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CN-122012600-A - Genetic transformation method of dendrobium nobile without genotype dependence and application

CN122012600ACN 122012600 ACN122012600 ACN 122012600ACN-122012600-A

Abstract

The invention relates to a genetic transformation method of dendrobium nobile without genotype dependence and application thereof, belonging to the technical field of genetic engineering. The method comprises the steps of taking embryogenic callus, protocorm or protocorm-like body of dendrobium candidum as an explant, introducing a binary vector carrying a target gene into agrobacterium to prepare an invasion solution, infecting the explant, co-culturing, transferring to a culture medium containing a screening agent to obtain a resistant explant, and differentiating and rooting culture to obtain a transgenic plant. According to the invention, through systematic optimization of plant growth regulator combinations, agrobacterium strain types, infection modes and auxiliary screening modes, a high-efficiency stable genetic transformation system which does not depend on specific genotypes and takes multi-type explants as receptors is established for the first time, the problems of strong genotype dependence and limited tissue types in the traditional method are solved, good applicability and repeatability are shown in a plurality of autumn dendrobium hybrid seeds, and a general technical platform is provided for gene function research and molecular breeding.

Inventors

  • LI YAMEI
  • YIN JUNMEI
  • LI CHONGHUI
  • Jiang Qionghai

Assignees

  • 中国热带农业科学院热带作物品种资源研究所
  • 中国热带农业科学院海口实验站

Dates

Publication Date
20260512
Application Date
20260327

Claims (10)

  1. 1. The genetic transformation method of the dendrobium nobile without genotype dependence is characterized by comprising the following steps of: (1) Preparing an explant, namely selecting embryogenic callus of dendrobium candidum, protocorm or protocorm-like as the explant; (2) Agrobacteria infection, namely introducing a binary vector carrying a target gene into agrobacteria, preparing agrobacteria infection liquid and infecting the explant; (3) Co-culturing, namely placing the infected explant into a co-culture medium for co-culturing; (4) Transferring the co-cultured explants to a screening medium containing a screening agent, and carrying out auxiliary screening by using a visual screening marker gene to obtain resistant explants; (5) Differentiation and rooting, namely transferring the eGFP expression resistance explant to a differentiation medium to induce bud differentiation, and transferring to a rooting medium to induce rooting, so as to obtain a transgenic plant.
  2. 2. The method of claim 1, wherein the agrobacterium in step (2) is a K599 strain and other types of agrobacterium rhizogenes.
  3. 3. The method of claim 1, wherein the infection in the step (2) is negative pressure assisted infection, the negative pressure condition is-0.08 to-0.10 MPa, and the infection time is 20-35 min.
  4. 4. The method of claim 1, wherein the OD 600 value of the agrobacterium infection solution in step (2) is 0.6-0.8.
  5. 5. The method of claim 1, wherein the screening agent in step (4) is G418 at a concentration of 30-50 mg/L.
  6. 6. The method according to claim 1, wherein the co-cultivation in step (3) is performed at a temperature of 22 to 23 ℃ for a co-cultivation time of 2 to 4 days.
  7. 7. The method of claim 1, wherein the screening medium in step (4) further comprises a bacteriostatic agent, timentin, at a concentration of 100-400 mg/L.
  8. 8. The method of claim 1, wherein the binary vector carries an eGFP reporter gene or other visual reporter gene and the positive explants are screened in step (4) by the assistance of eGFP fluorescent expression.
  9. 9. The method of claim 1, wherein the autumn dendrobium hybrid is a d. 'Sonia Hiasakul' or d. 'Noble Woman' or other dendrobium hybrid.
  10. 10. Use of the method according to any one of claims 1 to 9 in genetic engineering breeding, functional genetic verification or genome editing of dendrobium nobile.

Description

Genetic transformation method of dendrobium nobile without genotype dependence and application Technical Field The invention relates to the technical field of genetic engineering, in particular to a genetic transformation method of dendrobium nobile without genotype dependence and application thereof. Background Dendrobium nobile (Phalaenopsis-typeDendrobiumhybrids, den-Phals) is a horticultural cultivar obtained by cross-breeding of different species in the butterfly orchid group (sectionPhalaenopsis) of Dendrobium (Dendrobium). The genus Dendrobium is the second largest genus of Orchidaceae (Orchidaceae). The autumn dendrobium has gorgeous flower color, unique flower shape and long ornamental period after harvest, and the hybrid is widely used as cut flowers and potted ornamental plants, and is an important component of the global tropical flower industry. In addition, part of dendrobium hybrid has long history of application in Asian traditional herbal medicines, and the economic value of the dendrobium hybrid is further improved. However, with the rapid development of the flower industry, the main cultivated varieties in the market are still mainly of the type with single flower color and highly repeated genetic background, and the ornamental attraction and commercial competitiveness of the dendrobium candidum are severely restricted. Traditional breeding plays an important role in variety improvement, but due to factors such as long childhood of dendrobium candidum, limited spontaneous genetic variation, narrow germplasm resource gene library and the like, the conventional breeding means are difficult to realize efficient genetic improvement. In contrast, genetic engineering breeding has the advantages of targeted trait directional improvement and short breeding period, and is hopeful to break through the bottleneck. To date, although a great deal of research and report on transgenic breeding of dendrobium candidum exist, the problems of low transformation efficiency, complex operation, strong genotype dependence and the like are common. At present, only a few dendrobium species realize stable transformation and character improvement of exogenous genes. Successful application of either Gene Modification (GM) or CRISPR/Cas-based Gene Editing (GE) techniques is highly dependent on stable and efficient genetic transformation platforms. In the past three decades, although few studies have achieved transgenic improvement of the flower shape, flower shape and other traits of dendrobium candidum, genome targeting editing has been reported to be successfully applied to dendrobium candidum (d. Oficicle), d. 'Chao PRAYA SMILE' and phalaenopsis in recent years, CRISPR/Cas mediated genome editing has not been applied to dendrobium candidum, and the root cause is the lack of a high-efficiency and universal genetic transformation system. In recent years, a plurality of functional genes related to ornamental traits and stress response have been identified in dendrobium nobile, and preliminary functional analyses have been performed by means of multiple sets of chemical analysis, transient over-expression, dsRNA silencing, virus-induced gene silencing (VIGS), and the like. Although these methods provide a viable approach for gene function studies, the related genes are difficult to apply to practical breeding due to the lack of a stable genetic transformation system. At present, only a few dendrobium plants establish a genetic transformation system, and the transformation efficiency is generally low. It is noted that most studies use "resistant explant rate" rather than "transgenic line yield" as a statistical indicator of transformation efficiency, and the difference in resistance between different varieties is significant, e.g., 0.5% -18% for d.normal and 5% -46.2% for d.oficiole. In view of the high heterozygosity of the genetic background of the commercial dendrobium candidum, the establishment of a transformation system for each variety independently is neither realistic nor economical. Therefore, a set of genetic transformation method which is systematic, efficient and independent of genotype is constructed, and the method has important significance for promoting the molecular breeding research of dendrobium candidum. The research system optimizes key parameters of genetic transformation of the agrobacterium-mediated dendrobium candidum, including the type of an explant, the agrobacterium strain, the infection concentration and mode and a screening strategy, and successfully establishes a set of stable and repeatable genetic transformation system which does not depend on the explant, wherein the average transformation efficiency reaches about 20 percent (calculated by the acquisition rate of a transgenic strain). Through eGFP fluorescence observation, PCR analysis and NPTII protein detection, stable integration and expression of exogenous genes are verified. The establishment of the platform provides key technic