embryos of Lagerstroemia indica L.

A method to produce encapsulatable units for synthetic seeds was developed in L. indica. Somatic embryos were harvested from leaf derived embryogenic callus on Murashige and Skoog's basal medium supplemented with 2, 4-dichlorophenoxy acetic acid (2, 4-D, 0.5 mg/l), 6-benzyl amino purine (BAP, 1 mg/l) and ascorbic acid (AA, 50 mg/l). The embryos were encapsulated in alginate beads and dehydrated. Germination ability of the artificial seeds were investigated. The frequency of regeneration from the encapsulated embryos was significantly affected by (i) the concentration of alginate (ii) the duration of storage, and (iii) the effect of different types of media. A 2% sodium alginate concentration on MS salts resulted in significantly higher germination frequencies than at other concentrations. L. indica showed maximum germination on MS medium (93.84%) after 6 weeks of culture. The germinated synthetic seeds with well developed roots and shoots were transferred successfully to green house. This is the first report on artificial seeds in Lagerstroemnia indica.