System Optimization Of Isolated Microspore Culture And Screening Of Stress Tolerances To Salt And Low Nitrogen In Haploid Barley

In order to establish and optimize the practibility of microspore culture in barley improvement, in the present research, the effects of supplement of colchicines in the extacts, florets co-culture during the induction and the types and concentration of nitrogen complements were compared using barler cultivars or elite lines grown around the Shanghai area. The optimized system has significantly increased the green seedling production. To determine the relationship of Salt/Low nitrogen tolerances at the haploid level and the plant level, the grain yield and germination indexes of the plants grown under salt or low nitrogen stresses were compared with the callus yield of the microspres when cultured under the salt or low nitrogen stresses, and the results indicated their consistency for both traits. Based on the above results, mutants were screened for salt and low nitrogen tolerances, and materials with improved tolerances have been identified. The main results obtained were as following:1. The effect of colchicine concentration on the survival rate of microspores and embryoid induction rate. The effect of colchicine concentration on the survival rate of microspores and embryoid induction rate was investigated using barley varieties/elite lines which are adapted to the Shanghai and nearby barley growing areas. The supplement of10mg/L colchicines in the extract and pretreatment solution could both increase the survival rate of microspores and obviously reduce the death rate after being cultured for72h. However, the effects on different barley varieties/lines showed significant difference, e.g., the pretreatment was more effective on PDJ than on Shanghai No.8, and no obvious effect was observed for S22and Hua30. The colchicine supplement in the extract and pretreatment medium is especially important for those materials with low survival rates, such as PDJ, which had the lowest survival rate among the varieties/lines used in the present research. In addition, we also found that the supplement of colchicine in the extract and pretreatment improved the induction rate of ELS, promoted potential seedlings regeneration ability and significantly increased the green seedling differentiation rate. The inorganic salts in the the extract and pretreatment medium showed significant effects on the ELS production of the isolated microspore culture, and the extents of this effect was dependent on the barley genotype.2. The effect of co-culture with florets on isolated microspore culture. Three barley materials, i.e. SD1, A11and E2, were used for comparison of the effects of florets co-culture on the isolated microspore culture. The result indicated that after7d co-culture with florets the number of MCS were significantly increased, showing that the supplement of florets can promote the formation of MCS. We also observed more MCS and ELS in lines S23and SD1when co-cultured with florets from93A than with florets from themselves. Line93A is a tetraploid, we deduced that that the florets from tetraploid plant were more effective than ones from diploid plant for co-culture supplement. The combination of cold pretreatment of spikes and florets co-culture were further investigated by isolating microspores from SD1spikes which were cold treated for15d,20d or25d, and then co-cultured with florets from SD1and93A, respectively. The results showed that the effect of florets co-culture on the formation of both MCS and ELS was the most when cold pretreated for15d, and reduced as the pretreatment prolonged, and little effect was observed when pretreated for25d. Further comparison showed that when co-cultured with florets, the formation of ELS and the regeneration rate of green plants were better than all those controls, regardless the florets were taken from monokaryophase or dikaryophase, and and with or without cold pretreatment. Florets derived from tetraploid line93A were significantly more effective than those derived from diploid line S23, and produced the highest ELS (862.50±63.19) and green plant regeneration rate (43.00%). The treatment of microspores co-cultured with floret from materials which were cold treatmented for15days resulted in more ELS, however, no consistency result was for green plant regeneration rate. For both ELS yield and green plant regeneration rate, microspores co-cultured with monokaryophase florets were better.3. Nitrogen source and concentration on the callus yield and green seedling differentiation. A group of barley varieties/lines were used for the improvement of the callus yield and green seedling differentiation by supplement of different concentration of inorganic nitrogen (KNO3and (NH4)2SO4) and organic nitrogen (Glu and CH) in the induction medium. The results showed that callus yield could be significantly improved by reducing the inorganic nitrogen concentration and adding the Glu and CH. When the KNO3and (NH4)2SO4were present alone or together in the medium, the callus yield were very low for all the tested materials. However, when both the organic and inorganic nitrogen were present, regardless of KNO3or (NH4)SO4, the callus yield dramaticly increased. The callus yield for six of the eight materials significantly increased when the concentration of inorganic nitrogen reduced to1/2-1/4(KNO31415-707.5mg/L、(NH4)2SO4231.5115.5mg/L). The higher callus yields were also obtained by increasing the concentration of organic nitrogen (Glu and CH) from800mg/L to2000mg/L. The most optimized medium for callus formation was:1/4N6inorganic nitrogen (KNO3707.5mg/L.(NH4)2SO4115.5mg/L) combination with2000mg/L organic nitrogen. Little effect of organic nitrogen was observed on the green plantlet differentiation rate, the callus regeneration ability mostly depended on the genotype.4. The effects of concentration of organic nitrogen on the callus yield of microspore culture and the effects of nitrogen on the plant growth of barley. Four barley genotypes (Hua30, BR06-5, BI-45and BI-49) were uused to study:(1) the effects of organic nitrogen concentration on callus yield of microspore culture;(2) the effects of different concentration of NH4NO3on the growth of barley seedlings; and (3) the effects of normal N-supply and no N-supply on grain yields per plant in barley. The results showed that the decrease of nitrogen content in the medium significantly reduced the callus production of microspore culture. When the organic nitrogen concentration is reduced to400mg/L, callus production for all the materials decreased and there were significant differences among genotypes. Relative value of callus production can be divided into two types, one was