Anther Culture And Regeneration System Establishment Of Actinidia Arguta (Sieb.& Zucc) Planch.ex Miq.

This study mainly includes two parts.One is the anther culture of Actinidia arguta to obtain haploid plants that originated from the microspores;another one is the regeneration system establishment of Actinidia arguta cultivar ‘Kui Lv’ and ‘Jia Lv’ by studying the influence factors of callus induction and plantlet regeneration.The main research results are as follows:1.In order to explore the relationship between microspore development stage and bud morphological characteristics,we collected the Actinidia arguta ‘Kui Lv’ and ‘Lv Wang’ buds which microspores were at the stage of tetrad to mature period.The results showed that the stage of the microspore development from tetrad period to mature period is quite short time;the morphology of two cultivars buds was similar in the same developmental stage,but the diameter of ‘Kui Lv’ flower is larger than the ‘Lv Wang’.Microspores showed correlativity with bud diameter and morphological characteristics at the development stage.We can identify the developmental stage by observing the diameter of Microspores.2.We explored the effects of microspore development,4℃ low temperature pretreatment duration,basic medium,plant growth regulator and genetype on Actinidia argute anther callus induction by single factor experiment.The findings showed that: microspores at the late-uninucleate stage has the highest frequency of callus induction;The rate of callus induction by low temperature pretreatment at 4℃ for 5 days was higher than the treatment for 1d,3d,7d.The basic culture medium and the type and concentration of plant growth regulators play a key role on induction of anther callus.In the medium with MS+2,4-D 1mg/L+6-BA 5 mg/L +sucrose 30g/L + agar 5.5g/L showed the highest induction of anther callus,was 93.30%;the most suitable medium for ‘Kui Lv’ anther callus induction was MS+2,4-D 2mg/L+6-BA 5 mg/L with sucrose 30g/L and agar 5.5g/L,the induction rate was 90.91%.3.Adding different types and concentrations of plant growth regulator in the MS medium to induce the differentiation of anther callus adventitious bud based.The results showed that the type and concentration of plant growth regulator has very important influence on the regeneration of adventitious buds.ZT can induce adventitious bud of ‘Kui Lv’ to regenerate successfully,but cannot induce the ‘Lv Wang’ adventitious bud to regenerate.While,anther callus of neither ‘Kui Lv’ nor ‘Lv Wang’ has the differentiation of adventitious buds in the medium with 6-BA or TDZ.‘Kui Lv’ callus in the medium MS+IBA 0.1mg/L+ZT 3mg/L+sucrose 30g/L showed the highest adventitious shoot regeneration rate of 64.55%.The regeneration seedlings rooted in the medium of 1/2MS+IBA 0.01 mg/L,so we obtained the intact plants.4.The ploidy of 119 regenerated plants was identified using flow cytometry.The results showed that: the ploidy of the plantlets obtained by the vitro anther culture of Actinidia arguta was complex,with 3.36% haploid,56.3% tetraploid;4.20% hexaploid,36.13% octoploid.The morphology of different ploidy plants was different;the ploidy increased,and the organs(roots,stems and leaves)of regenerated plants increased.5.To establish the regeneration system of ‘Kui Lv’ and ‘Jia Lv’,the effect of explants types and plant growth regulator combination on the callus induction and plantlet regeneration of different cuitivars Actinidia arguta was studied.The results show that the reactions of different genotypes to the culture condition were different.The effect of 6-BA on the callus induction of Actinidia arguta was better than that of KT.Neither 6-BA nor TDZ can induce Actinidia arguta shoots differentiation,but ZT has a good effect on it.The best medium of ‘Kui Lv’ leaf callus induction was MS+2,4-D 0.5 mg/L+6-BA 2 mg/L,and the induction rate was 93.3%;the best medium of ‘Jia Lv’ leaf callus induction was MS+2,4-D 0.5 mg/L + 6-BA 1 mg/L with the 96.2% callus induction rate.The differentiation rate was the highest when ‘Kui Lv’ and ‘Jia Lv’ callus was in the medium of MS+NAA 0.05 mg/L +ZT 3 mg/L,and the differentiation rate was 72.0% and 37.8%.Under the same culture condition,the shoot differentiation rate of ‘Kui Lv’ was significantly higher than that of the ‘Jia Lv’.Different explants have the different ability of callus induction and plant regeneration.Stem without bud and leaf callus induction rates were higher than 90%;adventitious buds differentiation rate of leaf was highest,significantly higher than that of stem and petiole.Compared with stem and petiole,leaf was more suitable for used as explants of Actinidia arguta plant regeneration.The bud rooting rate on the 1/2MS+IBA 0.01 mg/L was 97%.