濒危植物三棱栎的种质保存及驯化生物学研究
郑艳玲
学位类型博士
导师李唯奇
2010-05
学位授予单位中国科学院研究生院
学位专业植物学
摘要本文对濒危植物三棱栎(Trigonobalanus doichangensis)的大孢子发生和雌配子体发育、种子形态与萌发、种子贮藏特性、离体培养及驯化生物学特性进行了研究。结合已发表的细胞学及分子遗传学等方面的研究结果,探讨了三棱栎种质保存与引种驯化的基础生物学及技术问题,为该植物种质保存和持续利用提供理论依据及技术指导。主要研究结果如下:1. 大孢子发生和雌配子体发育:三棱栎的柱头下方位置着生有雄蕊,在雌蕊发育后期,此花粉粒败育,因此,功能上的雌性花是形态上的两性花。胚珠倒生,双珠被,厚珠心,单孢原,大孢子四分体排列呈直线型,其中只有合点端一个发育成功能大孢子,雌配子体发育为蓼型。三棱栎子房中有6枚胚珠,发育正常的果实中会有1枚发育成种子,在三棱栎的大孢子发生和雌配子体发育过程中,有多个败育环节,93.3%的成熟胚囊都是败育的。2. 种子形态与萌发特征:三棱栎种子的形态指标(长、宽和千粒重)和萌发相关指标(萌发率、萌发指数和活力指数)的大部分变异都来源于居群内单株间的变异,而有胚种子百分数在居群间和居群内变异量相当,各占48%左右。以上指标在年间都差异不显著,且大部分变异来源于年内单株间的变异。种子形态指标彼此之间都存在显著相关性,但它们与有胚种子百分数以及萌发相关指数都没有显著相关性。三棱栎种子有深色种皮和浅色种皮之分,浅色种皮比深色种皮的种子发育显著要差。三棱栎种子在吸胀吸水的不同阶段对高温的敏感性是不同的。在每一个处理阶段,热驯后高温处理与直接高温处理间萌发率、萌发指数和苗鲜重并没有显著差异。种子吸胀时用SA(水杨酸)代替蒸馏水,热激后种子萌发率、萌发指数与对照无显著差异并且显著高于其它处理,而苗鲜重显著高于对照和其它处理。3. 种子的贮藏特性:三棱栎种子在贮藏特性上属于正常型种子,能忍受一定程度的脱水,低温和相对低的含水量有利于种子的保存。居群间种子活力丧失速率不同,保存前萌发率在居群间存在显著差异,而室温保存1年后居群间种子萌发率差异变为不显著。高温高湿比单纯高温对种子的伤害更严重,而且含水量低能使种子对高温有更强的耐受性。在种子活力检测上,电导率和脱氢酶活性与萌发率间无显著相关性。4. 离体繁殖体系的建立与种质离体保存:子叶节是一种有效的外植体。低盐培养基有利于芽的增殖和根的诱导。1/4 MS培养基中加入1 mg/L 6-BA和0.05 mg/L NAA能使一个子叶节在4个月内得到20-25个芽。生长素能促进根的诱导,但是考虑到愈伤组织的不利因素,IBA或低浓度的NAA效果更好。该研究成功建立了三棱栎离体繁殖体系,为该植物特有种质资源(结实率低和种子败育严重的单株)的离体保存提供了技术支撑。5. 驯化生物学特性:栓皮栎、滇青冈和三棱栎都是壳斗科植物,自然分布范围依次减小。3个种的半年生苗对温度的耐受范围依次变小,这与它们自然分布范围的大小相一致。三棱栎1年生苗的高温半致死温度约为49.5℃,低温半致死温度约为-5℃,提示其耐受温度胁迫的范围较大。高温和低温短期驯化并没有提高其对温度的耐受性,说明其可能缺乏对温度的诱导性抗性。在适当的条件下,ABA处理叶圆片能提高叶圆片的耐冻性,SA处理叶圆片,也能提高其耐热性。本文对濒危植物三棱栎(Trigonobalanus doichangensis)的大孢子发生和雌配子体发育、种子形态与萌发、种子贮藏特性、离体培养及驯化生物学特性进行了研究。结合已发表的细胞学及分子遗传学等方面的研究结果,探讨了三棱栎种质保存与引种驯化的基础生物学及技术问题,为该植物种质保存和持续利用提供理论依据及技术指导。主要研究结果如下:1. 大孢子发生和雌配子体发育:三棱栎的柱头下方位置着生有雄蕊,在雌蕊发育后期,此花粉粒败育,因此,功能上的雌性花是形态上的两性花。胚珠倒生,双珠被,厚珠心,单孢原,大孢子四分体排列呈直线型,其中只有合点端一个发育成功能大孢子,雌配子体发育为蓼型。三棱栎子房中有6枚胚珠,发育正常的果实中会有1枚发育成种子,在三棱栎的大孢子发生和雌配子体发育过程中,有多个败育环节,93.3%的成熟胚囊都是败育的。2. 种子形态与萌发特征:三棱栎种子的形态指标(长、宽和千粒重)和萌发相关指标(萌发率、萌发指数和活力指数)的大部分变异都来源于居群内单株间的变异,而有胚种子百分数在居群间和居群内变异量相当,各占48%左右。以上指标在年间都差异不显著,且大部分变异来源于年内单株间的变异。种子形态指标彼此之间都存在显著相关性,但它们与有胚种子百分数以及萌发相关指数都没有显著相关性。三棱栎种子有深色种皮和浅色种皮之分,浅色种皮比深色种皮的种子发育显著要差。三棱栎种子在吸胀吸水的不同阶段对高温的敏感性是不同的。在每一个处理阶段,热驯后高温处理与直接高温处理间萌发率、萌发指数和苗鲜重并没有显著差异。种子吸胀时用SA(水杨酸)代替蒸馏水,热激后种子萌发率、萌发指数与对照无显著差异并且显著高于其它处理,而苗鲜重显著高于对照和其它处理。3. 种子的贮藏特性:三棱栎种子在贮藏特性上属于正常型种子,能忍受一定程度的脱水,低温和相对低的含水量有利于种子的保存。居群间种子活力丧失速率不同,保存前萌发率在居群间存在显著差异,而室温保存1年后居群间种子萌发率差异变为不显著。高温高湿比单纯高温对种子的伤害更严重,而且含水量低能使种子对高温有更强的耐受性。在种子活力检测上,电导率和脱氢酶活性与萌发率间无显著相关性。4. 离体繁殖体系的建立与种质离体保存:子叶节是一种有效的外植体。低盐培养基有利于芽的增殖和根的诱导。1/4 MS培养基中加入1 mg/L 6-BA和0.05 mg/L NAA能使一个子叶节在4个月内得到20-25个芽。生长素能促进根的诱导,但是考虑到愈伤组织的不利因素,IBA或低浓度的NAA效果更好。该研究成功建立了三棱栎离体繁殖体系,为该植物特有种质资源(结实率低和种子败育严重的单株)的离体保存提供了技术支撑。5. 驯化生物学特性:栓皮栎、滇青冈和三棱栎都是壳斗科植物,自然分布范围依次减小。3个种的半年生苗对温度的耐受范围依次变小,这与它们自然分布范围的大小相一致。三棱栎1年生苗的高温半致死温度约为49.5℃,低温半致死温度约为-5℃,提示其耐受温度胁迫的范围较大。高温和低温短期驯化并没有提高其对温度的耐受性,说明其可能缺乏对温度的诱导性抗性。在适当的条件下,ABA处理叶圆片能提高叶圆片的耐冻性,SA处理叶圆片,也能提高其耐热性。
