Abstract:

Leymus chinensis (Trin.) Tzvel. is a perennial rhizomatous high quality species of the Poaceae family. It not only has high forage values and good palatability, but also has great tolerant to drought, cold and salt-alkaline conditions. This plant is a dominant grass species in Songnen Plain of Northern China, and also considered an ideal grass for rangeland use in the arid and semiarid regions. In recent years, with the development of animal husbandry and the great efforts on the ecological environment, artificial Leymus chinensis grasslands were constantly built. Therefore, the request on the quantity and quality of the seeds was also higher and higher. In this paper, we investigated seed development, dormancy characters and salt-alkaline tolerance of Leymus chinensis. On the one hand, we clearly determined the optimum seed harvest time of this species, providinga scientific basis for agricultural production and high quality seed harvest. On the other hand, we excavated the seed germinating potential, deeply analysed the stress tolerance mechanism, and provided a theoretical basis for the utilization rate and breeding new varieties of Leymus chinensis. The main results and conclusions in our experiments were listed as follows:
(1) Results of seed development of Leymus chinensis showed that, seed color changed from green to light green, yellow and heavy brown into the final along with the seed maturity. 1000-seed weight increased constantly, and reached highest at 33 d after peak anthesis. However, water content and electric conductivity of the seeds showed a declining trend, water content reached lowest at 36 d after peak anthesis, and the values of electric conductivity of the two experimental years were different, which reached minimum at 27 and 30 d after peak anthesis, respectively. The results of standard germination test showed that germination percentage was highest at 39 d after peak anthesis. At this time, germination starting days, 50% germination days and germination energy were all reached the optimal value. Although a slight difference was found between the standard germination test and accelerated aging test, seeds at 39 d after peak anthesis also had the strongest anti-aging capability. Above results shows that seeds at 39 d after peak anthesis have the highest vigor and best quality, and can be considered the optimum seed harvest time of Leymus chinensis.
(2) Response to burial depth and salt-alkaline stress of Leymus chinensis seeds at different maturation time were also greatly different. The ability of seedling emergence and growth was decreased with the increasing burial depth, and 1 cm was the most suitable planting depth. At this time, the seedling emergence rate was highest, time to seedling emergence was shortest, and the length and biomass of the shoot and root were also highest. In addition, the ability of seedling growth of the seeds at different maturation time was also different. Seeds at 39 d after peak anthesis had the highest vigor, the above index of seedling growth were optimum. Germination percentage and energy were significantly affected by seed maturation time, salt-alkaline stress and their interactions. Highest germination percentage and germination energy were occurred at 39 d after peak anthesis, especially at the highest salinity stress (400 mM). The recovery test showed that recovery percentage was also the highest at 39 d after peak anthesis. Above results show that although seeds at different maturation time have germination ability, the ability of seedling emergence and resistance to salt-alkaline stress is highest at 39 d after peak anthesis, which further support the view that 39 d after peak anthesis is the optimum seed harvest time of Leymus chinensis. In addition, shallow sowing is suitable for this species, and 1 cm is the most optimum planting depth.
(3) Artificial treatments can obviously break seed dormancy of Leymus chinensis. The results showed that many methods such as H₂SO₄, cold stratification, PEG, GA₃, KNO₃ and soaking in the water were all enhanced the germination percentage, germination rate, starting germination time, and 50% germination time. However, in production practice, both efficiency and economic cost should be considered, and combined with our results, we recommend the way of soaking seeds in water under lower temperature for 20 d in order to break seed dormancy and increase the germination percentage of Leymus chinensis.
(4) Lemmas are important factors inhibiting seed germination of Leymus chinensis, but it can also improve salt resistance of the seeds. We investigated the effects of lemmas on seed imbibition and dehydration, germination responses to various temperature regimes and the impact of different duration salt stress on seed germination. The results showed that lemmas could significantly enhance water absorption, and slowed down the dehydration rate under drought conditions. Lemmas, temperature regimes and their interactions significantly decreased germination percentage and germination rate, indicating that lemmas could inhibit germination process of Leymus chinensis. Under different duration of salt stress, recovery percentages of non-germinated seeds with lemmas were higher than that without lemmas, especially under long duration of salt stress, indicating that lemmas could enhance the salt resistance of the seed. Once precipitation and melting snow decreased salinity concentration in the soil, seeds with lemmas can geminate again.
(5) Germination percentages and rates were inhibited by either an increase or decrease temperature from the optimal temperature 20-30℃, and this temperature can alleviate the inhibitory effects of salt-alkaline stress on seed germination. With the increasing salinity and alkalinity, seed germination and germination rate were both decreased, and the reductions were much greater under alkaline stress. Under salt stress, when salinity < 200 mM, lower temperature was the main factor inhibiting seed germination. With the increasing salinity, higher temperature aggravated the adverse effects. While under alkali stress, germination percentage and germination rate were both decreased markedly at 25-35℃ even though the alkalinity was very low. Recovery percentage of non-germinated seeds increased with the increasing salinity, but increased at first and then declined under alkaline stress, and recovery percentages were lowest in both stresses at 25-35℃, especially under alkaline stress. Seedling growth had similar response to the interactions of temperature and salt-alkaline stress, 20-30℃ was also the optimum temperature. In addition, both of the two stresses inhibited root growth much stronger. Due to the above results, early July sowings in the field would be recommended, when temperature is appropriate and salinity-alkalinity concentrations are always reduced by the high rainfall.
(6) Under mixed salt-alkaline stresses, germination percentage and germination rate were decreased with the increasing salinity under all the stress treatments, and the reductions were greater under treatment which the proportion of alkaline salts was greater. At 250 mM salinity, germination percentage of treatment A that without alkalinity was 6.5%, but the other five treatments were 0. Seedling growth was also affected by salinity, pH and their interactions. However, radicle length decreased more markedly with the increasing salinity and pH. Stepwise regression analysis results showed that salinity was the dominant factor for seed germination under mixed salt-alkaline stress conditions. However, once radicl break through the seed coat, and pH changed into the dominant factor for seedling establishment. Above results indicates that mixed salt-alkaline stress has different impacts on germination and early seedling stages of Leymus chinensis. The interactions of high salinity and high pH have the strongest inhibition on seed germination and seedling growth.
(7) Both saline stress and alkaline stress were significantly decreased seedling length, fresh weight and water content, and the reductions were much greater under alkaline stress. The Na⁺ concentration, Na⁺/K⁺ ratio increased but K⁺ concentration decreased under both stresses, and the changes were greater under alkali stress. Under salt stress, shoots mainly accumulated Cl^- and little change was found in organic acids. While under alkali stress, the concentrations of Cl^-, NO₃^- and H₂PO₄^-♂ were all decreased, and organic acids were accumulated, especially malic acid and citric acid. In addition, soluble sugar was the same osmoregulation under the two stresses. Above results indicate that alkali stress inhibited early seedlings of Leymus chinensis much greater because of the high pH, different accumulation characters of Cl^- and organic acids indicate that different adaptive mechanism to saline and alkaline stresses are existing of Leymus chinensis during early seedling stage.

