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.

Figure 1

Standardization of tissue culture steps for regeneration of field pea plants. (A) Embryonic axis attached to cotyledon from immature seeds; (B) callus initiation from cotyledon and embryonic segments on the D medium in 2–3 weeks; (C) development and proliferation of green callus on DBC3; (D) failed green callus regeneration on LREG; (E) regenerating callus on KREG; and (F) plantlets with roots on rooting media.

Figure 2

Agrobacterium-mediated transformation in field pea. (A) Embryonic axis of immature seeds after 2 days of inoculation with Agrobacterium strain AGL1 on KREG-CO; (B) callus initiation of inoculated embryonic on KREG after 1 week; (C) GFP expression in 4 weeks old callus; (D) selection of transgenic calli on KREG using 100–150 mg/L kanamycin; (E) transgenic plants on rooting media; and (F) healthy transgenic plant.

Figure 3

PCR amplification of the KAN gene from plants pJP3502; (A) T₀ events. (B) T₁ plants. Lane 1: 1 kb plus ladder; lanes 2–17: transformants; lane 18: non-transgenic control; lane 19: water; lane 20: plasmid.

Figure 4

PCR amplification of the PAT gene from Greenfeast transformed with pJP3679. (A) T₀ events. Lane 1: Marker; lanes 2–16: transgenic events; lane 17: non-transgenic control; lane 18: +ve transgenic control; lane 19: water; lane 20: plasmid DNA. (B) T₁ plants. Lane 1: marker; lanes 2–17: transgenic plants; lane 18: non-transgenic control, lane 19: water; and lane 20: plasmid DNA.

Figure 5

PCR amplification of the KAN gene from CN 43016 transformed with particle gun bombardment. (A) T₀ events. Lane 1. Marker; lanes 2–11 transgenic events; lane 12: plasmid DNA: lane 13: non-transgenic control; lane 14: water. (B) Progeny of T₁ transgenic plants. Lane 1: marker; lanes 2–15 T₂ plants; lanes 16–17 T₁ transgenic plants; lane 18: non-transgenic control; lane 19: plasmid DNA; and lane 20: water.

Figure 6

Leaf paint assay of the transgenic events (pJP3679) showing Liberty herbicide tolerance, with a representative image showing the reaction of Liberty herbicide on pea leaves after 1 week. (A) Plants resistant to Basta and (B) susceptible (control).

Figure 7

Expression analysis of different genes in T₂ transgenic Greenfeast plants using real-time quantitative PCR; (A) expression of selectable marker PPT; (B): expression of the DGAT gene; and (C): expression of the OLEO gene. The statistical significance of relative expression in transgenic plants and Greenfeast wild control was determined with a student t-test (*p < 0.05).