In vitro regeneration of Bienertia sinuspersici

Abstract

This thesis presents a study on the development of plant regeneration protocols for Bienertia sinuspersici, one of three species of the family Chenopodiaceae that were discovered to perform C4 photosynthesis in individual chlorenchyma cells. Protocols for in vitro regeneration of B. sinuspersici were developed as follows: 1) vegetative propagation via adventitious root induction of cuttings was optimized; 2) direct organogenesis and plant regeneration using bud cultures was achieved; 3) plant regeneration via indirect organogenesis of stem-derived callus was established. All plant materials were obtained from mature, greenhouse grown B. sinuspersici plants and cultured in vitro on Murashige and Skoog (MS) basal media supplemented with phytohormones. Vegetative propagation by adventitious root formation of cuttings was examined using various strengths of MS medium as well as the effects of auxins [2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and α-napthalene acetic acid (NAA)] on root development. Half-strength MS medium and IBA were most effective for adventitious root induction. Histological analysis of root anatomy demonstrated the various effects of individual auxin on root development. Direct organogenesis via bud culture for shoot multiplication and plant regeneration was established. Shoot multiplication occurred on medium supplemented with a cytokinin, 6-benzylaminopurine (BAP). Elongation of multiplied shoots was achieved using medium containing a lower concentration of BAP or gibberellic acid. Rooting of elongated shoots was attempted using IBA in vitro or ex vitro, and through micrografting. Rooted shoots were transplanted to soil, acclimated in the greenhouse, and transferred to a growth chamber. Plant regeneration via indirect organogenesis was also established. Stem explants were cultured on medium containing kinetin and 2,4-D to induce callus formation. Shoot organogenesis from callus tissue was obtained on medium containing thidiazuron (TDZ). In vitro-derived shoots were transferred to medium containing BAP for shoot multiplication, and subsequently elongated and rooted as described above. Histological analysis of leaves of regenerated plants from both direct and indirect organogenesis methods showed chlorenchyma cells have the distinctive feature, intracellular compartmentalization of organelles, of the single-cell C4 system. Immunoblot analysis of proteins isolated from leaves of regenerated plants detected key C4 photosynthetic enzymes, providing further evidence that single-cell C4 photosynthesis is present. Cytological analysis of root tips from in vitro-derived plants via indirect organogenesis verified normal diploid chromosome number (2n=18) in dividing cells, suggesting neither polyploidy nor aneuploidy occurred in regenerated plants. Reproducible methods of in vitro regeneration of Bienertia sinuspersici have been established for vegetative propagation from cuttings, direct organogenesis via axillary bud culture, and indirect organogenesis. These in vitro techniques would serve as useful tools for multiplication and genetic transformation of this plant species.