Transformation of peas (Pisum sativum L.) using immature cotyledons

Abstract

A reliable Agrobacterium tumefaciens-mediated transformation method has been developed for peas (Pisum sativum) using immature cotyledons as the explant source. Transgenic plants were recovered from the four cultivars tested: Bolero, Trounce, Bohatyr and Huka. The method takes approximately 7 months from explant to seed-bearing primary regenerant. The binary vector used carried genes for kanamycin and phosphinothricin resistance. Transformed pea plants were selected on 10 mg/l phosphinothricin. The nptII and bar genes were shown to be stably inherited through the first sexual generation of transformed plants. Expression of the phosphinothricin-resistance gene in the transformed plants was demonstrated using the 'Buster' (='Basta') leaf-paint test and the phosphinothricin acetyl transferase enzyme assay.

Stable gene transformation in cowpea (Vigna unguiculata L. walp.) using particle gun method

“…The high mortality rate of T 1 , T 2 and T 3 plants under Basta spray does not necessarily indicate the non-integration of the bar gene or high application levels of the Basta. Plants that did not express the phosphinothricin-resistance gene in the leaf tissue but were shown to have the gene by Southern analysis have been reported for peas (Grant et al, 1995). The 0.2% of Basta applied is within the ranges reported for other crops even though different crop plants may have different responses to the herbicide (Christou et al, 1991;.…”

“…Most legume transformation studies have used Agrobacterium tumefaciens to generate for example transgenic soya bean (Hinchee et al, 1994), chickpeas (Fontana et al, 1993), peas (Puouti-Kaerlas et al, 1990;Schroeder et al, 1993;Grant et al, 1995) and cowpea (Muthukumar et *Corresponding author, Present address: CSIRO Plant Industry, Black Mountain Laboratories, G.P.O Box 1600, Canberra ACT 2601, Australia. Email: Joe.Ikea@csiro.au λ Present address: Mahyco Research Foundation, A.G. Heights, Road No.…”

Organogenesis and long-term micropropagatlon of Polish pea cultivars

“…We have chosen nine Polish pea cultivars of different utilization type and botanic features to select a genotype characterizing a high regeneration potential. We have used immature embryos as primary explants, according to their high regeneration potential showed by other authors (Grant et al 1995;Kosturkova et al 1997;Nadolska-Orczyk et al 1994;Natali and Cavallini 1987;Özcan et al 1992;Schroeder et al 1993;Tétu et al 1990). Both embryo axis (Kosturkova et al 1997;Natali and Cavallini 1987;Schroeder et al 1993) and the nodal part of cotyledons (Grant et al 1995;Nadolska-Orczyk et al 1994;Özcan et al 1992) were used as starting materials.…”

“…Shoots regenerated from immature embryos can be sufficiently micropropagated and subsequently efficiently rooted in vitro or grafted after pre-conditioning. Thus far, transgenic pea plants were mainly obtained after inoculation of immature embryos with Agrobacterium strains and subsequent organogenesis in callus (Grant et al 1995;Polowick et al 2000;Puonti-Kaerlas 1990;Schroeder et al 1993). Presented regeneration protocol seems to be useful and easily adaptable for pea transformation procedures.…”

“…The following methods of in vitro cultures were applied to regenerate peas: direct regeneration via meristem cultures, organogenesis, somatic embryogenesis and protoplast cultures (for review: Davies and Mullineaux 1993;Griga and Novak 1990). To obtain transgenic peas, mainly organogenesis de novo (Grant et al 1995;Polowick et al 2000;Puonti-Kaerlas, 1990;Schroeder et al 1993), but also meristem cultures (Bean et al 1997;Davies et al 1993;Nadolska-Orczyk and Orczyk 1999) have been successfully used. Unfortunately, the rate of pea transformation was low and varied considerably from a fraction of a percent up to several percent.…”