| Masson pine(Pinus massoniana Lamb.)belonging to the genus Pinus of Pinaceae family,is a highly adaptable fast-growing species with tolerance to drought and leanness.It has high economic and ecological values and is an important timber afforestation species in southern China.However,the forest productivity of Masson pine is highly restricted by shortage of the improved varieties,low production of seeds and unstable yield.To meet the urgent needs of improved Masson pine seedlings for forestry production,we applied tissue culture study using mature Masson pine embryos as explants and established a tissue culture rapid propagation system.The main results are shown as follows:1)A sterile system for the proliferation of Masson one mature embryo was constructed.Before culturing,the seed coats were removed and soaked in sterilized water for 0.5h and followed by soaking in 75%ethanol for 30s,rinsing in sterilized water for 3 times,soaking in 0.1%mercuric chloride for 6min,and rinsing again in sterilized water for 3 times.The browning rate and contamination rate are low,and the survival rate of the sterilized seeds was 86.11%.2)The optimum basic medium for inducing the adventitious buds of P.massoniana was WPM+30g/L sucrose+8.5g/L agar.3)Different regulator combinations have significant effects on the proliferation of adventitious buds of Masson pine.The best combination with the best proliferation effect was 1mg/L 6-BA+0.05mg/L NAA+0.25mg/L KT.After one month of the treatment,the proliferation coefficient was 5.12.4)The concentration of different growth regulators and their treatment duration significantly affected the proliferation of the mature embryos of Masson pine.When 6-BA and TDZ were used alone,the concentration has an interaction with the treatment duration on adventitious bud proliferation.High cytokinin concentration or long treatment duration was not conducive to bud elongation.At the same concentration,TDZ showed a stronger proliferation promoting effect than 6-BA.The optimum proliferation medium was WPM+0.5mg/L TDZ and following by the transplant to the hormone-free medium after 14 days of induction.The proliferation coefficient was 6.68,respectively,and the adventitious buds grew well.5)The concentration of different growth regulators and their treatment duration significantly affected the proliferation of adventitious buds of young hypocotyls of Masson pine.When 6-BA and TDZ are used alone,the concentration also has an interaction with the treatment duration.In addition,comparing to the adventitious buds directly induced by intact embryoids,the epicotyls precultured for 6 days were more stable in growth.The best proliferation medium was WPM+10mg/L TDZ,followed by the transplantation to hormone-free medium after 10 days of induction.The proliferation coefficient was 6.31,respectively.However,the process of pre-culture and embryo cutting was increased which indirectly increased the working difficulty.6)Natural organic compounds and activated carbon have significant effects on the growth of adventitious buds of Masson pine.The best elongation medium is WPM+200g/L banana puree+3g/L activated carbon+30g/L sucrose+7g/L agar.7)Different growth regulator combinations have significant effects on the proliferation of Masson pine stems.The optimum combination of growth regulators for stem proliferation of for Masson pine stems was 0.2mg/L TDZ+1mg/L 6-BA+0.2mg/L NAA.8)The basic medium,growth regulator and temperature have significant effects on the induction of adventitious roots of Masson pine.At 25℃,the optimum induction medium for adventitious roots of Masson pine was 1/2MS+4mg/L IBA+20g/L sucrose+6.5g/L agar.At this time,the rooting rate reached 76%,the average number of main roots was around 6,the number of fibrous roots is large,the rooting time is short,and the roots grew well.9)The most suitable transplanting substrate for Masson pine tissue culture seedlings is 1/2 peat soil+3/8 vermiculite+1/8 perlite(volume ratio),and the survival rate of transplanting was 76%.In this paper,the problems related to the establishment of rapid propagation system of pinus massoniana organs were discussed,aiming at the establishment of the industrial pilot production line,and the detailed discussion on how to improve the number of effective buds and shorten the culture cycle,etc.A short period and rapid propagation system was established from inoculation treatment,adventitious bud induction and elongation rooting culture.In the induction of adventitious buds of Masson pine,when the mature embryos were planted to the treatment with 0.5mg/L TDZ for 14 days,following by the transplant to the hormone-free medium,the proliferation coefficient was the highest.The adventitious buds grew well,the proliferation cycle was shortened with simple processing steps,and was more in line with the requirement of efficient,easy-to-operate industrialized rapid propagation. |
