Breeding Salt-Tolerant Materials With Elite Agronomic Traits For Beta Vulgaris L. By Bioengineering Techniques

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 ABAand 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 ⁶⁰Co- 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. Thesurvival 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, andwere 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...