利用生物工程技术创造甜菜耐盐新种质
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摘要
甜菜(Beta vulgaris L.)是重要的糖料和饲料作物,糖用甜菜提供了全球40%以上的食糖,饲料甜菜是家畜最佳的多汁饲料,块根肥大,营养丰富。甜菜具有耐盐潜质,能在盐分低于0.4%的土壤中正常生长。甜菜的一些近缘野生种起源于滨海,耐盐性较强。饲用甜菜虽生物量大,耐盐性却不高。因此,高耐盐性饲用甜菜的开发和利用,不仅可充分开发利用滨海盐碱地,推动养殖业发展,而且能有效改善生态环境。
采用常规育种技术培育甜菜耐盐品种周期长,见效慢,至今还没有培育出适合滨海盐碱地种植的品种。采用生物工程技术可以获得耐盐性大幅度提高的工程植株,并能缩短育种年限,这为甜菜耐盐育种开辟了新途径。通过耐盐细胞突变体筛选等技术,可创造出甜菜耐盐新种质。采用转基因技术,可将从细菌和盐生植物中克隆的耐盐基因导入甜菜,获得高度耐盐的优良甜菜新种质。本论文的目标就是综合利用细胞工程和基因工程技术,培育适合在我国中低度滨海盐碱地中种植的耐盐饲用甜菜新品系。
以14个甜菜品种的幼胚、叶柄和沙培小苗下胚轴切段为材料,分别诱导愈伤组织并继代培养。研究了影响愈伤组织诱导频率的因素,对愈伤组织继代培养及分化的条件也进行了探索。发现同一基因型甜菜的不同外植体的愈伤组织诱导率存在差异。幼胚较易产生愈伤组织,下胚轴、叶柄、叶片则较难。不同基因型的外植体,愈伤组织诱导率有明显差异。外植体的发育时期和生理状态对愈伤组织的诱导有明显影响。经过多次继代培养,从4个基因型的幼胚得到了颜色为乳白或淡黄、结构疏松、颗粒状的胚性愈伤组织。胚性愈伤组织在MS+5%蔗糖附加0.05mg/L ABA或附加0.05mg/L ZT+0.05mg/L ABA的分化培养基上通过胚状体发生途径再生植株。不同基因型材料再生植株的最适培养基组合有差异。从下胚轴和叶柄诱导的愈伤组织由于诱导率低且继代培养难度大,只得到了少量胚性愈伤组织,未能获得再生植株。
部分胚性愈伤组织经5～20Gy剂量的~(60)Co-Y射线辐照后,在附加不同浓度盐分(1.0%～2.5%NaCl)的培养基上连续筛选三代,获得稳定耐1.5%NaCl的耐盐
Salinity stress is one of the most serious worldwide factors retarding the growth of plants and crops productivity. In China, saline soils where the salt concentration is between 0.6-1.2 % are about 1.5 million hectares, furthermore, fresh water is lacked in these areas. Thus, the most valuable and economic strategy to utilize these bare soils is to screen and select salt-tolerant plants for cultivation. Sugar beet is a more able crop to tolerate salinity, and some wild genotypes origin from the seacoast. Traditional breeding is time-consuming and laborious, and has not resulted in salt-tolerant cultivars. Development of cell engineering techniques to screen salt-tolerant mutants and genetic manipulation to create salt-tolerant materials could decrease breeding time and create salt-tolerant cultivars of sugar beet. In this paper, the goal is to breed new strains of fodder beet that could grow luxuriantly in the saline soil containing low and middle concentrations of salts near the seashore in China via the comprehensive utilization ofboth cellular and genetic engineering techniques.Calli of 14 cultivars of sugar beet were induced and subcultured from immature embryos, petioles or hypocotyls derived from seedlings in pots of sand respectively. The factors influencing the induction frequency, subculture, and differentiation of calli were studied. The induction frequencies of calli differed among the explants of immature embryos, hypocotyls, petioles or leaves in same genotype, and immature embryos gave the highest percentage of induction. There were significant differences between the induction frequencies of the explants from different genotypes of beet. The development stage and physiologic state of explants also obviously influenced the induction frequency of calli. After several subcultures, opal or pale-yellow granular calli with loose constitution, inducing from immature embryos of four genotypes, were selected. Embryonic calli produced plantlets via embyogenesis pathway on MS media either supplemented with 5% sucrose and 0.05 mg/L ABA or with 5%sucrose, 0.05 mg/L ABA
and 0.05 mg/L ZT. The optimal compositions of medium for regeneration of plantlets varied from the genotypes. Only a little embryonic calli were selected from hypocotyls or petioles and no plantlets were regenerated.A part of embryonic calli were irradiated by 5-20 Gy of ~(60)Co- Y ray, then cultured on media supplemented with varied NaCl concentrations (1.0%-2.5%) continuously for 3 generations. Then, cell lines, which could actively proliferated on media containing 1.5% NaCl were obtained, and were induced to regenerate plantlets via embryogenesis pathway on the differentiation medium. Self-pollinated progenies (Ri) of regenerated plants were sown in pots of sand and irrigated with 2.0%-3.0% NaCl solution every day. There were 18 lines grown in 2.0% NaCl solution for long time, 6 lines germinated in solution of 2.5% NaCl, but the seedlings were dying soon after the germination from the damages of NaCl, whereas there were no survival seedlings from control seeds when irrigated with 2.0% NaCl solution. This provided a successful example for breeding salt-tolerant strains from calli for sugar beet by using radiation breeding and directional selection techniques.An efficient method has been developed in in vitro multiplication of sugar beet from the excised immature inflorescence tips of 20 genotypes on MS medium supplemented with 1 mg/L 6-BA. Multiple bud clumps were induced from segments of inflorescence tips after inoculation for 3 weeks, and the induction frequency was above 90%. The initial time and frequency of bud development varied from genotypes, generally bud clumps formed within 10-20 days after culture initiation, and large amount of bud clumps were produced and proliferated rapidly with further subculture. They were then cut into small bud clumps to subculture at 15-day intervals onto the unaltered medium and each bud could generally produce 5-8 new buds in each subculture. The proliferation rate usually reached 1:20-30 or more on the induction and proliferation media supplementedwith 1 mg/L 6-BA every month.Once, single buds had reached about 6-10 mm in length, they were cut down frommultiple bud clumps to inducing root on medium supplemented with 1 mg/L NAA. The rooting frequency was related with the age, height, genotype and physiologic statue of the buds. Generally, larger buds in rapid growth easily produced root, for instance, 50-80% white and strong roots formed within 2 weeks. The rooted cuttings were transplanted in pots and 1 month later the survivals were transplanted in the field. The
survival rates of transplanted plantlets from multiple bud clumps varied from genotypes, usually above 75% and a few of them up to 98%.Little buds were cut from multiple bud clumps and irradiated with 60Co- Y ray in dosage of 10-20 Gy, then were transferred to the medium containing 1.0%-2.5% NaCl for the selection of salt-tolerant buds. Buds tolerating 2.0% NaCl were selected to regenerate plantlets. After vernalization and self-pollination, the seeds (Ri) of the regenerated plants were sown in pots of sand and irrigated with solution of 2.0%-3.0% NaCl every day. Some of the seeds germinated and grew normally in the 2.0% NaCl solution, exhibiting higher salt-tolerance compared to the controls. When the seedlings after the saline selection were transplanted to soil, the plants grew normally and produced plump root tuber similar to controls. The seeds from two selected lines could germinate and grow for a few weeks in 2.5% NaCl solution before withered. In 0.9% NaCl saline soil, the tuber yields of the plants regenerated from three salt-tolerant lines were about 45-50 tons/hectare, approximately 2.6-2.9 times of the controls. It is concluded that salt-tolerant materials with good agronomic traits in sugar beet were obtained by using radiation method and directional selection techniques to multiple bud clumps. The study provided a successful approach to produce useful mutants for those genotypes difficult toregenerate plantlets from single cell or callus in vitro.1-3 mm small buds of sugar beet derived from the multiple bud clumps subcultured for 7 days were infected with agrobacterium LBA4404 harboring plasmid pCAMBIA1300-&e*/f-/*pr or pCAMBlAUOO-AtNHXl-hpt, or agrobacterium EHA105 harboring plasmid pROK2-asPLD r-als for 10-20 min. After co-culture for 2-4 days on induction medium, the buds were transferred to medium supplemented with 100 mg/L cefotaxime to inhibit the growth of agrobacterium for 10 days. In the period of co-culture and inhibiting agrobacterium, the buds developed out 2 or 3 leaves and produced multiple bud clumps. After three further 10-15 days subcultures on medium containing hygromycin B at the concentration of 10 mg/L or lvchuanglong (sulfonylurea herbicide) at the concentration of 0.01-0.05 mg/L, survival frequency of infected buds was about 7%- 23%, varied from genotypes and parameters for infection. Then, survival buds were transferred to medium without hygromycin B or lvchuanglong and proliferated to form multiple bud clumps. Larger buds from multiple bud clumps were rooted on the rooting medium, and
were transplanted into pots. Based on these results, we obtained the higher transformation efficiency when the bud meristems were infected for 5-10 minutes in agrobacterium suspension of OD6oo 0.3-0.5 and co-cultured for 2-4 days.DNA was extracted from leaves of transformed buds or plants for PCR. Primers were designed according to the sequences of hpt, als or PLD gene. DNA of the plants showing PCR positive were extracted and digested by EcoR I and HindlR for Southern blotting. Over 80% plants gave positive signal at Southern blotting, indicating that the betA, AtNHXlor anti-sense PLD rgene had been integrated into the genome of some transformed plants respectively.After proliferation, the salt-tolerance of transformed buds showing PCR positive were detected by culturing for 3 continuous generations at 15-day interval on media containing different concentrations of NaCl (1.0%-3.0%) respectively. On the media supplemented with NaCl, the survival rate of buds decreased following the increase of salt concentration. The transgenic buds of different genotypes showed significantly various improved salt-tolerance compared to the non-transgenic controls, even if the transgenic buds of same genotype existed the difference of salt-tolerance. Only part of transgenic buds displayed higher salt-tolerance and the means of the improvement of salt-tolerance was 1.0% NaCl.5-leaf stage transgenic plants were potted in sand and irrigated with solution containing different concentrations of NaCl (1.0%-3.0%). They could tolerate additional 1.0%-1.5% NaCl compared to the non-transgenic controls and only show minor symptoms of salinity damage. It was obvious that the differences of salt-tolerance existed among transgenic plants from same genotype and also existed among the genotypes. Under the same concentration of NaCl, transgenic plants were generally showed symptoms of damage from salinity 3-5 days later compared to controls. Watered with 3.0% concentration of NaCl for 30 days, about half of transgenic plants survived but only 4% controls were alive. There were not significant differences of salt-tolerance between transgenic buds and transgenic plants, in another word, the plants regenerated from themore salt-tolerant buds showed similar higher salt-tolerance.Self-pollinated seeds (Ti) of transgenic plants suffered the selection of salt solution inpots of sand, and seedlings that could tolerate 2.0%, 2.5% or 3.0% NaCl solution were selected and self-pollinated in the next spring.
