Primary Studies on Salt Tolerance of Canola'Nanyanyou1'and Its Transformation System
|School||Nanjing Agricultural College|
|Keywords||Brassica napus L. salt stress salt resistance seed germination regeneration Agrobacterium mediation|
Canola ’Nanyanyou1’(Brassica napus L.) was as the experimental material in this study, setting5(0,50,100,200and300mmol·L-1) NaCl treatment density. The experiments were studied on salt resistance of seed emergence and seedlings. In addition, using hypocotyl and the cotyledon with petiole for tissue culture studies in ’Nanyanyou1’, and optimizing its regeneration system, making sure the more suitable transgenic explant were carried in this study. Further, using hypocotyl as an explant, by Agrobacterium-mediated method and importing Na+/H+antiporter gene of Aeluropus sinensis (AINHX1) into ’Nanyanyou1’were also studied in this study. The results and conclusions showed that;1. Germination rate of canola seed was reduced with increasing NaCl concentration, and the seeds can hardly germinated under300mmol·L-1NaCl. However, after relieving stresses, seed germination rates were all increased significantly at varing degrees. Relative germination potential, relative germination rate, relative germination index, germination rate after relieving stress, and simplified vigor index of the canola seeds all presented a highly negative correlation with the NaCl concentration. The germination process and the relative water absorption of canola seeds had a close relationship with the level of the salt concentration; With the increase of NaCl concentration, the germinating of seeds was reduced in uniformity, and germination of time is also delayed. After all, salt stress significantly inhibited canola germination.2. Our objective was to reveal the relationship between photosynthetic traits and salt tolerance of canola (Brassica napus L.). We investigated the effects of different concentrations of NaCl (0,50,100,200,300mmol·L-1) on plant growth, photosynthetic pigment and photosynthesis trait in canola seedlings for10and30days. The results showed that plant dry weight was decreased significantly under NaCl stresses, especially under high salt stress and long time treatment. As NaCl concentration increased, chlorophyll (Chl) content and Chl a/Chl b ratio were ascended in first and descended followed, however, under treatment for10and30d, Chl content and Chl a/Chl b ratio both achieved to the highest values at the treatment of200mmol·L-1NaCl and300mmol·L-1NaCl. Pn, Ci and Ls in canola leaves grown under50and100mmol-L·1NaCl stress remains unaffected. When the NaCl concentration increased, Pn, Gs, Ci and Tr were markedly decreased, WUE and Ls were significantly increased. Correlation analysis indicated that the growth of canola was insignificantly related to Chl content and Chl a/Chl b ratio, very negatively related to Na, Cl content, WUE and Ls, and very positively related to R/S, K, Ca content, K/Na, Ca/Na ratio, SK.Na, Sca,Na, Pn, Gs, Ci and Tr, respectively. Our results implicate that stomatal limitation to canola photosynthesis was mainly affected by200mmol·L-1NaCl for10and30d, and300mmol-L"1NaCl for10d, while the reduction in its assimilation was due to both stomatal and non-stomatal limitations under300mmol·L-1NaCl for30d. Na, Cl, K, Ca content, WUE, Ls, R/S, K/Na, Ca/Na ratio, SK,Na, SCa,Na, Pn, Gs, Ci and Tr can be regarded as value indexes of salt adaptation of canola.3. Under salt stress, the ion distributed unevenly in various organs. With the increase in stress level and time, salt ion (Na+and Cl-) content in organs was increased, and K+and Ca2+content, Sk,Na and SCa,Na content were declined. N and P content distribution in the organs under salt stress was different. With the increase of stress concentration, N and P contents showed first increase and then decrease. Proline content of the canola plant was increased obviously under salt stress. Soluble sugar content in various organs of the canola, first increased and then decreased as the NaCl concentration increased, and in200mmol·L-1, all reached maximum. The content of soluble sugar in petioles was relatively higher than that in leaves and roots. In short, under NaCl stress, canola seedlings can maintain good ionic homeostasis, high N, P, Proline and soluble sugar content.4. The plant tissue culture was tried in the experiment, in which hypocotyl and the cotyledon with petiole of’Nanyanyou1’(Brassica napus L.) were used as materials. Effects of cross combination of the different pre-culture and differentiation culture on the canola plant regeneration were studied. The results showed that different concentrations of2,4-D,6-BA and AgNO3had varing degree of influences on explant differentiation, however, differentiated buds were all able to take root and grow into plant. The differentiation rate of hypocotyl achieved the peak value (95.23%) in the pre-culture (MS+0.5mg·L-12,4-D+1.0mg·L-16-BA) and differentiation (MS+4.5mg·L-16-BA+0.1mg·L-1NAA+5.0mg·L-1AgNO3); the cotyledon with petiole achieved the peak value (89.52%) in pre-culture (MS+0.5mg·L-12,4-D+1.0mg·L-16-BA) and differentiation (MS+4.5mg·L-16-BA+0.1mg·L-1NAA+5.0mg·L-1AgNO3). To sum up, compared with the cotyledon with petiole, hypocotyl may be more suitable as the explant in tissue culture research of’Nanyanyou1’.5. Agrobacterium culture resuspending in’MS+AS+Antibiotics’ medium for24h, then continuing to foster in’MS+AS’ medium for several hours gave the best transformation result. Agrobacterium was completely suppressed in the medium containing700mg·L-1carbenicillin, and such concentration antibiotics can also benefit explant growth. The medium containing15mg·L-1Kan that is more suitable as a screening concentration.