BI-49(0.60) and BR06-5(0.58) and the other was Hua30(0.44) and BI-45(0.40). Various indexes, such as the seedling growth, plant height, main root length, and dry weight were severely inhibited under the nitrogen stress. Significant difference was found among genotypes, all the relative values of BI-49and BR06-5were more than those of Hua30and BI-45. Effective panicles and grain yield per plant of barley without N-supply were lower than the control of normal N-supply; the relative value was BI-49> BR06-5>BI-45> Hua30. There were also differences among different genotypes. It was found that the relative value of callus yield was consistent with that of index, such as plant height, main root length, and root dry weight in the plant level. So there is a link between the haploid cell level and plant level for lower nitrogen tolerance.5. The consistency among grain yield, indexes at germination and callus production stages under the salt tolerance. In order to determine the consistency among grain yield, indexes at germination and callus production stages under the salt tolerance, three independent experiments were carried out using2barley cultivars, i.e.(1) effects of NaCl concentration on callus production in microspore culture;(2) effects of NaCl concentration on the traits related to seed germination at germination stage; and (3) effects of NaCl stress on grain yield per plant. The results showed that the callus production was reduced with the increase in NaCl concentration in the induction medium, and there were significant differences between the two barley cultivars in the reduction range. It was observed that the microspore of Hua30was quite sensitive to NaCl and the callus production was seriously reduced from133.5mg/pan to78.0mg/pan even at0.1g/L NaCl, while callus production of Hua11was not sensitive in microspore culture with0.1～0.3g/L NaCl about208.8mg/pan-181.3mg/pan. Germination rate, main root and coleoptile length were also reduced with increase in NaCl concentration, and the two barley cultivars showed differences in these parameters. In the pot experiment, it was found that the grain yield per plant was obviously reduced under salt stress compared to the control. Similar differences were noticed between the two barley cultivars. Greater decline was recorded in the grain yield per plant of Hua30from3.7g to2.9g (63.7%) than of Hua11from4.4g to1.6g (21.6%). Therefore, the results showed that the relative callus production was closely associated with relative traits at the germination stage and relative value of grain yield per plant. It can be concluded that there is also consistency between microspore and plant levels for salt tolerance in barley.6. Salt tolerance mutants screen based on barley haploid technology. Microspores, spikes and dry seeds from Hua30were treated with mutagens including EMS and bleomycin, and60Co γ-rays irradiation, respectively. Mutants were screened at the induction and differentiation phases under the pressure of NaCl in the isolated microspore culture. The results showed that EMS treated microspores (1-5mg/L,48h) and60Co γ-rays irradiated dry seeds (dose rate1GY/min, dosage for400-500GY) were obviously superior to bleomycin treated microspores (1-5mg/L,48h) and60Co γ-rays irradiated spikes (dose rate1GY/min, dosge for5-15GY). Callus yield from EMS treated microspores was about123.71mg/dish on the medium with300mg/L NaCl, and the green plantlet yield was about35.51plants/100mg callus under3g/L NaCl stress on the regeneration medium. The callus yield from microspores derived from60Co γ-rays irradiated dry seeds were109.68mg/dish on the medium with300mg/L NaCl, and the green plantlet yield was17.14plants/100mg callus under3g/L NaCl stress. Obviously, the NaCl content can be increased to some extent in the induction medium, but3%NaCl content is too high to cause a decline of the green seedling differentiation. Thus, the NaCl concentration need to be further optimized in the microspore culture. A series of indexes such as seed germination rate, seedling survival rate, plant tiller number, plant height and yield per plant were compared using seeds from16regenerated plants by seeds irradiation under0.5%NaCl stress. Under this germination condition, the germination rate of Hua30is0, while the14plants were higher than Hua30and they are considered to be salt tolerance mutants. Among them,4materials were much more salt tolerant than Hua30. The expression patterns and quantity of Na+/H+antiporter gene (NHX1, NHX2and NHX3) and betaine aldehyde dehydrogenase gene (BBD1and BBD2) were studied and we found that the enhancement of salt tolerance were related to the changed expression pattern of these genes.7. Low nitrogen tolerance mutants screening based on barley haploid technology. In order to induce the low nitrogen tolerance mutants of barley variety Hua30, the effects on callus yield and the green seedlings differentiated from the microspores treated with EMS, bleomycin or the microspores from irradiated spikes and dry seeds were first investigated. After immersion in EMS for48h, callus yield obtained from isolated microspore of Hua30was82.35mg/dish when treated with1/10inorganic nitrogen,400mg/L Glu and CH. The green plantlet yield of the obtained callus was126.76plants/100mg callus in the differentiation medium with1/10inorganic nitrogen. Similarly, under the same nitrogen stress, callus yield from isolated microspore of Hua30after bleomycin treatment for48h, was only36.75mg/dish, and the green plantlet yield was10.49plants/100mg callus. Under the nitrogen stress with1/10inorganic nitrogen,400mg/L Glu and CH, the callus yield from isolated microspores derived from irradiated spike and dry seeds of Hua30were37.65mg/dish,67.08mg/dish, and the green plantlet yield was18.23/100mg callus,96.99/100mg callus, respectively. So, the callus yield from isolated microspores treated by EMS and isolated microspores derived from irradiated dry seeds by60Co y were obviously better than that from microspores treated by bleomycin and irradiated spike by60Co γ. Using the plant regeneration seeds obtained from EMS mutagenic microspores, the tiller number, ear number, plant height and yield per plant were compared under the normal and no fertilizer treatments. The results showed that, most of the obtained regenerated plants had higher tiller numbers, effective spikes and yield per plant than wildtype Hua30. Their self-fertilized progenies also showed increased yield, higher tiller number, plant height than wildtype Hua30. We found that the number of superior mutants were more than the deteriorate mutants, indicating that the the induction of mutation at the microspore level combined with nitrogen stress is an effective way to improve the nitrogen use efficiency.