| Lagerstroemia indica ’Zijingling’ is a new variety of L.indica selected by Hunan Academy of Forestry,with purple-blue flowers and lush blooms;long flowering period,high ornamental value and high quality Seedlings are in great demand.Plant tissue culture propagation speed,short seedling cycle,can produce a large number of seedlings in a short period of time,to meet the growing demand for ’Zijingling’ yew seedlings.At present,the efficient genetic transformation system of L.indica has not been established at home and abroad,and the efficient induction and differentiation of healing tissue is an important way to construct an efficient genetic transformation system of L.indica.In this paper,single-factor,two-factor and orthogonal experimental designs were used to select stem parts,sterilize HgCl2,select basic medium and phytohormone types and concentrations,induce healing tissues and differentiate adventitious buds and the content of endogenous hormones in tissue culture.In order to screen the most suitable combination of medium for primary culture and secondary proliferation and rooting culture and transplanting,and to screen the suitable medium for healing tissue induction and differentiation,and to investigate the main regulatory role and mechanism of endogenous hormones in the culture process,as well as the effect of the addition of plant growth regulator on their content changes,in order to provide a theoretical basis for fast tissue culture of L.indica and the establishment of an efficient genetic transformation system.The main results are as follows:(1)In the selection of stem segment,the best explants were the middle stem segment of semi-lignified twigs,and the survival rate reached 86.67%after sterilization with 0.1%HgCl2 for 7 min.Plant growth regulator added to the starter medium had an effect on the germination rate of stem segment,and the best starter medium combination was DKW+6-BA1.5 mg·L-1+NAA0.2 mg·L-1+sucrose 30.0 g·L-1+AGAR5.0 g·L-1.(2)The results of subculture and proliferation culture with buds germinating from stem segments with axillary buds as explants showed that different basic media,different types and concentrations of cytokinin and auxin concentration had significant effects on proliferation and growth.The suitable medium combination for the sub generation and proliferation of L.indica ’Zijingling’ was MS+6-BA 2.0 mg·L-1+NAA 0.4 mg·L-1+ sucrose 30.0 g·L-1+AGAR 5.0 g·L-1.The coefficient of increment was 6.15.(3)In the rooting induction experiment with tissue culture seedlings as explants showed that the type and concentration of basic medium and auxin were the main factors affecting rooting.The suitable medium combination for rooting induction was 1/2MS+IBA0.6 mg·L-1+sucrose 15.0 g·L-1+AGAR5.0 g·L-1+activated carbon 200 mg·L-1.The rooting rate was 92.5%.The results of paraffin section showed that the initiation of adventitious roots was the induced root primordia type,and the rooting mode was the direct type.(4)The results of callus induction and differentiation with leaves of tissue culture seedlings as explants showed that MS basic medium promoted callus induction significantly,and plant growth regulator affected callus induction rate.The suitable medium combination for callus induction was MS+6-BA1.5 mg·L-1+2,4-D1.2 mg·L1+sucrose 30.0 g·L-1+AGAR5.0 g-·-1+coconut juice 10%.The induction rate was up to 93.55%.The effect of 6-BA concentration on callus differentiation was significant,with the highest differentiation rate at 4.0 mg·L-1 and the best adventitious shoot growth at 1.0 mg·L-1.The best differentiation medium combination was MS+6-BA 4.0 mg·L1+NAA0.1 mg·L-1+sucrose 30.0 g·L-1+AGAR 5.0 g·L-1.The differentiation rate was 13.33%.(5)The results showed that endogenous hormone GA3 was the most abundant endogenous hormone in the process of proliferation and rooting,followed by IAA,and less ZR and ABA.Plant growth regulators could significantly increase the content of endogenous hormones,in which ZR,GA3,IAA and ABA increased by 48.21%,11.34%,130.74%and 47.66%respectively compared with the control.The hormone ratios of IAA/ZR,GA3/ZR and GA3/ABA fluctuated greatly,indicated that they jointly regulated the proliferation and growth of L.indica ’Zijingling’.Auxin was the main hormone to promote rooting.GA3 and IAA were more endogenous hormones in the rooting process,while ABA and ZR were less.IAA was the endogenous hormone with greater variation,indicated that endogenous IAA was the main factor in inducing rooting of tissue cultured seedlings.Exogenous IBA can significantly increase the content of IAA and decrease the content of ZR,and promote the rooting of ’Zijingling’.The high IAA/ZR ratio promoted the differentiation and growth of root primordia and adventitious root elongation.(6)In callus induction,higher endogenous GA3 and ABA content and lower ZR and IAA content were required.ABA and ZR content changed significantly,indicated that they play an important role in callus induction.IAA/ZR value was an important influencing factor in callus induction.Each hormone and its ratio jointly regulated callus induction and formation of L.indica ’Zijingling’.After 30 days of callus differentiation,its endogenous hormone content indicated that GA3 was the most abundant,followed by ABA,and the contents of IAA and ZR were lower,so GA3 and ABA were the main factors inducing differentiation and growth.To summarize the above results,we identified the middle stem section of’Zijingling’ as the most suitable explant,screened out the suitable primary medium,secondary multiplication medium,rooting medium,healing tissue induction and differentiation medium;established the tissue culture fast propagation system and preliminary research on differentiation and regeneration of adventitious buds,which is the basis for the tissue culture and efficient genetic transformation system of L.indica.We have also established a tissue culture fast breeder system and preliminary research on regeneration of adventitious shoots,which will lay the foundation for tissue culture and efficient genetic transformation system of L.indica. |
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