WO2021103165A1 - 一种利用氨基寡糖素促进杉木体胚发生的方法 - Google Patents

一种利用氨基寡糖素促进杉木体胚发生的方法 Download PDF

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WO2021103165A1
WO2021103165A1 PCT/CN2019/125010 CN2019125010W WO2021103165A1 WO 2021103165 A1 WO2021103165 A1 WO 2021103165A1 CN 2019125010 W CN2019125010 W CN 2019125010W WO 2021103165 A1 WO2021103165 A1 WO 2021103165A1
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chinese fir
amino
somatic
somatic embryogenesis
embryos
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陈金慧
施季森
成铁龙
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南京林业大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A01H4/006—Encapsulated embryos for plant reproduction, e.g. artificial seeds

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  • the invention belongs to the technical field of plant tissue culture, and more specifically, relates to a method for promoting Chinese fir somatic embryogenesis by using amino-oligosaccharins.
  • Chinese fir (Cunninghina lanceolata) belongs to the Chinese fir genus. It is one of the important timber species in my country. It grows fast, has a straight and round stem, is light and soft, easy to process, beautiful in texture, has a special aroma, does not warp or crack, Moth-resistant and corrosion-resistant, it is an excellent raw material for construction, furniture, bridges, shipbuilding, papermaking and wood fiber industries.
  • Chinese fir seedlings are mainly propagated through three methods: seeds, cuttings and traditional tissue culture [Wen Yafeng. Research on the induction and subgeneration of Chinese fir embryonic tissue based on immature zygotic embryos [D].
  • Somatic embryogenesis belongs to asexual reproduction technology.
  • the seedlings produced have little genetic background differences and the seedlings are neat. Compared with cuttings and traditional tissue culture, its scale is larger and the cost is lower.
  • somatic embryogenesis and angiosperm somatic embryogenesis have completely different pathways and mechanisms. It is very difficult for mature seeds and plants developed from seeds to transform vegetative cells into embryogenic cells.
  • some of the nucellar cells are in the undifferentiated stage during the development of the female gametophyte into seeds after fertilization. Under proper culture conditions, the characteristics of fission and polyembryonic can be used to induce the formation of somatic embryos. Gupta et al.
  • the technical problem to be solved by the present invention is to provide a method for promoting Chinese fir somatic embryogenesis by using amino-oligosaccharins, which can improve the induction efficiency of Chinese fir somatic embryos.
  • a method for promoting Chinese fir somatic embryogenesis by using amino-oligosaccharins Take Chinese fir embryonic embryonic stalk mass and add it to the somatic embryo induction medium, and conduct dark culture in an incubator to induce and promote Chinese fir somatic embryogenesis; among them, somatic embryo induction Medium: DCR is used as the basic medium, supplemented with amino-oligosaccharin with a concentration of not more than 8mg/L.
  • the somatic embryo induction medium also includes: 1mg/L VC, 0.45g/L glutamine, 0.5g/L hydrolyzed casein, 2 ⁇ 4g/L Ac, 2.8g/L crystal agar, 0.2g/L L asparagine, 0.2g/L proline, 100 ⁇ 200g/L PEG, 1 ⁇ 5mg/L GA, 2 ⁇ 8mg/L ABA.
  • the concentration of the amino oligosaccharin is 6 mg/L.
  • the culture temperature of the dark culture is 23°C, and the culture time is 60-100 days.
  • the method for obtaining the embryonic stalk mass of Chinese fir is: taking the immature cones of Chinese fir to obtain the seeds of fir, sterilizing the seeds of the fir in an ultra-clean workbench, removing the seed coat under a microscope, cutting the endosperm to take out the seed embryo, Put the seed embryos in a callus induction medium and culture in the dark at a culture temperature of 23°C for 25-30 days to obtain a Chinese fir embryonic stalk mass; wherein the callus induction medium is DCR medium Add 0.5mg/L KT, 0.5mg/L BA, 1mg/L VC, 1 ⁇ 2mg/L 2,4-D, 0.45g/L glutamine, 0.5g/L hydrolyzed casein, 2.5g/L Ac, 2.3g/L crystal agaric.
