| Polyploids derived from chromosome doubling are important materials to create citrus seedless germplasm through interploidy hybridization. In the present paper,colchicine and oryzalin were used to treat protoplasts and/or callus of Citrus and Fortunella. The major results are as follows:1. The influences of two antimitotic agents, colchicine and oryzalin, on protoplast viability were investigated. Fortunella crassifolia cv. Meiwa protoplasts were treated by 0.01%, 0.05 %, 0.1% colchicine and 0.005%, 0.01%, 0.02% oryzalin for 24 h.The results showed that oryzalin was less toxic to the cells than colchicine. Protoplasts viability was gradually decreased with higher concentrations in both colchicine and oryzalin. But the viability of protoplasts treated by oryzalin was higher than that treated by colchicine. The optimum concentrations of oryzalin and colchicine were 0.005 % and 0.01%, respectively,which gave viability up to 90.51% and 88.23%, respectively. Protoplasts of Valencia were treated by 0.01% , 0.05% , 0.1% colchicine for 8 h. The optimum concentration was 0.05%, with viability up to 85.85%. But protoplast viability dramatically dropped when they were treated for more than 8 h.2. Embryogenic callus of Frost navel orange(C. sinensis cv. Frost) were treated with 0.01 %, 0.05%, 0.1 % colchicine for 2d, 4d, 8d, respectively. One and five tetraploid embryoids were regenerated respectively in treatments 0.1%col-4 d and 0.1%col-8 d, one plantlet was obtained in each treatments. In addition,one 2X + 4X chimeric embryoid was revealed in 0.1%col-8 d treatment.3. Meiwa Kumquat protoplasts were treated with 0.01 % , 0.05 % , 0.1 % colchicine for 8h,16h,24h. The highest rate of inducing tetraploid single-cell-derived sibling lines was 30.77% in treatment 0.05%col-24 h, followed by 0.1%col-8 h and 0.01%col-24 h, 19.23% and 17.71%, respectively. Only callus were regenerated in treatment 0.05% col-24 h, and many abnormal embryoids were obtained in treatment 0.1%col-16 h, whereas buds were obtained in treatment 0.1%-8 h. No tetraploid embryoids were regenerated from Meiwa Kumquat protoplasts treated by 0.005%, 0.01%, 0.02% oryzalin for 24 h and 48 h. Valencia protoplasts were treated by 0.01 % , 0.05 % , 0.1 % colchicine for 2 h and 4 h, respectively. Only tetraploid single-cell-derived sibling lines were obtained with inducing rate up to 22.8% in treatment 0.01 %col-4 h.4. Diameters of diploid and tetraploid cells of Meiwa Kumquat, Guoqing No.1 andValencia were tested. The diploid cell diameter of Meiwa Kumquat, Guoqing No.l, Valencia were 20.40±2.47?m > 21.53l2.87/mi > 21.50±3.27^m, respectively. While diameter of tetraploid cell were 29.50±2.62/um > 33.00+3.15/tfn > 29.75±2.88^m, respectively.5. Cell cycle of diploid and tetraploid Meiwa Kumquat, Guoqing No.l, Valencia were tested. Mitotic index of the three diploid peaked on the 4th, 6th, 4th day, respectively,with the mitotic index 25.5±1.5%s 25.3±0.6%> 31.8+1.0%, respectively. Mitotic index of the three tetraploid reached their peaks on the 6th, 10th, 4th day,with the values of 22.7±1.0%> 21.2+1.2%, 28.8±2.7%, respectively.6. Twenty four primer pairs were used to detect the genetic variation between diploid and colchicine-induced autotetraploid embryoid or callus of Meiwa Kumquat and Guoqing No.l, respectively. No difference between Guoqing No.l diploid and colchicine-induced autotetraploid was detected, whereas E06-MC01 primer pair demonstrated that 5 bands were absent in colchicine-induced autotetraploid Meiwa Kumquat compared with diploid. |
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