WO2019134331A1 - 一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法 - Google Patents

一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法 Download PDF

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WO2019134331A1
WO2019134331A1 PCT/CN2018/088620 CN2018088620W WO2019134331A1 WO 2019134331 A1 WO2019134331 A1 WO 2019134331A1 CN 2018088620 W CN2018088620 W CN 2018088620W WO 2019134331 A1 WO2019134331 A1 WO 2019134331A1
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baishanzu
immature
embryo
seedlings
fir
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French (fr)
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陈利萍
刘柯
陈德良
吴友贵
向珣
于明坚
曹丽雯
于宁宁
许大明
周荣飞
王挺进
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Baishanzu Administration Office Of Fengyangshan-Baishanzu National Nature Reserve Zhejiang
Zhejiang University
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Baishanzu Administration Office Of Fengyangshan-Baishanzu National Nature Reserve Zhejiang
Zhejiang University
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Priority to JP2020501141A priority Critical patent/JP6876193B2/ja
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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
    • A01H4/002—Culture media 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
    • A01H4/008—Methods for regeneration to complete plants

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  • the invention relates to a method for rapidly cultivating the seedlings of Baishanzu fir by the embryo rescue technique, thereby achieving the purpose of expanding the number of endangered species.
  • Abiesbeshanzuensis is an Abies plant of Pinaceae, an evergreen tree with a height of 17 meters and a breast diameter of 80 cm.
  • Autumn fruit has a cylindrical shape with short stems, 7 to 12 cm long and 3.5 to 4 cm in diameter. It is light brown or pale brown when mature. Flowering in May and ripening in November. It is an ancient relict plant unique to Zhejiang Province. It is widely distributed in the Tertiary Period of the provinces of Jiangsu, Zhejiang, Fujian and Guizhou, and the only rare species that survived in the local area after the Quaternary Ice Age. Baishanzu fir is a national first-class protected plant.
  • Baishanzu fir is naturally distributed in the narrow sheltered valley of the Baishanzu Nature Reserve at an altitude of about 1,740 meters. It is the only species of the genus Abies in the southeastern part of China. It is a living fossil preserved during the Quaternary glacial period. For geography and climate. Studies in other disciplines are of great significance.
  • Baishanzu fir cones Under natural conditions, the Baishanzu fir cones have a long development time and low seed setting rate. When a cone matures, a large number of embryos are aborted, resulting in a very small number of normally developing seeds. At the same time, Baishanzu fir is affected by the ecological environment, causing problems such as difficulty in natural regeneration and weak ability to continue to reproduce. Therefore, there is an urgent need to establish a simple and rapid method for cultivating the seedlings of Baishanzu fir. Embryo rescue technology is an effective means to overcome embryo abortion or dysplasia, improve germination rate and early germination, and shorten plant development time in breeding research. Using embryo rescue technology to rapidly cultivate Baishanzu fir seedlings can effectively prevent the endangered plant Baishanzu fir from extinction due to difficulties in breeding under natural conditions.
  • the object of the present invention is to provide a method for rapidly cultivating the seedlings of Baishanzu fir by the embryo rescue technique in view of the deficiencies of the prior art.
  • the object of the present invention is achieved by the following technical solution: a method for rapidly cultivating a seedling of Baishanzu fir by an embryo rescue technique, comprising the following steps:
  • the immature cone shape characteristic is that the cone is green.
  • the surface sterilization procedure is: rinsing the immature seeds under running water for 3 hours, rinsing and soaking and oscillating with a 70% ethanol aqueous solution on a clean bench. 30s, rinse 3 times with sterile water for 1 min each time. After immersing in an aqueous solution of mercury chloride having a mass fraction of 0.1% and shaking for 10 minutes, it was washed 5 times with sterile water for 1 min each time. Finally, use sterile filter paper to blot excess water on the surface of the seed.
  • the method for stripping the immature embryos is to fix the immature seeds with a sterile forceps for the left hand, and the right hand with a sterile scalpel at a distance of 0.5 mm from the edge of the seed coat for slitting. And cross-cutting, using seedlings to separate the seed coat from the immature embryo and its endosperm.
  • the medium is DCR (Gupta and Durzan medium) as a basic medium, and sucrose, agar, hydrolyzed casein, etc., sucrose, agar, hydrolyzed casein are additionally added.
  • the concentrations were 20 g/L, 8 g/L, and 500 mg/L, respectively, and the pH of the medium was 5.8.
  • the embryo germination morphology is characterized in that the radicle or cotyledon protrudes from the endosperm.
  • the method for separating the seedlings is to separate the seedlings from the endosperm after longitudinally cutting the endosperm with a sterilized scalpel.
  • the invention has the advantages that the explants used in the invention are immature embryos, are cultured by the embryo rescue technique, overcome the defects of seed embryo abortion under natural conditions, and can quickly obtain more Baishanzu in a short period of time. Fir seedlings.
  • the method has strong operability, simple and high efficiency, low pollution rate, and is of great significance for the rapid cultivation of the world's endangered plant seedlings.
  • Figure 1 is an immature cone on a wild plant of Baishanzu fir.
  • Figure 2 is a diagram showing the explants of the Baishanzu fir explants - immature embryos and endosperm;
  • Figure 3 is a germination diagram of immature embryos of Baishanzu fir
  • Figure 4 is a graph showing the growth of germinated seedlings.
  • MATERIALS In this example, immature embryos and endosperm of Baishanzu fir were used as culture explants.
  • Step (1) Take the material
  • Step (3) Isolation and inoculation of immature embryos
  • the medium is based on DCR (Gupta and Durzan medium), and sucrose, agar, hydrolyzed casein, etc., and the concentrations of sucrose, agar and hydrolyzed casein are 20g/L, 8g/L, 500mg/L, respectively.
  • the pH of the base is 5.8.
  • Step (4) dark culture and heterotrophic culture
  • the immature embryos and their endosperm after inoculation were cultured for 3 days under dark conditions of 20 ⁇ 2°C, and then germinated for 10 days under low light conditions.
  • the low light illumination conditions were: light intensity 20 ⁇ mol ⁇ m -2 ⁇ s -1 and the illumination time is 12h/d.
  • the seedlings are then separated from the endosperm and inoculated onto the medium for autotrophic culture and preservation.
  • the medium is based on DCR (Gupta and Durzan medium), and sucrose, agar, hydrolyzed casein, etc., sucrose, agar,
  • the concentrations of hydrolyzed casein were 20 g/L, 8 g/L, and 500 mg/L, respectively, and the pH of the medium was 5.8.
  • the culture conditions were temperature 20 ⁇ 2° C., light intensity 80 ⁇ mol ⁇ m -2 ⁇ s -1 , and illumination time 12 h/d.
  • the seedlings after 15 days of culture are shown in Fig. 3.
  • a relatively large number of sterile seedlings of Baishanzu fir can be obtained for rapid cultivation of the endangered plant Baishanzu fir in a short period of time.

