Biological Characterization And Purification Of Taller And Leafy Mutants Of Tobacco

November 2005, a gigantic mutant of tobacco was found in the tobacco field in the county of Qianjiang, Chongqing province. Compared with the contemporary control plants, it expressed several dominant growth characteristics. It is taller, stronger and has more leaves.In this experiment, tissue culture were used to clone the mutant and the control plants which then rapidly propagated and planted in the tobacco field later. The morphological comparison, cytological observation, physical-biochemical and molecular biology characterization were also applied to mutant of cloned mutant plants. While it was flowering, Doubled Haploid population shall be established though anther culture and chromosome-doubling with colchicines.The morphological comparison showed that the cloned mutant plants were quite different from the control. For example, the mutant plants grew vigorously, and were taller with abundant deep green leaves. At early June, the control plants appeared flowers (being planted in the field for 70 days or so), however, until the middle of October, the mutant plants appeared flowers, and at that time, the average height of mutant plants was normally 179 cm with more than 86 leaves.The cytological observation showed that the mutant plants were diploid (2n=48), almost the same as the controls. But the chloroplasts number of the guard cells of stoma was different between the mutant and the control. The mutant plant had 22.2 chloroplasts on average, while the control had 19.2 chloroplasts.The content of chlorophyll a, b and total chlorophyll in the mutant plants were higher than in the control. The content of the soluble protein in the mutant was 1.48 times as much as in the control. Peroxidase isozyme electrophoresis analysis indicated that the mutant plant has 1 more band in than the control in expression of enzyme. The soluble protein electrophoresis showed that 4 new bands appeared in the mutant while 3 bands disappeared in the control. All of these showed that there were differences in the physiology and biology chemistry between the mutant and the control.The mutant and the control's DNA were investigated by RAPD and SRAP analysis. The RAPD research showed that 7 out of 20 arbitrary primers produced distinctive bands, and in the 7 primers, 4 primers were polymorphic; The SRAP research showed that 82 out of 88 arbitrary primers produced distinctive bands, and in the 82 primers, 24 primers were polymorphic; The result revealed that the mutant might be changed in the molecular level of DNA.The flowering time of the mutant plants was more than 3 months later than the control. Because of its late-flowering, the mutant plants prolonged the vegetative period, which resulted in the fact that the mutant plant was taller, stronger and had more leaves. The above research initially suggested that the mutant plant might be a genetic mutant of a gigantic late-flowering type.Compared to the other mediums, the most appropriate medium for anther culture was H + 0.1% AC, which promoted tobacco anther culture emergence and accelerated tobacco seedlings' growing. It showed that activated carbon (AC) is better than IAA for increasing the rate of emergence of tobacco anther culture.The colchicines soaking method and the colchicines medium method were also used in doubling. The result showed: by means of colchicines medium, application 0.4%colchicine for 72 hours, we could get the highest inducing rate at 28.6%; in the way of colchicines soaking, application 0.4%colchicine for 48 hours, we could get the highest inducing rate at 60.0%. So the method of soaking young plantlets in colchicines was chosen to construct DH population.The chloroplasts number of the guard cells of stoma showed that the chloroplasts number of the haploid plants is between 9 and 11, about 10.3 chloroplasts on average; while the number of the diploid plants is between 20 and 22, about 21.1 chloroplasts. This method showed that each of the haploid plants without colchicines working has 24 chromosomes (2n=24); while each of the pure diploid plants through colchicines working has 48 chromosomes (2n=48).