| Malus prunifolia(Willd.)Borkh also known as Begonia fruit,Haihong,Kaempferia.The emergence rate of M.prunifolia seeds is very low,the seedlings from seeds grow unevenly,the separation of offsprings are serious as well.a mature technology makes it possible to induce seedling from one cell,which is called reproduction in vitro,has great effects on genetic engineering and mutagenesis studies.The induction of polyploidy by colchicine is of great significance to increase the quality and value of fruit.In this study,tissue culture seedlings of M.prunifolia were used as materials.Firstly,the efficient in vitro regeneration system of M.prunifolia was established.secondly,in vitro polyploid induction system of M.prunifolia was established by using regeneration in vitro system,and more than140 tetraploid lines were identified.The main results are as follows:1.Establishment of high-efficient in vitro regeneration system of M.prunifolia.In this experiment,tissue culture seedlings of M.prunifolia were used as test materials.According to the regeneration medium formula of M.prunifolia,different explant types were used in the experiments,the dates of dark culture,orientation of explants and wound modes of explants,the efficient regeneration system of M.prunifolia was obtained: using the formula above,putting the leaves in reverse position after transection,the average regeneration frequency reached 5.4 after dark-culturing for three weeks,and the single leaf regeneration buds reach 15.2.Establishment of polyploid induction system of M.prunifolia in vitro.2% DMSO is beneficial to colchicine induced polyploid experiment.The optimum induction concentration of colchicine was 0.01% after 5-days cultivation,germination rate was 8.70 ± 2.48%,regeneration rate was 18.01 + 3.19%,and induction rate was the highest 7.45%.When0.05% colchicine was mixed for 2 days,the best result was obtained.The germination rate,regeneration rate and induction rate were 15.7±0.32%,33.82±0.64% and 4.83±0.73%.Under the condition of no significant difference in induction rate,the concentration of colchicine induced by the best stem segment was 0.02%,the survival rate was 10.56±1.47%,the proliferation coefficient was 0.78±0.06,and the induction rate was 13.33±0.88%.More than 140 tetraploid and 1 aneuploid lines were obtained from M.prunifolia cultivation with colchicine and DMSO.3.Screening and identification the polyploidy samples among all M.prunifolia.Stomatal observation can be used as first step of polyploidy screening,which is more referential than the results of morphological identification.The first screened potential polyploids were identified by flow cytometry,thus polyploids were identified eventually.4.Biological effects of chromosome doubling in M.prunifolia.Polyploid rooting rate of M.prunifolia is high,the elongated roots are few,with the characteristics of thick,brittle,easy to break,and many lateral roots grow on the main roots.The leaf length of polyploid plants decreases,while the width increases,the leaf shape index decreases,and the leaf shape changes from spindle of diploid plants to ellipse;the plant height decreases,and the growth potential is not as good as those of diploid plants.The contents of chlorophyll a,chlorophyll b,carotenoids and total chlorophyll in four polyploid lines were significantly lower than those in diploid lines.There was no significant difference in photosynthetic efficiency among polyploids,between polyploids and diploids,and individual plants showed significant higher photosynthetic efficiency than a certain line.The stomatal length,width and stomatal aperture of four polyploid lines were significantly higher than those of diploid lines,and their density was significantly lower than that of diploid lines.Diploid stomata are more rounded and evenly located.The ratio of length to width of polyploid stomata is bigger.It is long ellipse,uneven in distribution,uneven in size and occasionally super-large stomata. |
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