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CN-121992017-A - Tissue culture-independent genetic transformation method for high-altitude layering of rubber tree

CN121992017ACN 121992017 ACN121992017 ACN 121992017ACN-121992017-A

Abstract

The application relates to a high-altitude layering genetic transformation method of a rubber tree independent of tissue culture, and relates to the technical field of plant genetic engineering. The method comprises the following steps of S1, providing agrobacterium rhizogenes K599 bacteria liquid containing target genes, wherein the target genes are loaded in a pCAMBIA2301 carrier, S2, preparing an infection material (infection liquid or lawn) by using the agrobacterium rhizogenes K599, S3, carrying out girdling treatment on stems of rubber seedlings and pretreatment on girdling wounds, S4, carrying out infection on the pretreated girdling wounds by using the infection material, S5, covering and mounting the girdling wounds by using a moist culture medium to keep the wounds moist and incubating strains, and wherein the target genes are GUS genes, RUBY genes or JcFT genes. The method can bypass a series of complex operations such as tissue culture and the like, is simple, convenient, quick, low in cost, free from season limitation, easy to operate and independent of genotype, and particularly, can obtain higher positive rate by utilizing the method.

Inventors

  • TANG MINGYONG
  • DING HUCHENG
  • Zheng si
  • BAI XUE
  • SHU YUANXIN
  • Bi ting

Assignees

  • 中国科学院西双版纳热带植物园

Dates

Publication Date
20260508
Application Date
20260122

Claims (10)

  1. 1. A method for genetic transformation of a high-altitude layering of a rubber tree independent of tissue culture is characterized by comprising the following steps: S1, providing agrobacterium rhizogenes K599 bacterial liquid containing target genes, wherein the target genes are loaded in a pCAMBIA2301 vector; s2, preparing an infection material by using the agrobacterium rhizogenes K599 bacterial liquid; S3, carrying out girdling treatment on the stems of the rubber saplings, and carrying out pretreatment on girdling wounds to remove fresh latex secreted at the wounds; S4, infecting the pretreated girdling wound by using the infecting material; S5, covering and mounting the girdling wound by adopting a wet culture medium so as to keep the wound moist and incubate strains; Wherein the target gene is GUS gene, RUBY gene or JcFT gene.
  2. 2. The method according to claim 1, wherein in step S2, the infesting material is agrobacterium rhizogenes K599 infested solution or agrobacterium rhizogenes K599 lawn.
  3. 3. The method according to claim 1, wherein the Agrobacterium rhizogenes K599 bacterial liquid OD600 is 0.4-0.8, and the infection method is a bacteria wrapping method or an injection method.
  4. 4. The method of claim 1, wherein the Agrobacterium rhizogenes K599 bacterial liquid OD600 is 0.6-0.8, and the infection method is an injection method.
  5. 5. The method according to claim 1, wherein in step S3, the girdling process is performed by cutting a phloem on a roll of the stem at a distance of 50 cm from the ground.
  6. 6. The method of claim 1, wherein the pre-treatment in step S3 comprises wiping latex secreted from the girdling wound, followed by washing.
  7. 7. The method of claim 6, wherein the washing is performed using a mixed solution containing 5% sucrose and 30 mM CaCl 2 %.
  8. 8. The method according to claim 1, wherein in step S5, the culture medium is machine-made soil.
  9. 9. The method of claim 1, wherein in step S5, the culture medium is packed in an overhead layering propagation cassette to effect the covering and Bao Biao of the girdling wound.
  10. 10. The method according to any one of claims 1 to 9, wherein the rubber tree seedling variety is GT1, PR107 or RRIM600.

Description

Tissue culture-independent genetic transformation method for high-altitude layering of rubber tree Technical Field The application relates to the technical field of plant genetic engineering, in particular to a high-altitude layering genetic transformation method of a rubber tree without tissue culture dependence. Background The natural rubber produced by the rubber tree (Hevea brasiliensis) is a vital industrial raw material and strategic material. However, the traditional crossbreeding period is as long as 30-50 years, so that excellent characters such as high yield, stress resistance and the like are difficult to be efficiently polymerized, and the variety updating is severely restricted. The modern molecular breeding means represented by the transgenic technology provides possibility for realizing accurate control of the rubber tree genome and greatly shortening the breeding period, and is also a key way for carrying out gene function identification. Currently, the application of rubber tree transgenic technology is highly dependent on tissue culture systems. The system has the common problems of high cost, long period, complex operation and the like, and the callus induction and somatic embryogenesis processes of the rubber tree have strict genotype dependence. The current situation results in that a stable genetic transformation system can be established in a few easily regenerated varieties, while important cultivars in China such as GT1, tian Ying 31-45 and other key parent materials are difficult to induce somatic embryos, so that high-efficiency genetic transformation is difficult to realize through a tissue culture way, and the method becomes an important bottleneck for comprehensively applying molecular breeding technology. To break through this bottleneck, tissue-independent genetic transformation methods are becoming an important research direction. For example, the patent with publication number CN119752983A, a genetic transformation method of rubber tree roots independent of tissue culture, uses agrobacterium rhizogenes to directly infect the root-removed rubber seedlings, successfully bypasses the complex tissue culture regeneration process, and provides a new idea for realizing genetic operation of difficult-to-transform varieties. However, the method has significant limitations in practical application, namely firstly, the high-efficiency transformation (the positive rate can reach 63.7%) is only under the specific condition of the GT1 variety, the transformation rate suddenly drops under other conditions such as poor temperature seedling age, the universality is insufficient, secondly, the rooting-removed seedling is taken as an explant, the material acquisition is limited by seasons, the transformed plant is weak, the subsequent maintenance is difficult, and the technical stability and the operability are required to be improved. Therefore, the development of a high-efficiency non-tissue culture genetic transformation method which is not limited by seasons, is applicable to a wider variety of rubber trees and can obtain a robust transformation plant has urgent practical significance for accelerating the molecular breeding process of the rubber trees, and is also a target for continuous pursuit in the field. Disclosure of Invention In order to solve or partially solve the problems in the related art, the application provides a genetic transformation method for a high-altitude layering of a rubber tree, which is not dependent on tissue culture. The application provides a high-altitude layering genetic transformation method of a rubber tree independent of tissue culture, which comprises the following steps: S1, providing agrobacterium rhizogenes K599 bacterial liquid containing target genes, wherein the target genes are loaded in a pCAMBIA2301 vector; s2, preparing an infection material by using the agrobacterium rhizogenes K599 bacterial liquid; S3, carrying out girdling treatment on the stems of the rubber saplings, and carrying out pretreatment on girdling wounds to remove fresh latex secreted at the wounds; S4, infecting the pretreated girdling wound by using the infecting material; S5, covering and mounting the girdling wound by adopting a wet culture medium so as to keep the wound moist and incubate strains; Wherein the target gene is GUS gene, RUBY gene or JcFT gene. Further, in step S2, the infesting material is agrobacterium rhizogenes K599 infesting solution or agrobacterium rhizogenes K599 lawn. Further, the OD600 of the agrobacterium rhizogenes K599 bacterial liquid is 0.4-0.8, and the infection method is a bacteria wrapping method or an injection method. Further, the OD600 of the agrobacterium rhizogenes K599 bacterial liquid is 0.6-0.8, and the infection method is an injection method. Further, in the step S3, the girdling treatment is to cut off a circle of phloem on the stems at the position which is 50 cm from the ground. Further, in step S3, the pretreatment includes wipin