大麦游离小孢子培养技术的优化及单倍体耐盐、耐低氮胁迫筛选体系的建立
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摘要
为了使大麦小孢子培养技术更具有实用性,以大田种植的大麦品种/品系为供试材料,研究了提取液中添加秋水仙碱、诱导培养时采用完整小花共培养以及优化了诱导培养基中的氮含量(有机氮和无机氮),极大地提高了大麦游离小孢子培养的绿苗产量；为了确定大麦单倍体细胞水平与植株水平的耐盐/低氮性之间是否一致,研究了盐/低氮胁迫下大麦籽粒产量和萌发期生长指标(苗期植株生长量)与小孢子培养阶段的盐、/低氮胁迫下愈伤组织产量与对照之间的差异,单倍体细胞水平的耐盐/低氮性与植株水平的耐盐/低氮性是一致的；在此基础上,开展了小孢子水平诱变和盐/低氮胁迫筛选研究,利用小孢子培养技术快速获得了耐盐/低氮性优于原始品种的、纯合的变异体材料。主要研究结果如下：
为了提高大田种植材料的小孢子培养效果,以大田生长的、适合上海及周边地区种植的优良品种/品系为材料,研究了秋水仙碱对大麦离体小孢子活力以及胚状体诱导率的影响。提取液和预处理液中添加10mg/L的秋水仙碱不仅可以提高小孢子提取时的存活率,还能明显降低小孢子在培养72h后的死亡率,但小孢子存活率提高的幅度和小孢子死亡率下降的幅度在供试材料之间相差很大,品系PDJ的预处理效果好于沪麦8号,对S22和花30的处理效果不明显,由于PDJ在提取时的存活率最低,所以认为在提取液和预处理液中添加秋水仙碱对小孢子活力低的材料更为必要。提取液和预处理液中添加秋水仙碱还能提高胚状体的诱导率,促进胚状体的成苗潜力,绿苗分化率显著提高。提取液和预处理液中的无机盐成分对供试材料小孢子培养的胚状体产量存在明显的影响,但影响程度因基因型的不同而有所差异。
为了研究小花共培养对大麦离体小孢子培养的影响,进行了一下4个方面的研究。首先,以SD1、A11和E2为材料,小孢子培养时采用自生小花共培养,在培养7d时统计多细胞结构(MCS)的数量,结果表明共培养处理的多细胞结构数量明显高于对照,相对值都达到1.5,表明添加小花可以促进多细胞结构的形成。其次,研究了不同倍性小花共培养的效果,以S23和SD1为材料,共培养时分别采用自身小花和93A小花共培养来比较不同共培养材料对小孢子培养多细胞结构和胚状体形成的影响,采用93A小花共培养时形成多细胞结构和胚状体的数量多于采用自身小花共培养的,由于93A是个四倍体材料,因而推测采用四倍体材料的共培养效果优于采用二倍体的。第三,研究了小孢子培养材料低温预处理时间对共培养效果的影响,以SD1为供试材料,离体穗经15、20和25天低温预处理后,收集小孢子进行培养,培养时分别添加SD1和93A的小花进行共培养,统计多细胞结构和胚状体数量,小花共培养的效果在低温预处理15天时最为明显,随着低温预处理时间的延长而有所削弱,在低温预处理达到25天时共培养的效果已不明显。第四,进一步比较了共培养材料小花的发育时期和低温预处理时间对供试材料S23游离小孢子培养中胚状体形成和绿苗率的影响,结果表明,不管小花取自单核或双核期,也不管是否经过低温预处理,小花共培养的所有结果皆优于对照；93A小花共培养的效果又优于S23的,获得了最高的胚状体产量(每个培养皿的胚状体数为862.50±63.19)和最高的绿苗分化率(43.00%)；用经低温处理15天的小花共培养得到了比未经低温处理的更多的胚状体产量,但在绿苗频率上表现并不一致；单核期小花共培养的胚状体产量和绿苗率优于双核期的。从胚状体产量上看,单核期小花的培养效果优于双核期的,小花低温预处理后的效果也明显优于未经低温预处理的。从绿苗率上看,大致也是单核期小花的共培养效果比双核期的好,但小花低温预处理的效果不明显,且略有下降。
