| D.is a deciduous tree of the persimmon family(Ebenacene)of the genus Diospyros L.,its fruit is rich in nutrients and has extremely high medicinal value,and the seedlings cultivated with its seeds are excellent grafting rootstocks.In recent years,with the increase of global temp?rature,great climate change and uneven rainfall,the demand of persimmon plants for stress-resistant rootstocks has increased.At present,stress-resistant rootstocks are mainly obtained through seed propagation,and the acquisition of stress-resistant rootstocks through vegetative propagation and molecular breeding is still in its infancy.There are still many problems in the tissue culture of D.lotus.,such as:severe browning of explants,weak leaf growth of sterile seedlings,poor regeneration efficiency and poor rooting culture effect,etc.These key problems seriously limit the research on genetic transformation of D.lotus..In this paper,by studying the effects of different disinfection methods,basal culture medium and plant growth regulators on the growth of tissue culture seedlings of D.lotus.,the tissue culture regeneration system of D.lotus.was successfully established.On this basis,the preliminary study on the genetic transformation of D.lotus.was carried out,it provides a theoretical basis for persimmon molecular breeding.The main results are as follows:1.Primary generation and proliferation bud induction of D.lotus.:(1)The seed embryo of D.lotus.was used as the explant to study the effects of different disinfection methods,seed pretreatment and culture medium formula on seed germination of D.lotus..The results showed that:The seeds of D.lotus.were disinfected in 75%ethanol for 4 minutes,then sterilized with 0.1%HgCl2 for 8 minutes,and then rinsed with sterile water for 3-5 times,and the sterilized seeds were sealed in a glass bottle with sterile water and treated in a 50℃ water bath for 4 hours,and then inoculated into 1/2MS+1.0 mg/L GA3+1.0 g/L activated carbon culture medium,cultured in a 30℃ light culture room.The seed embryo germination rate was 88.05%,and the pollution rate is 11.6%.The tissue culture seedlings germinated from the embryo of seed of D.lotus.were inoculated into 1/2MS+2.0 mgL ZT+2.0 mgL 2ip+0.05 mg/L IAA culture medium.After 25 days of culture,the average plant height of the tissue culture seedlings was 3.4 cm and the average proliferation coefficient was 6.5.(2)The semi-lignified bud bearing stem segment of D.lotus.was taken as the explant to study the effects of different disinfection methods and culture medium formulas on axillary bud induction of shoot segments with buds.The results showed that:The bud bearing stem segments of D.lotus.were disinfected with 75%absolute ethanol for 25 seconds,then disinfected with 0.1%HgCl2 for 25 minutes,washed with sterile water for 3-5 times,and inoculated into 1/2MS+2.0 mg/L 6-BA+0.5 mg/L NAA culture medium.The axillary bud germination rate was 67.1%,the pollution rate was 11.7%,and the average germination time was 17.2 days.The axillary bud induced by D.lotus.with sprouts and stem segments were inoculated in 1/2MS+2.0 mg/L ZT+2.0 mg/L 2ip+0.05 mg/L IAA culture medium,the average height of the axillary bud was 3.7 cm,and the proliferation coefficient was 7.6.(3)Using the D.lotus.bud stem segment and seed as explants,to compare the effect of the callus tissue induced by the two explants,The results showed that:The effect of callus induction by explants obtained from stem segments with buds is better than that of seed embryos,which were inoculated to 1/2MS+2.0 mg/L 6-BA+0.5 mg/L NAA culture medium.The callus induction rate of the leaves of the tissue culture seedlings with bud stem segments as explants was 82.3%,and the callus induction rate of the leaves of the tissue culture plantlets proliferated and cultured with seed embryos as explants was 70.5%,and the ability of callus induced with bud stem segments to differentiate adventitious buds in the later stage was also better than that of seed embryos.2.Establishment of leaf regeneration system of D.lotus.:The leaves of aseptically proliferating axillary buds which were obtained from the tissue culture seedlings cultured from the stem segments with buds as explants were used as materials,to study the effects of different sucrose concentrations,plant auxin regulator and dark culture time on leaf regeneration.The results showed that:(1)The basal culture medium suitable for callus induction of D.lotus,leaves was 1/2MS culture medium,and the concentration of sucrose was 40 g/L.The time required for leaf callus induction was the shortest when cultured in dark for 48 hours,and the induction time was 29 days.The highest callus induction rate was 93.6%(2)The optimal culture medium formula for inducing adventitious buds from the leaf callus of D.lotus.was:1/2MS+2.0 mg/L TDZ+0.5 mg/L NAA+2.0 mg/L 2ip+40 g/L sucrose.The induction rate of adventitious buds was 75.2%,and the average number of adventitious buds was 7.9.The height of 3-5 adventitious buds could reach more than 2 cm after 25 days of continuous culture.(3)The ability of callus to differentiate adventitious buds decreased from the 4th generation,and the callus could not continue to induce adventitious buds from the 6th generation.From the external observation,there was no difference in callus between the callus and the normal callus,and it was observed that the inner texture was loose and frothywhen the callus was cut.(4)The robust adventitious buds with a height of 2-3 cm·induced by callus were inoculated into 1/2MS+1.0 mg/L IB A + 0.5 mg/L NAA culture medium.After dark culture for 5 days and light culture for 40 days,the average number of roots could reach 9.6 and rooting rate of D.lotus.was 70.2%.(5)After refining the rooted tissue culture seedlings of D.lotus.were transplanted into the matrix with the ratio of "vermiculite:perlite:nutrient soil=2:2:1",and the survival rate was 89.3%after 25 days of culture under glass cover.3.Preliminary study on genetic transformation of D.lotus.:Based on the establishment of leaf regeneration system of D.lotus.,the conditions suitable for genetic transformation of D.lotus.The results showed that:The callus of D.lotus.was infected in Agrobacterium(GENE-Chr2.507)suspension with a bacterial conc entration of OD600 0.5 for 10 minutes,and then co-cultured in LB+1.0 mg/L ZT+0.5 mg/L NAA culture medium for 3 days.After co-culture,the callus was inoculated into 1/2MS+2.0 mg/L 2ip+2.0 mg/L TDZ+0.5 mg/L KT+50 mg/L Kan culture medium for dark culture for 7 days,and then the callus was inoculated into 1/2MS+2.0 mg/L 2ip+2.0 mg/L TDZ+0.5 mg/L KT+50 mg/L Kan culture medium for light culture for 10 days.Finally,it was inoculated into 1/2MS+2.0 mg/L 2ip+2.0 mg/L TDZ+0.5 mg/L NAA+50 mg/L Kan culture medium for adventitious bud induction.30 callus were randomly selected from 100 callus,and the adventitious buds produced by these 30 callus were cultured for strong seedlings,and then 11 adventitious buds were obtained.After confirmed by PCR,3 adventitious buds were positive,and the genetic transformation system of D.lotus.was preliminarily established. |
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