A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD(600) congruent with 0.6 and diluted to a cell density of 10(9) cells ml(-1) for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 mug ml(-1)). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l(-1) indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T(1) seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.

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Inheritance of resistance to herbicide (300 mg/l glufosinate ammonium) up to the third (T3) seed generation was compared in two populations of transgenic lettuce (Lactuca sativa L. cv 'Evola') harbouring a T-DNA containing the bar gene, linked to either the Cauliflower Mosaic Virus (CaMV) 35S promoter, or a -784-bp plastocyanin promoter from pea (petE). Only 2.5% (4/163) of CaMV 35S-bar plants, selected by their kanamycin resistance(T0 generation), transmitted herbicide resistance at high frequency to their T3 seed generation compared with 97% (29/30) for kanamycin resistant petE-bar plants. In the case of 35S-bar transformants, only 16% (341/2,150) of the first seed generation (T1) plants, 22% (426/1,935) T2 plants and 11% (1,235/10,949) T3 plants were herbicide-resistant. In contrast, 63% (190/300) T1 plants, 83% (2,370/2,845) T2 plants and 99% (122/123) T3 petE-bar transformed plants were resistant to glufosinate ammonium. The T-DNAs carrying the petE-bar and CaMV 35S-bar genes also contained a CaMV 35S-neomycin phosphotransferase (nptII) gene. ELISA showed that NPTII protein was absent in 29% (45/156) of the herbicide-resistant T2 plants from 8/19 herbicide-resistant petE-bar lines. This indicated specific inactivation of the CaMV 35S promoter on the same T-DNA locus as an active petE promoter. The choice of promoter and T-DNA construct are crucial for long-term expression of transgenes in lettuce.

A simple and efficient protocol for high frequency plant regeneration of a grain legume grasspea (Lathyrus sativus L.) is described. Of different explant types tested epicotyl segments were most responsive. Murashige and Skoog's (1962) medium augmented with 17.76 μM 6-benzyladenine + 10.74 μM α-naphthaleneacetic acid showed the highest percentage of direct shoot regeneration. Among cultivars IC-120487 showed the highest regeneration frequency (80 %) with maximum shoot numbers (8.2 shoots per explant) and maximum average shoot length (4.1 cm). About 78 % of the regenerated shoots were rooted in half-strength MS medium containing 2.85 μM indole-3-acetic acid. After primary hardening the plantlets were established in soil with a survival rate of 75 %.

A reproducible procedure is described for adventitious shoot organogenesis in epicotyl segments resulting in prolific plant regeneration of a grain legume grasspea (Lathyrus sativus L.). Among seedling explant types examined, epicotyl segments were most responsive. The highest percentage of direct shoot regeneration was elicited on Murashige–Skoog (MS) medium augmented with 4.0 mg L–1 6-benzyladenine (BA) + 2.0 mg L–1 α-naphthaleneacetic acid (NAA). Compared with four other genotypes examined, IC-120487 showed the highest shoot regeneration frequency (approximately 80%) with maximum shoot numbers (averaging eight shoots per explant) and longest average shoot length (approximately 4 cm). Rhizogenesis was induced in ~78% of the regenerated shoots in half-strength MS medium containing 0.5 mg L–1 indole-3-acetic acid (IAA). Plantlets were acclimated in vermi-compost and 75% of those transferred to soil survived and set viable seeds.