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  • Life Sciences & Earth Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

本发明公开了一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，以百山祖自然保护区中百山祖冷杉野生植株的未成熟球果为起始材料，表面消毒后剥取其未成熟胚及胚乳，接种到培养基上进行暗培养、异养期培养和自养期培养，通过胚拯救技术快速培育百山祖冷杉野生植株后代，短时间内获得数量较多的百山祖冷杉幼苗。

Description

一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法 技术领域
本发明涉及一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，从而达到扩大濒危物种数量的目的。
背景技术
百山祖冷杉(Abiesbeshanzuensis)是松科(Pinaceae)冷杉属(Abies)植物，常绿乔木，高17米，胸径达80厘米。秋果圆柱形，有短梗，长7～12厘米，直径3.5～4厘米，成熟时淡褐色或淡褐黄色。5月开花，11月球果成熟。是浙江省特有的古老孑遗植物。在苏、浙、皖、闽等省第三纪时广泛分布，第四纪冰期后在局部地段唯一生存至今的珍稀物种。百山祖冷杉为国家一级重点保护植物，1987年被世界自然保护联盟物种生存委员会(SSC)公布列为世界最濒危的12种植物之一。百山祖冷杉自然分布于百山祖自然保护区海拔1740米左右的狭小避风谷地，是我国大陆东南部唯一的一种冷杉属植物，是第四纪冰川期保存下来的活化石，对于地理学和气候学等学科研究具有十分重要的意义。
自然条件下，百山祖冷杉球果发育时间长、结实率低。球果成熟时大量的胚存在败育的现象，导致正常发育的种子数量极少。同时，百山祖冷杉受到生态环境影响，造成天然更新困难、持续繁衍能力极弱等问题。因此，迫切需要建立一种简单的、快速的培育百山祖冷杉幼苗的方法。胚拯救技术是育种研究中克服胚败育或者发育不良，提高萌发率以及提早萌发，缩短植物发育时间的有效手段。运用胚拯救技术快速培育百山祖冷杉幼苗，可有效的防止濒危植物百山祖冷杉因在自然条件下繁殖困难而灭绝。
发明内容
本发明的目的是针对现有技术的不足，提供一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法。
本发明的目的是通过以下技术方案实现的：一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，包括如下步骤：
(1)采集百山祖冷杉植株上的未成熟球果；
(2)剥取未成熟种子并进行表面灭菌；
(3)用解剖刀切开未成熟种子，剥取未成熟胚并接种到培养基上，将其水平放置。20±2℃黑暗条件下培养3d；
(4)暗培养后，在弱光条件下(光照强度为20μmol·m -2·s -1、光照时间为12h/d)培养，直到胚萌发；
(5)胚萌发后，在正常光照条件下(光照强度为80μmol·m -2·s -1、光照时间为12h/d)培养10d后，将幼苗从胚乳中分离并接种到培养基上进行保存，培养条件为温度20±2℃、光照强度为80μmol·m -2·s -1、光照时间为12h/d。
进一步地，在所述步骤(1)中，所述未成熟球果形态特征为球果呈现绿色。