资助项目Trigonobalanus doichangensis is an endangered plant. In this paper, the megasporogenesis and development of female gametophyte, seed morphological traits and seed germination, seed conservation, micropropagation and acclimatization of this species were studied. Combined with the published results of cytology, molecular genetics and other researches,the mechanisms of extinction, basic biology and technology of germplasm conservation and acclimatization of T. doichangensis were discussed. The main results are summarized as follows:1. Megasporogenesis and development of female gametophyte,Stamens exist under the stigma of T. doichangensis, and the pollen is aborted on the later development stage of pistil, therefore, the pistillate flower in function is hermaphrodite flower in morphology. The ovule is anatropous, bitegmic and crassinucellate. The primary archesporium is hypodermal and single-celled and the sporogenous cell of the nucellus functions directly as a megaspore mother cell which goes meiosis to form a linear tetrad. The chalazal megaspore of the tetrad is functional. The development of embryo sac conforms to the polygonum type. There are six ovules in the ovary of T. doichangensis, and only one develops into a seed in normal fruits. In the process of megasporogenesis and development of female gametophyte, there are several links of abortion, and 93.3% of mature embryo sacs is aborted.2. Morphological characters and germination of seeds,Most of the variation occurred among individual trees within populations in seed morphological traits (length, width and 1000-seed weight) and germination-related indices (germination percentage, germination index and vigor index). In addition, the variation in percentage of well-developed seeds among populations and among individual trees within populations is equal, each accounting for 48%. Each of seed morphological traits has significantly positive correlation with each other (p < 0.01), but they have no significant correlation with percentage of well-developed seeds and germination-related indices. In the same batch of seeds of T. doichangensis, there are light-colored and dark-colored seed coats, and development of light-colored seeds is significantly poorer than that of dark-colored seeds.The sensitivity of seeds to high temperature varys in different stages of seed imbibition. In each stage, heat acclimatization don’t increase germination percentage, germination index and fresh weight of seedlings. If the distilled water is substituted by solution of SA during seed imbibition, seed germination and germination index after heat shock are not significantly different from control, but they are significantly