为了进一步提高供试材料的愈伤组织产量,优化了诱导培养基中的氮源,以KN03和(NH4)2SO4作为无机氮,谷氨酰胺(Glu)和水解干酪素(CH)作为有机氮,比较了诱导培养基中无机氮和有机氮浓度对一批大麦品种/品系小孢子培养愈伤组织产量和绿苗分化的影响。结果表明降低诱导培养基中无机氮浓度、添加Glu和CH可以显著地提高愈伤组织产量。培养基中KN03和(NH4)2SO4单独或共同存在时,所有供试材料的愈伤组织产量都很低：无论是KNO3还是(NH4)2SO4,只要和有机氮同时存在,愈伤组织产量就会显著提高；降低培养基中无机氮浓度至原含量的1/2-1/4(KN031415.0-707.5mg/L、(NH4)2SO4231.5-115.5mg/L),8份供试材料中有6份的愈伤组织产量明显提高；培养基中有机氮浓度(Glu和CH)从各800mg/L提高至2000mg/L,所有供试材料的愈伤组织产量随着有机氮浓度的增加而提高；1/4N6的无机氮(KN03707.5mg/L、(NH4)2SO4115.5mg/L)结合2000mg/L有机氮(Glu和CH)的使用最适宜于愈伤组织的形成。研究还表明有机氮的浓度对绿苗分化率影响不大,诱导培养基中有机氮浓度尽管对愈伤组织产量有着非常明显的影响,但对愈伤组织分化成苗能力的影响较小,愈伤组织的再生能力在很大程度上取决于基因型。
为了确定低氮胁迫下大麦籽粒产量和植株苗期生长量与小孢子培养阶段低氮胁迫下愈伤组织产量在供试品种内变化趋势是否一致,以4份(花-30、BR06-5、BI-45和BI-49)大麦品种／品系为供试材料,研究了培养基中有机氮使用量对小孢子培养愈伤组织产量、营养液中NH4NO3不同添加量对大麦苗期生长量以及盆栽时正常施氮与不施氮处理对大麦单株产量的影响。培养基中有机氮含量的下降明显降低小孢子培养愈伤组织产量,当培养基中有机氮浓度降至400mg/L时,所有供试材料愈伤组织产量极显著下降,不同基因型间存在明显的差异,4个供试材料愈伤产量的相对值明显分成二类,BI-49和BR06-5为一类,相对值为0.60和0.58；花-30和BI-45则为另一类,相对值为0.44和0.40。营养液中的氮素胁迫严重抑制供试材料苗期的生长,降低植株高度、主根长度和植株、根干重,不同基因型之间存在明显的差异,BI-49和BR06-5的所有苗期统计指标的相对值都大于花-30和BI-45。盆栽时不施氮处理的大麦有效穗和单株产量低于正常施氮的对照,不同基因型间有差异,有效穗和单株产量的相对值都是BI-49>BR06-5>BI-45>花-30。氮素胁迫下,4个供试材料小孢子培养愈伤组织产量的相对值与苗期植株高度、茎叶干重、主根长度和根干重的相对值以及单株产量相对值的变化在品种内具有相同的趋势,证实了单倍体细胞水平与植株水平的耐低氮性之间的相关性。
为了确定盐胁迫下大麦籽粒产量和萌发期生长指标与小孢子培养阶段的盐胁迫下愈伤组织产量在供试品种内变化趋势是否一致性,以2份(花11和花30)大麦品种为供试材料,研究了诱导培养基中NaCl含量对小孢子培养愈伤组织产量的影响、萌发液中NaCl含量对大麦种子萌发期生长指标的影响和NaCl胁迫处理对大麦单株产量的影响。结果表明,诱导培养基中NaCl含量提高可降低小孢子培养愈伤组织产量,但2份品种的降幅存在明显的差异,花30小孢子培养过程对NaCl相当敏感,诱导培养基中NaCl含量达到0.1g/L时就严重影响愈伤组织的形成,愈伤组织产量从133.5mg/皿下降至78.0mg/皿；而花11的小孢子则对诱导培养基中0.1-0.3g/LNaCl不敏感,愈伤组织产量在208.8mg/皿-181.3mg/皿,没有明显下降。萌发液中NaCl含量提高可降低种子发芽率、主根长度和胚芽鞘长度,2份品种间降幅上也存在明显的差异,花30的下降幅度大于花11。盆栽条件下NaCl胁迫处理的大麦单株产量明显低于无NaCl的对照,2份品种间差异显著,花11的单株籽粒产量从3.7g下降至2.9g,下降幅度为21.6%,花30的单株籽粒产量从4.4g下降至1.6g,下降幅度高达63.7%,花30的下降幅度明显大于花11。NaCl胁迫下,2份供试材料小孢子培养愈伤组织产量的相对值与萌发期的相对性状以及成熟期单株产量的相对值变化趋势品种内是相同的,品种间是有差异的。
