Gibberellic acid promotes in vitro regeneration and shoot multiplication in Lotus corniculatus L.

Abstract

Shoots of Lotus corniculatus L., previously transformed with Agrobacterium tumefaciens LBA4404/pTOK233, were grown in gibberellic acid (GA(3))-containing media in an attempt to improve their growth and multiplication. Nodal stem segments of four poorly multiplying clones, a clone with very good multiplication, and two non-transformed clones were incubated in media containing GA(3) for 3 weeks, and then returned to a hormone-free medium for a further 3-5 subculture periods. Gibberellic acid increased the number of axillary buds from one in controls to 2-13 shoots during the first 3 weeks of subculture in GA(3)-containing media. The multiplied buds, which apparently derived from additional axillaries, continued to multiply in hormone-free medium, producing bunches of more than 50 shoots after 9 weeks. In addition, many nodal segments also produced callus tissues at the basal end, or along the internode below the node, in which abundant shoot meristems formed de novo. Histological examination confirmed their origin via organogenesis. About 25% of regenerated shoots rooted spontaneously. The application of auxins significantly improved rooting in more than 75% of all clones. Well-developed plantlets were produced upon the transfer of rooted shoots to pots. No differences in responses to hormones were observed between LBA4404/pTOK233-transformed clones and non-transformed clones, indicating that the reaction to GA(3) was not the consequence of transformation events. The results point to a possible involvement of GA(3) in lateral branching. They might also be recommended as a procedure suitable for increasing the production of transformed plants in L. corniculatus.