  • the advantages of the present invention are: the method for promoting Chinese fir somatic embryogenesis by using amino-oligosaccharins provided by the present invention uses DCR as the basic medium with additional amino acids whose concentration does not exceed 8 mg/L.
  • the embryonic stalk mass of Chinese fir was inoculated on the somatic embryo induction medium, and then cultured in the dark in an incubator at a temperature of 23°C to induce and promote somatic embryogenesis of Chinese fir, cultured for 60-100 days,
  • the number of somatic embryos up to 2 times higher than that of the control can be obtained, which greatly improves the induction efficiency of somatic embryos of Chinese fir, thus establishing a high-efficiency embryogenesis system of Chinese fir immature embryos, which is beneficial to speed up the fast-growing and excellent seedlings of Chinese fir
  • the development of industrialization has good practicability.
  • Figure 2 is a diagram showing the developmental status of Chinese fir A somatic embryos under different culture time and different concentration of amino-oligosaccharin under stereo microscope; a to d are the control group (without amino-oligosaccharin) at 0, 10, 20, and 30 days Embryogenic stalk mass development process; e ⁇ h are the embryogenic stalk mass development process when 6mg/L aminooligosaccharin is added at 0, 10, 20, and 30 days; j ⁇ n are respectively 0, 2 when added at 45 days 4. The development of embryogenic stalk mass of aminooligosaccharin at 6, 8 mg/L, the scale is 2 mm;
  • Figure 3 is a statistical diagram of the occurrence of mature cotyledon embryo B under different concentrations of aminooligosaccharin
  • Figure 4 shows the developmental status of Chinese fir somatic embryo B under different culture times and different concentrations of amino-oligosaccharin under stereomicroscope; a to d are the control group (without amino-oligosaccharin) at 0, 10, 20, and 30 days Embryogenic stalk mass development process; e ⁇ h are the embryogenic stalk mass development process when 6mg/L aminooligosaccharin is added at 0, 10, 20, and 30 days; j ⁇ n are respectively 0, 2 when added at 45 days 4.
  • the development of embryogenic stalk mass of aminooligosaccharin at 4, 6, 8 mg/L, the scale is 2 mm.
  • the raw materials used in the following examples are immature cones randomly collected from Yangkou Forest Farm in Fujian, and marked, and the embryogenic stalk masses induced by immature zygotic embryos (labeled A, B, randomly from two different Chinese fir) is the starting material.
  • Somatic embryo induction medium Chinese fir somatic embryo induction is the process of inducing Chinese fir callus to form somatic embryo.
  • the medium formula is DCR basic medium with 1mg/L VC, 0.45g/L glutamine, 0.5g/L hydrolyzed casein, 2 ⁇ 4g/L Ac, 2.8g/L crystal agar, 0.2g/L Asparagine, 0.2g/L proline. 100 ⁇ 200g/L PEG, 1 ⁇ 5mg/L GA, 2 ⁇ 8mg/L ABA.
  • Somatic embryo induction add embryonic embryo stalk masses A and B to different concentrations (0, 2, 4, 6, 8 mg/L) of amino-oligosaccharin somatic embryo medium, so as not to add exogenous amino-oligosaccharin (0mg/L)
  • the somatic embryo material induced by the culture medium was used as the control. Each dish was inoculated with 6 calli, at least 3 dishes were repeated, cultured in dark, and the culture temperature was 23°C.
  • the control group It is significantly different from the control group, and the other components are not significantly different; at 100 days, the average number of mature cotyledon embryos per dish of 6mg/L aminooligosaccharin is about 28.3, which is about 2.02 times that of the control group.
  • the control group has significant differences, and there is no significant difference among the remaining groups. It can be seen that the 6mg/L aminooligosaccharin has the strongest ability to induce somatic embryos of Chinese fir.