higher than that of other treatments. Moreover, when the seeds are treatmented with SA, the fresh weight of seedlings is significantly higher than that of control and other treatments.3. Seed conservation,Seeds of T. doichangensis belong to orthodox seeds which can tolerate certain level of dehydration. The condition of low temperature and low water content of seeds is conducive to seed conservation.Germination of fresh seeds shows significant variation among populations, howerer, germination of the seeds after storage for one year in room temperature shows no significant variation among populations.High temperature and high relative humidity damages the seeds more severely than high temperature does. In addition, low water content of seeds enable the seeds to be more tolerant to high temperature.The electrical conductivity, dehydrogenase activity and germination percentage have no significant correlation with each other.4. Micropropagation and in vitro conservation,Cotyledonary nodes are a kind of efficient explants. Low salt media are conducive to shoot propagation and root induction.The maximum multiplication rate (20-25 shoots/explant within 4 months) is achieved on quarter-strength Murashige and Skoog (1/4 MS) medium supplemented with 1 mg·L-1 6-benzyladenine (6-BA) and 0.05 mg·L-1 α-naphthaleneacetic acid (NAA).Rooting is promoted by auxins, however, IBA alone or low concentrations of NAA are preferable due to small amount of callus induced. The research has established an efficient protocol for micropropagation of T. doichangensis, and it provides technology support for in vitro conservation of special germplasm of the species.5. Acclimatization,Quercus variabilis, Cyclobalanopsis glaucoides and T. doichangensis belong to the family of Fagaceae, and the natural distribution ranges of the 3 species are decreasing in turn. The research suggests that the ranges of temperature tolerance of the 3 species are decreasing corresponding to their distribution ranges.The high and low semi-lethal temperature of one-year old T. doichangensis is 49.5℃ and -5℃ respectively. It suggests that T. doichangensis has a wide range of basic temperature tolerance. Short-term heat and cold acclimatization cannot expand the range of temperature tolerance. It can be inferred that T. doichangensis may lack induced tolerance to temperature. Under proper conditions, ABA can increase the cold tolerance, and SA can increase the heat tolerance of leaf discs of T. doichangensis.
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/16014
专题昆明植物所硕博研究生毕业学位论文
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郑艳玲. 濒危植物三棱栎的种质保存及驯化生物学研究[D]. 中国科学院研究生院,2010.
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