Study On Tetraploid Mutagensis Of "Legacy" Vaccinium L.

Vaccinium, also called blueberry, is a genus of perennial deciduous or evergreen shrubs belonging to the Ericaceae originating from North America. Its fruits are blue or red. Because of its riched in multiple vitamins, microelements and pharmacologically active compounds, it is of great value in nutritional and medicinal. It has a good taste and very popular among consumers. Because of different species or genus, the plant height are different. It has alternate simple leaves. The distribution of root system is shallow in soil. As a sun-loving plant, it grows in acid soil with pH 4.3-5.5,such as moist coniferous forest in mountain, broadleaf mixed coniferous forest, shrub or wet land.Currently the varieties of Vaccinium and the cultivated area in northern China are more than in the South. The varieties of Vaccinium adapted the climate in South China are small berries, but growing prosperous. The’legacy’ with the genes of V. austral and V. corymbosum, is a big berry, suitable for fresh food and convenient transportation. It introduced into Chongqing for five years. The plant displays good fruit quality and big berry, but middling disease resistance and growth vigor.In the study, plant regeneration system of Vaccinium L.’Legacy’ was established by the stem segments and studied the effect of cold pretreatment on the contamination and survival rate in vitro rapid propagation and screened the concentration of ZT in initial medium. Colchicine was used in the experiment to induce Vaccinium L.’Legacy’ with the immersion method, to determine the best concentration and time. Through the morphological observation and chromosome counting, we can get variant plants from ploidy identification. The variant plants were chimaeras that in order to get tetraploid with separating. Comparative tests between chimaeras and diploid plants involving plant morphology, cytology, physiological indicators provided basic data for breeding new varieties of strong growth and stress resistance.The main research results are as follows:1. Cold pretreatment can effectively reduce contaminate rate and increase survival rate. Explants disinfection best combination is cold pretreatment(4℃) for 24h,75% alcohol disinfection for 20s and 0.15%HgCl2 for 8min. Its contamination rate is 26.7% and survival rate 66.7%.2. Add the low concentration ZT in initial medium can contribute to axillary bud, increasing the number of branches. But add the high concentration ZT can restrain to axillary bud, delay branches growth or die. The best initial medium in this study is modified WPM medium with 0.75mg/L ZT.3. The soaking shoot tip method can induce chimaeras in vitro. It showed that:the best mutagenesis condition is to soak shoot tip for 24h with 0.10% colchicines in the immersion method. The morphology variation rate 16% and the variation rate is 10%. The chromosome number count results show that the chromosome number of diploid plant is 2n=2x=24, the tetraploid cells 2n=4x=48.The variant plants are chimaeras whose tetraploid cells rate is 49.06%.4. With 0.05% colchicines to soak shoot tip for 24h, the multiplication rate of subculture has a very significant difference between the control group and treated groups. According to the cytological study, the chromosome number of the plants is not change. When these plants subcultured four generation, the multiplication rates were 6.38,6.66,6.68,6.78. These plants grow strong. This phenomenon is not receded by increasing generation. When transplanting these plants, they display growth strong, enhancing ability to branch, changing leaves growth. Plant height and the number of branch increase respectively 10.93%,150% and have a very significant difference between the control group and treated groups.5. Basing on the morphological features and anatomic study, the variant plants showed typical huge organ, deeper leaf color, more hair of top stem and leaf epidermal. Variants of the stem diameter, leaf length, thickness were controlled strain of 141.50%, 170.00%,105.72%,124.11% respectively. The variant has larger guard cells and less stomata, as well as palisade and sponge tissue. Those were controlled strain of 152.2%, 149.5%,219.7%,143.4% respectively. Those changes all have a very significant difference between the control group and treated groups.The changes of morphological features and anatomic could be used to identify diploid and polyploid in early.6. Comparative tests containing physiological indicators between variant plants and diploid showed that soluble sugar and chlorophyll were higher than diploid, increased respectively 11.42%,14.29%, but the malondialdehyde and electrolyte permeability were lower than diploid, reduced respectively 26.03%,12.41%. These have a very significant difference between the control group and treated groups. So we the inferred variant plants have higher stress resistance than control group.