Use of sonication and vacuum infiltration for Agrobacterium – mediated transformation of an Indian lentil (Lens culinaris Medik.) cultivar

Abstract

The regeneration and transformation protocol for lentil employing both sonication and vacuum infiltration of whole seed explants is described. This protocol overcomes the non-competence of whole seed explants to Agrobacterium mediated transformation though the regeneration potential of this explant is high as compare to other agro-competent explants. The decoated seeds of four genotypes were cultured on regeneration media that consisted of Murashige and Skoog (MS) salts and Gamborg (B5) vitamins (MSB5V) supplemented with different concentrations of 6-benzylaminopurine (2.5–20μM) and thidiazuron (0.1–10μM) separately. Best regeneration response with an average of 9 shoots per explant was observed in 95% of the cultures on MSB5V medium fortified with 5.0μM thidiazuron. The protocol was found independent to the effect of genotype. Among the effects of physical treatments, it was found that sonication at 20KHz for 60s followed by vacuum infiltration at −660mm of Hg for 5min resulted in highest transient GUS expression (68%) with no significant effect on regeneration. Rooting was observed in 45% of shoots on rooting medium containing half strength MS salts and full strength MS vitamins supplemented with 2.5μM IBA. Regenerated plantlets were established in soil with 50% survival rate and grown to maturity. GUS activity in leaves and roots of primary transformants recovered on kanamycin containing selection media confirmed the expression of transgene. These plants were tested and found positive for nptII gene by PCR and Southern analysis with an overall transformation frequency of 0.66%. PCR analysis of T0 progeny revealed the inheritance of transgene.