进行了利用大麦单倍体技术诱导筛选耐盐变异体研究。以EMS、平阳霉素和60Coγ-射线作为诱变剂,诱变花30的离体小孢子、离体穗和干种子,在小孢子培养的诱导和分化阶段进行NaCl的胁迫培养和筛选。结果表明,EMS诱变离体小孢子(1-5mg/L/48h)和60Co γ-射线辐照干种子(剂量率1GY/min、剂量为400-500GY)诱变后小孢子培养效果明显优于平阳霉素诱变小孢子(1-5mg/L、48h)和60Co γ-射线辐照离体穗(剂量率1GY/min、剂量为5-15GY)。在诱变剂和诱变方法适宜的条件下(EMS诱变小孢子和60Co γ-射线辐照干种子),胁迫筛选压的确立对获得再生植株的数量至关重要。EMS诱变后在300mg/L NaCl胁迫下愈伤产量为123.71mg/皿,这种愈伤组织在含3g/L NaCl的胁迫分化培养基上绿苗产量为35.51株/100mmg愈伤组织。花30干种子60Co γ-射线辐照后的植株的小孢子在300mg/L NaCl胁迫下愈伤产量为109.68m/皿,愈伤组织在含3g/L NaCl的胁迫分化培养基上绿苗产量为17.14株/100mg。很明显诱导培养基中NaCl含量还可以提高些,但分化培养基中的3%NaCl含量太高了,导致绿苗分化率下降太多,所以本试验获得的耐盐变异体数量太少。因而小孢子培养中NaCl浓度和氮胁迫浓度仍需进一步优化。以16份源于种子辐照处理的再生植株的自交一代种子为供试材料,比较了在0.5%NaCl胁迫下种子的发芽率和幼苗的成活率以及植株的分蘖数、株高和单株产量,花30发芽率为0,供试的16份耐盐变异体中,有14份材料NaCl胁迫下的发芽率优于花30,鉴定出4份耐盐性明显优于花30的变异体材料。选择耐盐变异体作为供试材料,测定了变异体中Na+/H+逆向转运蛋白基因NHX1、NHX2和NHX3和甜菜碱醛脱氢酶基因BBD1和BBD2的表达模式和表达量,结果表明变异体耐盐性的提高与这些基因的表达量存在联系。
进行了利用大麦单倍体技术诱导筛选耐低氮变异体研究。以大麦品种花30作为供试材料,比较了EMS和平阳霉素处理小孢子,60Coγ-射线辐照处理离体穗和干种子,对游离小孢子在氮胁迫培养下诱导的愈伤组织产量和愈伤组织在氮胁迫下分化的绿苗数量的影响。花30的离体小孢子经过EMS处理48h后在1/10无机氮、Glu和CH400mg/L的氮胁迫下愈伤产量为82.35mg/皿,这种愈伤组织在含1/10无机氮的胁迫分化培养基上绿苗产量为126.76株/100mg愈伤。花30的离体小孢子经过平阳霉素处理48h后在1/10无机氮、Glu和CH400mg/L的氮胁迫下愈伤产量只有36.75mg/皿,这种愈伤组织在含1/10无机氮的胁迫分化培养基上绿苗产量为10.49株/100mg愈伤。花30离体穗经过60Co γ-射线辐照后小孢子在1/10无机氮、Glu和CH400mg/L的氮胁迫下愈伤产量为37.65mg/皿,这种愈伤组织在含1/10无机氮的胁迫分化培养基上绿苗产量为18.23株/100mg愈伤。花30干种子60Co γ-射线辐照后的植株的小孢子在1/10无机氮、Glu和CH400mg/L的氮胁迫下愈伤产量为67.08mg/皿,这种愈伤组织在含1/10无机氮的胁迫分化培养基上绿苗分化率为96.99株/100mg愈伤。EMS处理离体小孢子和60Co γ-射线辐照干种子的愈伤组织产量和绿苗数量明显优于平阳霉素处理小孢子和60Co γ-射线辐照离体穗。以140份源于EMS诱变小孢子后培养获得的再生植株自交一代种子为供试材料,播种后采用正常施肥和不施肥两种处理,比较了变异体材料的分蘖数、成穗数、株高和单株产量。结果表明,大部分自交一代材料的分蘖数、有效穗和单株产量均高于原始品种花30。变异体自交一代中的产量性状以及与产量性状相关的一些性状如分蘖数、胁迫下的株高优于原始品种花30的变异体数量远远多于产量性状劣于花30的变异体数量,即发生正向变异材料的数量远远多于负向变异材料的数量,表明小孢子水平的诱变和氮胁迫筛选是有效的,提高了自交一代植株的氮素利用率。
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.
