CN-121992024-A - Genetic transformation method based on pear body cell embryogenesis

CN121992024ACN 121992024 ACN121992024 ACN 121992024ACN-121992024-A

Abstract

The invention discloses a genetic transformation method based on pear somatic embryogenesis, and belongs to the technical fields of genetic engineering and molecular breeding. Aiming at the problems of low transformation efficiency, high chimera rate, long period, difficulty in obtaining gene editing regeneration plants and the like in the conventional pear genetic transformation, the invention provides two paths, namely, firstly, a immature zygotic embryo of 30-60d after pear flowers is used as an explant, a resistant plant is directly induced by a somatic embryo regeneration path, the transformation efficiency reaches 73.91 in 40-60d, secondly, the embryogenic callus with consistent genetic background is used as the explant for infection, and the embryogenesis is maintained and the proliferation efficiency is improved by optimizing a culture medium formula, so that the resistant plant with consistent genetic background can be obtained within 30-50 d. Overcomes the defect that the existing callus transformation is difficult to obtain regenerated plants, obviously shortens the breeding period, has high transformation efficiency, low chimera rate and controllable consistency of genetic background, and provides technical support for pear functional genomics research and molecular breeding.

Inventors

YANG YINGJIE
GUAN YUJUAN
ZHANG HUIYING
LI DINGLI
LIU JIANLONG
CUI ZHENHUA
SONG JIANKUN
MA WEIZHEN
ZHANG JIYAO

Assignees

青岛农业大学

Dates

Publication Date: 20260508
Application Date: 20260226

Claims (9)

1. A genetic transformation method based on pear somatic embryogenesis, comprising the steps of: (a) Preparing an explant, namely taking immature zygotic embryos of pears or embryogenic callus obtained by induction of the immature zygotic embryos of pears as a transformation explant; (b) The preparation of agrobacterium infection solution, namely, culturing and resuspending the agrobacterium strain containing the target carrier to prepare the agrobacterium infection solution; (c) Immersing the transformed explant of the step (a) into the agrobacterium infection solution of the step (b) for infection, and transferring the explant to a co-culture medium for co-culture after the infection is finished; (d) And (3) screening and regenerating, namely transferring the co-cultured explant into a culture medium for primary screening culture and secondary screening culture to obtain a transformed plant.
2. The genetic transformation method according to claim 1, wherein in the step (a), the immature zygotic embryos are obtained by collecting pear fruits 30-60 d after flowers, washing and sterilizing, washing with sterile water, splitting the fruits, taking out immature seeds, picking up seed coats, and extruding the immature zygotic embryos.
3. The genetic transformation method according to claim 1 or 2, wherein in step (a), the embryogenic callus is obtained from immature zygotic embryos by induction culture, subculture, comprising the following steps: inoculating the immature zygotic embryo into an induction culture medium, and placing the culture medium in the dark at 25 ℃ for induction culture for 30 d to obtain embryogenic callus; and (3) crushing embryogenic callus with excellent selection state, transferring the crushed embryogenic callus into a subculture medium, and performing subculture at 25 ℃ in darkness to obtain embryogenic callus with consistent genetic background.
4. The genetic transformation method according to claim 3, wherein the induction medium is MS medium +2.0 mg/L2, 4-D +0.5 mg/L KT +30 g/L sucrose +7 g/L agar, pH=5.82; the secondary culture medium is MS culture medium +1.0 mg/L NAA +0.5 mg/L TDZ +2.0mg/L ABA +30 g/L sucrose +7 g/L agar, pH=5.82.
5. The genetic transformation method according to claim 1, wherein the period of infection in step (C) is 15min, during which time the somatic embryos are contused or embryogenic callus are broken up with forceps and the invasive solution is shaken frequently, the co-culture medium is MS medium +1.0 mg/L NAA +0.5 mg/L TDZ +30 g/L sucrose +7 g/L agar, pH=5.82, and the co-culture conditions are 25℃and co-culture under darkness 3 d.
6. The genetic transformation method according to claim 1, wherein the culture medium of the preliminary screening culture in step (d) is MS culture medium +1.0 mg/L NAA +0.5 mg/L TDZ +25 mg/L kanamycin +200 mg/L cefotaxime +30 g/L sucrose +7 g/L agar, pH=5.82, and the preliminary screening culture is carried out under conditions that the culture is carried out under 25 ℃ in darkness, and when cotyledonary embryos appear on the explants, the explants are transferred to light for culture until candidate resistant plants are obtained.
7. The genetic transformation method according to claim 1, wherein the medium for the double screening culture in the step (d) is MS medium +1.5 mg/L6BA+0.2 mg/LIBA+25 mg/L kanamycin +200 mg/L cefotaxime +30 g/L sucrose +7 g/L agar, pH=5.82, and the condition for the double screening culture is that the medium is placed at 25℃and the light intensity is 100. Mu. Mol.m 2 ·s 1 The strain which can grow and proliferate normally on the culture medium is the positive plant after 16h illumination/8 h dark culture.
8. Use of the method of any one of claims 1-7 in pear gene functional research or genetic breeding.
9. Embryogenic callus subculture medium for genetic transformation of pears, characterized in that the medium takes MS as basic medium and is added with 1.0 mg/L NAA, 0.5 mg/L TDZ, 2.0 mg/L ABA, 30g/L sucrose and 7 g/L agar, pH=5.82.

