| Pinus massoniana Lamb,one of the main wood species,is an unique native tree specie in south of China.As an important material for building and paper making due to its high quality and long fiber,Masson pine also has high economic value and ecological benefits.However,the breeding research of Masson pine has problems such as long breeding cycle,low breeding rate of varieties.Therefore,in order to significantly increase the yield of forest land and the quality of forest trees to improve the economic efficiency of the Masson pine industry,it is an urgent work to establish a rapid propagation technology system for Masson pine to provide urgently needed high-quality seedlings with excellent traits,stable inheritance and high gains for production.Using endosperm as material for tissue culture is expected to obtain haploid materials.Haploid breeding has the advantages of quickly obtaining homozygous genes,shortening breeding years and improving breeding efficiency,which can be combined with a variety of breeding techniques to speed up breeding research.In this paper,the megagametophytes of Masson pine was used as the material for tissue culture research,and the main research results are as follows:(1)The suitable disinfection method for masson pine endosperm material was using 75%alcohol for 30 s then 2% Na Cl O for 15 min and the optimal medium for callus induction was DCR+NAA2mg/L+ 6-ba 1mg/L,induction rate was 68.75%.The callus induction rate could be increased effectively by adding the 6-BA soaked explants into the medium without cytokinin,and the highest induction rate was 87.77% when the 6-BA concentration reaching 1mg/ml.While other kinds of cytokinins like TDZ and KT repressing the callus induction at the concentrating ranging from 0.5mg/ml to 1mg/ml.The study also found that the megagametophytes callus of Masson pine was difficult and slow to develop,and the callus began to form gradually at the fourth week.In the first three weeks,there would be pseudocallus,and the projection on the cutting surface looked like callus,but in essence,it was only the phenomenon of energy consumption and enlargement in megagametophytes cells.(2)The optimal conditions for callus induction of Masson pine were as follows: a)The optimal concentration of sucrose in the medium is 20g/L ～ 30 g/L;b)The optimal pH was 5.5,if the pH was too high or too low,it would decrease the callus induction rate;c)The agar concentration in the medium had an impact on the texture of the medium,and the optimal agar concentration was 7g/L;d)Mashed potato and banana had an inhibitory effect on callus induction of masson pine megagametophytes,and should not be added in the medium.Coconut milk had a certainly promoting effect on callus induction when the concentration was about 10-70 m L /L,and the best result was getting when the concentration was 70 m L /L;e)Heat shock treatment can accelerate the time of callus breaking and callus formation in masson pine megagametophytes,and the callus induction is higher after heat shock.The optimal heat shock treatment is 45℃ for10 min.(3)The optimal subculture medium for masson pine megagametophytes callus was WPM+NAA 0.1mg /L+ 2,4-D 0.1mg /L+6-BA 2.0mg /L.Under this condition,the callus was light green with higher antioxidant enzyme activity and stronger differentiation ability.The callus of masson pine megagametophytes callus were difficult to differentiate to adventitious shoot,and only using medium with WPM+NAA 0.1mg/L + KT 2mg/L could induce adventitious shoot forming.6-BA played a key role in the proliferation and elongation of adventitious shoot.When the concentration of 6-BA was 0.1mg/L,the adventitious shoot prolifulated,while the elongation was not obvious.When the concentration of 6-BA increased to 0.5mg/L,the adventitious shoot began to elongate.(4)The number of subculture had a significant effect on the callus induction of masson’ pine megagametophytes.With the increase of culture time,the proportion of haploid cells in masson pine megagametophytes callus decreased gradually,while t diploid cells proportion increased gradually,even polyploid cells appeared.2,4-D had a significant effect on chromosome variation in masson pine megagametophytes callus.With the increase of 2,4-D concentration,the proportion of chromosome number variation also increased.In addition,the higher the concentration of total auxin,the greater the degree of chromosome variation.(5)Through the cultivation of the megagametophytes of Masson pine,the adventitious shoot was successfully induced,and the chromosome ploidy of the adventitious shoot was identified,and its chromosome ploidy showed a naturally doubled diploid. |