Analysis of the salt-tolerance and inherit stability of betA in the transgenic progeny indicated that the salt-tolerance improvement of the progeny was correlated with the expression of betA gene, and in some of the transgenic lines hpt and betA gene segregated as dominant Mendelian traits (3:1 or 15:1). In 58 plant lines assayed, no more than 20% ones showed segregation ratio of 3:1 or 15:1. This result indicated that non-mosaic transgenic plants could be obtained from the buds infected by agrobacterium, but the frequency was low.From the results of the selection with 2% NaCl solution and yields in the saline field, 276 plants with betA were chosen from 24 transgenic plant lines (Ti), and the self-pollinated seeds (T2) of 243 plants were harvested. In the saline field of Dongying district in Shandong province where the salt concentration was 0.6%, 1.0% or 1.5% respectively, some of the T2 transgenic progenies exhibited significantly improved salt-tolerance and much better yields than the controls. In the field containing 1.0% salt concentration, yields of root tubers were 582-978 Kg/100 m2, as much as 225.8%-338.4% yields of non-transgenic controls. The plant from 109 plant lines with better salt tolerance and agronomic traits were chosen for producing T3 seeds. T3 plants were also selected according to their salt-tolerance and agronomic traits exhibition in saline field. These results demonstrated that we had bred salt-tolerant sugar beet strains by genetic transformation with betA.218 Ti seedlings with AtNHXl gene that tolerated 2.0%-2.5% NaCl were selected. In some transgenic lines AtNHXl gene inherited as Mendilian segregation. Some of progeny (T2) of Ti transgenic lines were sown in the saline field of seashore where the salt concentration was 0.6%, 1.0% or 1.5% respectively. The plants with good salt-tolerance and agronomic traits were chosen from the T2 transgenic plants for the production of seeds (T3). T3 plants from a number of plant lines germinated evenly and grew normally in the field of 1% salt components, and produced the yield of 568-1020 Kg root tubers /100 m2, that were about 168%-368% comparing to controls. These results revealed that it. was feasible to improve the salt-tolerance of beets by the introduction of AtNHXl gene into cultured buds and to obtain the salt-tolerant beet cultivars by genetic transformation with AtNHXl.Among the salt-tolerant materials with good agronomic traits produced by cellular engineering techniques, plants of the most salt-tolerant line could tolerate the stress of 2.0% NaCl solution, and produce high yields in the saline field of 0.9% salt
采用常规育种技术培育甜菜耐盐品种周期长,见效慢,至今还没有培育出适合滨海盐碱地种植的品种。采用生物工程技术可以获得耐盐性大幅度提高的工程植株,并能缩短育种年限,这为甜菜耐盐育种开辟了新途径。通过耐盐细胞突变体筛选等技术,可创造出甜菜耐盐新种质。采用转基因技术,可将从细菌和盐生植物中克隆的耐盐基因导入甜菜,获得高度耐盐的优良甜菜新种质。本论文的目标就是综合利用细胞工程和基因工程技术,培育适合在我国中低度滨海盐碱地中种植的耐盐饲用甜菜新品系。
以14个甜菜品种的幼胚、叶柄和沙培小苗下胚轴切段为材料,分别诱导愈伤组织并继代培养。研究了影响愈伤组织诱导频率的因素,对愈伤组织继代培养及分化的条件也进行了探索。发现同一基因型甜菜的不同外植体的愈伤组织诱导率存在差异。幼胚较易产生愈伤组织,下胚轴、叶柄、叶片则较难。不同基因型的外植体,愈伤组织诱导率有明显差异。外植体的发育时期和生理状态对愈伤组织的诱导有明显影响。经过多次继代培养,从4个基因型的幼胚得到了颜色为乳白或淡黄、结构疏松、颗粒状的胚性愈伤组织。胚性愈伤组织在MS+5%蔗糖附加0.05mg/L ABA或附加0.05mg/L ZT+0.05mg/L ABA的分化培养基上通过胚状体发生途径再生植株。不同基因型材料再生植株的最适培养基组合有差异。从下胚轴和叶柄诱导的愈伤组织由于诱导率低且继代培养难度大,只得到了少量胚性愈伤组织,未能获得再生植株。
部分胚性愈伤组织经5～20Gy剂量的~(60)Co-Y射线辐照后,在附加不同浓度盐分(1.0%～2.5%NaCl)的培养基上连续筛选三代,获得稳定耐1.5%NaCl的耐盐
Salinity stress is one of the most serious worldwide factors retarding the growth of plants and crops productivity. In China, saline soils where the salt concentration is between 0.6-1.2 % are about 1.5 million hectares, furthermore, fresh water is lacked in these areas. Thus, the most valuable and economic strategy to utilize these bare soils is to screen and select salt-tolerant plants for cultivation. Sugar beet is a more able crop to tolerate salinity, and some wild genotypes origin from the seacoast. Traditional breeding is time-consuming and laborious, and has not resulted in salt-tolerant cultivars. Development of cell engineering techniques to screen salt-tolerant mutants and genetic manipulation to create salt-tolerant materials could decrease breeding time and create salt-tolerant cultivars of sugar beet. In this paper, the goal is to breed new strains of fodder beet that could grow luxuriantly in the saline soil containing low and middle concentrations of salts near the seashore in China via the comprehensive utilization ofboth cellular and genetic engineering techniques.Calli of 14 cultivars of sugar beet were induced and subcultured from immature embryos, petioles or hypocotyls derived from seedlings in pots of sand respectively. The factors influencing the induction frequency, subculture, and differentiation of calli were studied. The induction frequencies of calli differed among the explants of immature embryos, hypocotyls, petioles or leaves in same genotype, and immature embryos gave the highest percentage of induction. There were significant differences between the induction frequencies of the explants from different genotypes of beet. The development stage and physiologic state of explants also obviously influenced the induction frequency of calli. After several subcultures, opal or pale-yellow granular calli with loose constitution, inducing from immature embryos of four genotypes, were selected. Embryonic calli produced plantlets via embyogenesis pathway on MS media either supplemented with 5% sucrose and 0.05 mg/L ABA or with 5%sucrose, 0.05 mg/L ABA
and 0.05 mg/L ZT. The optimal compositions of medium for regeneration of plantlets varied from the genotypes. Only a little embryonic calli were selected from hypocotyls or petioles and no plantlets were regenerated.A part of embryonic calli were irradiated by 5-20 Gy of ~(60)Co- Y ray, then cultured on media supplemented with varied NaCl concentrations (1.0%-2.5%) continuously for 3 generations. Then, cell lines, which could actively proliferated on media containing 1.5% NaCl were obtained, and were induced to regenerate plantlets via embryogenesis pathway on the differentiation medium. Self-pollinated progenies (Ri) of regenerated plants were sown in pots of sand and irrigated with 2.0%-3.0% NaCl solution every day. There were 18 lines grown in 2.0% NaCl solution for long time, 6 lines germinated in solution of 2.5% NaCl, but the seedlings were dying soon after the germination from the damages of NaCl, whereas there were no survival seedlings from control seeds when irrigated with 2.0% NaCl solution. This provided a successful example for breeding salt-tolerant strains from calli for sugar beet by using radiation breeding and directional selection techniques.An efficient method has been developed in in vitro multiplication of sugar beet from the excised immature inflorescence tips of 20 genotypes on MS medium supplemented with 1 mg/L 6-BA. Multiple bud clumps were induced from segments of inflorescence tips after inoculation for 3 weeks, and the induction frequency was above 90%. The initial time and frequency of bud development varied from genotypes, generally bud clumps formed within 10-20 days after culture initiation, and large amount of bud clumps were produced and proliferated rapidly with further subculture. They were then cut into small bud clumps to subculture at 15-day intervals onto the unaltered medium and each bud could generally produce 5-8 new buds in each subculture. The proliferation rate usually reached 1:20-30 or more on the induction and proliferation media supplementedwith 1 mg/L 6-BA every month.Once, single buds had reached about 6-10 mm in length, they were cut down frommultiple bud clumps to inducing root on medium supplemented with 1 mg/L NAA. The rooting frequency was related with the age, height, genotype and physiologic statue of the buds. Generally, larger buds in rapid growth easily produced root, for instance, 50-80% white and strong roots formed within 2 weeks. The rooted cuttings were transplanted in pots and 1 month later the survivals were transplanted in the field. The
