| Quercus chungii,belonging to the genus of Cyclobalanopsis in the Fagaceae family,is an evergreen broad-leaved tall tree species in subtropical region of South China,also known as Yellowwood,and Zhongshi Quercus.Its wood is reddish brown and there is no distinctness between the heartwood and sapwood.It is a high-quality hardwood of the Redwood family,always ranking the first among the precious and precious tree species of Redwood.It is a top traditional furniture material with high-valuable in the exploration of precious timber species.Currently,however,there are limit Q.chungii germplasm resources and lack of seedling for afforestation,which seriously restrict the development of Q.chungii precious timber resources.The clone propagation approaches,including cutting and tissue culture,are the effective ways to rapid propagation limited germplasm resources to produce a large number of seedlings to meet up the urgent demand of seedlings in afforestation.It is worthy of developing the Q.chungii clone propagation technique for rapid propagation of excellent materials of precious timber tree species in afforestation.In this paper,the exploration of key techniques of clone propagation（including cutting and tissue culture）with three-month-old and two-year-old seedlings were carried out,and the effects of different factors on the cutting rooting and different stages in tissue culture（including sterilization,callus induction with leaf,induction of axillary buds in stem segments,bud proliferation and rooting）were discussed.Regeneration plantlets were obtained using the cutting and tissue culture technique.The main findings were as following:1)The plantlet regeneration system was established using cutting propagation in Q.chungii.There were significant effects in the rooting ratios among different types of plant growth regulators（PGR）（K-IBA,ABT1,Guoguang rooting powder）and concentrations(1000 mg·L-1,3000 mg·L-1,8000 mg·L-1)in the cutting of Q.chungii,and the generated adventitious roots were mainly from phloem.The ranking order in the rooting effects with three different PGRs in Q.chungii cutting process was following:K-IBA>Guoguang rooting powder>ABT1,with the rooting rates ranged from 9.38%to 37.50%.The largest rooting ratio was observed in the treatment of K-IBA with the concentration of 3000 mg·L-1,while the largest rooting ratios in the ABT1 and Guoguang rooting powder were achieved under the treatment of 1000 mg·L-1,with 18.75%and 25.00%,respectively.However,there was no significant difference among the other rooting parameters（average number of roots,root length,and rooting index）in the maximum rooting ratios obtained in different PGRs.Nevertheless,the largest numbers of 2.13,2.78cm,and 2.08 in average number of roots,root length,and rooting index in K-IBA treatment with a concentration of 3000 mg·L-1 had obtained,respectively,which were better than the other two treatments in Guoguang rooting powder and ABT1.Thus the K-IBA treatment with a concentration of 3000 mg·L-1 was recommended as the best treatment for rooting in two-year-old seedlings of Q.chungii cutting.2)A suitable disinfection and sterilization system was established using different sources and types of explants for Q.chungii tissue culture.It was found that the explants sterilization should be disinfected differently according to the original sources and types of explants,for example,the materials obtained in different seasons and physiological ages should be treated with different considerations in sterilization process.The treatment of 75%alcohol 30 s+0.1%mercuric chloride 8 minutes+Tween 20 with two drops)was the optimal disinfection for stem segments of seedlings with three-month-year-old,and obtained a lower pollution rate of.33.33%;while the treatment of（0.1%carbendazim,30 min+75%alcohol 30 s+0.1%mercuric chloride 8 min+Tween 20 with two drops）was the the most suitable disinfection treatment for the stem segments taken in August from the two-year seedlings,and a lower pollution rate was obtained with 19.45%;For the sprouted leaves explants from the two-year-old seedlings,the related suitable disinfection treatment was 75%alcohol 15 s+0.1%mercuric chloride 6 minutes,and