  • Fig. 4 It can be seen from Fig. 4 that at 0d, the callus of embryogenic stalk mass B has more water, the color of embryogenic stalk mass is transparent, whitish, and the tissue is relatively loose (Fig. 4, a, e); at 10d, the control The content of the group and the 6mg/L amino-oligosaccharin concentration increased significantly, the embryo head turned yellow, and the embryogenic embryonic stalk mass began to arrange tightly; at 20d, the embryogenic embryonic stalk mass continued to develop, and the base of the embryo body became bright yellow, and the embryo The head turns red and begins to form a transparent meristem, transforming into a columnar embryo.
  • the control group ( Figure 4c) is more convex than the experimental group ( Figure 4g); at 30 days, columnar embryos form and the embryo head turns green , The meristems began to differentiate to form cotyledons. Observed by stereoscopic observation, it was found that the number of columnar embryos in the control group (3d) was less, while the number of columnar embryos in the experimental group was larger (4h); at 45d, cotyledon embryos were produced at each concentration ( Figure 4, j ⁇ n).

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  • Developmental Biology & Embryology (AREA)
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Abstract

本发明公开了一种利用氨基寡糖素促进杉木体胚发生的方法，属于植物组培技术领域。本发明所提供的方法具体为：以DCR为基本培养基，附加有浓度不超过8mg/L的氨基寡糖素作为杉木体胚诱导培养基；通过杉木未成熟球果获得未成熟种胚，将其放在在愈伤诱导培养基上暗培养25～30天获得杉木胚性胚柄团；将所得杉木胚性胚柄团放入杉木体胚诱导培养基中，于培养箱中进行暗培养，诱导促进杉木体胚发生，23℃条件下，培养60～100天，即可得到比对照数目最高可达2倍以上的体胚，大大提高了杉木体胚的诱导效率，具有很好的实用性。

Description

一种利用氨基寡糖素促进杉木体胚发生的方法 技术领域
本发明属于植物组培技术领域，更具体地说，涉及一种利用氨基寡糖素促进杉木体胚发生的方法。
背景技术