进一步地，在所述步骤(2)中，所述表面灭菌程序为：将未成熟种子置于流水下冲洗3h，冲洗后在超净工作台上用体积分数为70％乙醇水溶液浸泡并振荡30s，无菌水冲洗3次，每次1min。再用质量分数为0.1％的氯化汞的水溶液浸泡并振荡10min后，用无菌水冲洗5次，每次1min。最后用无菌滤纸吸干种子表面多余的水分。
进一步地，在所述步骤(3)中，所述剥取未成熟胚的方法为左手用无菌镊子固定未成熟种子，右手用无菌解剖刀在距种皮边缘0.5mm，分别进行纵切和横切，用镊子将种皮从未成熟胚及其胚乳上分离。
进一步地，在所述步骤(3)和步骤(5)中，培养基以DCR(Gupta and Durzan medium)为基本培养基，另外添加蔗糖、琼脂、水解酪蛋白等，蔗糖、琼脂、水解酪蛋白的浓度分别为20g/L、8g/L、500mg/L，培养基的pH为5.8。
进一步地，在所述步骤(4)中，所述胚萌发形态特征为胚根或者子叶从胚乳中伸出。
进一步地，在所述步骤(5)中，所述分离幼苗的方法为用灭菌的解剖刀将胚乳纵向切开后，将幼苗从胚乳中分离。
本发明的优点是：本发明采用的外植体为未成熟胚，利用胚拯救技术对其进行培养，克服自然条件下种子胚败育的缺陷，可在短期内快速获得较多的百山祖冷杉幼苗。该方法可操作性强，简单高效，污染率低，对世界濒危植物幼苗的快速培育具有重要意义。
附图说明
图1是百山祖冷杉野生植株上的未成熟球果
图2是用于培养的百山祖冷杉外植体—未成熟胚及胚乳图；
图3是百山祖冷杉未成熟胚培养后萌发图；
图4是萌发幼苗生长图。
具体实施方式
下面结合实施例对本发明进一步说明。
实施例1：
材料：本实例以百山祖冷杉未成熟胚及胚乳为培养外植体。
步骤(1)：取材
7月下旬至8月上旬于百山祖自然保护区收集百山祖冷杉未成熟球果，颜色绿色，这种绿色的未成熟果子表面种鳞、苞鳞排列紧密。可放于密封袋中，4℃低温中贮藏备用。
步骤(2)：种子表面灭菌
用镊子分离种鳞和种子，将种子置于流水下冲洗3h，在超净工作台上用70％乙醇处理30s，无菌水冲洗3～5次。再用0.1wt％氯化汞处理10min后，用无菌水冲洗3～5次并用无菌滤纸吸干表面水分。
步骤(3)：未成熟胚分离与接种
将冲洗后的种子置于铺有灭菌滤纸的培养皿中，左手用无菌镊子固定未成熟种子，右手用无菌解剖刀在距种皮边缘0.5mm，分别进行纵切和横切，用镊子将种皮从未成熟胚及其胚乳上分离(图1)。剥取未成熟胚及其胚乳并接种到培养基上，将其水平放置。培养基以DCR(Gupta and Durzan medium)为基本培养基，另外添加蔗糖、琼脂、水解酪蛋白等，蔗糖、琼脂、水解酪蛋白的浓度分别为20g/L、8g/L、500mg/L，培养基的pH为5.8。
步骤(4)：暗培养与异养期培养
接种后的未成熟胚及其胚乳先于20±2℃黑暗条件下培养3d，再弱光光照条件下培养10天可见种子萌发，所述弱光光照条件为：光照强度为20μmol·m -2·s -1、光照时间为12h/d。
步骤(5)：自养期培养
未成熟胚培养后，萌发率可达95％以上(图2)。然后幼苗从胚乳中分离并接种到培养基上进行自养期培养与保存，培养基以DCR(Gupta and Durzan medium)为基本培养基，另外添加蔗糖、琼脂、水解酪蛋白等，蔗糖、琼脂、水解酪蛋白的浓度分别为20g/L、8g/L、500mg/L，培养基的pH为5.8。培养条件为温度20±2℃、光照强度为80μmol·m -2·s -1、光照时间为12h/d。培养15天后的幼苗如图3所示，由此可见，通过本方法可在短期内获得数量比较多的百山祖冷杉无菌幼苗用于濒危植物百山祖冷杉的快速培育。

