Development of a simple and efficient protocol for chickpea (Cicer arietinum L.) transformation by floral injection method
By: Syed Hassan Shahid, Mahmood ul Hassan, Irfan Iqbal, Noreen Fatima, Tayyuba Kousar, Zahid Ikram, Shaukat Ali
Key Words: Chickpea, inplanta transformation, Agrobacterium-mediated transformation, Floral injection method.
Int. J. Biosci. 8(5), 50-59, May 2016.Certificate
Agrobacterium-mediated transformation has been widely used in various crops for development of varieties better in quality and adaptation against biotic and abiotic stresses. Chickpea plant shows limitation towards in vitro regeneration during course of transgenic plant development therefore it is the need of hour to develop a protocol independent of pre-culture, co-cultivation and regeneration steps. Therefore, in the present study a modification has been done in floral dip method of transformation in chickpea to examine the effect of genotypes, optical density of bacterial culture and floral bud stage. Six varieties (Parbat, Punjab-2008, Dasht, CM-2008, NCS-0709 and Noor-2009) were grown. Overnight culture of Agrobacterium tumefaciens strain LBA4404 harboring binary vector pCAMBIA1301 containing GUS gene was injected into the floral buds at two different stages. Transformed seeds were harvested at plant maturity and plants were subjected to GUS assay. Effect of genotypes, culture concentrations and floral bud stages on transformation efficiency was calculated. Analysis of Variance (ANOVA) and Tukey’s Multiple Range Test (TMRT) were applied to data by using MSTAT-C software. Among all of the genotypes studied; DASHT genotype at hooded floral bud stage with Agrobacterium cell density of 0.5 OD600 has exhibited the highest transformation efficiency percentage.This method of plant transformation is more economical and feasible in chickpea because it doesnot involve regeneration stages.