杉木(Cunninghina lanceolata)隶属于杉科杉木属，是我国重要用材树种之一，其生长快、干形通直圆满，材质轻软细致，易加工，纹理美观，有特殊香气，不翘不裂，抗蛀耐腐，是建筑、家具、桥梁、造船、造纸及木纤维等工业的优良原材料。目前，主要通过三种途径繁殖杉木种苗：种子、扦插和传统组织培养[温亚锋.基于未成熟合子胚的杉木胚性组织诱导和继代的研究[D].福建农林大学，2011.]，但是良种种子产量、种子播种品质不稳定，第2代以上高级良种供应不足，且种子播种品质差，尤其是种子涩粒多，通常占重量的40％～60％[郑仁华.杉木遗传育种研究进展与对策[J].世界林业研究，2005(03)：63-65.]；而扦插育苗则受到母树穗条的多少、场地空间及生根率等方面的限制，其规模很难满足现代林业生产对杉木种苗的大量需求；此外，传统的组织培养根的诱导较为困难，诱导出的根数量少且弱，在一定程度上影响了苗木质量和造林的成功率。因此建立高效率的杉木未成熟胚的胚胎发生体系，有利于加快杉木速生优苗的产业化发展。
体细胞胚发生属于无性繁殖技术，生产出的种苗遗传背景差异小，苗木整齐。与扦插和传统组织培养相比，其规模更大、成本更低。然而，作为裸子植物的杉木，体胚的发生与被子植物的体细胞胚发生有着完全不同的途径和机理，成熟的种子以及由种子发育而来的植株，营养细胞转变为胚性细胞十分困难。但在针叶树种中，雌配子体受精后发育成种子的过程中有一部分珠心细胞处于未分化的阶段，在适当的培养条件下，可以利用裂生多胚特性，诱导体胚形成。Gupta等(1985，1995)报道过利用这一生物学特性，诱导出花旗松(Pseudotsuga menziesii)和湿地松(Pinuselliottii)的体胚，其大致可以分为四个步骤：胚性胚柄细胞团(ESM：Embryogenesissuspensor mass)的诱导、保持和增殖、体胚的成熟、萌发和植株再生，并证明该技术体系具有达到产业化的前景。
然而，目前虽有采用杉木未成熟胚诱导体胚发生的报道，但是只能在外植体上形成个别体细胞胚，存在诱导率低、数量少的问题，难以在生产上应用。因此建立高效率的杉木未成熟胚的胚胎发生体系，有利于加快杉木速生优苗的产业化发展。已有研究表明氨基寡糖素具有调控植物生长发育、促进植物繁殖、增加植物的防病抗病性等功能，但目前未见将其应用于杉木体胚发生的报导。
发明内容
针对现有技术存在的上述问题，本发明所要解决的技术问题在于提供一种利用氨基寡糖素促进杉木体胚发生的方法，能够提高杉木体胚诱导效率。
为了解决上述技术问题，本发明所采用的技术方案如下：
一种利用氨基寡糖素促进杉木体胚发生的方法：取杉木胚性胚柄团加入体胚诱导培养基中，于培养箱中进行暗培养，诱导促进杉木体胚发生；其中，体胚诱导培养基：以DCR为基本培养基，附加有浓度不超过8mg/L的氨基寡糖素。
所述的体胚诱导培养基还包括：1mg/L VC，0.45g/L谷氨酰胺，0.5g/L水解酪蛋白，2～4g/L Ac，2.8g/L水晶洋菜，0.2g/L天冬酰胺，0.2g/L脯氨酸，100～200g/L PEG，1～5mg/L GA，2～8mg/L ABA。
所述的氨基寡糖素的浓度为2～8mg/L。
所述的氨基寡糖素的浓度为6mg/L。
所述暗培养的培养温度为23℃，培养时间为60～100天。
所述杉木胚性胚柄团的获取方法为：取杉木未成熟球果得到杉木种子，在超净工作台中让杉木种子先灭菌后，在显微镜下去除种皮，切开胚乳取出种胚，将种胚放入愈伤诱导培养基中，暗培养，培养温度为23℃，培养25～30天，即得杉木胚性胚柄团；其中，所述的愈伤诱导培养基为DCR培养基中加入0.5mg/L KT，0.5mg/L BA，1mg/L VC，1～2mg/L 2,4-D，0.45g/L谷氨酰胺，0.5g/L水解酪蛋白，2.5g/L Ac，2.3g/L水晶洋菜。
有益效果：相比于现有技术，本发明的优点为：本发明所提供的利用氨基寡糖素促进杉木体胚发生方法，是以DCR为基本培养基，附加有浓度不超过8mg/L氨基寡糖素，取杉木胚性胚柄团接种到所述的体胚诱导培养基上，于培养箱中进行暗培养，培养温度为23℃，诱导促进杉木体胚发生，培养60～100天，即可得到比对照数目最高可达2倍以上的体胚，大大提高了杉木体胚的诱导效率，由此 建立了高效率的杉木未成熟胚的胚胎发生体系，有利于加快杉木速生优苗的产业化发展，具有很好的实用性。