survival rates of transplanted plantlets from multiple bud clumps varied from genotypes, usually above 75% and a few of them up to 98%.Little buds were cut from multiple bud clumps and irradiated with 60Co- Y ray in dosage of 10-20 Gy, then were transferred to the medium containing 1.0%-2.5% NaCl for the selection of salt-tolerant buds. Buds tolerating 2.0% NaCl were selected to regenerate plantlets. After vernalization and self-pollination, the seeds (Ri) of the regenerated plants were sown in pots of sand and irrigated with solution of 2.0%-3.0% NaCl every day. Some of the seeds germinated and grew normally in the 2.0% NaCl solution, exhibiting higher salt-tolerance compared to the controls. When the seedlings after the saline selection were transplanted to soil, the plants grew normally and produced plump root tuber similar to controls. The seeds from two selected lines could germinate and grow for a few weeks in 2.5% NaCl solution before withered. In 0.9% NaCl saline soil, the tuber yields of the plants regenerated from three salt-tolerant lines were about 45-50 tons/hectare, approximately 2.6-2.9 times of the controls. It is concluded that salt-tolerant materials with good agronomic traits in sugar beet were obtained by using radiation method and directional selection techniques to multiple bud clumps. The study provided a successful approach to produce useful mutants for those genotypes difficult toregenerate plantlets from single cell or callus in vitro.1-3 mm small buds of sugar beet derived from the multiple bud clumps subcultured for 7 days were infected with agrobacterium LBA4404 harboring plasmid pCAMBIA1300-&e*/f-/*pr or pCAMBlAUOO-AtNHXl-hpt, or agrobacterium EHA105 harboring plasmid pROK2-asPLD r-als for 10-20 min. After co-culture for 2-4 days on induction medium, the buds were transferred to medium supplemented with 100 mg/L cefotaxime to inhibit the growth of agrobacterium for 10 days. In the period of co-culture and inhibiting agrobacterium, the buds developed out 2 or 3 leaves and produced multiple bud clumps. After three further 10-15 days subcultures on medium containing hygromycin B at the concentration of 10 mg/L or lvchuanglong (sulfonylurea herbicide) at the concentration of 0.01-0.05 mg/L, survival frequency of infected buds was about 7%- 23%, varied from genotypes and parameters for infection. Then, survival buds were transferred to medium without hygromycin B or lvchuanglong and proliferated to form multiple bud clumps. Larger buds from multiple bud clumps were rooted on the rooting medium, and
were transplanted into pots. Based on these results, we obtained the higher transformation efficiency when the bud meristems were infected for 5-10 minutes in agrobacterium suspension of OD6oo 0.3-0.5 and co-cultured for 2-4 days.DNA was extracted from leaves of transformed buds or plants for PCR. Primers were designed according to the sequences of hpt, als or PLD gene. DNA of the plants showing PCR positive were extracted and digested by EcoR I and HindlR for Southern blotting. Over 80% plants gave positive signal at Southern blotting, indicating that the betA, AtNHXlor anti-sense PLD rgene had been integrated into the genome of some transformed plants respectively.After proliferation, the salt-tolerance of transformed buds showing PCR positive were detected by culturing for 3 continuous generations at 15-day interval on media containing different concentrations of NaCl (1.0%-3.0%) respectively. On the media supplemented with NaCl, the survival rate of buds decreased following the increase of salt concentration. The transgenic buds of different genotypes showed significantly various improved salt-tolerance compared to the non-transgenic controls, even if the transgenic buds of same genotype existed the difference of salt-tolerance. Only part of transgenic buds displayed higher salt-tolerance and the means of the improvement of salt-tolerance was 1.0% NaCl.5-leaf stage transgenic plants were potted in sand and irrigated with solution containing different concentrations of NaCl (1.0%-3.0%). They could tolerate additional 1.0%-1.5% NaCl compared to the non-transgenic controls and only show minor symptoms of salinity damage. It was obvious that the differences of salt-tolerance existed among transgenic plants from same genotype and also existed among the genotypes. Under the same concentration of NaCl, transgenic plants were generally showed symptoms of damage from salinity 3-5 days later compared to controls. Watered with 3.0% concentration of NaCl for 30 days, about half of transgenic plants survived but only 4% controls were alive. There were not significant differences of salt-tolerance between transgenic buds and transgenic plants, in another word, the plants regenerated from themore salt-tolerant buds showed similar higher salt-tolerance.Self-pollinated seeds (Ti) of transgenic plants suffered the selection of salt solution inpots of sand, and seedlings that could tolerate 2.0%, 2.5% or 3.0% NaCl solution were selected and self-pollinated in the next spring.
Analysis of the salt-tolerance and inherit stability of betA in the transgenic progeny indicated that the salt-tolerance improvement of the progeny was correlated with the expression of betA gene, and in some of the transgenic lines hpt and betA gene segregated as dominant Mendelian traits (3:1 or 15:1). In 58 plant lines assayed, no more than 20% ones showed segregation ratio of 3:1 or 15:1. This result indicated that non-mosaic transgenic plants could be obtained from the buds infected by agrobacterium, but the frequency was low.From the results of the selection with 2% NaCl solution and yields in the saline field, 276 plants with betA were chosen from 24 transgenic plant lines (Ti), and the self-pollinated seeds (T2) of 243 plants were harvested. In the saline field of Dongying district in Shandong province where the salt concentration was 0.6%, 1.0% or 1.5% respectively, some of the T2 transgenic progenies exhibited significantly improved salt-tolerance and much better yields than the controls. In the field containing 1.0% salt concentration, yields of root tubers were 582-978 Kg/100 m2, as much as 225.8%-338.4% yields of non-transgenic controls. The plant from 109 plant lines with better salt tolerance and agronomic traits were chosen for producing T3 seeds. T3 plants were also selected according to their salt-tolerance and agronomic traits exhibition in saline field. These results demonstrated that we had bred salt-tolerant sugar beet strains by genetic transformation with betA.218 Ti seedlings with AtNHXl gene that tolerated 2.0%-2.5% NaCl were selected. In some transgenic lines AtNHXl gene inherited as Mendilian segregation. Some of progeny (T2) of Ti transgenic lines were sown in the saline field of seashore where the salt concentration was 0.6%, 1.0% or 1.5% respectively. The plants with good salt-tolerance and agronomic traits were chosen from the T2 transgenic plants for the production of seeds (T3). T3 plants from a number of plant lines germinated evenly and grew normally in the field of 1% salt components, and produced the yield of 568-1020 Kg root tubers /100 m2, that were about 168%-368% comparing to controls. These results revealed that it. was feasible to improve the salt-tolerance of beets by the introduction of AtNHXl gene into cultured buds and to obtain the salt-tolerant beet cultivars by genetic transformation with AtNHXl.Among the salt-tolerant materials with good agronomic traits produced by cellular engineering techniques, plants of the most salt-tolerant line could tolerate the stress of 2.0% NaCl solution, and produce high yields in the saline field of 0.9% salt
引文
