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CN-122012369-A - Genetic transformation method of China rose

CN122012369ACN 122012369 ACN122012369 ACN 122012369ACN-122012369-A

Abstract

The invention relates to a genetic transformation method of China rose, belonging to the field of plant tissue culture. Specifically, the invention develops a genetic transformation method of the China rose variety kala.

Inventors

  • ZHANG WEI
  • TIAN MIAO
  • ZHANG YUEXIN
  • TONG WANTING
  • NING GUOGUI

Assignees

  • 华中农业大学

Dates

Publication Date
20260512
Application Date
20260129

Claims (6)

  1. 1. A somatic embryo induction method of 'kala' rose, which is characterized by comprising the following steps: i. obtaining tissue culture seedlings; ii. Obtaining somatic embryos: (1) Slightly scratching undeployed small leaves at the top of the tissue culture seedling obtained in the step (i), inoculating the small leaves onto a callus induction culture medium, and culturing in the dark until callus is obtained, wherein the callus induction culture medium comprises MS+4.0 mg/L2.4-D+0.05 mg/L KT+30 g/L glucose+3.0 g/L PHYTAGEL; (2) Inoculating the callus obtained in the step (1) onto a somatic embryo induction culture medium, and culturing in the dark until somatic embryos are obtained, wherein the somatic embryo induction culture medium comprises MS+1.0 mg/L2.4-D+0.1 mg/L KT+30 g/L glucose+3.0 g/L PHYTAGEL.
  2. 2. The method according to claim 1, wherein the method for obtaining tissue culture seedlings comprises the following steps: (1) Cutting a healthy and growing top branch with consistent growth vigor into a stem segment with 1-2 axillary bud points, sequentially sterilizing 0.1% (w/v) carbendazim, 75% (v/v) alcohol and 0.1% mercuric chloride, washing with sterile water, and inoculating to a bud initiation culture medium after the sterile filter paper is dried to obtain axillary buds induced by the stem segment, wherein the bud initiation culture medium comprises MS+1.0 mg/L6-BA+0.01 mg/L NAA+0.1 mg/L GA 3 +30 g/L sucrose+7.5 g/L Agar; (2) Inoculating the axillary buds obtained in the step (1) onto a tissue culture seedling multiplication medium, and culturing to obtain the tissue culture seedlings, wherein the formulation of the tissue culture seedling multiplication medium is MS+0.05 mg/L6-BA+0.05 mg/L NAA+30 g/L sucrose+7.5 g/L Agar.
  3. 3. The method of claim 2, wherein the 0.1% mercuric chloride disinfection time is 18 min.
  4. 4. A somatic embryo proliferation method of 'kala' rose is characterized in that somatic embryos obtained by the method of any one of claims 1-3 are inoculated on a somatic embryo proliferation culture medium, and cultured in the dark to proliferate somatic embryos, wherein the somatic embryo proliferation culture medium has a formula of MS+0.5 mg/L2, 4-D+0.05 mg/L KT+30 g/L glucose+3.0 g/L PHYTAGEL.
  5. 5. The tissue culture method of the 'kala' rose is characterized by comprising the following steps: (1) Inoculating somatic embryos obtained by the method according to any one of claims 1-4 onto a differentiation medium, and culturing under light until the somatic embryos differentiate into adventitious buds, wherein the differentiation medium has a formula of MS+1.0 mg/L TDZ+0.5 mg/L GA 3 +0.01 mg/L NAA+30 g/L glucose+3.0 g/L PHYTAGEL; (2) Inoculating the somatic embryos differentiated into the adventitious buds obtained in the step (1) onto an adventitious bud elongation culture medium, and culturing by illumination until seedlings are obtained, wherein the adventitious bud elongation culture medium has a formula of MS+0.5 mg/L6-BA+0.05 mg/L NAA+30 g/L sucrose+7.5 g/L Agar; (3) Inoculating the seedling obtained in the step (2) onto a rooting medium, and culturing by illumination until a complete regenerated plant is obtained, wherein the rooting medium is prepared from 1/2MS+0.05 mg/L NAA+30 g/L sucrose+7.5 g/L Agar.
  6. 6. The genetic transformation method of the 'kala' rose is characterized by comprising the following steps: (1) Infecting somatic embryos obtained by the method of any one of claims 1-4 with agrobacterium tumefaciens bacteria liquid carrying a target gene expression vector, and inoculating the infected somatic embryos to a co-culture medium, and culturing in darkness for 3D, wherein the formula of the co-culture medium is MS+0.5 mg/L2.4-D+0.05 mg/L KT+100 mu mol/L As+30 g/L glucose+3.0 g/L PHYTAGEL; (2) Inoculating the somatic embryos cultured in the step (1) onto a selection medium, and culturing in darkness for 2-3 months, wherein the selection medium comprises the formula of MS+0.5 mg/L2.4-D+0.05 mg/L KT+80 mg/L Kan+300 mg/L Cef+30 g/L glucose+3.0 g/L PHYTAGEL; (3) Inoculating the somatic embryos cultured in the step (2) onto a selective germination medium, and culturing by illumination until adventitious buds are differentiated, wherein the selective germination medium is prepared from MS+1.0 mg/L TDZ+0.5 mg/L GA 3 +0.01 mg/L NAA+80 mg/L Kan+300 mg/L Cef+30 g/L glucose+3.0 g/L PHYTAGEL; (4) Inoculating the somatic embryos cultured in the step (3) onto a selection strong bud culture medium, and carrying out illumination culture until the somatic embryos are differentiated into seedlings, wherein the selection strong bud culture medium comprises the formula of MS+0.5 mg/L6-BA+0.5 mg/L NAA+50 mg/L Kan+150 mg/L Cef+30 g/L sucrose+7.5 g/L Agar; (5) Inoculating the seedlings cultured in the step (4) to a rooting medium, and culturing by illumination until complete positive plants are obtained, wherein the rooting medium is prepared from 1/2MS+0.05 mg/L NAA+25 mg/L Kan+50 mg/L Cef+30 g/L sucrose+7.5 g/L Agar.