附图说明
图1为不同浓度氨基寡糖素处理下杉木A成熟子叶胚发生数统计图；
图2为体视镜下不同培养时间不同浓度氨基寡糖素处理下杉木A体胚发育状态图；a～d是对照组(没有添加氨基寡糖素)在0、10、20、30天的胚性胚柄团发育过程；e～h是添加6mg/L氨基寡糖素在0、10、20、30天的胚性胚柄团发育过程；j～n分别是45d时添加0、2、4、6、8mg/L氨基寡糖素胚性胚柄团发育情况，标尺为2毫米；
图3为不同浓度氨基寡糖素处理下成熟子叶胚B发生数统计图；
图4为体视镜下不同培养时间不同浓度氨基寡糖素处理下杉木体胚B发育状态图；a～d是对照组(没有添加氨基寡糖素)在0、10、20、30天的胚性胚柄团发育过程；e～h是添加6mg/L氨基寡糖素在0、10、20、30天的胚性胚柄团发育过程；j～n分别是45d时添加0、2、4、6、8mg/L氨基寡糖素胚性胚柄团发育情况，标尺为2毫米。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
以下实施例中所使用的原材料为福建洋口林场随机采集的未成熟球果，并做好标记，通过未成熟合子胚诱导的胚性胚柄团(标记为A、B，随机来自不同的两棵杉木)为起始材料。
实施例1
母液配制：氨基寡糖素易溶于水，称取1.0g氨基寡糖素粉末于烧杯中用超纯水溶解，并定容至1L，配置成1mg/mL的氨基寡糖素母液，最后将其转移到4℃冰箱中，用棕瓶保存备用。
胚性胚柄团诱导和增殖培养基：杉木胚性胚柄团诱导是杉木种子诱导形成愈伤的过程，培养基配方为DCR培养基中加入0.5mg/L KT，0.5mg/L BA，1mg/L VC，1～2mg/L 2,4-D，0.45g/L谷氨酰胺，0.5g/L水解酪蛋白，2.5g/L Ac，2.3g/L水晶洋菜。
体胚诱导培养基：杉木体胚诱导是杉木愈伤诱导形成体胚的过程。培养基配方为DCR基本培养基中加入1mg/L VC，0.45g/L谷氨酰胺，0.5g/L水解酪蛋 白，2～4g/L Ac，2.8g/L水晶洋菜，0.2g/L天冬酰胺，0.2g/L脯氨酸。100～200g/L PEG，1～5mg/L GA，2～8mg/L ABA。
胚性胚柄团诱导与增殖：在超净工作台中让杉木种子先灭菌后，在显微镜下去除种皮，切开胚乳取出种胚，将胚放入愈伤诱导培养基中，胚和培养基要尽可能接触，这样有助于愈伤组织的形成。杉木愈伤继代周期为25～30天，暗培养，培养温度为23℃。
体胚诱导：将胚性胚柄团A和B分别加入不同浓度(0、2、4、6、8mg/L)的氨基寡糖素体胚培养基中，以不添加外源氨基寡糖素(0mg/L)培养基诱导的体胚材料为对照，每皿接种6块愈伤，至少3皿重复，暗培养，培养温度为23℃。
采用One Way ANOVA、多重比较检验法进行数据统计和分析，分析软件为SPSS 16.0，显著性水平设为α＝0.05，采用GraphPad对SPSS软件分析结果进行绘图。
(1)胚性胚柄团A的体胚发生
由图1可知，60天时，随着氨基寡糖素浓度的升高，杉木的成熟子叶胚数量增加，到6mg/L时，成熟子叶胚个数最多，8mg/L时成熟子叶胚数量下降，每皿的体胚平均数为4.67、6、9.33、15.3、8.33个从(2～8mg/L)，6mg/L的氨基寡糖素每皿成熟子叶胚个数约为对照组的2.37倍，与对照组具有显著差异，其余各组件并没有显著差异；在100天时，6mg/L氨基寡糖素平均每皿的成熟子叶胚的个数约为28.3个，约为对照组的2.02倍，与对照组具有显著差异，其余组间并没有显著差异。由此可以看出，6mg/L的氨基寡糖素对杉木体胚诱导能力最强。