1.中国农业科学院甜菜研究所主编。《中国甜菜栽培学》,农业出版社,北京,1982,P23-56,P72-80,P81-114
2. Margara, J. Neoformation de bourgeons in vitro chez la Betterave sucriere (Beta vulgaris L.) culture in vitro. C. R. Acad. Sci. (Paris), Ser. D, 1970, 285:1041-1044
3. Greef W D, Jacobs M. In vitro culture of the sugarbeet: Description of a cell line with high regeneration capacity. Plant Sci Lett, 1979, 17: 55-61
4. Krens F A, Jamar D. The role of explants source and culture conditions on callus induction and shoot regeneration in sugarbeet (Beta vulgaris L.). J Plant Physiol, 1989, 134: 651-655
5.石建明,王伏雄,朱至清。甜菜幼胚培养中的植株再生。植物学通报,1991,8:57-59
6.张剑峰,李天然,邓香兰。高效诱导甜菜再生植株的研究,生物工程学报,1997,13:273-276
7.马龙彪,郭九峰,陆国庆。甜菜基因型的筛选及胚性细胞系建立的研究。中国甜菜,1994(3):8-11
8.卫群,张剑峰。不同基因型甜菜叶柄的组织培养及植株的再生。中国糖料,1998(4):21-24
9.张悦琴,邵明文。花药、未授粉胚珠培养技术及其在甜菜育种中的应用。甜菜糖业,1992(5):6-11
10.张悦琴,邵明文。甜菜花粉植株的诱导及其观察。中国甜菜,1984(3):1-8
11.王宇清,罗翠娥。甜菜(Beta vulgaris L.)花药培养的研究。甜菜糖业(甜菜分册),1983(1):16-19
12.张悦琴、邵明文。甜菜胚珠培养诱导单倍体植株的研究。中国甜菜,1986(3):9-13
13.罗翠娥等。糖甜菜(Beta vulgaris L.)胚珠培养的研究。甜菜糖业(甜菜分册),1987(1):11-13
14.邵明文,张悦琴,黄彩云。甜菜未授粉胚珠培养的研究。中国甜菜,1988(2):4-13
15.邵明文,张悦琴,黄彩云。甜菜花药、胚珠培养及其在育种上应用。中国农业科学,1993(2):56-62
16.马龙彪,张悦琴,邵明文等。甜菜花培新品种——中甜花培1号的选育。中国糖料,2002(4):12-14
17.黄彩云。甜菜远缘杂交胚培养及植株再生。中国甜菜糖业,1997,4:1-3
18. Freytag A H, Wrather J A, Erichsen A W. Salt tolerant sugarbeet progeny from tissue cultures challenged with multiple salts. Plant Cell Rep, 1990, 8: 647-650
19.张悦琴,马龙彪,陆国庆。利用细胞工程育种方法选育甜菜抗褐斑病品系初报。中国糖料,1997,4:20-23
20.马龙彪,张悦琴,吴则东。抗甜菜褐斑病体细胞无性系变异的研究。中国糖料,2001(1):1-5
21. Lisu-Nam, Landova B, Landa Z. Isolation of protoplasts from sugar beet leaves. Biologia Plantrum, 1976, 18: 389-392
22. Bhat S R, Ford-loyd B V, Callow J A et al. Isolation of protoplasts and regeneration of callus from suspension cultures of cultivated beets. Plant Cell Rep, 1985, 4: 345-350
23. Krens F A, Jamer D, Rouwendal G J A et al. Transfer of cytoplasm from new Beta CMS sources to sugar beet by asymmetric fusion, Ⅰ. Shoot regeneration from mesophyll protoplasts. Theor Appl Genet, 1990, 79: 390-396
24.李兴锋.陈惠民,邵明文等。甜菜原生质体培养直接产生体细胞胚。植物学报,1992,34(5):402-404
25. Hall R D, Pederson C, Krens F A. Improvement of protoplast culture protocols for Beta vulgaris L.(sugar beet). Plant Cell Rep. 1993, 12: 339-342
26.邵明文,马龙彪。甜菜原生质体培养再生愈伤组织。植物学报,1993,35:727-729
27.郭九峰,邵明文,马龙彪等。甜菜原生质体培养再生植株的研究。中国甜菜,1994,(4):6-9
28. Lenzener S, Zoglauer K, Schider O. Plant regeneration from protoplasts of sugar beet(Beta vulgaris L.). Physiol. Plant, 1995, 94: 342-350
29. Hall R D, Rikson B T, Weyens G J et al. A high efficiency technique for the regeneration of transgenic sugar beet from stomatal guard cells. Nature Biotechnology, 1996, 14: 1133-1138
30. Sevenier R, Hall R D, Ingfid M et al. High level fruetan accumulation in a transgenic sugar beet. Nature Biotechnology, 1998, 16: 843-846
31. Hussey G, Hepher A. Clonal propagation of sugar beet plants and the formation of polyploida by tissue culture. Ann Bot, 1978, 42: 477
32. Saunders J W. A flexible in vitro shoot culture propagation system for sugarbeet that includes rapid floral induction of ramets. Crop Sci., 1982, 22: 1102-1105
33. Saunders, J W, Mahoney M D. Benzyladenine induces foliar adventitious shoot formation on young plants of two sugarbeet (Beta vulgaris L.) cultivars. Euphytica, 1982, 31: 801-804
34. Miedema P. A tissue culture technique for vegetative propagation and low temperature preservation of Beta vulgaris. Euphytica, 1982,31: 635-643
35. Tetu T, Sangwan R S, Sangwan-Norreel B S. Hormonal control of organogenesis and somatic embryogenesis in Beta vulgaris callus. J Exp Bot, 1987,38: 506-517
36. Detrez C, Tetu T, Sangwan R S. Direct organogenesis from petiole and thin cell layer explants in sugar beet cultured in vitro. J Exp Bot, 1988,204:917-926
37. Doley W P, Saunders J W. Hormone-free medium will support callus production and subsequent shoot regeneration from whole plant explant in some sugar beet, Beta vulgaris L. populations. Plant Cell Rep, 1989, 8: 222-225
38. Goska M, Rogozinska J. Micropropagation of sugar beet and possibilities of using it in breeding. Hodowla Roslin Aklinat. Nasien, 1990, 32(5-6): 87-94
39. Snezana M, Sibila J, Kovacer L. Vegetative propagation of sugar beet from floral ramets. J Sugaberet Res, 1990, 27:90-96
40. Zhong Z X, Smith H G, Thomas T H. In vitro culture of petioles and intact leaves of sugarbeet. Plant Growth Regulation, 1993, 12: 59-66.
41. Zhong Z X, Smith H G, Thomas T H. Micropropagation of wild beet inflorescence pieces. Plant Growth Regulation, 1993, 12: 53-66.
42. Konwar B K. Plant regeneration of three genotypes of sugarbeet. Crop Improvement, 1993, 20(1): 88-97
43. Potyondi L. Callus induction and plant regeneration in haploid sugar beet, Beta vulgaris L. lines. Acta agron. Ovariensis, 1993,35:215-220
44. Sullivan C F, Finch I, Dix P J et al. Studies of in vitro propagation systems for sugar beet. Irish J Agr Food Res, 1993,32:27-35
45. Toldi O, Gabor Gyulai, Jozsef K et al. Antiauxin enhanced microshoot initiation and plant regeneration from epicotyl-originated thin-layer explants of sugarbeet. Plant Cell Rep, 1996, 15: 851-854
46. Krens F A, Adelina Trifonjova L C, Paul K et al. The effect of exogenously-applied
phytohormones on gene transfer efficiency in sugarbeet(Beta vulgaris L.). Plant Science, 1996, 116: 96-106
47. Rady M R. In vitro mulplication of Beta vulgaris L. throughout excised shoot tips. Biologia Plantrum, 1997, 40(4): 515-522
48.王关林,方宏筠。植物基因工程原理与技术。1998,科学出版社,北京,P179-181
49. Paul H, Zijlstra C, Leeuwaugh J E et al. Reproduction of the beet cyst nematode Heterodera schachtii on transformed root cultures of Beta vulgaris L. Plant Cell Repo 1987, 6: 379-381
50. Krens F A, Janar Zijistra C, Van der Molen W et al. Transformation and regeneration in sugar beet induced by shooter mutants of Agrobacterium tumefaciens. Euphytica, 1988, 185-194
51. Lindsey K, Jones M G K. Stable transformation of sugar beet protoplast by electroporation. Plant Cell Rep, 1987, 10: 43-52
52. Lindsey K, Jones M G K. Transient gene expression in electroporated protoplast intact cell of sugar beet. Plant Mol Bio, 1989, 8: 71-74
53. Hamill J D, Prescott A, Martin C. Assessment of the efficiency of cotransformation of T-DNA of disarmed binary vectors derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes. Plant Mol Bio, 1987, 9: 573-584
54. Harpster M H, Townsend J A, Jones J D G et al. Relative strengths of the 35S cauliflower mosaic virus, 1', 2' and nopaline synthase promoters in transformed tabacco, sugar beet and oilseed rape callus tissue. Mol Genet, 1988, 212: 182-190
55. Lindsey K, Gallois P. Transformation of Sugarbeet(Beta vulgaris L.) by Agrobacterium tumefaciens. J Exp Bot, 1990, 41(226): 529-536
56. Paul H, van Deelen J E M, Henken B et al. Expression in vitro of resistance to Heterodera schachtii in hairy roots of an alien monotelosomic addition plant of Beta vulgaris L., transformed by Agrobacterium rhizogenes. Euphitica, 1990, 48: 153-157
57. Steen P, Pedersen H C. Gene transfer for herbicide resistance. J Sugar Beet Res, 1993, 30(4): 267-274
58. Jacq B, Lesobre O, Sangwan R Set al. Factors influencing T-DNA transfer in Agobacterium-mediated transformation of sugarbeet. Plant Cell Rep, 1993, 12: 621-624
59. Marie M, Britt-Louise L, Paul T. Reduced titer of BNYVV on transgenic sugarbeets expressing the BNYVV coat protein. Euphytica, 1996, 90: 293-299
60. Joersbo M, Donaldson I, Kreiberg J et al. Analysis of mannose selection used for transformation of sugar beet. Mol Breed, 1998, 4: 111-117
61. Kifle S, Shao M, Jung C et al. An improved transformation protocol for studying gene expression in hairy roots of sugar beet(Beta vulgaris L.). Plant CellRep, 1999, 18: 514-519
62. Ingersoll J C, Huette T M, Owens L D. Effect of promoter-leader sequences on transient expression of reporter gene chimeras biolistically transferred into sugarbeet(Beta vulgaris L.) suspension cells. Plant Cell Rep, 1996, 15: 836-840
