Induction And Identification Of Autotetraploid Of Akebia Trifoliata

As the third generation of new diet health fruit,the cultivated area of Akebia trifoliata is expanding day by day,and the downstream processing industry is developing rapidly.The traditional wild domestication of Akebia trifoliata has many seeds and low edible rate,which can no longer meet the needs of industrial development.It is urgent to cultivate new varieties with fewer seeds and high edible rate.The practice shows that chromosome engineering breeding is an important means to realize fruit denuclearization,and the induction and identification of autotetraploid is the key content of chromosome engineering breeding.In order to enhance the development fruit value of trifoliata and its market potential,this study used the natural population of1418 Akebia trifoliata to screen superior lines of Akebia trifoliata.The high efficiency induction technology system of autotetraploid is integrated with seeds of superior lines.A method system for rapid detection and identification of autotetraploid by flow cytometry was established.A batch of autotetraploid materials were screened,and the morphological indexes and physiological and biochemical parameters of these materials were studied.The results are as follows:1.29 superior lines were preliminarily screened out from natural population of Akebia trifoliata by the disease-resistance,the skin texture,the single fruit size,the ripening time and the fruit skin color.In addition,the weight of fruit association attributes{fruit weight,soluble sugar content,soluble protein content,edible rate and seed number,Seed proportion}={0.32,0.19,0.05,0.29,0.06,0.09}was comprehensively determined by APH stratification analysis and entropy method.Four lines suitable for fruit development were selected from 29 candidate lines by fuzzy comprehensive evaluation method.The correlation analysis show that there is positive correlation between the size of fruits and soluble protein contents(P=0.05).2.The efficient induction of autotetraploid technology system was integrated.It was found that the colchicine treated subjects had a great influence on the formation probability of autotetraploid,and the order from easy to difficult was seedling at cotyledon extension stage>germination seed>ungerminated seed.The seedlings at cotyledon stretch stage were most likely to double into autotetraploid under the induction of 400μmol/L colchicine,and the induction probability was 4.17%.Although the probability of autotetraploid formation increased with the increase of colchicine concentration,the tolerance threshold of colchicine was 500μmol/L,above which samples could hardly survive.3.A method system was established to identify autotetraploid by flow cytometry.In this study,the maximum nucleation efficiency was found under the action of LB01,Plant ploidy can be detected by nuclear suspension within 90 min.4.A batch of breeding materials with chromosome ploidy level changes were screened out.In this study,18 chimeras and 16 tetraploids of Akebia trifoliata were identified from 2088 materials treated with different colchicine concentrations.5.Materials with different ploidy levels of chromosomes showed significant differences in morphological characteristics.It was observed that all plants treated with colchicine showed slow growth,uneven leaf margin,increased leaf width,and three compound leaves of some leaves fused into single leaf.The cell content and morphology of tetraploid leaves change significantly,the leaf length-width ratio decreased significantly:diploid,chimera,and tetraploid body length-width ratio were147.27%,127.83%,105.82%.The stomatal density of tetraploid was 96.43 no/mm~2,which was significantly lower than that of chimera(128.40 no/mm~2)and diploid(133.94 no/mm~2).The stomatal area of tetraploid(233.33μm~2)was significantly higher than that of chimera(168.10μm~2)and diploid(150.54μm~2).6.The results showed that the chlorophyll content of tetraploid and chimeric plants was 1.08 mg/g and 1.18 mg/g respectively,which was significantly higher than that of diploid plants(0.53 mg/g).The contents of total soluble protein,total soluble sugars,flavonoids,total phenols and flavonoids in tetraploid leaves are 19.43 mg/g,6.35 mg/g,8.60 mg/g,23.12 U and 31.82 U,which are significantly higher than those in diploid plants.The POD activity and SOD activity of tetraploid plants are 31.98 U and 9.48 U,higher than those of diploid plants,CAT activity(7.29 U)of tetraploid plants lower than that of diploid plants.