如图2，a～d和e～h是对照组(没有添加氨基寡糖素)和添加6mg/L氨基寡糖素在0、10、20、30天的胚性胚柄团发育过程，0d时，对照组和实验组愈伤组织形态相似，胚性胚柄团颜色较白，偏透明，内含物少，胚头胚柄不能很好的区分(图2，a、e)；在10d时，对照组和6mg/L氨基寡糖素处理下细胞变的致密且呈黄色，在体式显微镜下成“颗粒状”(图2，b、f)。与对照组相比，6mg/L氨基寡糖素处理下的胚性胚柄团在体视镜下胚体结构明显；在20d时，在各个浓度中，内含物继续增加，胚性胚柄团开始转变为刺状凸起，胚头、胚轴部位开始变红，少量发育较快的胚头开始变绿，形成透明的顶端分生组织(图2，c、g)即柱状胚，其中6mg/L氨基寡糖素相比对照组柱状胚较多。在30d时，胚柄伸 长，对照组大量柱状胚开始形成(图2d)，而6mg/L氨基寡糖素的胚头已经分化为早期子叶原基，变成早期胚(图2h)；在45d时，由图可知，每个浓度均能产生早期子叶胚产生(图2，j、n)，而4～6mg/L的氨基寡糖素开始向成熟胚子叶胚转变(图2，n、d)，综上所述，氨基寡糖素能调控胚性胚柄团A杉木体胚发生过程，促进体胚生长。
(2)胚性胚柄团B的体胚发生
由图3可知，在60天时，6mg/L氨基寡糖素子叶胚个数最高，平均每皿的子叶胚个数为20.3个，其次为8mg/L，平均每皿的成熟子叶胚个数为16.3，而对照组平均每皿的子叶胚个数为12.3个；6mg/L氨基寡糖素成熟子叶胚的个数与对照组具有显著差异，其余组间并没有显著差异；在100天时，成熟子叶胚个数上升，6mg/L的成熟子叶胚数量最多，平均每皿的子叶胚个数为56个，对照组成熟子叶胚平均每皿个数为27.3，除了与8mg/L没有差异外，与其余各个组间均具有显著差异，说明6mg/L的氨基寡糖素能有效促进胚性胚柄团B的体胚发生；在8mg/L时，氨基寡糖素与成熟子叶胚个数成负相关，平均每皿成熟子叶胚45个，但仍然比对照组要高。
由图4可知，在0d时，胚性胚柄团B的愈伤组织水分较多，胚性胚柄团颜色透明，偏白，组织较为松散(图4，a、e)；10d时，对照组和6mg/L氨基寡糖素浓度内含物明显增多，胚头变黄，胚性胚柄团开始排列紧密；20d时，胚性胚柄团继续发育，胚体基部变为亮黄色，胚头变红并开始形成透明的分生组织，向柱状胚转变，对照组(图4c)与实验组(图4g)相比实验组凸起较多；30d时，柱状胚形成，胚头变绿，分生组织开始分化形成子叶，通过体视镜观察发现对照组(3d)的柱状胚数量较少，而实验组柱状胚数量较多(4h)；45d时，各个浓度都有子叶胚产生(图4，j～n)。

Claims (6)

  1. 一种利用氨基寡糖素促进杉木体胚发生的方法，其特征在于，取杉木胚性胚柄团加入体胚诱导培养基中，于培养箱中进行暗培养，诱导促进杉木体胚发生；其中，体胚诱导培养基：以DCR为基本培养基，附加有浓度不超过8mg/L的氨基寡糖素。
  2. 根据权利要求1所述的利用氨基寡糖素促进杉木体胚发生的方法，其特征在于，所述的体胚诱导培养基组分还包括：1mg/L VC，0.45g/L谷氨酰胺，0.5g/L水解酪蛋白，2～4g/L Ac，2.8g/L水晶洋菜，0.2g/L天冬酰胺，0.2g/L脯氨酸，100～200g/L PEG，1～5mg/L GA，2～8mg/L ABA。
  3. 根据权利要求2所述的利用氨基寡糖素促进杉木体胚发生的方法，其特征在于，所述的氨基寡糖素的浓度为2～8mg/L。
  4. 根据权利要求3所述的利用氨基寡糖素促进杉木体胚发生的方法，其特征在于，所述的氨基寡糖素的浓度为6mg/L。
  5. 根据权利要求1～4任一所述的利用氨基寡糖素促进杉木体胚发生的方法，其特征在于，所述暗培养的培养温度为23℃，培养时间为60～100天。
  6. 根据权利要求1～4任一所述的利用氨基寡糖素促进杉木体胚发生的方法，其特征在于，所述杉木胚性胚柄团的获取方法为：取杉木未成熟球果得到杉木种子，在超净工作台中让杉木种子先灭菌后，在显微镜下去除种皮，切开胚乳取出种胚，将种胚放入愈伤诱导培养基中，暗培养，培养温度为23℃，培养25～30天，即得杉木胚性胚柄团；其中，所述的愈伤诱导培养基为DCR培养基中加入0.5mg/L KT，0.5mg/L BA，1mg/L VC，1～2mg/L 2,4-D，0.45g/L谷氨酰胺，0.5g/L水解酪蛋白，2.5g/L Ac，2.3g/L水晶洋菜。
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