The Optimization Of Tissue Culture And The Induction Of Callus Of Mytilaria Laosensis

Being an evergreen broad-leaved tree belonging to Hamamelidaceae,Mytilaria laosensis is an excellent fast-growing and high-yielding timber tree species in the south subtropical region of China.With the increase of M.laosensis afforestation area,efficient cultivation of fine seedlings has become an important part of the development of plantation.However,due to the tissue culture research of M.laosensis is still in the laboratory stage,the effect of different factors on the proliferation of subsequent generations was unclear,and the low reproduction efficiency and high cost of the rapid propagation of M.laosensis in tissue culture,it has greatly restricted the promotion and application of tissue culture seedlings in production,resulting in the construction of M.laosensis plantation is still dominated by normal seedlings.How to propagate selected excellent varieties effectively and asexually by improving the multiplication ratio and production efficiency of tissue culture is an important task to be solved for the development of M.laosensis plantation.Therefore,it is necessary to carry out the research on the optimization of tissue culture and the induction of callus on the basis of the previous establishment of tissue culture and rapid propagation system of M.laosensis,in an attempt to provide technical support for further improving the tissue culture and propagation system and promoting the development of M.laosensis plantation.In this study,the best explant disinfection method and refrigeration time were screened out,and the optimal basic medium and hormone formula for the subculture proliferation were determined by orthogonal test design,and the optimal auxin concentration for rooting culture was selected through double factor test,taking the sprouts of M.laosensis saplings as the experimental material;in addition,suitable substrate ratio for transplanting tissue culture seedlings was also decided using a single-factor randomized block design.On the other hand,the best hormone type and concentration were determined for inducing callus induction in the stem segments,leaves and petioles of M.laosensis by single factor experiment,and a preliminary study on callus induction was conducted.The main results were as follows.（1）Among the three disinfection methods,the highest success rate of disinfection was71.25%when treated with 75%alcohol 30 S+2%sodium hypochlorite for 10 min+0.1%mercury for 8 min,which was significantly higher than the other two treatments;There was no significant difference in explant browning rate and axillary bud induced germination rate among different treatments.The explants inoculated after different refrigeration time had no significant difference in pollution rate,browning rate and germination rate of axillary buds,and the seedling state was best after refrigeration for 2 days.Generally,the highest disinfection success rate and the best condition of axillary bud seedling could be obtained by collecting shoots in direct sunlight,keeping them with leaf removal in the refrigerator at 4℃for 0-2days,disinfecting with 75%alcohol 30 S+2%sodium hypochlorite for 10 min+0.1%mercury for 8 min,and inoculating in MS+6-BA 1.0 mg/L+NAA 0.2 mg/L medium,the disinfection success rate reached 75%.（2）The order of the influence of various factors on the proliferation times of the secondary culture was basal medium>NAA>6-BA>GA₃;the basal medium had a very significant effect on the proliferation times while NAA and 6-BA had a significant effect on the multiplication factor,and GA₃ had no significant effect on the multiplication factor of M.laosensis.The medium with the highest proliferation times of M.laosensis was WPM+6-BA0.5 mg/L+NAA 0.1 mg/L.The order of the influence of various factors on the height of proliferation seedlings was basal medium>NAA>GA₃>6-BA,the basal medium had a very significant effect on the height of proliferation seedlings;NAA had a significant effect on the height of proliferation seedlings,and GA₃ and 6-BA had no significant effect on the height of proliferation seedlings.The most suitable medium for the growth of secondary seedlings was WPM+6-BA 0.2 mg/L+NAA 0.1 mg/L+GA₃0.2 mg/L.According to the comprehensive test results,the culture medium mixed with WPM+6-BA 0.5 mg/L+NAA 0.1 mg/L medium was suitable for the propagation of M.laosensis,and the multiplication rate was 4.29times and seedling height reached 4.79 cm after one-month multiplication culture.（3）NAA and IBA had significant effects on rooting rate and average root number,but the effect of NAA was greater than that of IBA.When NAA was not added,there was no callus at the base of the plant,but a small number of slender roots could grow directly..The addition of NAA in the medium helped to form callus at the base of the plant and effectively increased the rooting rate and the average number of roots.However,different ratio of IBA and NAA resulted in different callus and root states.The formula of 1/2 MS+IBA 0.4 mg/L+NAA0.2 mg/L was the most suitable medium combination for rooting tissue cultured seedling among nine medium treatments,which induced light green and active callus at the base of tissue culture seedlings,and promoted the highest rooting rate of 96.67%and the maximum root number of 16.20 pieces per plant for tissue cultured seedlings.（4）The survival rate of tissue culture seedlings was significantly affected by different matrix ratio;the highest survival rate of transplantation was 83.33%for the seedlings transplaned in 100%yellow soil substraed,and the second was 81.11%done 60%yellow soil+40%peat soil.With the increasing of light substrate proportion in the transplanted substrate for tissue culture seedlings,the biomass and seedling quality index of M.laosensis seedlings increased first and then decreased,reaching the maximum when seedlings was transplanted in60%yellow soil+40%peat soil.Therefore,the substrate mixed with 60%yellow soil+40%peat soil was the ideal substrate for transplanting seedlings of M.laosensis.（5）Single cytokinin could not induce the adventitious buds of M.laosensis explants through direct organogenesis,but only a small number of callus and adventitious roots,and its overall differentiation rate was low.MS+IBA could hardly induce the formation of callus of M.laosensis while MS+NAA 1.0 mg/L was the best for the callus induction of three explants of stem segment,leaf and petiole.Among the mediums with MS+NAA 1.0 mg/L+different concentrations of cytokinin,cytokinin concentrations of 6-BA 1.0 mg/L was most favorable for callus formation because callus induction rates for stem segment,leaf and petiole were 95.00%,98.33%and 85.00%respectively,significantly higher than those of the other 11treatments,and the induced callus had good quality chareacterized with light green,obvious and sticky granular,uniform texture and fast growth.For different explants,the callus induction rate of the petiole was the lowest and easy to brown and die,while that of leaf vein was highest and the callus appeared in about 15 days,better than stem segment and petiole callus.On the whole,MS+NAA 1.0 mg/L+6-BA 1.0 mg/L was the most suitable for callus induction of M.laosensis,and the most preferred explant material was leaf veins.