为了提高大田种植材料的小孢子培养效果,以大田生长的、适合上海及周边地区种植的优良品种/品系为材料,研究了秋水仙碱对大麦离体小孢子活力以及胚状体诱导率的影响。提取液和预处理液中添加10mg/L的秋水仙碱不仅可以提高小孢子提取时的存活率,还能明显降低小孢子在培养72h后的死亡率,但小孢子存活率提高的幅度和小孢子死亡率下降的幅度在供试材料之间相差很大,品系PDJ的预处理效果好于沪麦8号,对S22和花30的处理效果不明显,由于PDJ在提取时的存活率最低,所以认为在提取液和预处理液中添加秋水仙碱对小孢子活力低的材料更为必要。提取液和预处理液中添加秋水仙碱还能提高胚状体的诱导率,促进胚状体的成苗潜力,绿苗分化率显著提高。提取液和预处理液中的无机盐成分对供试材料小孢子培养的胚状体产量存在明显的影响,但影响程度因基因型的不同而有所差异。
为了研究小花共培养对大麦离体小孢子培养的影响,进行了一下4个方面的研究。首先,以SD1、A11和E2为材料,小孢子培养时采用自生小花共培养,在培养7d时统计多细胞结构(MCS)的数量,结果表明共培养处理的多细胞结构数量明显高于对照,相对值都达到1.5,表明添加小花可以促进多细胞结构的形成。其次,研究了不同倍性小花共培养的效果,以S23和SD1为材料,共培养时分别采用自身小花和93A小花共培养来比较不同共培养材料对小孢子培养多细胞结构和胚状体形成的影响,采用93A小花共培养时形成多细胞结构和胚状体的数量多于采用自身小花共培养的,由于93A是个四倍体材料,因而推测采用四倍体材料的共培养效果优于采用二倍体的。第三,研究了小孢子培养材料低温预处理时间对共培养效果的影响,以SD1为供试材料,离体穗经15、20和25天低温预处理后,收集小孢子进行培养,培养时分别添加SD1和93A的小花进行共培养,统计多细胞结构和胚状体数量,小花共培养的效果在低温预处理15天时最为明显,随着低温预处理时间的延长而有所削弱,在低温预处理达到25天时共培养的效果已不明显。第四,进一步比较了共培养材料小花的发育时期和低温预处理时间对供试材料S23游离小孢子培养中胚状体形成和绿苗率的影响,结果表明,不管小花取自单核或双核期,也不管是否经过低温预处理,小花共培养的所有结果皆优于对照；93A小花共培养的效果又优于S23的,获得了最高的胚状体产量(每个培养皿的胚状体数为862.50±63.19)和最高的绿苗分化率(43.00%)；用经低温处理15天的小花共培养得到了比未经低温处理的更多的胚状体产量,但在绿苗频率上表现并不一致；单核期小花共培养的胚状体产量和绿苗率优于双核期的。从胚状体产量上看,单核期小花的培养效果优于双核期的,小花低温预处理后的效果也明显优于未经低温预处理的。从绿苗率上看,大致也是单核期小花的共培养效果比双核期的好,但小花低温预处理的效果不明显,且略有下降。
为了进一步提高供试材料的愈伤组织产量,优化了诱导培养基中的氮源,以KN03和(NH4)2SO4作为无机氮,谷氨酰胺(Glu)和水解干酪素(CH)作为有机氮,比较了诱导培养基中无机氮和有机氮浓度对一批大麦品种/品系小孢子培养愈伤组织产量和绿苗分化的影响。结果表明降低诱导培养基中无机氮浓度、添加Glu和CH可以显著地提高愈伤组织产量。培养基中KN03和(NH4)2SO4单独或共同存在时,所有供试材料的愈伤组织产量都很低：无论是KNO3还是(NH4)2SO4,只要和有机氮同时存在,愈伤组织产量就会显著提高；降低培养基中无机氮浓度至原含量的1/2-1/4(KN031415.0-707.5mg/L、(NH4)2SO4231.5-115.5mg/L),8份供试材料中有6份的愈伤组织产量明显提高；培养基中有机氮浓度(Glu和CH)从各800mg/L提高至2000mg/L,所有供试材料的愈伤组织产量随着有机氮浓度的增加而提高；1/4N6的无机氮(KN03707.5mg/L、(NH4)2SO4115.5mg/L)结合2000mg/L有机氮(Glu和CH)的使用最适宜于愈伤组织的形成。研究还表明有机氮的浓度对绿苗分化率影响不大,诱导培养基中有机氮浓度尽管对愈伤组织产量有着非常明显的影响,但对愈伤组织分化成苗能力的影响较小,愈伤组织的再生能力在很大程度上取决于基因型。
