Studies On Tetraploid Induction Of Cassava In Vitro And Evaluation Of The Mutagenic Material

Cassava (Manihot esculenta Crantz) is belong to Euphorbiaceae family. Cassava is with high tolerance to drought and barren, and it is widely planted in Americas, Asia and Africa such as more than one hundred countries. Cassava of wide uses not merely only can providing food but also can be processed into the a variety of industrial products like starch and ethanol. The yield of cassava is unable to fill the international needed although the area of planting cassava in China is about 7 millions mu and 10 million tons of fresh cassava producted. The demand for cassava in our country is rising with the devolpment of industry of ethanol, starch and feed. But lacking of the high yield and qulity cassava variety, less planting area of elite cultivar, and low comparative economic benefit is the main problem of restricting devolpment of the cassava industry. So, elite cultivar breeding is important for devolpment of the cassava industry.Polyploid breeding is the popular method of plant breeding. In our study, to exolore the potencial of the cassava tetraploid whether it could become an elite cultivar, the colchicine was used forinducing the cassava tetraploid. After inducing, Ploidy level of cassava plant was analyzed by flow cytometry and chromosome counting in root tips. And then, the tetraploid and the diploid were compared of morphology, anatomy, physiological and biochemical characteristics, genomic differences and yield. The main results were as following:1. The most of seedlings is dead when the colchicine concentration is 0.1 g/L and when the colchicine concentration is above 0.25 g/L, all seedlings completely died. For three cassava variety, survival rate of seedlings in SC 205 is the lowest; survival rate of seedlings in NZ199 is the highest. In this experiment,0.05 g/L is the appropriate concentration of inducing cassava tetraploids.2. After demonstrated by flow cytometry and chromosome counting, 20 cassava homozygous tetraploid variants and 18 mixoploidy variants were obtained; fluorescence intensity of the tetraploid is two times higher than the diploid’s.3. The cassava tetraploid and the diploid were compared in morphology. The results showed that leaves of the tetraploid were wider and thicker, with deeper colour and lower leaf index; there is no difference in number of middle lobe, the stem diameters, the inner and outside skin color of the stem and the tuber, the color of the tuber.4. Through dissecting the leaves of the diploid and the tetraploid, it turned out that the tetraploid have smaller stomatal density, increase of the number of chloroplasts, the wider guard cell, thicker palisade tissue, spongy tissue, upper epidermis, lower epidermis, which were significantly different from that of the diploid.5. The results of compassion of the physiological and biochemical characteristics showed that the Chlorophyll content and water content of the tetraploid is higher than the diploid; starch content of the tetraploid is lower than the diploid. The results of photosynthesis measurement showed that the tetraploid’s net photosynthetic rate, stomatal conductance is higher than the diploid’s; the difference in intercellular CO2 concentration between the tetraploid and the diploid is not significant.6. An optimal SCoT-PCR reaction system for cassava was established, that is, the total volume of the reaciton is 20 uL with DNA template 60 ng; primers concentrations 0.3 umol/L; Taq DNA polymerase 1.5 U; Mg2+ 1.5 mmol/L; dNTPs 0.25 mmol/L. Nineteen primers were screened from 36 SCoT primers, based on the optimal SCoT-PCR reaction system.7. The genomic difference between the tetraploid and the diploid was explored by SSR molecular markers technology and SCoT molecular markers technology. The results showed that no polymorphic bands obtained, that is the genomic difference has not found.8. The yield of the tetraploid and the diploid was compared. The harvest index and number of the tuber of the tetraploid is equivalent to the diploid; the tuber weight of the tetraploid decreased by 20% compared to the diploid.