Studies On Cross Breeding, RAPD Markers Assisted Selection And Tissue Culture Of Pansy (Viola Wittrockiana Gam.)

Pansy (Viola wittrockiana) is an important ornamental plant used in the winter and early spring. However, most of the cultivars of pansy in China are importation with high price, so it is necessary to develop our own breeding system of pansy for more color range, long bloom duration and fitness to hot climate.In this study, the pollination biology of pansy was investigated. Diallel analysis was used to study the heredity of some ornamental traits. And melocluar markers were developed to indentify the genetic diversity of inbred lines and to predict the heterosis of some crosses. Finally, anther culture was carried out to obtain haploid plants, and the regeneration system via petiole callus was established Tor further transgenic research. The results were presented as following:1. The pollination biology of pansy. High concentration of sucrose was needed for the pollen germination of pansy while that exceed 30% could inhibit the growth of pollen tubes. High concentration of NAA could promote the pollen germination. The ratios of germination of pollen in the forenoon were higher than those in the afternoon, and the ratios in the sunny day were also higher than that in the cloudy day. The ratios were different among the different accessions, and there was no significant correlation between the ratio of pollen germination and the ratio of fruits setting. The viability of pollen decreased quickly in ordinary environment. Moreover, the growth of ovaries was inhibited if castrations were performed too early.2. Inheritance of ornamental traits. No significant degradation was observed for 7 main ornamental traits after inbred lines of pansy were self-crossed for several generations. Inheritance of 10 traits was studied by diallel cross analysis (Griffing 2) performed within 5 inbred lines using a MINQUE method (additive-dominance-epistatic model). Dominance effects were main effects for most of the traits, however, additive effects were main effects for plant height and number of flowers, while additive and additive epistatic effects for plant size and thickness of petals. The narrow heritalities of different traits ((h~2)_N) ranged from 0.33 to 0.87 with an averge of 0.53, and the broad heritalities ((h~2)_B) ranged from 0.10 to 0.53 with an averge of 0.33. Both (h~2)_N and (h~2)_B were large for number of flowers. Flower size and number of flowers of hybrid 12×18 showed the higher predicted values of domaince effect, suggesting that this hybrid has high potential for heterosis use. Correlation analysis showed that significant positive correlation existed between flower size and number of flowers. No significant correlation were observed between flower size andvegetative traits, such as plant height, plan size, number of branches, leaf area. Significant correlations existed between number of flowers and plant size, plant height, while no significant correlations were observed between number of flowers and number of branches or leaf area. No significant correlations existed within the other traits.In the study on the inheritance of flower color, phenotypic effects, paper chromatography and UV-Vis absorption spectrum were used. The pigments of hybrid 11X 12> 11X15 were similar to the parental inbred line 11, suggesting that violet color showed dominance effect to blue color or red color. Both of flavonoides and carotenoides of hybrid 4X9 were similar to the parental lines 4, suggesting that yellow color showed a partial dominance effect to white color in pansy. Flavonol was the main pigment of inbred lines 4 and hybrid 4X9. Flavanones or flavanonols and flavones might compose the main pigments of inbred line 9.3. Five methods for DNA extracting of pansy were compared and the optimal method was adopted. The factors affecting PCR reaction, such as Taq DNA polymerase, primer, dNTPs and DNA concentration etc, were optimized. RAPD was used to analyse the genetic diversity between 18 inbred lines of pansy, and 5 inbred lines were selected and used in a diallel analysis to study the correlations between the genetic distances based on RAPD and the heterosis of 9 traits of pansy. Twenty-one random primers were selected and 127 polymorphic bands out of 167 ones were obtained, UPGMA cluster analysis was performed, and 5 main groups could be observed, which were consistent with the flower size types and the original places of the inbred lines. No significant correlations were observed between the genetic distance based on RAPD and heterosis in all traits except for flower numbers at 0.1 level. The results indicated that it was presently unsuitable to predict heterosis of these traits expect for flower number using RAPD genetic distance in pansy.4. The tissue culture of pansy. In the study of anther culture Nitsch or 1/4MS was found to be the best basal medium. 0.3 mg/1 2,4-D and 4mg/l NAA were effective to induce higher anther response. High concentration of sucrose inhibit callus induction. The best sampling period was when the flower was about to blossom. The duration of chilling pretreatment at 4°C should be 2-3 days. There was significant difference within genotypes, and the anther response of hybrids was not better than that of inbreds lines. In the study of plant regeneration of pansy, we found that subculture on appropriate medium and the application of TDZ were important for successfulregeneration, and a optimized protocol was established as following: callus induction on a half-strength MS medium supplemented with O.lmgA 2,4-D plus 2.0 mg/1 BA, shoot subculture on medium F (1/2MS with 1.0mg/l 2,4-D, 0.5mg/l NAA and O.lmg/1 BA) and then on medium T4 (1/2MS with l.Omg/1 2,4-D, 0.5mg/l NAA and 0.5mg/l BA), shoots regeneration on medium D6 (MS media supplemented with lmg-1'1 GA3, 4 rng-1"1 AgNO3, 0.06% active carbon andl.O mg-1"1 TDZ), shoots multiplication on medium M (half-strength MS medium containing NAA 0.2 mg/1, 2,4-D 2.0mg/l and GA3 3mg/l), and then shoots elongation and rooting on medium R(MS medium supplemented with 0.2mg/l NAA and 0.25 mg/1 BA).