【作者】 曾少华；

【导师】 刘继红；

【作者基本信息】 华中农业大学 ， 果树学， 2005， 硕士

【摘要】 通过染色体加倍创造多倍体是开展倍性杂交获得柑橘无核新种质的一个重要途径,也是柑橘育种主要目标之一。本研究用colchicine(简写col)和oryzaline简写ory)处理柑橘原生质体和/或愈伤组织,主要结果如下: 1、用0.01%、0.05%、0.1%的colchicine和0.005%、0.01%、0.02%的oryzalin处理宁波金柑(Fortunella crassifolia cv. Meiwa)原生质体24h,结果表明:随着浓度的提高,原生质体活力降低,但oryzalin比colchicine的毒性低,前者处理的原生质体活性较高;原生质体处理后活性最高的处理是0.005%ory和0.01%col,分别达到90.51%和88.23%。用0.01%、0.05%、0.1%的col处理伏令夏橙(Citrus sinensis cv. Valencia)原生质体8h后,原生质体活性急剧下降;当处理时间为8h,浓度为0.05%时,活力达到最高85.85%。 2、用0.01%、0.05%、0.1%的colchicine处理佛罗斯特(C. sinensis cv. Frost)脐橙愈伤组织2d、4d、8d,结果表明:在0.1%col-4d和0.1%col-8d处理中分别检测到1个和5个四倍体胚状体,目前各获得1棵四倍体植株。此外,在0.1%col-8d处理中检测到1个嵌合体胚状体。 3、用0.01%、0.05%、0.1%的colchicine分别处理宁波金柑原生质体8h、16h、24h,其中0.05%col-24h处理的四倍体单细胞系诱导率最高(30.77%),其次是0.1%col-8h和0.01%col-24h,诱导率分别为19.23%和17.71%。其中处理0.05%col-24h再生愈伤组织,处理0.1%col-16h再生胚状体,而处理0.1%col-8h获得的愈伤组织成功再生出芽。用0.005%、0.01%、0.02%的oryzalin处理宁波金柑原生质体24h和48h,未检测到四倍体胚状体。0.01%、0.05%、0.1%的colchicine处理伏令夏橙原生质体2h和4h,结果只有处理0.01%col-4h得到四倍体单细胞系,诱导率为22.86%,四倍体愈伤组织再生胚状体,但未能再生成芽。 4、测定了宁波金柑、国庆1号(C. unshiu ’Guoqing No.1’)和伏令夏橙的二倍体和四倍体细胞直径。三个品种的二倍体细胞直径分别为20.40±2.47μm、21.53±2.87μm、21.50±3.27μm;四倍体细胞直径分别为29.50±2.62μm、33.00±3.15μm、29.75±2.88μm。 5、测定了宁波金柑、国庆1号和伏令夏橙二倍体和四倍体细胞周期。三个品种的二倍体细胞分裂高峰分别出现在继代培养后第4d、6d、4d,细胞分裂指数分别为25.5±1.5%、25.3±0.6%、31.8±1.0%;而四倍体细胞分裂高峰分别出现在继代培养后第6d、10d、4d细胞分裂指数分别为22.7±1.0%、21.2±1.2%、28.8±2.7%。 6、用24对AFLP引物对宁波金柑胚状体和国庆1号愈伤组织的二倍体和四倍更多还原

【Abstract】 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.更多还原