A method for highly synchronized somatic embryogenesis and plant regeneration of pine wood nematode-resistant Pinus pine

A technology for pine wood nematode disease and somatic embryos, applied in the field of highly synchronized anti-pine wood nematode disease pine somatic embryogenesis and plant regeneration

Active Publication Date: 2018-09-25
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few domestic research reports on somatic embryogenesis of Pinus chinensis (Wu Jing et al., 2015)

Method used

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  • A method for highly synchronized somatic embryogenesis and plant regeneration of pine wood nematode-resistant Pinus pine
  • A method for highly synchronized somatic embryogenesis and plant regeneration of pine wood nematode-resistant Pinus pine
  • A method for highly synchronized somatic embryogenesis and plant regeneration of pine wood nematode-resistant Pinus pine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] ABA concentration (10, 15, 20mg / L) and PEG (polyethylene glycol) 8000 concentration (100, 140, 180g / L) were subjected to a two-factor randomized block test. will be 22 # The embryogenic suspensor mass (ESM) of -1 was transferred to the designed medium (basic medium LP, additionally inositol 8g / L, maltose 60g / L, MES 250mg / L, CH 500mg / L , VC 10mg / L, glutamine 450mg / L, activated carbon (AC) 2.0g / L, plant gel 3g / L, pH 5.8), 30 pieces of ESM per treatment, repeated 3 times, real-time observation of somatic embryo maturation Happening. Medium. The medium was sterilized by autoclaving at 121°C for 20min.

[0023] Induction, proliferation and maturation experiments of somatic cell embryos of disease-resistant Scots pine were cultured in the dark in a tissue culture room with a culture temperature set at 23±2°C, and observed once every two weeks. The number of normal somatic embryos is the number of somatic embryos formed per gram of callus (piece / g) = the number of normal s...

Embodiment 2

[0029] will be 22 #-1 ESM was transferred to LP as the basic medium, adding different types of sugar (maltose, sucrose) and different concentrations (20, 30, 45, 60, 70g / L), 30 pieces of ESM per treatment, repeated 3 times , real-time observation of somatic embryo maturation. The medium was supplemented with ABA 15mg / L, PEG8000 140g / L, inositol 8g / L, MES 250mg / L, CH 500mg / L, VC 10mg / L, glutamine 450mg / L, activated carbon (AC) 2.0g / L, Plant gel 3g / L, pH5.8. The medium was sterilized by autoclaving at 121°C for 20min. Somatic embryo culture and measurement are the same as in Example 1.

[0030] The results showed that the effects of sugar type and concentration on the maturation of somatic embryos of resistant Pinus spp. were significantly different, and maltose was more effective than sucrose in increasing the number of somatic embryos of resistant Scots pine ( figure 1 ). When the concentration of maltose was 60g / L, the number of somatic embryos of the disease-resistant P...

Embodiment 3

[0032] will be 22 # -1 ESM was transferred to LP basic medium, adding inositol 0, 2, 4, 6, 8, 10, 16g / L, 30 ESMs per treatment, repeated 3 times, and observed the maturation of somatic embryos in real time. Added ABA 15mg / L, PEG8000 140g / L, MES250mg / L, CH 500mg / L, VC 10mg / L, glutamine 450mg / L, activated carbon (AC) 2.0g / L, plant gel 3g / L, pH5.8. The medium was sterilized by autoclaving at 121°C for 20min. Somatic embryo culture and measurement are the same as in Example 1.

[0033] the result shows( figure 2 ), in the absence of inositol, although the resistant pine can produce cotyledon embryos, the cotyledon embryo head is enlarged, the embryo stalk is short and even cannot develop normally, the embryo body is short and thick, the color is yellowish, and the growth state is poor, all of which are deformed embryo; and the color of the callus is brownish, the structure is gradually compacted, and the growth status obviously declines ( figure 2 -J1). When adding inosito...

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Abstract

The invention discloses a high-synchronization bursaphelenchus xylophilus resistant pinus densiflora somatic embryogenesis and plant regeneration method. The high-synchronization bursaphelenchus xylophilus resistant pinus densiflora somatic embryogenesis and plant regeneration method comprises the steps of inducement and proliferation of embryonic callus as well as maturation, germination and transformation of somatic embryos; and the maturation culture of the somatic embryos is carried out by using a liquid-solid proliferation-solid maturation method. According to the high-synchronization bursaphelenchus xylophilus resistant pinus densiflora somatic embryogenesis and plant regeneration method, with immature zygotic embryos of bursaphelenchus xylophilus resistant pinus densiflora as explants, mature somatic embryos and regenerated plants are successfully obtained by carrying out tests, such as somatic embryo maturation, germination and plant regeneration, on the embryonic callus obtained by successfully inducing the immature zygotic embryos of bursaphelenchus xylophilus resistant pinus densiflora; the mature somatic embryos and the regenerated plants are transplanted and survive; and the germination rate and plant transformation rate of the somatic embryos generated on a maturation culture medium are high and are respectively 67.2% and 46.5%; therefore, the high-synchronization bursaphelenchus xylophilus resistant pinus densiflora somatic embryogenesis and plant regeneration method provides an important scientific basis and technical support for large-scale propagation and factory production of the bursaphelenchus xylophilus resistant pinus densiflora.

Description

technical field [0001] The invention belongs to the technical field of tree breeding, and in particular relates to a highly synchronized method for somatic embryogenesis and plant regeneration of pine wood nematode-resistant Pinus pine. Background technique [0002] Red pine (Pinus densiflora), mainly distributed in Japan, North Korea, southeastern Russia and eastern my country, can be used as a tree species in gardens and barren hills for afforestation, but it is easily infected with pine wood nematode (Bursaphelenchus xylophilus) and dies in large numbers, causing serious damage to forestry production and ecological environment. severe damage. Due to the low reproductive coefficient of conventional seedlings, it is difficult to breed disease-resistant materials to meet the needs of large-scale forestry production. In order to rapidly multiply and select excellent disease-resistant genotypes and realize large-scale production in a short period of time, it is particularly im...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/005A01H4/008
Inventor 吴小芹许建秀叶建仁吴静朱丽华潘珺
Owner NANJING FORESTRY UNIV
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