63. Fry J E, Barnason A R, Hinchee M. Genotype-independent transformation of sugar beet using Agrobacterium tumefaciens in molecular biology and plant development. Third international congress of the ISPMB, Tucson, USA, 1991, P84
64. Ben-Tahar S, Gerents D, Kallerhoff J et al. Transforming Beta vulgaris cells with Agrobacterium-contag. vector which imparts e.g. resistance to virus, by contacting bacteria with dispersion or suspension of callus tissue. 1991, Patent No: W O 9113159
65. D'Halluin K, Bossut M, Bonne E et al. Transformation of sugar beet(Beta vulgaris L.) and evaluation of herbicide resistance in transgenic plants. Bio/Technology, 1992, 10: 309-314
66. Marie M, Tuvesson S, Steen P et al. Transgenie sugar beet tolerant to glyphosate. Euphytica, 1997, 94: 83-91
67.姚华建,李大伟,于嘉林等。甜菜坏死黄脉病毒外壳蛋白基因在甜菜转基因植株中的表达。生物工程学报,1997,13(4):440-442
68.李天然,张剑峰,李旭刚。抗冷冻蛋白基因转化甜菜的研究。内蒙古大学学报,1998,29(1):144-146
69. Snyder G W, Ingersoll J C, Smigocki A C et al. Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugarbeet(Beta vulgaris L.) by Agrobaeterium or particle bombardment. Plant Cell Rep, 1999, 18: 829-834
70. Dewar A, May M. GM sugar beet-the present situation. British Sugar Beet Rev, 2000, 68(2): 22-27
71.马龙彪,徐香玲,张悦琴。几丁质酶基因转化甜菜的初步研究。中国糖料,2000,(3):18-20
72. Ivic S D, Sicher R C, Smigocki A C. Growth habit and sugar accumulation in sugarbeet(Beta vulgaris L.) transformed with a cytokinin biosynthesis gene. Plant Cell Rep, 2001, 20: 770-773
73.郝秀英,于嘉林,王燕飞等。新疆甜菜品种(系)基因转化和再生体系的建立。中国糖料,2002(4):5-7
74.徐德昌,刘巧红,江莉萍。甜菜转基因植株抗性表现及种子获得。中国甜菜糖业,2002(4):3-11
75. Hasegawa P M, Bressan R A, Zhu J K. Plant cellular and molecular responses to high salinity. Annu Rev Plant Physio. Plant Mo. Biol, 2000, 51: 463-399
76. Netting A G. pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations. J Exp Bot, 2000, 51(343): 147-158
77. Xiong L, Zhu J K. Salt tolerance. In: The Arabidopsis Book. 2002, 1-22. Electronic Book ed. by American Society of Plant Biologists
78. Zhang J, Ngnyen H T, Blum A. Genetic analysis of osmotic adjustment in crop plants. J Exp Bot, 1999, 50(322): 291-302
79. Liu Q, Kasuga M, Sakuma Y et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 1998, 10: 1391-1406
80. Matsuoka D, Nanmori T, Sato K et al. Activation of AtMEK1, an Arobidopsis mitogen-activated protein kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings. Plant J, 2002, 29: 637-647
81. Tarczynski M C, Jensen R G, Bohnert H J. Stress protection of transgenic tobacco by production of the osmolyte mannitoi. Science, 1993, 259(22): 508-510
82.刘俊君,彭学贤,王海云等。转基因烟草的甘露醇合成和耐盐性。生物工程学报,1994,12(2):206-210
83. Kasuga M, Liu Q, Miura S et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnol, 1999, 17: 287-291
84.刘风华,孙仲序,崔德才等。细菌mtl-D基因的克隆及在转基因八里庄杨中的表达。遗传学报,2000,27(5):428-433
85.戴秀玉,吴大鹏,周坚。大肠杆菌海藻糖合成酶基因的克隆和表达。遗传学报,2000,27(2):158-164
86.戴秀玉,王忆琴.周坚等。大肠杆菌海藻糖的代谢调控。生物工程进展,2000,20(6):26-29
87. Lilius G, Holmberg N, Bulow L. Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase. Bio/Technology, 1996, 14: 177-180
88. Apse M P, Aharon G S, Snedden W A et al. Salt tolerance conferred by overexpression of a vacuolar Na~+/H~+ antiport in Arabidopsis. Science, 1999, 285: 1256-1258
89. Holmstrom K O, Somersalo S, Mandal A et al. Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine, J Exp Bot, 2000, 51(343): 177-185
90. Zhang H X, Blumwald E. Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat. Biotechnol. 2001, 19: 765-768
91. Boncompagni E, Magne (?)ster(?)s, Poggi M C et al. Occurrence of choline and glycine betaine uptake and metabolism in the family Rhizobiaceae and their roles in osmoprotection. Appl Envir Microbio, 1999, 65(5): 2072-2077
92. Hayashi H, Alia Mustardy L et al. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinbetaine and enhanced tolerance to salt and cold stress, Plant J, 1997,12: 133-142.
93. Takabe T, Hayashi Y, Tanaka A. Evaluation of glycinebetaine accumulation for stress tolerance in transgenic rice plants. Proceedings of International Workshop on Breeding and Biotechnology for Environmental Stress in Rice. 1998, Hokkaido Nationaal Agricultural Experiment Station and Japan International Science and Technology Exchange Center, Sapporo, Japan, P63-68
94.梁峥,骆爱玲,赵原等。干旱和盐胁迫诱导甜菜叶中的甜菜碱醛脱氢酶的积累。植物生理学报,1996,22(2):161-164
95. Huang J, Hirji R, Adam L et al. Genetic Engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations. Plant Physiol, 2001, 122: 747-756
96.刘凤华,郭岩,谷冬梅等。转甜菜碱醛脱氢酶基因植物的耐盐性研究。遗传学报,1997,24(1):54-58
97.郭岩,张莉,肖岗等。甜菜碱醛基脱氢酶基因在水稻中的表达及转基因植株的耐盐性研究。中国科学,1997,27(2):151-155
98.郭北海,张艳敏,李洪杰等。甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达。植物学报,2000,42(3):279-283
99.何锶洁,董伟,李慧芬等。转甜菜碱醛脱氢酶基因玉米及其耐盐性的研究。高技术通讯,1999,2:50-52
100.李银心,常凤启,杜立群等。转甜菜碱醛脱氢酶基因豆瓣菜的耐盐性。植物学报,2000,42(5):480-484
101.张慧,董伟,周骏马等。果聚糖蔗糖转移酶基因的克隆及耐盐转基因烟草的培育。生物工程学报,1998,14(2):181-186
102. Pilon-Smits E A, Terry N, Sears T et al. Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress. J Plant Physiol, 1998, 152: 525-532
103.王宝山,邹琦,赵可夫。液泡膜转运蛋白与植物耐盐性研究进展。植物学通报,1996,13(3):30-36
104. Assmann SM. From proton pump to proteome. Twenty-five years of research on ion transport in higher plants. Plant Physiol, 2001, 25: 139-141
105. Serrano R, Mulet J M, Rios G et al. A glimpse of the mechansms of ion homeostasis during salt stress. J Exp Bot, 1999, 50: 1023-1036
106. Martinoia E, Massouneau A, Frangne N. Transport processes of solutes across the vacuolar membrane of higher plants. Plant Cell Physiol, 2000, 41: 1175-1186
107. Nass R, Cunningham K W, Rao R. Intracellular sequestration of sodium by a novel Na~+/H~+ exchanger in yeast is enhanced by mutations in the plasma membrane H~+-ATPase. J Biol Chem, 1997, 272: 26145-26152
108. Gaxiola R A, Rao R, Sherman A et al. The Arabidopsis thaliana proton transporters, AtNHX1 and Avpl, can function in cation detoxification in yeast. Proc Natl Acad Sci USA, 1999, 96: 1480-1485
109. Hamada A, Shono M, Xia T et al. Isolation and characterization of a Na~+/H~+ antiporter gene from the halophyte Atriplex gmelin. Plant Mol Biol, 2001, 46: 35-42
110. Kiegerl S, Cardinale F, Siligan C et al. SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK. Plant Cell, 2000, 12: 2247-2258
111. Tena G, Asai T, Chiu W L et al. Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol, 2001, 4: 392-400
112. Zwerger K, Hirt H. Recent advances in plant MAP kinase signalling. Biol Chem, 2001, 382: 1123-1131
113. Zhang S, Klessig D F. MAPK cascades in plant defense signaling. Trends Plant Sci, 2001, 6: 520-527
114. Asai T, Tena G, Plotnikova J et al. MAP kinase signaling cascade in Arabidopsis innate immunity. Nature, 2002, 415: 977-983
115. Ren D, Yang H, Zhang S. Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis. J Biol Chem, 2002, 277: 559-565
116. Wang X. The role of phospholipase D in signaling cascades. Plant Physiol, 1999, 120: 645-652
117. Wang X. Molecular analysis of phospholipase D. Trends inplant, 1997, 2(7): 261-266
118. Munnik T, Irvine R F, Musgrave A. Phospholipid signaling in plants. B B A, 1998, 1389(3): 222-272
119. Munnik T, Harold J G M, Riet B et al. Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate. Plant J, 2000, 22(2): 147-154
120. Fan L, Zheng S, Cui D et al. Subcellular distribution and tissue expression of phospholipase D_α, D_β, and D_γ inArabidospsis. PlantPhysiol, 1999, 119: 1371-1378
121. Shi H and Zhu J K. SOS4, a pyridoxal kinase gene, is required for root hair development in Arabidopsis. Plant Physiol, 2002, 129: 585-593,
122. Shi H., Xiong L, Stevenson B et al. The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for vitamin B6 in plant salt tolerance. Plant Cell, 2002, 14: 575-588
123.梁峥,骆爱玲。甜菜碱和甜菜碱合成酶。植物生理学通讯,1995,31(1):1-8
124. Hall J L, Harvey D M, Flowers T J. Evidence for the cytoplasmic localization of betaine in leaf cells ofSuaeda maritima. Planta, 1978, 140: 59-6298.
