Studies On Physiological Changes And Genetic Variation During In Vitro Morphogenesis In Narcissus Tazetta Var. Chinensis

The slivers of bulbs and slices of ovaries were used as explants to carry out studies on the highly-efficient culture in vitro system, physiological changes during callus differentiation especially, as well as genetic variation during in vitro morphogenesis in Narcissus tazetta var. chinensis. The main results were described as follows:1. Establishment of the highly-efficient culture in vitro system of Narcissus tazetta var. chinensisThe slivers of bulbs were used as explants to carry out studies on optimizing conditions of in vitro culture in Narcissus tazetta var. chinensis. The results showed that the bulbs of Narcissus tazetta var. chinensis were soaked in 3% carbendazim for 6 hours, which was the key measure to establish the aseptic system. As the best natural additive, 5% banana slurry was favorable for inducing bulblets directly. 200 mg·L^-1 was the optimal concentration of betaine for bulblet induction. Both the MS medium supplemented with 2.0 mg·L^-1 2,4-D and 1.0 mg·L^-16-BA and the MS medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 KT were suitable for callus induction. Both the MS medium supplemented with 3.0 mg·L^-16-BA, 2.0 mg·L^-1 KT and 0.1 mg·L^-1 NAA and the MS medium supplemented with 3.0 mg·L^-1 6-BA and 5.0 mg·L^-1 KT were facilitated to callus differentiation.The studies on the establishment of the efficient regeneration system of Narcissus tazetta var. chinensis from slices of ovaries were carried out. The results showed that in the initial culture, slices of ovaries that had shorter development time could induce bulblets directly on the MS medium supplemented with 15.0 mg·L^-1 6-BA and 1.0 mg·L^-1 NAA. Three different types of calli were induced (TypeⅠcallus was white and its surface had strumae; TypeⅡcallus was light yellow and its surface had no obvious granular; TypeⅢcallus was also light yellow and its surface had obvious granular.). The induction rates of the three different types of calli reached highest respectively on the MS medium supplemented with 0.5 mg·L^-1 2,4-D and 1.0 mg·L^-1 KT, the MS medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA and the MS medium supplemented with 2.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Both TypeⅠcallus and TypeⅡcallus cultured on the MS medium supplemented with 5.0 mg·L^-16-BA, 5.0 mg·L^-1 KT and 0.1 mg·L^-1 NAA had better differentiation effects. The MS medium supplemented with 3.0 mg·L^-1 6-BA and 5.0 mg·L^-1 KT was facilitated to differentiation of TypeⅡcallus and TypeⅢcallus.In this study, the method of subculture multiplication of bulblets in vitro following with bulblet expansion culture was used firstly, which broke the routine cultural method and specially could provide strong bulbs in order to have better results after subculture multiplication of bulblets in vitro. The single-factor test results showed that when the concentration of PP333 was 3.0 mg·L^-1 , bulblet expansion culture had the best effects; the multi-factor test results showed that the effect of different factors on bulblet expansion culture was as the order——PP333> 2,4-D> NAA>2,4-D× NAA. The best medium which was selected for bulblet expansion culture was MS medium with 1.0 mg·L^-12,4-D and 2.0 mg·L^-1 NAA and 3.0 mg·L^-1 PP333. The double-factor test results showed that the medium with 3.0 mg·L^-1 6-BA and 30 g·L^-1 white granulated sugar was in favor of subculture multiplication of bulblet in vitro; the 1/2 MS medium supplemented with 0.5 g·L^-1 Ac was most suitable for shoot growth and rooting of bulblets in vitro; the survival rate of bulblets in vitro was up to 91.67% after being transferred into the mixture of rural soil and peat soil (2:1) , which was without any treatment but most facilitated to transplantation.2. The histiocytic observation of callus induced by slices of ovaries of Narcissus tazetta var. chinensis by paraffin sectionThe results indicated that the microstructural characteristics of the cells of callus induced by slices of ovaries that had longer development time were loose, large and irregular. In contrast, the cells of the three types of calli induced by slices of ovaries that had shorter development time were closer and more regular: The tightness of cells was as the order——TypeⅢ> TypeⅡ> Type; the size of cells was as the order——TypeⅠ> TypeⅡ> TypeⅢ; the homogeneous degree of cells was as the order——TypeⅢ> TypeⅠ> TypeⅡ.3. Some physiological changes during differentiation of callus from Narcissus tazetta var. chinensisBy means of studies on the changes of POD, SOD and CAT activities and the contents of soluble proteins, glucose, fructose, sucrose and starch during differentiation of callus from Narcissus tazetta var. chinensis, the results showed that when types and sources of callus were different, the changes of every physiological index during differentiation existed some differences. As the differentiation time past, the starch contents of the TypeⅠcallus induced from slices of ovaries that had shorter development time trend toward firstly decrease then increase, on the contrary, the rest of the physiological indexes trend toward firstly increase then decrease. During the TypeⅡcallus differentiation, both SOD activity and starch contents presented the changing curve of"W"shape, whereas, the soluble protein contents decreased at first and increased subsequently then decreased, and all the other physiological indexes changed as a single peak curve. During the TypeⅢcallus differentiation, the POD activity changed as a single peak curve, whereas, the CAT activity and starch contents both showed a double-peak curve, both glucose and fructose contents trend toward firstly decrease then increase, and the rest of the physiological indexes presented the changing curve of"W"shape. During differentiation of callus induced from slices of ovaries that had longer development time, the POD activity was totally descending trend, but the SOD activity showed a double-peak curve, the glucose contents presented the changing curve of"W"shape, the contents of soluble proteins and starch both decreased at first and increased subsequently then decreased, whereas, the rest of physiological indexes changed as a single peak curve. During differentiation of callus induced from slivers of bulbs, the contents of fructose and starch both decreased at first and increased subsequently then decreased, whereas, both CAT activity and glucose contents presented the changing curve of"V"shape, and the rest of physiological indexes changed as a single peak curve.4. The detection of genetic stability during in vitro morphogenesis of Narcissus tazetta var. chinensisThe root tip cells of bulblets, which were induced from slivers of bulbs and slices of ovaries by different in vitro morphogenesis pathways and had been completed shoot growth and rooting culture, were used to investigate the chromosome number. The results showed that there were some variations at a certian degree. The non-triploids from direct organogenesis and indirect organogenesis ranged from 3.92% to 4.95% and from 10.38% to 18.10% respectively. Using ISSR markers, with regard to donor plants, the genetic integrity of cultures that derived from slivers of bulbs and slices of ovaries by way of direct organogenesis at different culture stages was validated. In the way of indirect organogenesis, there were some abnormal bands in calli; the cultures at the rest of culture stages had rare abnormal bands; at the final stage, the variant rates of cultures only ranged from 0.00 to 1.69%. In a word, the genetic stability of indirect organogenesis was lower than that of direct organogenesis during in vitro morphogenesis of Narcissus tazetta var. chinensis. At the same time, this study showed that DNA pattern of ISSR was not entirely consistent between vitrified bulblets and normal bulblets which both derived from callus of slivers of bulbs. On the DNA level, the degree of genetic stability of the three types of calli induced from slices of ovaries was followed by TypeⅡ> TypeⅢ> TypeⅠ; the DNA pattern was not entirely consistent among vitrified bulblets, bulblets with buds and normal bulblets which all derived from TypeⅠcallus; both vitrified bulblets and bulblets with buds had some variations in the DNA pattern as compared with their donor plants and the normal bulblets were diploid mostly.