Description

Genetic transformation method of China rose Technical Field The invention relates to a genetic transformation method of China rose, belonging to the field of plant tissue culture Background China rose (Rosa hybrid) is one of the most important ornamental plants worldwide, and has extremely high economic and cultural values. Among them, cut rose has a huge market, and 'kala' (r. 'carola') is a global classical cut rose variety, which occupies a central position in industry due to its excellent properties such as beautiful flower shape, pure color, long bottle insertion period, etc. However, most commercial varieties of China rose are heterozygotes with complex genetic background, the traditional breeding period is long, and due to factors such as gene linkage and the like, specific characters (such as disease resistance, flower color, aroma, postharvest life and the like) are difficult to be directionally introduced or improved while the original excellent comprehensive characters are maintained through sexual hybridization. The development of modern molecular biology and genetic engineering technology provides a revolutionary approach for the directional improvement of China rose traits. Exogenous target genes are directly introduced into a plant genome through genetic transformation technologies such as an agrobacterium-mediated method, so that the reproductive isolation limit of species can be broken, and the precise and efficient improvement of the characters can be realized. Therefore, the establishment of a high-efficiency and stable genetic transformation system is a precondition and key for developing China rose gene function research and molecular breeding. Although the establishment of genetic transformation systems has been reported in some China rose cultivars (such as 'Samantha', 'Yuehuang' and the like, a few cultivars which are easy to regenerate), the technology has significant genotype dependence. The in vitro regeneration and genetic transformation efficiency of China rose is deeply influenced by the genotype of the variety, and a mature system established on one variety is often difficult to be directly applied to other varieties. At present, for the global important commercial variety 'kala', the stable and efficient genetic transformation system is not reported in the published materials. The technical bottlenecks of difficult somatic embryo regeneration, low screening and regeneration rate after transformation and the like seriously obstruct the gene function research and molecular design breeding process aiming at the variety. Therefore, aiming at the specific and important commercialized genotype of 'kala', developing a set of efficient and repeatable genetic transformation system from explant selection, disinfection, tissue culture seedling culture, callus induction, somatic embryo regeneration, agrobacterium infection, co-culture to resistant bud screening, regeneration and complete plant regeneration becomes a key technical problem to be broken through in the field. The establishment of the system not only can provide an indispensable technical tool for analyzing the molecular mechanism of important agronomic characters of 'Kaola', but also can lay a solid foundation for directly utilizing the genetic engineering technology to improve the variety and cultivate new excellent China rose varieties with independent intellectual property rights. Disclosure of Invention In order to achieve the above purpose, the invention adopts the following technical scheme: The invention provides a somatic embryo induction method of 'kala' China rose, which is characterized by comprising the following steps: i. obtaining tissue culture seedlings; ii. Obtaining somatic embryos: (1) Slightly scratching undeployed small leaves at the top of the tissue culture seedling obtained in the step (i), inoculating the small leaves onto a callus induction culture medium, and culturing in the dark until callus is obtained, wherein the callus induction culture medium comprises MS+4.0 mg/L2.4-D+0.05 mg/L KT+30 g/L glucose+3.0 g/L PHYTAGEL; (2) Inoculating the callus obtained in the step (1) onto a somatic embryo induction culture medium, and culturing in the dark until somatic embryos are obtained, wherein the somatic embryo induction culture medium comprises MS+1.0 mg/L2.4-D+0.1 mg/L KT+30 g/L glucose+3.0 g/L PHYTAGEL. In some embodiments, the method for obtaining the tissue culture seedling in the step i is as follows: (1) Cutting a healthy and growing top branch with consistent growth vigor into a stem segment with 1-2 axillary bud points, sequentially sterilizing 0.1% (w/v) carbendazim, 75% (v/v) alcohol and 0.1% mercuric chloride, washing with sterile water, and inoculating to a bud initiation culture medium after the sterile filter paper is dried to obtain axillary buds induced by the stem segment, wherein the bud initiation culture medium comprises MS+1.0 mg/L6-BA+0.01 mg/L NAA+0.1 mg/L GA 3 +30 g/L sucrose+7.