125.杨传平,刘桂丰,梁宏伟等。耐盐基因betA转化小黑杨的研究。林业科学,2001,37(6):34-38
126. Hayashi A H, Sakamoto A et al. Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of glycinebetaine, Plant J, 1998, 16(2): 155-161
127. Sakamoto A, Murata N. The use of bacterial choline oxidase, a glycinebetaine synthesizing enzyme, to create stress-resistant transgenic plants. Plant Physiol, 2001, 125: 180-188
128. Wyn Jones R G, Storey R. Betaines. In: Paleg L G, Aspinall D (eds). The physiology and Biochemistry of Drought Resistance in Plants. Sydney: Academic press, 1981, P171-204
129. Wyn Jones RG, Storey R. Betaines. In: Paleg LG, Aspinall D (eds). The physiology and Biochemistry of Drought Resistance in Plants. Sydney: Academic press, 1981, 171-204.
130. Blumwald E, Aharaon G S, Apse M P. Sodium transport in plant cells. B B A, 2000, 1465: 140-51
131. Niu X, Bressan R, Hasegawa P M et al. Ion homeostasis in NaCl stress environments. Plant Physiol, 1995, 109: 735-742
132. Staal M, Maathuis F, Elzenga T, Overbeek J, Prins H. (1991) Na~+/H~+ antiport activity of the salt-tolerant Plantago maritime and the salt-sensitive Plantago media. Physiol. Plant. 82: 179-184
133. Barlda B J, Zingarelli L, Blumwald E et al. Tonoplast Na~+/H~+ antiport activity and its energization by the vacuolar H~+-ATPase in the halophytic plant Mesembryanthemum crystallimum L. Plant Physiol, 1995, 109: 549-556
134. Zhang H., Hodson J N, Williams J P et al. Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. PNAS, 2001, 98: 12832-12836.
135. Wang X, Xu L, Zheng L. Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L. J Biol Chem, 1994, 269: 20312-20317
136. Fan L, Zheng S, Wang X. M. Antisense suppression of phospholipase D retards abscisic acid and ethylene promoted senescence of postharvest arabidopsis leaves. Plant Cell, 1997, 9: 2183-2196
137. Zhou H E, Zheng S, Lu F et al. Low temperature tolerance in transgenic tobacco and Arabidopsis thaliana expressing sense and antisense phospholipase D gene. Plant physiol, 1997, 114(supplement): 128
138. Lee S H, Chae S H, Lee T K et al. Ethylene mediated phospholipid catabolism pathway in glucose-starved carrot suspension cells. Plant Physiol, 1998, 116: 223-229
139. Munnik T, Katja Kerkmann et al. Water deficit triggers phospholipase D activity in the resurrection plant Craterostigma plantagfneum. Plant Cell, 2000, 12: 111-123
140.刘斌,李红双,王其会等。反义磷脂酶D_γ基因转化毛白杨的研究。遗传,2002,24(1):40-44
141. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 1962, 15: 473-497
142. Gardiner J M, Coe E H, Melia-Hancock S et al. Development of a core RFLP map in maize using an immortalized F_2 population. Genetics, 1993, 134: 917-930
143. Salzman R A, Fujita T, Zhu-Salzman K et al. An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Molt Biol Rep,
2. Margara, J. Neoformation de bourgeons in vitro chez la Betterave sucriere (Beta vulgaris L.) culture in vitro. C. R. Acad. Sci. (Paris), Ser. D, 1970, 285:1041-1044
3. Greef W D, Jacobs M. In vitro culture of the sugarbeet: Description of a cell line with high regeneration capacity. Plant Sci Lett, 1979, 17: 55-61
4. Krens F A, Jamar D. The role of explants source and culture conditions on callus induction and shoot regeneration in sugarbeet (Beta vulgaris L.). J Plant Physiol, 1989, 134: 651-655
5.石建明,王伏雄,朱至清。甜菜幼胚培养中的植株再生。植物学通报,1991,8:57-59
6.张剑峰,李天然,邓香兰。高效诱导甜菜再生植株的研究,生物工程学报,1997,13:273-276
7.马龙彪,郭九峰,陆国庆。甜菜基因型的筛选及胚性细胞系建立的研究。中国甜菜,1994(3):8-11
8.卫群,张剑峰。不同基因型甜菜叶柄的组织培养及植株的再生。中国糖料,1998(4):21-24
9.张悦琴,邵明文。花药、未授粉胚珠培养技术及其在甜菜育种中的应用。甜菜糖业,1992(5):6-11
10.张悦琴,邵明文。甜菜花粉植株的诱导及其观察。中国甜菜,1984(3):1-8
11.王宇清,罗翠娥。甜菜(Beta vulgaris L.)花药培养的研究。甜菜糖业(甜菜分册),1983(1):16-19
12.张悦琴、邵明文。甜菜胚珠培养诱导单倍体植株的研究。中国甜菜,1986(3):9-13
13.罗翠娥等。糖甜菜(Beta vulgaris L.)胚珠培养的研究。甜菜糖业(甜菜分册),1987(1):11-13
14.邵明文,张悦琴,黄彩云。甜菜未授粉胚珠培养的研究。中国甜菜,1988(2):4-13
15.邵明文,张悦琴,黄彩云。甜菜花药、胚珠培养及其在育种上应用。中国农业科学,1993(2):56-62
16.马龙彪,张悦琴,邵明文等。甜菜花培新品种——中甜花培1号的选育。中国糖料,2002(4):12-14
17.黄彩云。甜菜远缘杂交胚培养及植株再生。中国甜菜糖业,1997,4:1-3
18. Freytag A H, Wrather J A, Erichsen A W. Salt tolerant sugarbeet progeny from tissue cultures challenged with multiple salts. Plant Cell Rep, 1990, 8: 647-650
19.张悦琴,马龙彪,陆国庆。利用细胞工程育种方法选育甜菜抗褐斑病品系初报。中国糖料,1997,4:20-23
20.马龙彪,张悦琴,吴则东。抗甜菜褐斑病体细胞无性系变异的研究。中国糖料,2001(1):1-5
21. Lisu-Nam, Landova B, Landa Z. Isolation of protoplasts from sugar beet leaves. Biologia Plantrum, 1976, 18: 389-392
22. Bhat S R, Ford-loyd B V, Callow J A et al. Isolation of protoplasts and regeneration of callus from suspension cultures of cultivated beets. Plant Cell Rep, 1985, 4: 345-350
23. Krens F A, Jamer D, Rouwendal G J A et al. Transfer of cytoplasm from new Beta CMS sources to sugar beet by asymmetric fusion, Ⅰ. Shoot regeneration from mesophyll protoplasts. Theor Appl Genet, 1990, 79: 390-396
24.李兴锋.陈惠民,邵明文等。甜菜原生质体培养直接产生体细胞胚。植物学报,1992,34(5):402-404
25. Hall R D, Pederson C, Krens F A. Improvement of protoplast culture protocols for Beta vulgaris L.(sugar beet). Plant Cell Rep. 1993, 12: 339-342
26.邵明文,马龙彪。甜菜原生质体培养再生愈伤组织。植物学报,1993,35:727-729
27.郭九峰,邵明文,马龙彪等。甜菜原生质体培养再生植株的研究。中国甜菜,1994,(4):6-9
28. Lenzener S, Zoglauer K, Schider O. Plant regeneration from protoplasts of sugar beet(Beta vulgaris L.). Physiol. Plant, 1995, 94: 342-350
29. Hall R D, Rikson B T, Weyens G J et al. A high efficiency technique for the regeneration of transgenic sugar beet from stomatal guard cells. Nature Biotechnology, 1996, 14: 1133-1138
30. Sevenier R, Hall R D, Ingfid M et al. High level fruetan accumulation in a transgenic sugar beet. Nature Biotechnology, 1998, 16: 843-846
31. Hussey G, Hepher A. Clonal propagation of sugar beet plants and the formation of polyploida by tissue culture. Ann Bot, 1978, 42: 477
32. Saunders J W. A flexible in vitro shoot culture propagation system for sugarbeet that includes rapid floral induction of ramets. Crop Sci., 1982, 22: 1102-1105
33. Saunders, J W, Mahoney M D. Benzyladenine induces foliar adventitious shoot formation on young plants of two sugarbeet (Beta vulgaris L.) cultivars. Euphytica, 1982, 31: 801-804
34. Miedema P. A tissue culture technique for vegetative propagation and low temperature preservation of Beta vulgaris. Euphytica, 1982,31: 635-643
35. Tetu T, Sangwan R S, Sangwan-Norreel B S. Hormonal control of organogenesis and somatic embryogenesis in Beta vulgaris callus. J Exp Bot, 1987,38: 506-517
36. Detrez C, Tetu T, Sangwan R S. Direct organogenesis from petiole and thin cell layer explants in sugar beet cultured in vitro. J Exp Bot, 1988,204:917-926
37. Doley W P, Saunders J W. Hormone-free medium will support callus production and subsequent shoot regeneration from whole plant explant in some sugar beet, Beta vulgaris L. populations. Plant Cell Rep, 1989, 8: 222-225
38. Goska M, Rogozinska J. Micropropagation of sugar beet and possibilities of using it in breeding. Hodowla Roslin Aklinat. Nasien, 1990, 32(5-6): 87-94
39. Snezana M, Sibila J, Kovacer L. Vegetative propagation of sugar beet from floral ramets. J Sugaberet Res, 1990, 27:90-96
40. Zhong Z X, Smith H G, Thomas T H. In vitro culture of petioles and intact leaves of sugarbeet. Plant Growth Regulation, 1993, 12: 59-66.