Claims (7)

  1. 一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，其特征在于，包括如下步骤：
    (1)采集百山祖冷杉植株上的未成熟球果；
    (2)剥取未成熟种子并进行表面灭菌；
    (3)用解剖刀切开未成熟种子，剥取未成熟胚并接种到培养基上，将其水平放置。20±2℃黑暗条件下培养3d；
    (4)暗培养后，在光照强度为20μmol·m -2·s -1、光照时间为12h/d的弱光条件下培养，直到胚萌发；
    (5)胚萌发后，在光照强度为80μmol·m -2·s -1、光照时间为12h/d的正常光照条件下培养10d后，将幼苗从胚乳中分离并接种到培养基上进行保存，培养条件为温度20±2℃、光照强度为80μmol·m -2·s -1、光照时间为12h/d。
  2. 根据权利要求1所述的一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，其特征在于：在所述步骤(1)中，所述未成熟球果形态特征为球果呈现绿色。
  3. 根据权利要求1所述的一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，其特征在于：在所述步骤(2)中，所述表面灭菌程序为：将未成熟种子置于流水下冲洗3h，冲洗后在超净工作台上用体积分数为70％乙醇水溶液浸泡并振荡30s，无菌水冲洗3次，每次1min。再用质量分数为0.1％的氯化汞的水溶液浸泡并振荡10min后，用无菌水冲洗5次，每次1min。最后用无菌滤纸吸干种子表面多余的水分。
  4. 根据权利要求1所述的一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，其特征在于：在所述步骤(3)中，所述剥取未成熟胚的方法为左手用无菌镊子固定未成熟种子，右手用无菌解剖刀在距种皮边缘0.5mm，分别进行纵切和横切，用镊子将种皮从未成熟胚及其胚乳上分离。
  5. 根据权利要求1所述的一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，其特征在于：在所述步骤(3)和步骤(5)中，培养基以DCR(Gupta and Durzan medium)为基本培养基，另外添加蔗糖、琼脂、水解酪蛋白等，蔗糖、琼脂、水解酪蛋白的浓度分别为20g/L、8g/L、500mg/L，培养基的pH为5.8。
  6. 根据权利要求1所述的一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，其特征在于：在所述步骤(4)中，所述胚萌发形态特征为胚根或者子叶从胚乳中伸出。
  7. 根据权利要求1所述的一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法，其特征在于：在所述步骤(5)中，所述分离幼苗的方法为用灭菌的解剖刀将胚乳纵向切开后，将幼苗从胚乳中分离。
PCT/CN2018/088620 2018-01-04 2018-05-28 一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法 Ceased WO2019134331A1 (zh)

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