In Vitro Adventitious Shoot Multiplication And Regeneration Of The Tea Plant (Camellia Sinensis Cv. Longjing 43)

A technical approach for in vitro clonal propagation has been developed in tea plants. In this paper, axillary buds of tea plant (Camellia sinensis cv. Longjing 43) were cultured in vitro. Initiation of culture, shoot multiplication system, plantlet regeneration system and rooting were investigated. The results were shown as follows:(1) Initiation of cultureAxillary buds excised from fresh tea shoots in field were cultured on half–strength M- urashige and Skoog medium (1/2MS) supplemented with 8.88μmo1-L^-1 6-Benzylade- nine (BA), 0.49μmo1-L^-1 Indole-3-butyric acid (IBA), and 8.66μmo1-L^-1 gibberellic acid (GA₃) as initiation culture. Nodal segments with dormant axillary buds of either juvenile or adult origin of current year growth are suitable for explants for tea micropr- opagation. We found that the explants with 2.0 g-L^-1 polyvinylpyrolidone aqueous solution for 30 min, and cultured in 8℃for 16h highly prevented explants browning.(2) Multiplication of shootMaximum number of shoots were developed on half–strength MS (1/2 MS) medium containing 0.05 or 0.1μmo1-L^-1 Thidiazuron (TDZ), and supplemented with 0.25 or 0.49μmo1-L^-1IBA. With the increase of TDZ concentrations, the multiplication rate of tea proliferation declined. Number of shoots formed in explants decreased when successive subculture on medium containing 0.1μmo1-L^-1 TDZ and failed to develop into shoots. Shoot elongation was achieved when shoots were cultured on hormone-free 1/2 MS medium.(3) Plantlet regenerationAt 0.1, 0.5, 1.0μmo1-L^-1 concentrations of TDZ, with different combinations of IBA like 0, 0.49, 0.98, 1.97μmo1-L^-1 were tested for callus induced response of nodal segments, leaf and shoot on 1/2MS medium. We observed at 0.1μmo1-L^-1 TDZ and 0.49μmo1-L^-1 IBA, maximum rate (100%) of the callus responded for nodal segments and leaf in1/2MS medium. However, no response was observed when TDZ was used beyond the concentrations of 0.5μmo1-L^-1.The shoots produced compact, nodular callus from the basal of shoots on all combinations of plant growth regulator. When shoots connected with callus were transferred to 1/2 MS medium supplemented with 8.88μmo1-L^-1 BA, 0.49μmo1-L^-1 IBA, the callus germinated into plantlets, optimal regeneration rates of about 52.6% after 12weeks.Experiments were repeated three times, regeneration rates of callus were 67.4%, 53.6%, 45.3%, 49.6% respectively. We excised single regeneration buds from the callus, then were inoculated into the medium containing TDZ or BA, the buds were hardly growing and prone to necrosis; conversely, the regeneration buds that constantly growing on callus were able to good elongation, after 6 weeks culture on 1/2MS+ BA 8.88μmo1-L^-1+ IBA 0.49μmo1-L^-1, the frequency of the shoot height beyond1cm up to 64.3%.(4) Rooting of shootsFor rooting, tea shoots were treated with 2450μmo1-L^-1IBA solution for 5 minutes and transferred directly to hormone-free 1/4 MS medium. After 6 weeks in 1/4 strength MS basal medium, 70.8% of them produced roots, mean number of root per micro-shoots was 3.8. The rooted plants were transferred to a sterile mixture of peat and soil (1:1) in bottles, irrigated with sterile water. After 30 days, the frequency of survival was 63.6%.