Androgenic Haploid Plant Development Via Embryogenesis With Simultaneous Determination of Bioactive Metabolites in Cambod Tea (Camellia Assamica ssp. Lasiocalyx)
Abstract This pioneering work reports successful androgenic plant development via embryogenesis from microspore calluses in anther cultures and estimation of bioactive metabolites in in vitro regenerants and parent plant (control) of Cambod tea, Camellia assamica ssp. lasiocalyx (Planch MS) cultivar TV19. Anthers bearing microspores at early-to-late uni-nucleate stage were selected to initiate androgenesis. A pre-treatment of 50 C for five days in the dark was most effective to initiate profusely growing white callusing from microspores within 10 weeks of culture on MS medium (6% sucrose) supplemented with high cytokinins/ auxin ratio maintained by benzyl adenine (BAP) and 2,4-dichlorophenxyacetic acid (2, 4-D). Nodular structures on the callus surface differentiated into embryos. Further developement of the embryos occurred on embryogenesis medium but, with ten times reduced concentration of growth regulators and additives. Germination of embryos into complete plantlets was achieved when major salts in medium were reduced to half MS (½ MS) and augmented with BAP, GA3 and IBA along with glutamine and serine. Cytological examination of the root-tip cells revealed that regenerated plantlets were haploids (2n=x=15), which was further confirmed through flow cytometry. The hot-water extracts from in vitro haploid calluses, embryos and field-grown donor plant were utilized for quantification of (+)-catechin, (-)-epicatechin, (-)-epigallocatechin gallate, caffeine and theophylline. Our findings revealed that the metabolite profile of in vitro regenerated haploid cultures is comparable to that of the mother plant, thereby presenting them as potential source for genome duplication and development of genetically stable homozygous pure breeding lines.