41. Zhong Z X, Smith H G, Thomas T H. Micropropagation of wild beet inflorescence pieces. Plant Growth Regulation, 1993, 12: 53-66.
42. Konwar B K. Plant regeneration of three genotypes of sugarbeet. Crop Improvement, 1993, 20(1): 88-97
43. Potyondi L. Callus induction and plant regeneration in haploid sugar beet, Beta vulgaris L. lines. Acta agron. Ovariensis, 1993,35:215-220
44. Sullivan C F, Finch I, Dix P J et al. Studies of in vitro propagation systems for sugar beet. Irish J Agr Food Res, 1993,32:27-35
45. Toldi O, Gabor Gyulai, Jozsef K et al. Antiauxin enhanced microshoot initiation and plant regeneration from epicotyl-originated thin-layer explants of sugarbeet. Plant Cell Rep, 1996, 15: 851-854
46. Krens F A, Adelina Trifonjova L C, Paul K et al. The effect of exogenously-applied
phytohormones on gene transfer efficiency in sugarbeet(Beta vulgaris L.). Plant Science, 1996, 116: 96-106
47. Rady M R. In vitro mulplication of Beta vulgaris L. throughout excised shoot tips. Biologia Plantrum, 1997, 40(4): 515-522
48.王关林,方宏筠。植物基因工程原理与技术。1998,科学出版社,北京,P179-181
49. Paul H, Zijlstra C, Leeuwaugh J E et al. Reproduction of the beet cyst nematode Heterodera schachtii on transformed root cultures of Beta vulgaris L. Plant Cell Repo 1987, 6: 379-381
50. Krens F A, Janar Zijistra C, Van der Molen W et al. Transformation and regeneration in sugar beet induced by shooter mutants of Agrobacterium tumefaciens. Euphytica, 1988, 185-194
51. Lindsey K, Jones M G K. Stable transformation of sugar beet protoplast by electroporation. Plant Cell Rep, 1987, 10: 43-52
52. Lindsey K, Jones M G K. Transient gene expression in electroporated protoplast intact cell of sugar beet. Plant Mol Bio, 1989, 8: 71-74
53. Hamill J D, Prescott A, Martin C. Assessment of the efficiency of cotransformation of T-DNA of disarmed binary vectors derived from Agrobacterium tumefaciens and the T-DNA of A. rhizogenes. Plant Mol Bio, 1987, 9: 573-584
54. Harpster M H, Townsend J A, Jones J D G et al. Relative strengths of the 35S cauliflower mosaic virus, 1', 2' and nopaline synthase promoters in transformed tabacco, sugar beet and oilseed rape callus tissue. Mol Genet, 1988, 212: 182-190
55. Lindsey K, Gallois P. Transformation of Sugarbeet(Beta vulgaris L.) by Agrobacterium tumefaciens. J Exp Bot, 1990, 41(226): 529-536
56. Paul H, van Deelen J E M, Henken B et al. Expression in vitro of resistance to Heterodera schachtii in hairy roots of an alien monotelosomic addition plant of Beta vulgaris L., transformed by Agrobacterium rhizogenes. Euphitica, 1990, 48: 153-157
57. Steen P, Pedersen H C. Gene transfer for herbicide resistance. J Sugar Beet Res, 1993, 30(4): 267-274
58. Jacq B, Lesobre O, Sangwan R Set al. Factors influencing T-DNA transfer in Agobacterium-mediated transformation of sugarbeet. Plant Cell Rep, 1993, 12: 621-624
59. Marie M, Britt-Louise L, Paul T. Reduced titer of BNYVV on transgenic sugarbeets expressing the BNYVV coat protein. Euphytica, 1996, 90: 293-299
60. Joersbo M, Donaldson I, Kreiberg J et al. Analysis of mannose selection used for transformation of sugar beet. Mol Breed, 1998, 4: 111-117
61. Kifle S, Shao M, Jung C et al. An improved transformation protocol for studying gene expression in hairy roots of sugar beet(Beta vulgaris L.). Plant CellRep, 1999, 18: 514-519
62. Ingersoll J C, Huette T M, Owens L D. Effect of promoter-leader sequences on transient expression of reporter gene chimeras biolistically transferred into sugarbeet(Beta vulgaris L.) suspension cells. Plant Cell Rep, 1996, 15: 836-840
63. Fry J E, Barnason A R, Hinchee M. Genotype-independent transformation of sugar beet using Agrobacterium tumefaciens in molecular biology and plant development. Third international congress of the ISPMB, Tucson, USA, 1991, P84
64. Ben-Tahar S, Gerents D, Kallerhoff J et al. Transforming Beta vulgaris cells with Agrobacterium-contag. vector which imparts e.g. resistance to virus, by contacting bacteria with dispersion or suspension of callus tissue. 1991, Patent No: W O 9113159
65. D'Halluin K, Bossut M, Bonne E et al. Transformation of sugar beet(Beta vulgaris L.) and evaluation of herbicide resistance in transgenic plants. Bio/Technology, 1992, 10: 309-314
66. Marie M, Tuvesson S, Steen P et al. Transgenie sugar beet tolerant to glyphosate. Euphytica, 1997, 94: 83-91
67.姚华建,李大伟,于嘉林等。甜菜坏死黄脉病毒外壳蛋白基因在甜菜转基因植株中的表达。生物工程学报,1997,13(4):440-442
68.李天然,张剑峰,李旭刚。抗冷冻蛋白基因转化甜菜的研究。内蒙古大学学报,1998,29(1):144-146
69. Snyder G W, Ingersoll J C, Smigocki A C et al. Introduction of pathogen defense genes and a cytokinin biosynthesis gene into sugarbeet(Beta vulgaris L.) by Agrobaeterium or particle bombardment. Plant Cell Rep, 1999, 18: 829-834
70. Dewar A, May M. GM sugar beet-the present situation. British Sugar Beet Rev, 2000, 68(2): 22-27
71.马龙彪,徐香玲,张悦琴。几丁质酶基因转化甜菜的初步研究。中国糖料,2000,(3):18-20
72. Ivic S D, Sicher R C, Smigocki A C. Growth habit and sugar accumulation in sugarbeet(Beta vulgaris L.) transformed with a cytokinin biosynthesis gene. Plant Cell Rep, 2001, 20: 770-773
73.郝秀英,于嘉林,王燕飞等。新疆甜菜品种(系)基因转化和再生体系的建立。中国糖料,2002(4):5-7
74.徐德昌,刘巧红,江莉萍。甜菜转基因植株抗性表现及种子获得。中国甜菜糖业,2002(4):3-11
75. Hasegawa P M, Bressan R A, Zhu J K. Plant cellular and molecular responses to high salinity. Annu Rev Plant Physio. Plant Mo. Biol, 2000, 51: 463-399
76. Netting A G. pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations. J Exp Bot, 2000, 51(343): 147-158
77. Xiong L, Zhu J K. Salt tolerance. In: The Arabidopsis Book. 2002, 1-22. Electronic Book ed. by American Society of Plant Biologists
78. Zhang J, Ngnyen H T, Blum A. Genetic analysis of osmotic adjustment in crop plants. J Exp Bot, 1999, 50(322): 291-302
79. Liu Q, Kasuga M, Sakuma Y et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 1998, 10: 1391-1406
80. Matsuoka D, Nanmori T, Sato K et al. Activation of AtMEK1, an Arobidopsis mitogen-activated protein kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings. Plant J, 2002, 29: 637-647
81. Tarczynski M C, Jensen R G, Bohnert H J. Stress protection of transgenic tobacco by production of the osmolyte mannitoi. Science, 1993, 259(22): 508-510
82.刘俊君,彭学贤,王海云等。转基因烟草的甘露醇合成和耐盐性。生物工程学报,1994,12(2):206-210
83. Kasuga M, Liu Q, Miura S et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnol, 1999, 17: 287-291
84.刘风华,孙仲序,崔德才等。细菌mtl-D基因的克隆及在转基因八里庄杨中的表达。遗传学报,2000,27(5):428-433
85.戴秀玉,吴大鹏,周坚。大肠杆菌海藻糖合成酶基因的克隆和表达。遗传学报,2000,27(2):158-164
86.戴秀玉,王忆琴.周坚等。大肠杆菌海藻糖的代谢调控。生物工程进展,2000,20(6):26-29
87. Lilius G, Holmberg N, Bulow L. Enhanced NaCl stress tolerance in transgenic tobacco expressing bacterial choline dehydrogenase. Bio/Technology, 1996, 14: 177-180
88. Apse M P, Aharon G S, Snedden W A et al. Salt tolerance conferred by overexpression of a vacuolar Na~+/H~+ antiport in Arabidopsis. Science, 1999, 285: 1256-1258
89. Holmstrom K O, Somersalo S, Mandal A et al. Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine, J Exp Bot, 2000, 51(343): 177-185
90. Zhang H X, Blumwald E. Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat. Biotechnol. 2001, 19: 765-768
91. Boncompagni E, Magne (?)ster(?)s, Poggi M C et al. Occurrence of choline and glycine betaine uptake and metabolism in the family Rhizobiaceae and their roles in osmoprotection. Appl Envir Microbio, 1999, 65(5): 2072-2077
92. Hayashi H, Alia Mustardy L et al. Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinbetaine and enhanced tolerance to salt and cold stress, Plant J, 1997,12: 133-142.