为了确定低氮胁迫下大麦籽粒产量和植株苗期生长量与小孢子培养阶段低氮胁迫下愈伤组织产量在供试品种内变化趋势是否一致,以4份(花-30、BR06-5、BI-45和BI-49)大麦品种／品系为供试材料,研究了培养基中有机氮使用量对小孢子培养愈伤组织产量、营养液中NH4NO3不同添加量对大麦苗期生长量以及盆栽时正常施氮与不施氮处理对大麦单株产量的影响。培养基中有机氮含量的下降明显降低小孢子培养愈伤组织产量,当培养基中有机氮浓度降至400mg/L时,所有供试材料愈伤组织产量极显著下降,不同基因型间存在明显的差异,4个供试材料愈伤产量的相对值明显分成二类,BI-49和BR06-5为一类,相对值为0.60和0.58；花-30和BI-45则为另一类,相对值为0.44和0.40。营养液中的氮素胁迫严重抑制供试材料苗期的生长,降低植株高度、主根长度和植株、根干重,不同基因型之间存在明显的差异,BI-49和BR06-5的所有苗期统计指标的相对值都大于花-30和BI-45。盆栽时不施氮处理的大麦有效穗和单株产量低于正常施氮的对照,不同基因型间有差异,有效穗和单株产量的相对值都是BI-49>BR06-5>BI-45>花-30。氮素胁迫下,4个供试材料小孢子培养愈伤组织产量的相对值与苗期植株高度、茎叶干重、主根长度和根干重的相对值以及单株产量相对值的变化在品种内具有相同的趋势,证实了单倍体细胞水平与植株水平的耐低氮性之间的相关性。
为了确定盐胁迫下大麦籽粒产量和萌发期生长指标与小孢子培养阶段的盐胁迫下愈伤组织产量在供试品种内变化趋势是否一致性,以2份(花11和花30)大麦品种为供试材料,研究了诱导培养基中NaCl含量对小孢子培养愈伤组织产量的影响、萌发液中NaCl含量对大麦种子萌发期生长指标的影响和NaCl胁迫处理对大麦单株产量的影响。结果表明,诱导培养基中NaCl含量提高可降低小孢子培养愈伤组织产量,但2份品种的降幅存在明显的差异,花30小孢子培养过程对NaCl相当敏感,诱导培养基中NaCl含量达到0.1g/L时就严重影响愈伤组织的形成,愈伤组织产量从133.5mg/皿下降至78.0mg/皿；而花11的小孢子则对诱导培养基中0.1-0.3g/LNaCl不敏感,愈伤组织产量在208.8mg/皿-181.3mg/皿,没有明显下降。萌发液中NaCl含量提高可降低种子发芽率、主根长度和胚芽鞘长度,2份品种间降幅上也存在明显的差异,花30的下降幅度大于花11。盆栽条件下NaCl胁迫处理的大麦单株产量明显低于无NaCl的对照,2份品种间差异显著,花11的单株籽粒产量从3.7g下降至2.9g,下降幅度为21.6%,花30的单株籽粒产量从4.4g下降至1.6g,下降幅度高达63.7%,花30的下降幅度明显大于花11。NaCl胁迫下,2份供试材料小孢子培养愈伤组织产量的相对值与萌发期的相对性状以及成熟期单株产量的相对值变化趋势品种内是相同的,品种间是有差异的。
