Highly Efficient and Reproducible Genetic Transformation in Pea for Targeted Trait Improvement.

ABSTRACT

A reproducible tissue culture protocol is required to establish an efficient genetic transformation system in highly recalcitrant pea genotypes. High-quality callus with superior regeneration ability was induced and regenerated on optimized media enriched with copper sulfate and cytokinins, 6-benzylaminopurine and indole-3-acetic acid. This successful regeneration effort led to the development of a highly efficient transformation system for five pea genotypes using immature and mature seeds. The new transformation protocol included the addition of elevated glucose and sucrose concentrations for cocultivation and inoculation media to improve callus induction and regeneration, thus resulting in consistent transformation frequencies. Using the Agrobacterium strain AGL1, a transformation frequency of up to 47% was obtained for the pea genotype Greenfeast, using either of two different selection marker genes, PAT or NPT, sourced from two different vectors. Sixty-two transgenic pea events were able to survive kanamycin and phosphinothricin selection. A total of 30 transgenic events for Greenfeast, 15 for CN 43016, 9 for snap pea, and 5 for CN 31237 are reported herein. Two additional transgenic events were recovered from particle gun bombardment experiments. Quantitative RT-PCR analysis confirmed the transgenic status of pea plants, indicating elevated expression of relevant genes cloned into the transformation constructs.