93. Takabe T, Hayashi Y, Tanaka A. Evaluation of glycinebetaine accumulation for stress tolerance in transgenic rice plants. Proceedings of International Workshop on Breeding and Biotechnology for Environmental Stress in Rice. 1998, Hokkaido Nationaal Agricultural Experiment Station and Japan International Science and Technology Exchange Center, Sapporo, Japan, P63-68
94.梁峥,骆爱玲,赵原等。干旱和盐胁迫诱导甜菜叶中的甜菜碱醛脱氢酶的积累。植物生理学报,1996,22(2):161-164
95. Huang J, Hirji R, Adam L et al. Genetic Engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations. Plant Physiol, 2001, 122: 747-756
96.刘凤华,郭岩,谷冬梅等。转甜菜碱醛脱氢酶基因植物的耐盐性研究。遗传学报,1997,24(1):54-58
97.郭岩,张莉,肖岗等。甜菜碱醛基脱氢酶基因在水稻中的表达及转基因植株的耐盐性研究。中国科学,1997,27(2):151-155
98.郭北海,张艳敏,李洪杰等。甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达。植物学报,2000,42(3):279-283
99.何锶洁,董伟,李慧芬等。转甜菜碱醛脱氢酶基因玉米及其耐盐性的研究。高技术通讯,1999,2:50-52
100.李银心,常凤启,杜立群等。转甜菜碱醛脱氢酶基因豆瓣菜的耐盐性。植物学报,2000,42(5):480-484
101.张慧,董伟,周骏马等。果聚糖蔗糖转移酶基因的克隆及耐盐转基因烟草的培育。生物工程学报,1998,14(2):181-186
102. Pilon-Smits E A, Terry N, Sears T et al. Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress. J Plant Physiol, 1998, 152: 525-532
103.王宝山,邹琦,赵可夫。液泡膜转运蛋白与植物耐盐性研究进展。植物学通报,1996,13(3):30-36
104. Assmann SM. From proton pump to proteome. Twenty-five years of research on ion transport in higher plants. Plant Physiol, 2001, 25: 139-141
105. Serrano R, Mulet J M, Rios G et al. A glimpse of the mechansms of ion homeostasis during salt stress. J Exp Bot, 1999, 50: 1023-1036
106. Martinoia E, Massouneau A, Frangne N. Transport processes of solutes across the vacuolar membrane of higher plants. Plant Cell Physiol, 2000, 41: 1175-1186
107. Nass R, Cunningham K W, Rao R. Intracellular sequestration of sodium by a novel Na~+/H~+ exchanger in yeast is enhanced by mutations in the plasma membrane H~+-ATPase. J Biol Chem, 1997, 272: 26145-26152
108. Gaxiola R A, Rao R, Sherman A et al. The Arabidopsis thaliana proton transporters, AtNHX1 and Avpl, can function in cation detoxification in yeast. Proc Natl Acad Sci USA, 1999, 96: 1480-1485
109. Hamada A, Shono M, Xia T et al. Isolation and characterization of a Na~+/H~+ antiporter gene from the halophyte Atriplex gmelin. Plant Mol Biol, 2001, 46: 35-42
110. Kiegerl S, Cardinale F, Siligan C et al. SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK. Plant Cell, 2000, 12: 2247-2258
111. Tena G, Asai T, Chiu W L et al. Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol, 2001, 4: 392-400
112. Zwerger K, Hirt H. Recent advances in plant MAP kinase signalling. Biol Chem, 2001, 382: 1123-1131
113. Zhang S, Klessig D F. MAPK cascades in plant defense signaling. Trends Plant Sci, 2001, 6: 520-527
114. Asai T, Tena G, Plotnikova J et al. MAP kinase signaling cascade in Arabidopsis innate immunity. Nature, 2002, 415: 977-983
115. Ren D, Yang H, Zhang S. Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis. J Biol Chem, 2002, 277: 559-565
116. Wang X. The role of phospholipase D in signaling cascades. Plant Physiol, 1999, 120: 645-652
117. Wang X. Molecular analysis of phospholipase D. Trends inplant, 1997, 2(7): 261-266
118. Munnik T, Irvine R F, Musgrave A. Phospholipid signaling in plants. B B A, 1998, 1389(3): 222-272
119. Munnik T, Harold J G M, Riet B et al. Hyperosmotic stress stimulates phospholipase D activity and elevates the levels of phosphatidic acid and diacylglycerol pyrophosphate. Plant J, 2000, 22(2): 147-154
120. Fan L, Zheng S, Cui D et al. Subcellular distribution and tissue expression of phospholipase D_α, D_β, and D_γ inArabidospsis. PlantPhysiol, 1999, 119: 1371-1378
121. Shi H and Zhu J K. SOS4, a pyridoxal kinase gene, is required for root hair development in Arabidopsis. Plant Physiol, 2002, 129: 585-593,
122. Shi H., Xiong L, Stevenson B et al. The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for vitamin B6 in plant salt tolerance. Plant Cell, 2002, 14: 575-588
123.梁峥,骆爱玲。甜菜碱和甜菜碱合成酶。植物生理学通讯,1995,31(1):1-8
124. Hall J L, Harvey D M, Flowers T J. Evidence for the cytoplasmic localization of betaine in leaf cells ofSuaeda maritima. Planta, 1978, 140: 59-6298.
125.杨传平,刘桂丰,梁宏伟等。耐盐基因betA转化小黑杨的研究。林业科学,2001,37(6):34-38
126. Hayashi A H, Sakamoto A et al. Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of glycinebetaine, Plant J, 1998, 16(2): 155-161
127. Sakamoto A, Murata N. The use of bacterial choline oxidase, a glycinebetaine synthesizing enzyme, to create stress-resistant transgenic plants. Plant Physiol, 2001, 125: 180-188
128. Wyn Jones R G, Storey R. Betaines. In: Paleg L G, Aspinall D (eds). The physiology and Biochemistry of Drought Resistance in Plants. Sydney: Academic press, 1981, P171-204
129. Wyn Jones RG, Storey R. Betaines. In: Paleg LG, Aspinall D (eds). The physiology and Biochemistry of Drought Resistance in Plants. Sydney: Academic press, 1981, 171-204.
130. Blumwald E, Aharaon G S, Apse M P. Sodium transport in plant cells. B B A, 2000, 1465: 140-51
131. Niu X, Bressan R, Hasegawa P M et al. Ion homeostasis in NaCl stress environments. Plant Physiol, 1995, 109: 735-742
132. Staal M, Maathuis F, Elzenga T, Overbeek J, Prins H. (1991) Na~+/H~+ antiport activity of the salt-tolerant Plantago maritime and the salt-sensitive Plantago media. Physiol. Plant. 82: 179-184
133. Barlda B J, Zingarelli L, Blumwald E et al. Tonoplast Na~+/H~+ antiport activity and its energization by the vacuolar H~+-ATPase in the halophytic plant Mesembryanthemum crystallimum L. Plant Physiol, 1995, 109: 549-556
134. Zhang H., Hodson J N, Williams J P et al. Engineering salt-tolerant Brassica plants: Characterization of yield and seed oil quality in transgenic plants with increased vacuolar sodium accumulation. PNAS, 2001, 98: 12832-12836.
135. Wang X, Xu L, Zheng L. Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L. J Biol Chem, 1994, 269: 20312-20317
136. Fan L, Zheng S, Wang X. M. Antisense suppression of phospholipase D retards abscisic acid and ethylene promoted senescence of postharvest arabidopsis leaves. Plant Cell, 1997, 9: 2183-2196
137. Zhou H E, Zheng S, Lu F et al. Low temperature tolerance in transgenic tobacco and Arabidopsis thaliana expressing sense and antisense phospholipase D gene. Plant physiol, 1997, 114(supplement): 128
138. Lee S H, Chae S H, Lee T K et al. Ethylene mediated phospholipid catabolism pathway in glucose-starved carrot suspension cells. Plant Physiol, 1998, 116: 223-229
139. Munnik T, Katja Kerkmann et al. Water deficit triggers phospholipase D activity in the resurrection plant Craterostigma plantagfneum. Plant Cell, 2000, 12: 111-123
140.刘斌,李红双,王其会等。反义磷脂酶D_γ基因转化毛白杨的研究。遗传,2002,24(1):40-44
141. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 1962, 15: 473-497
142. Gardiner J M, Coe E H, Melia-Hancock S et al. Development of a core RFLP map in maize using an immortalized F_2 population. Genetics, 1993, 134: 917-930
143. Salzman R A, Fujita T, Zhu-Salzman K et al. An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Molt Biol Rep,