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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Publication number
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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WIPO (PCT)
Prior art keywords
baishanzu
immature
embryo
seedlings
fir
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PCT/CN2018/088620
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English (en)
French (fr)
Chinese (zh)
Inventor
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
Original Assignee
浙江大学
浙江凤阳山百山祖国家级自然保护区百山祖管理处
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Application filed by 浙江大学, 浙江凤阳山百山祖国家级自然保护区百山祖管理处 filed Critical 浙江大学
Priority to JP2020501141A priority Critical patent/JP6876193B2/ja
Publication of WO2019134331A1 publication Critical patent/WO2019134331A1/zh

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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

Definitions

  • 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)
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 WO2019134331A1 (zh)

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JP2020501141A JP6876193B2 (ja) 2018-01-04 2018-05-28 胚救助技術による百山祖冷杉の苗を短い時間で得ることが出来る方法

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CN201810008078.6A CN108552056B (zh) 2018-01-04 2018-01-04 一种通过胚拯救技术快速培育百山祖冷杉幼苗的方法
CN201810008078.6 2018-01-04

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CN109601377B (zh) * 2018-11-29 2022-05-06 浙江大学 一种通过未成熟胚快速培育水松幼苗的方法
CN112021139B (zh) * 2019-05-14 2022-09-16 浙江大学 一种快速人工栽培冷杉属植物幼苗的方法
CN111670703B (zh) * 2020-06-23 2022-04-26 浙江大学 一种百山祖冷杉高效嫁接的方法
CN114467750B (zh) * 2022-02-16 2023-05-02 浙江大学 一种大盘山榧未成熟种子看护培养方法
CN115136889B (zh) * 2022-04-14 2023-01-13 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） 一种番木瓜未成熟种子萌发方法
CN115125261B (zh) * 2022-06-21 2024-04-30 浙江大学 百山祖冷杉蔗糖合成酶基因和产品及用途

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CN101849505B (zh) * 2010-02-10 2012-07-18 南京林业大学 诱导马尾松未成熟种子胚性胚柄细胞团再生植株的方法
CN104620982B (zh) * 2015-01-27 2016-05-18 浙江大学 一种通过未成熟种子的培养繁育蛛网萼后代的方法

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ZHANG, QIANYUM ET AL.: "Influence Factores And Application of Plant Embryo Rescue Techniques", JOURNAL OF NANJING FORESTRY UNIVERSITY (NATURAL SCIENCES EDITION, vol. 38, no. 5, 30 September 2014 (2014-09-30), pages 143 - 148 *

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CN108552056B (zh) 2020-08-14
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