| Dioscorea zingiberensis is a perennial herbal plant distributed in China, and the dry turmeric roots usedas medicine. The main medicinal ingredient is diosgenin which called "medicinal gold " due to its effectson reducing blood lipid, anti-aging, anti-tumor, anti-bacterium, protecting the liver and so on. Dioscoreazingiberensis is an important medicinal plant. Excessive excavation without plan and rationality leads toresource reserves depletion. During breeding, the roots and stems of Dioscorea zingiberensis are used asvegetative forms leading to a low reproductive rate, slow growth quality degradation. In order to solve theabove problems, endosperm culture method was systematically studied including the triploid plantletsidentification and rapid propagation system of Dioscorea zingiberensis establishment. The results were asfollowed:1. Selection of the suitable medium for endosperm callus induction and subculture. The suitableinduction medium was MS+2,4-D2mg/L+6-BA0.5mg/L.and the light yellow callus could beinducted after72day cultivation; The suitable subculture medium was MS+NAA1.0mg/L andproliferation capacity and growth rate was34.73g/L and16g/L, respectively with proliferation of2.8times of the initial inoculum (12.5g/L).2. Study of the differentiated way and the suitable medium for the regenerated plants from endospermcallus. Cultivation of endosperm callus on the differentiation medium for180day could differentiate theadventitious buds and protocorm-like bodies. The suitable medium for differentiated adventitious buds wasMS+6-BA2.0mg/L+NAA0.1mg/L inducing differentiation rate of57.06%; the suitable medium fordifferentiated protocorm-like bodies was MS+6-BA3.0mg/L+NAA0.2mg/L+KT0.5mg/L inducingdifferentiation rate of72.25%.3. Identification of ploidy plantlets induced by Dioscorea zingiberensis endosperm.(1) Identification ofthe chromosome number. The growing point of axillary bud was used as material combined with theconventional slice production method of chromosome. The result showed that the cells with20,21-29and30chromosomes occupied10%,16%and74%of total observed cells, respectively and the regeneratedplants with20,21-29and30chromosomes occupied10%,50%and40%of total observed plants. Based onthe above, the triploid plantlets were finally obtained.(2) Morphological characterization. Compared to the diploid plantlets, and triploid plantlets were shorter, stronger stem, larger and thicker leaves with deepercolor and other features. The height of diploid plantlets increased23.02%than triploid with a significantdifference. The stem diameter, leaf length, leaf width and leaf thickness of triploid plantlets were generallyhigher than the diploid an increasing77.08%,48.06%,46.53%,29.82%, respectively with a significantdifference.(3)Cytological identification. The stomata density of triploid plantlets was significantly lowerthan the diploid, reducing by64.98%; chloroplast number in each single guard cell in triploid plantlets wasconsiderably higher than the diploid, increasing by115.25%. The length and width of stomata and stomataapparatus in triploid leaf were higher than the diploid increasing25.98%,4.1%,26.32%;4.11%,respectively with a significant difference between the length of stomata and stomata apparatus, but withouta difference between the width of stomata apparatus and stomata.(4) Physiological and biochemicalidentification. Compared with diploid plantlets, the content of chlorophyll b and carotenoids in the triploidplantlets averagely increased by60.71%, and9.1%, respectively reaching significant levels. Chlorophyll acontent in both plantlets did not reach significance levels although triploid plantlets increased by13.63%;The contents of soluble sugar, soluble protein, POD and SOD activity in both plantlets reached a significantlevels, among which the contents of soluble sugar and protein contents increased by18.67%and8.31%,and the activity of POD and SOD increased by50%and17.3%, respectively in triploid plantlets.(5)Isozyme identification. In SOD isozyme profile, there were3and2bands in the samples of the triploid anddiploid plantlets with both sharing1band; In POD isozyme profile, there were4and2bands in samples ofthe triploid and diploid plantlets with both sharing2bands.4. The technology system of triploid plant rapid propagation was established. The rapid suitablepropagation medium for triploid plantlets was MS+6-BA2.0mg/L+KT1.0mg/L+NAA0.1mg/L,and the propagation coefficient reached4.75by30days; the suitable rooting medium was1/2MS+NAA0.3mg/L, and the rate was90%. The rooting transplanting survival rate reached80%after one-monthacclimatization management.In this study, a technical system on plant regeneration, ploidy identification and triploids rapidpropagation of Dioscorea zinipponica endosperm were established, which was the base of innovation andbreeding of new varieties of germplasm resources. |
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