In Vitro Culture Of Black Pepper (Piper Nigrum Linn.) And Selection Of Somaclones Resistant To Foot Rot

Experiments on surface-sterilization methods carried out with various explants collected from field-grown black pepper (Piper nigrum Linn.) variety, Type Lampong, extensively cultivated in China. Due to endogenous contaminants, contamination of all types of explants except for seeds was not yet effectively controlled. In vitro clonal propagation of black pepper with shoot tips excised from aseptic seedlings used as explants through multiple-shoot multiplication methods is successfully achieved. The best establishment and proliferation of shoot tips was obtained on MS (1/2MS or B5) basal medium supplemented with 1-1.5 mg/1 BA and 0-0.2 mg/1 IAA (or NAA). Compared with those shoot tips placed vertically on the media in the establishment of first culture, horizontal orientation of shoot tips was found to be more suitable for axillary bud proliferation. As the concentration of BA increased, the multiplication rate increased, but the quality of newly forming shoots was affected as the concentration of BA above 2 mg/1. Addition of adenine sulphate had no notably influence on proliferation of multiple shoots. Excised microshoots were rooted in vitro on 1/2MS in the presence of 1 mg/1 IB A and 0.5 mg/1 IAA with the optimum rooting results. Plantlets had been successfully acclimatized and transferred to the greenhouse conditions.Complete plants were grown from mature and immature zygotic embryos of black pepper incubated on filter papper bridges in test tubes containing liquid SH (MS or B5) basal medium with no any growth regulators, and calli were induced with different combinations of auxins and cytokinins. Subculture of those calli onto the multiplication medium and differentiation medium led to browning and death finally, and no plant regeneration occurred. The morphogenetic potential of other explants such as leaf pieces and hypocotyl segments from aseptic seedlings was also investigated in vitro. Callus induction was relatively easy on MS (1/2MS, B5 or SH) basal medium fortified with a wide range of auxin-cytokinin combinations, but most attempts to regenerate plants from the calli were unsuccessful due to serious browning occurred during the subculture onto the multiplication medium and differentiation medium.Based on the in vitro shoot-tip multiplication technique described above, application of in vitro selection in selection of black pepper somaclones resistant to the foot rot (or quick wilt) disease caused by the fungus Phytophthora capsici (Phytophthora palmivora var. piperis or Phytophthora palmivora (Butl.) Butler MF4) was carried out with a large-leaf variety, Type Lampong, widely cultivated in Hainan but highly susceptible to P. capsici, used as explant resources. The results demonstrated that sterilization methods had a significantly influence on the toxicity of the culture filtrate of P. capsici. The fungal filtrate toxicity could be maintained using filtrate sterilization upon addition of the fungal filtrate into selective medium in stead ofautoclave sterilization after the addition. The percentage of shoot tips and multiple shoots survived and newly initiated shoots decreased with increased concentrations of the fungal culture filtrate. The remaining cultures blackened and died finally, seriously formed calloid or remained dormant which their growth were arrested. After 2 subculture onto the same selective medium, microshoots were rooted in vitro and transferred to the greenhouse conditions and screened for resistance to the fungus P. capsici. The plants exhibited no external symptoms of the disease during three successive times of resistance assay once three weeks were considered to be resistant. As the concentrations of fungal culture filtrate increased, the total number of regenerated plants obtained declined but the frequencies of plants resistant to P. capsici increased. 1 (1.54%), 4 (20.00%) and 3 (42.86%) disease resistant somaclonal variants were obtained with 25%, 50% and 75% fungal culture filtrate as selective agents, with the total number of 8 (2.35%) plants resistant to P. ca...