Androgenic haploid plant development via embryogenesis with simultaneous determination of bioactive metabolites in Cambod tea (Camellia assamica ssp. lasiocalyx)

Key message This is first report on haploids in out-breeding tree, Cambod tea. It's a significant achievement towards generating homozygous lines, which is impossible using conventional methods. Haploids showed consistent metabolite production. 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 5 degrees C for 5 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 cytokinin/auxin ratio maintained by 6-benzylaminopurine (BAP) and 2,4-dichlorophenxoyacetic acid. 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 (1/2 MS) and augmented with BAP, GA(3) 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.

Automated summary

This study reports the successful generation of haploids in the out-breeding tree Cambod tea through androgenesis and embryogenesis techniques. The metabolite profile of the in vitro regenerated haploid cultures is comparable to that of the mother plant, suggesting their potential as a source for developing genetically stable homozygous pure breeding lines.