Studies On Tissue Culture, Leaf Cutting And Semi-lethal Temperature Of Three China Native Wild Flowers Of Chirita Species

Gesneriaceae has high ornamental and medicinal value. Its speculiar plant morphology, gorgeous flower color and unique shade tolerance appeal to many flower fans. In recent years, the habitats of large number of wild Gesnerianceae were threatened by human disturbance and natural habitat degradation, leading to sharp decrease of their population number and size. It’s therefore urgent to build up proprogation and breeding systems to conserve these valuable rescources. In this study, we selected three Chirita species with high ornamental value, e.g. Chirita laxiflora, C. baishouensis and C. wangiana, as experiental materials to establish their rapid proprogation systems based on tissue culture and leaf cutting. We also investigate the genetic stability of tissue cultured seedling based on chromosome counting and genome size. Moreover, semi- lethal temperature of tissue cultured seedlings were also examinated to aid our understanding of the temperature limitation of the three species.The main results were as follows:1. Leaf explants of three Chirita species were suitable to initial culture, while the most optimal medium of inducing were all MS + 0.5 mg/L 6-BA + 0.1 mg/L NAA + 30 g/L sucrose+6.4 g/L agar. The effect of subculture MS medium respectively consisted of 0.5 mg/L 6-BA, 0.3 mg/L 6-BA + 0.05 mg/L NAA and 0.1 mg/L 6-BA + 0.01 mg/L NAA were best for C. laxiflora、C. baishouensis and C. wangiana. 1/2 MS +0.3 mg/L IBA+15 g/L sucrose+ 6.4 g/L agar was the best rooting culture medium of C. laxiflora. while the best medium for the rootage of plantlets of C. baishouensis and C. wangiana was 1/2 MS +0.1 mg/L IBA+15 g/L sucrose+ 6.4 g/L agar. The histological observation indicated that adventitious shoot regeneration on the medium containing 6-BA and NAA with lower hormone concentrations used in this experiment, the propagation and regenation system via direct shoot organogenesis.2. The chromosome number of all three Chirita species were 2n = 36. Flow cytometry analysis of ploid determination along with the parent showed that they all had one identical peak, indicating that the variant lines, like the parent, were diploid. The genome size(2C-Value) of C. laxiflora, C. baishouensis and C. wangiana detected by external standard method were 1.26±0.0096 pg,1.64±0.0328 pg and 1.69±0.0119 pg. While by internal standard method, the genome size(2C-Value) of C. laxiflora, C. baishouensis and C. wangiana were 1.26±0.0133 pg,1.82±0.0705 pg and 1.71±0.0343 pg.3. The result of the leaf cutting types showed the survival rates of all leaf, two and three hypomere segment cutting types were hight. the suivival rates of two and three upper section of leaf cutting types were low, but upper section leaf cutting types can produce more adventitious buds than those all leaf, two and three hypomere segment.4. The results of semi- lethal temperature showed that the high semi- lethal temperature of C. laxiflora, C. baishouensis and C. wangiana were 42.27℃, 44.5℃ and 42.6℃, While the low semi- lethal temperature of three Chirita species were 3.9℃,-2.13℃ and-1.99℃, respectively.In conclusion, the three Chirita species were all sensitive to 6-BA/NAA, generating roots easily and suitable to tissue culture and leaf cutting. The histological observation showed the adventitious shoot formed in direct organogenesis way. Flow cytometry analysis showed that tissue culture did not make it variation. Semi- lethal temperature can provide reference for cultivation mana gement, however, the defense mechanism of the three Chirita species under semi- lethal temperature was not clear and needs further research.