A study of shoot regeneration in leaf disc cultures of two solanaceous plants
Thesis DisciplinePlant Biotechnology
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The behaviour of Solanum laciniatum and "Mitchell" Petunia leaf discs in culture was examined. In both systems shoots were formed in response to an appropriate auxin/cytokinin balance via a caulogenic pathway involving the epidermal tissues with no intermediate callus. Petunia explants could also be induced to form roots, which were found to have an endogenous origin quite different to that observed for shoots. The shoot inducing hormone balance was found to be required for only a short window in the shoot formation timecourse. The hormone balance required to make explants competent for shoot initiation, was less specific than that required for them to become determined for shoot development. Development itself would occur on a medium devoid of growth regulators. The effect of culturing explants on ungelled medium was also examined. The number of shoots initiated was dramatically increased over those on agar gelled media for both species. Liquid cultured Petunia explants showed an increase in both the number of shoots developing per explant and the rate of development. The effects of a number of reagents on shoot formation from Solanum laciniatum and Petunia explants was examined as was the effect of the nitrogen source on Petunia explants. The response of explants of both species to 2,3,5 triiodobenzoic acid (TIBA) , ribose, sorbitol and acetylsalicylic acid (ASA) was similar although the sensitivities varied. TIBA was found to promote shoot formation at low concentrations, but inhibit it at higher levels. Ribose, sorbitol and ASA inhibited shoot formation from explants of both species, and partial substitution of Murashige and Skoog nitrogen with NH4Cl inhibited shoot formation from explants of Petunia. Changes in gene expression accompanying shoot, root and callus production in Petunia explants were monitored by two dimensional polyacrylamide gel electrophoresis of both total extractable protein, and labelled newly synthesised proteins (for shoot and callus forming cultures). In addition the protein profiles of shoot forming cultures inhibited with ASA, TIBA and NH4Cl were also examined. A single silver stainable protein specific to shoot forming cultures, and 4 proteins specific to root formation were discovered. In general, protein changes in the first 4 days of culture greatly exceeded subsequent changes for all three culture systems. The patterns of proteins observed from explants cultured on the three inhibitors were similar. The inhibitors did not prevent the formation of the shoot specific protein, but did result in other changes in protein profile. The most significant of these was the occurrence of a labelled protein which was also found to occur in the nonshoot-forming central portion of uninhibited explants.