Optimization Of The Regeneration System Of Elaeagnus Mollis In Vitro Culture

The regeneration system of endangered plant Elaeagnus mollis has been initially established, but many problems still remained to resolve, for example: plantlets Vitrification, culture browning, leaf yellowing and defoliation on root seedlings, lower rate of rooting and so on , these problems seriously restricted the application of this technology in production, Therefore, further optimizing its plant regeneration system, has important practical significance.Elaeagnus mollis is a unique rare and endangered plant, sterile germination seedling was used as material, epicotyl segments was selected as excellent explants in this study, different plant regulators such as NAA and cytokinins such as 6-BA,KT,ZTand GA3,PP333, AgNO3were added in media to study their effects on adventitious bud differentiation, clustered bud proliferation and shoots elongation (organ-type channels occurred); furthermore ,the effects of different growth regulators and cultured conditions and cultured methods on rooting of seedling were specialy studied, rooting media and methods was found, regeneration system of Elaeagnus mollis was further optimized, these work layed the foundament for further research of the Elaeagnus mollis genetic transformation. Main culture procedure and results are as follows:1. Selection of explants: After removed the hard mesocarp and endocarp, obtained uncovered kernel,after rutine disinfection, they were inoculated on 1/2MS medium, under light, about 30 days, epicotyl segments, hypocotyl, petiole, vein were selected as explant in vitro culture, the results show that epicotyl is best explant, not only frequency of adventitious buds is high(92.6%),but also regeneration of plantlets Vitrification and browning rate is low.2. Differentiation of adventitious buds: Epicotyl segments about 0.5-0.6cm in size ,established on differentiation medium and cultured in the dark for a week, effectively reduced the browning of explants.In different seasons, epicotyl segment were culutred in the same differentiation medium,the result showed that the frequency of bud differentiation and the degree of browing of cultures were obviously different, the spring was the best season for shoots formation ; The concentration of 6-BA play an important role in the differentiating of adventitious buds, too high or too low concentration are not in favor of the emergence of adventitious buds, 0.5mg/L 6-BA was option for differentiation. the appropriate conditions of Elaeagnus mollis epicotyl for adventitious bud differentiation as bellow: epicotyl segments MS+BA0.5mg/L +NAA 0.05 mg/L+LH300 mg/L+VitC150 mg/L +sucrose30g/L+Agar8g/L as the medium, for a week in dark , then were transferred to in the light, adventitious buds appear about 25 days.3.Adventitious bud proliferation and shoots elongation: Different concentrations of 6-BA and NAA have significant effect on the elongation of adventitious buds;lower 6-BA concentration can effectively reduce Vitrification; adding with 0.2mg/L of GA3 ,promote adventitious buds elongation, but the seedlings are slim and fragile; 0.005mg/L and 0.01mg/L of TDZ can increase bud numbers, the buds are not easy elongate and are not growing well; adding with 0.2mg/L AgNO3,compared to the control ,were found have no different on yellowing and abscission of leaf.Adventitious Buds on epicotyl was cutted into small pieces, they was cultured on medium MS supplemented with BA 0.25mg/L+NAA0.05mg/L+LH300mg/L+VitC150 mg/L+sucrose30g/L+Agar8g/L for 20 days, proliferation of buds are better; Adventitious Buds on epicotyl was cultured on medium MS supplemented with MS+ BA 0.25mg/L +NAA 0.125 mg/L+LH300mg/L + VitC150 mg/L+sucrose 30g/L+Agar 8g/Lfor 20 days,buds elongated to shoots in 2-3cm high.4. Rooting culture: Trough studying effects of four kinds of basic media (1/2MS,2/3MS,1/2WPM,WPM) on rooting and growing of the seedlings ,was found 1/2MS is best medium.Adopt 1/2MS as the basic medium,using two kinds of rooted methods to root culture .Step rooting method,2-3cm seedlings was cultured on medium 1/2MS supplemented with IBA1.0mg/L+NAA0.02mg/L+LH300mg/L+VitC150 mg/L+sucrose20g/L+Agar8g/L, after 10 days in dark, then were transferred to in the light , the highest rate of rooting was 50% after 45 days ; two-step rooting method, 2-3cm seedlings was soaked in 200mg/L of IBA solution for 30 min, then was vertically inoculated on medium 1/2MS supplemented with LH300mg/L+VitC150mg/L+ sucrose 20g/L+Agar8g/L, after 7 days in the dark, then were transferred to in the light, the highest rate of rooting was 66.67% after 45 days .