Optimization Of Propagation Technology Of Mussaenda Anomala In Plants With Very Small Population In Guizhou

Mussaenda anomala is a very small population of plants in Guizhou,its distribution area is narrow,the population is very small,and there are serious breeding obstacles to selfpollination,resulting in endangered predicament,and it is urgent to carry out research on the conservation of M.anomala.Therefore,by exploring the effects of different explant types,different collection times,disinfection treatment,antibrowning agent concentration,basic medium,plant growth regulators and additives on their regeneration,the suitable conditions for the regeneration of M.anomala were screened.These results were as follow.（1）The results of sterile explant experimental screening showed that the top buds collected in May were suitable explants,the pollution rate was controlled below 1.97%,the survival rate could reach 96.07%,the optimal disinfection treatment was 0.2%Hg Cl₂ for 8 min,and 0.2 g/L PVP could effectively prevent the browning of the top buds.In April,the leaves were the best explants induced by callus of M.anomala,treated with 0.1%Hg Cl₂for 10 min,the survival rate of leaves was the highest（49.29%）,the pollution rate was the lowest（45.78%）.（2）In different combinations of plant growth regulator,it was found that the top bud grew vigorously in the combination of 6-BA（2.0 mg/L）+IBA（0.05 mg/L～0.4 mg/L）,and the leaves were thick green and there were clumps of buds.The combination of 6-BA（3.0 mg/L）+IBA（0.2mg/L）+GA₃（0.7 mg/L）had a significant effect on the high growth,proliferation rate and proliferation factor of clumped buds,which were 3.0cm,100%and 7.2,respectively.IAA combined with IBA was conducive to explants rooting,IBA（0.5 mg/L）+IAA（0.5 mg/L）combination was the best,the rooting rate was 88.33%,the average number of roots was12.00,and the average root length was 5.01 cm.In the callus pathway,the induction rate of 6-BA（0.2 mg/L）+NAA（2.0 mg/L）was as high as 89.21%,and the average callus weight was 1.12g.The callus of M.anomala was differentiated and induced in the medium,but no adventitious buds were induced,and the callus was non-embryonic callus after cell morphology.（3）Through the screening experiment of basic medium,the results showed that N6 medium had the best effect on the induction of apical bud germination,followed by B5 and 1/2MS,WPM wasn’t conducive to the growth of M.anomala tissue culture seedlings;MS medium is conducive to the proliferation of clumpy buds,and clumped buds have the best rooting effect on 1/2B5 medium.The medium suitable for leaf callus induction was MS medium,with an induction rate of 89.21%,good callus texture,milky white and dense structure.（4）The effect of carbon source on the growth of M.anomala explants showed that when the glucose concentration was 20 g/L,the primary cluster buds grew the best,with a plant height of 3.18 cm,followed by 20g/L sucrose.In the proliferation culture,the proliferation rate of clumped buds was the highest（94.21%）when 30 g/L sucrose was added,and the proliferation factor was 6.62.Low concentration of sucrose（20 g/L）was conducive to clump shoots and rooting.Fructose wasn’t conducive to the growth of M.anomala explants.（5）In the tissue culture experiment of M.anomala,the additional glutamine,casein hydrolysate and lactoalbumin hydrolysate were added,and it was found that the addition of nitrogen source inhibits the growth of M.anomala explants,with the increase of nitrogen source concentration,the more obvious the inhibitory effect,the plant is short,abnormal development,leaves are yellow or even dead.（6）In the seedling transplanting experiment,the best cultivation substrate is:V（perlite）:V（vermiculite）=1:1,the survival rate of tissue culture seedlings can reach 97.17%,and the plant growth is robust.In summary,the suitable conditions for primary induction culture of M.anomala were:N6+2.0 mg/L 6-BA+（0.05 mg/L～0.4 mg/L）IBA+20 g/L glucose+0.2 g/L PVP,and the suitable conditions for callus-induced culture of leaf leaves were:MS+0.2 mg/L 6-BA+2.0 mg/L NAA+30 g/L sucrose+0.1 g/L PVP;The suitable conditions for secondary proliferation were:MS+3.0 mg/L 6-BA+0.2 mg/L IBA+0.7 mg/L GA₃+30 g/L sucrose;The suitable conditions for rooting culture were:1/2B5+0.5 mg/L IBA+0.5 mg/L IAA+20 g/L sucrose;The best substrate for refining seedlings:V（perlite）:V（vermiculite）=1:1.