进行了利用大麦单倍体技术诱导筛选耐盐变异体研究。以EMS、平阳霉素和60Coγ-射线作为诱变剂,诱变花30的离体小孢子、离体穗和干种子,在小孢子培养的诱导和分化阶段进行NaCl的胁迫培养和筛选。结果表明,EMS诱变离体小孢子(1-5mg/L/48h)和60Co γ-射线辐照干种子(剂量率1GY/min、剂量为400-500GY)诱变后小孢子培养效果明显优于平阳霉素诱变小孢子(1-5mg/L、48h)和60Co γ-射线辐照离体穗(剂量率1GY/min、剂量为5-15GY)。在诱变剂和诱变方法适宜的条件下(EMS诱变小孢子和60Co γ-射线辐照干种子),胁迫筛选压的确立对获得再生植株的数量至关重要。EMS诱变后在300mg/L NaCl胁迫下愈伤产量为123.71mg/皿,这种愈伤组织在含3g/L NaCl的胁迫分化培养基上绿苗产量为35.51株/100mmg愈伤组织。花30干种子60Co γ-射线辐照后的植株的小孢子在300mg/L NaCl胁迫下愈伤产量为109.68m/皿,愈伤组织在含3g/L NaCl的胁迫分化培养基上绿苗产量为17.14株/100mg。很明显诱导培养基中NaCl含量还可以提高些,但分化培养基中的3%NaCl含量太高了,导致绿苗分化率下降太多,所以本试验获得的耐盐变异体数量太少。因而小孢子培养中NaCl浓度和氮胁迫浓度仍需进一步优化。以16份源于种子辐照处理的再生植株的自交一代种子为供试材料,比较了在0.5%NaCl胁迫下种子的发芽率和幼苗的成活率以及植株的分蘖数、株高和单株产量,花30发芽率为0,供试的16份耐盐变异体中,有14份材料NaCl胁迫下的发芽率优于花30,鉴定出4份耐盐性明显优于花30的变异体材料。选择耐盐变异体作为供试材料,测定了变异体中Na+/H+逆向转运蛋白基因NHX1、NHX2和NHX3和甜菜碱醛脱氢酶基因BBD1和BBD2的表达模式和表达量,结果表明变异体耐盐性的提高与这些基因的表达量存在联系。
进行了利用大麦单倍体技术诱导筛选耐低氮变异体研究。以大麦品种花30作为供试材料,比较了EMS和平阳霉素处理小孢子,60Coγ-射线辐照处理离体穗和干种子,对游离小孢子在氮胁迫培养下诱导的愈伤组织产量和愈伤组织在氮胁迫下分化的绿苗数量的影响。花30的离体小孢子经过EMS处理48h后在1/10无机氮、Glu和CH400mg/L的氮胁迫下愈伤产量为82.35mg/皿,这种愈伤组织在含1/10无机氮的胁迫分化培养基上绿苗产量为126.76株/100mg愈伤。花30的离体小孢子经过平阳霉素处理48h后在1/10无机氮、Glu和CH400mg/L的氮胁迫下愈伤产量只有36.75mg/皿,这种愈伤组织在含1/10无机氮的胁迫分化培养基上绿苗产量为10.49株/100mg愈伤。花30离体穗经过60Co γ-射线辐照后小孢子在1/10无机氮、Glu和CH400mg/L的氮胁迫下愈伤产量为37.65mg/皿,这种愈伤组织在含1/10无机氮的胁迫分化培养基上绿苗产量为18.23株/100mg愈伤。花30干种子60Co γ-射线辐照后的植株的小孢子在1/10无机氮、Glu和CH400mg/L的氮胁迫下愈伤产量为67.08mg/皿,这种愈伤组织在含1/10无机氮的胁迫分化培养基上绿苗分化率为96.99株/100mg愈伤。EMS处理离体小孢子和60Co γ-射线辐照干种子的愈伤组织产量和绿苗数量明显优于平阳霉素处理小孢子和60Co γ-射线辐照离体穗。以140份源于EMS诱变小孢子后培养获得的再生植株自交一代种子为供试材料,播种后采用正常施肥和不施肥两种处理,比较了变异体材料的分蘖数、成穗数、株高和单株产量。结果表明,大部分自交一代材料的分蘖数、有效穗和单株产量均高于原始品种花30。变异体自交一代中的产量性状以及与产量性状相关的一些性状如分蘖数、胁迫下的株高优于原始品种花30的变异体数量远远多于产量性状劣于花30的变异体数量,即发生正向变异材料的数量远远多于负向变异材料的数量,表明小孢子水平的诱变和氮胁迫筛选是有效的,提高了自交一代植株的氮素利用率。
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.
引文
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