Key words: L. chinensis seeds, Seed dormancy, Salt-alkaline stress, Lemmas, Germination

摘要：

羊草(Leymus chinensis)是禾本科赖草属根茎型优质禾草,不但营养价值高、适口性好,而且具有较强的耐旱、耐寒和耐盐碱性。羊草既是东北松嫩平原的优势种,也是干旱与半干旱地区建植人工草地的优良草种。近年来,随着畜牧业的发展及生态环境治理力度的加大,人工羊草草地不断建植,为此,人们对羊草种子的需求量与品质的要求也越来越高。本文通过研究羊草种子的发育进程、休眠特性及与盐碱耐性的关系,一方面明确羊草种子的最适宜收获时间,为农业生产上收获高品质羊草种子提供科学依据,另一方面挖掘羊草种子的发芽潜能、深入解析抗逆机理,为提高其利用率及抗逆新品种的选育提供理论基础。主要研究结果与结论如下:(1)通过对羊草种子发育动态的研究结果表明,羊草种子在发育过程中,随着成熟度的不断提高,种子的颜色由绿色变为浅绿色,再变成黄色,最后变为棕黑色。种子千粒重不断增加,在盛花后33 d达到最大值,之后趋于恒定。含水量与种子浸出液电导率则呈下降趋势,含水量在盛花后36 d达到最小值,而2个试验年份电导率值有所差异,分别在盛花后27和30 d达到最小值。标准发芽试验结果显示,羊草种子在盛花后39 d发芽率最高,此时种子的开始发芽时间、50%种子发芽时间、发芽势等指标均为最优。尽管加速老化试验的发芽指标与标准发芽试验略有差异,但是盛花后39 d的种子同样具有最强的抗老化能力。上述试验结果表明,盛花后39 d羊草种子活力最高,品质最佳,是种子最适宜的收获时间。
(2)不同成熟度的羊草种子对土壤埋深与盐碱胁迫具有不同的响应方式。羊草种子出苗与其后的幼苗生长能力随着种埋深度的增加而降低,1 cm是最适宜的播种深度,此时的出苗率最高、出苗时间最短,并且叶片与根系长度与生物量最大;另外不同成熟度的羊草种子表现出不同的出土成苗能力,盛花后39 d的羊草种子活力最大,其上述各项幼苗生长指标均为最优。种子的成熟度与盐、碱胁迫及其交互作用显著影响羊草种子的发芽率与发芽势,盛花后39 d的羊草种子在胁迫下具有最高的发芽率与发芽势,特别是在高浓度(400 mM)盐胁迫下,尤为明显。复萌试验结果显示,盛花后39 d的羊草种子在盐碱胁迫(特别是高盐环境)解除后同样具有最高的发芽率。上述结果表明,尽管不同成熟度的羊草种子均具有发芽能力,但是盛花后39 d的羊草种子出苗及抵御盐碱胁迫伤害的能力最强,这也进一步支撑了39 d是羊草种子适宜收获时间这一结论。另外羊草适宜浅播,1 cm是其最适宜的播种深度。
(3)通过人工手段处理可以明显打破羊草种子的休眠特性。研究结果表明,除了热水浸种处理外,其余方法如浓硫酸、冷层积、PEG,GA₃,KNO₃及清水浸种均能一定程度上提高羊草种子的发芽率,发芽速率、开始发芽时间及50%种子发芽时间。但是在生产实际中,既要考虑高效性也要考虑经济耗费,结合本试验的研究结果,我们推荐在生产中采用低温浸种20 d的方法来打破羊草种子的休眠,提高其发芽率。