the lower pollution rate is 38.46%.3)A set of protocol on preventing browning of leaves,leaf callus induction and proliferation were obtained in Q.chungii tissue culture using leas explant.Adding 2000 mg·L-1 PVP to the medium or soaking the leaves in 1000 mg·L-1 PVP solution for 10 minutes resulted in a minimum browning rate of 32.67%.The optimal medium for inducing leaf callus was MS+1.00 mg·L-1 6-BA+1.00 mg·L-1 NAA,with an induction rate of 40.94%;The degree of leaf integrity also affects callus induction.The treatment of scratching or taking half of the leaves resulted in higher leaf callus induction rates,reaching 42.05%and 41.67%,respectively;The optimal medium for the proliferation of leaf callus tissue is MS+1.00 mg·L-1 6-BA+0.10 mg·L-1 NAA,with a proliferation coefficient of 2.04.The callus tissue has good growth potential and strong vitality,and forms spherical protrusions on the surface.The obtained callus was similar to embryonic callus under the microscopic observation.4)The induced axillary bud was obtained from stem segments in Q.chungii tissue culture induction stage.There were significant differences（P<0.05）among the three basic media of WPM,1/2MS,and MS in axillary bud induction from the stem segments of Q.chungii.The WPM was the best medium for axillary buds linduction,with the highest bud induction rate（33.33%）and robust bud growth.It was found that there were significant effects on the axillary bud induction from stem segments of three-month seedlings with the interaction effects between 6-BA hormone concentrations(2.00 mg·L-1,4.00 mg·L-1,8.00 mg·L-1)and induction duration（8 d,16 d,24 d）.The axillary bud induction rate was ranged between 11.11%to 36.09%,and the highest induction rate of 36.09%was obtained with low concentration of 6-BA(2.00 mg·L-1)under long duration（24 d）,the lowest induction rate of 11.11%was obtained with high concentration of 6-BA(8.00 mg·L-1)under long duration（24 d）and callus were observed at the base segment.Meanwhile,low concentration under short duration obtained a lower axillary bud induction rate（13.89%）;The combination of.NAA and 6-BA in the induction medium(0.10 mg·L-1 NAA+2.00 mg·L-1 6-BA)had better bud induction effect than the only use of 2.00 mg·L-1 6-BA,with a bud induction rate of 38.89%with better bud growth.For the axillary buds induction from stem segments in two-year-old seedlings,the induced axillary buds were obtained with a wide range of 6-BA concentrations from 0.10 mg·L-1 to 2.00 mg·L-1 and the highest induction rate of 50.00%was observed with the treatment of 6-BA 0.5 mg · L-1;Similarly,the treatment with a combination of 0.01 mg·L-1 NAA and 0.50 mg·L-1 6-BA increased a higher induction rate of 54.63%with more robust bud growth.5)A set of culture system for axillary bud proliferation from stem segment was established.The optimal concentration levels of required PGRs for the axillary bud proliferation varied according to the different cultured axillary bud original sources.The optimal medium for the axillary bud proliferation from three-month-old seedlings was WPM+2.00 mg·L-1 6-BA and 0.01 mg·L-1 NAA,with a proliferation coefficient of 2.47;The optimal medium for axillary bud proliferation from two-year-old seedlings was WPM+1.00 mg·L-1 6-BA and 0.01 mg·L-1 NAA,with a proliferation coefficient of 2.87.6)A rooting system for the stem buds was established,although the rooting rate was relatively low.In different rooting treatments,a rooting rate of 3.30%and 6.70%was obtained when the stem buds were soaked in 500 mg·L-1 or 1000 mg·L-1 DBA solution for 30 s,respectively.Another higher rooting rate of 10%was obtained with 0.50 mg·L-16-BA+0.50 mg·L-1 NAA in the rooting medium.The induced root was strong and the plantlet regeneration grew well.In summary,A complete plantlet regeneration with axillary bud induction,proliferation and rooting from stem segment was established in Q.chungii tissue culture,as well as the cutting propagation and rooting for the first time so far.These findings would facilitate the clone propagation of superior Q.chungii seedlings for the outstanding precious timber tree afforestation in the future. |
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