Establishment Of Tissue Culture And Plant Regeneration System Of Akebia Trifoliate Var. Australis

Akebia trifoliate var. australis, belonging to the family Lardizabalaceae and the genus Akebia, is cultivated as medicinal lianas for its roots, vine stems, leaves, seeds, fruits. Data display:current researches on Akebia quinata mainly focus on its chemical composition, physical and chemical properties of seeds, distribution of resources and identification of species etc. Its reproductive modes contain sowing, cutting and layering etc. However, studies on tissue culture of Akebia quinata start late, and most of the plants itself contains viruses and bacteria, which influence its productivity and fruit quality. In order to improve its fruit quality and yield effectively, studies on tissue culture of Akebia quinata are imperative to obtain virus-free seedlings. This essay aims at the study on tissue culture of Akebia quinata with study results as follows:1. Using stems with axillary buds as explants, studying the effects of drawing locations, drawing parts, and drawing season of explants on induction rate, contamination rate and browning rate, the results showed:1) The better disinfection method for stems with axillary buds was using 75% alcohol immersion 30 s, then using 5% NaClO immersion 15 min, and the contamination rate was only 4.25%, at the same time the browning rate could be controlled within 6.33%; 2) The drawing locations, drawing parts, and drawing season had certain influence on the browning and induction, and stem segments from the indoor potted seedlings were the best experimental materials with a 73.5% induction rate and a low contamination rate. Besides, induction rate of stem segments from the scion orchard was only 43.1% but the contamination rate was high; 3) The best drawing part was the middle part, and April was the best season for the tissue culture of Akebia quinata with a 12.4% contamination rate and a 14.1% browning rate as well as its induction rate was up to 73.5%.2. In the primary culture of stem segments, making comparison with the effects of basic medium and pH on axillary bud induction, and studied the impact of plant growth regulators on in vitro culture and plant regeneration, the results showed:1) The best original culture medium of stem was WPM for the axillary bud with induction rate up to 80.0% while MS is poor and its induction rate was only 61.7%, and the most appropriate pH of the MS medium was 5.5 because pH was not unfavorable for the growth of axillary buds when it was too high or too low.2) The fittest medium for germination of stem axillary bud was WPM+1.0 mg·L-1 6-BA+0.5 mg·L-1 IAA+2.0 mg·L-1 GA3, with a 81.27% germination rate; 3) The best proliferation medium was WPM+3.0 mg·L-16-BA+0.1 mg·L-1IBA, the multiplication coefficient could reach 4.26; 4) The best medium for seedling multiplication was WPM+0.5 mg·L-1 NAA+3.0 mg·L-1 GA3, the height was up to 2.88 cm; 5) The best rooting medium was 1/2MS+1.0 mg·L-1BA+0.5 mg·L-1 NAA+1.0 mg·L-1 GA3, with a 92.18% rooting rate. After the culture-bottle seedlings were transplanted into the matrix composed of peat soil, perlite and vermiculite (2:1:1), the survival rate was over 82%. This research supplied a new way of Akebia trifoliate rapid propagation.3. Using seed embryos of Akebia trifoliate var. australis as experimental materials, studying the effects of storage methods, sterilizing time, pH and light condition on seedlings of Akebia trifoliate var. australis, the results showed:1) Among three kinds of storage methods, the germination rate of the seed embryos storaged in sand was up to 65.6% while embryos storaged in refrigerator with a 32.6% germination rate, however, when storaged at room temperature, the seed embryos lost a large number of water and its germination rate was 0; 2) The better disinfection method for embryos was using 75% alcohol immersion 1 min, then using 5% HgCl2 immersion 20 min, and the contamination rate was 18.2%, at the same time when placed under natural conditions was 6.8%; the most appropriate pH of medium was 5.5 because pH was not unfavorable for its normal growth of embryos when it was too high, besides, compared to natural light, shading was beneficial to embryos’ germination.3) The appropriate medium of embryo vaccination was WPM+5.0mg/L GA3 for seedling rate up to 65.6%; The seedling rate was 12.8% by adding 0.5mg/L IAA in the culture mediums, but it was 0 when adding no plant growth regulators, which was not suitable for regeneration system establishment of Akebia trifoliate var. australis.4. Using leaves and hypocotyls of virus-free seedlings of Akebia trifoliate var. australis to induce callus and adventitious buds, the results showed:1) The fittest medium for leaves callus induction was WPM+0.5 mg·L-16-BA+1.5 mg·L-12,4-D +2.0 mg·L-1 NAA including callus induction rate up to 53.2% with no adventitious buds in later period; 2) The appropriate medium for hypocotyls callus induction was WPM+1.0 mg·L-1 6-BA+0.5 mg·L-1 NAA for callus induction rate up to 48.0% with no adventitious buds yet.