(4)稃是抑制羊草种子萌发的重要因素,但同时也一定程度提高了种子的抗盐性。通过测定稃对羊草种子吸水、脱水、不同温度条件下的发芽响应以及不同持续时间盐胁迫对种子发芽的影响,结果发现稃可以显著提高羊草种子的吸水量,并同时减缓种子在干旱环境下的脱水速率,使种子不会过度脱水而死亡。稃、不同温度处理及两者交互作用显著降低羊草种子的发芽率与发芽速率,表明稃对羊草种子萌发具有一定的抑制作用。在不同持续时间的盐胁迫处理下,未萌发的带稃种子复萌率均高于去稃种子,特别是在长时间及高盐胁迫下尤为明显,表明稃对羊草种子耐盐性起着重要的调节作用,一旦雨水、融雪等条件降低了土壤盐浓度,带稃种子就可以继续萌发出土。
(5)20~30℃是羊草种子最适宜的发芽温度,高温、低温均显著降低种子的发芽率与发芽速率,并且此温度可一定程度上减缓盐胁迫与碱胁迫对种子发芽的抑制效应。随着盐、碱胁迫浓度的增加,羊草种子发芽率与发芽速率均呈下降趋势,且在碱胁迫下的下降幅度更大。在盐胁迫下,当盐浓度<200 mM时,低温是影响种子萌发的主要因素,随着盐浓度的不断增加,高温则更加剧了盐胁迫对种子萌发的抑制作用;而在碱胁迫下,即使碱浓度较低,高温与其交互作用也大大加剧了对种子发芽的抑制。盐胁迫下未萌发的羊草种复萌率随盐浓度增加而增加,而在碱胁迫下则随着碱浓度增加呈先上升后下降的趋势,高浓度碱胁迫使羊草种子失去活力而死亡,并且碱胁迫下种子的复萌率低于盐胁迫,25~35℃同样不利于种子的复萌。幼苗生长对温度与盐碱胁迫交互作用的响应方式与发芽阶段相似,20~30℃同样是最适宜温度;另外,盐碱胁迫均对羊草幼苗根生长的抑制作用更强。基于以上研究结果,我们建议在初夏(7月上旬),高降雨过后,温度与土壤条件适宜的情况下进行播种,以提高羊草种子的发芽率,更好的达到恢复退化草地的效果。
(6)在混合盐碱胁迫下,羊草种子的发芽率与发芽速率均随着盐浓度的增加不断下降,且碱性盐比例越大下降越明显。在250 mM盐浓度下,无碱性盐的A组处理发芽率为6.5%,而其余5组处理发芽率均为0。羊草幼苗生长阶段同样受盐浓度、pH及2者交互作用影响,并且根系对胁迫伤害更敏感,所受抑制作用更强。逐级回归分析结果表明,在种子萌发阶段,盐浓度是羊草种子在混合盐碱胁迫下能否萌发的决定性因素,而一旦胚根突破种皮进入幼苗生长阶段,pH就转变为主导因素。上述研究表明,混合盐碱胁迫对羊草种子萌发与早期幼苗生长阶段的抑制机理有所不同,其中高盐浓度与高pH的交互作用对种子萌发与幼苗生长的抑制效应最强。
(7)盐胁迫与碱胁迫均显著降低羊草幼苗的长度、鲜重与含水量,且碱胁迫抑制作用更强。2种胁迫均造成羊草幼苗Na⁺浓度与Na⁺/K⁺升高,并且K⁺浓度下降,但是在碱胁迫下,Na⁺ 浓度、Na⁺/K⁺上升幅度与K⁺下降幅度均大于盐胁迫。另外,在盐胁迫下,羊草幼苗大量积累Cl^-,有机酸含量变化不大;而在碱胁迫下,Cl^-,NO₃^-与H₂PO₄^-均呈下降趋势,而有机酸则大量积累,其中苹果酸、柠檬酸是主要的有机酸组分,可溶性糖是羊草幼苗在两种胁迫下共同的渗透调节物质。上述结果表明,碱胁迫由于具有高pH,对羊草早期幼苗生长的抑制作用更强,Cl^-与有机酸积累特征的差异表明羊草早期幼苗在盐胁迫与碱胁迫下具有不同的适应策略。

关键词: 羊草种子, 发育动态, 种子休眠, 盐碱胁迫, 温度, 稃, 萌发, 幼苗生长