Description

Genetic transformation method based on pear body cell embryogenesis Technical Field The invention relates to the technical field of genetic engineering and molecular breeding, in particular to a genetic transformation method based on pear somatic embryogenesis. Background Pear (Pyrus spp) is an important deciduous fruit tree in China, and has large industrial scale and remarkable economic value. However, pear is used as perennial woody plant, traditional crossbreeding faces the bottleneck of long breeding period, difficult breaking of character linkage and the like, and severely restricts the rapid and directional breeding of key agronomic characters such as disease resistance, dwarfing, quality improvement, stress resistance and the like. Compared with the traditional time-consuming crossbreeding, the genetic engineering breeding has the remarkable advantages that the specific character can be accurately improved, the target genes are efficiently introduced into the genetic background of the good variety, the linkage encumbrance of a large number of bad genes is effectively avoided, the interference of childhood on the breeding process can be avoided to a certain extent, and therefore the target transgenic material is rapidly obtained. In genetic transformation studies, a highly efficient, stable receptor regeneration system is critical in determining the success of transformation. Somatic embryogenesis (Somatic Embryogenesis, SE) refers to the process by which somatic cells develop under specific conditions to form embryoid structures similar to zygotic embryos and can ultimately be regenerated into whole plants. The technology has the outstanding advantages of uniform genetic background, low rate of regenerated chimera, high propagation coefficient, suitability for large-scale operation and the like, and is recognized as an ideal receptor system for realizing efficient genetic transformation of perennial fruit trees such as pears and the like. Based on the method, a pear efficient somatic embryogenesis system is combined with a genetic transformation and CRISPR/Cas9 gene editing technology, so that the pear efficient somatic embryogenesis system becomes a core biotechnology path for breaking through the breeding cycle barriers of woody fruit trees and realizing accurate improvement of target traits. However, the existing pear genetic transformation technology still has a plurality of defects and shortcomings. The method is characterized in that genetic transformation efficiency is generally low, the proportion of chimeras in regenerated plants is high, the screening workload of target genotype homozygosity or purification is high, meanwhile, false positive somatic cells are increased in the transformation process, the complexity and cost of later-stage identification are increased, and the whole breeding period is still longer. In addition, the currently reported pear gene editing technology is mostly dependent on callus as a transformation receptor, and the currently reported pear gene editing technology is difficult to efficiently induce and obtain complete gene editing regeneration plants, so that the application potential of the gene editing technology in pear functional genomics research and precise breeding is limited. In view of the above, the prior art cannot meet the actual demands of efficient molecular breeding and large-scale trait improvement of pears, and there is a need to establish a set of stable genetic transformation methods with efficient somatic embryogenesis as a core, which can be simultaneously applied to gene overexpression and gene editing operations. Disclosure of Invention In view of the above, the invention provides a genetic transformation method based on pear body cell embryogenesis, which has the advantages of short period, high transformation efficiency and less chimera. In order to solve the technical problems, the application adopts the following technical scheme: The first object of the present application is to provide a genetic transformation method based on pear somatic embryogenesis, comprising the steps of: (a) Preparing an explant, namely taking immature zygotic embryos of pears or embryogenic callus obtained by induction of the immature zygotic embryos of pears as a transformation explant; (b) The preparation of agrobacterium infection solution, namely, culturing and resuspending the agrobacterium strain containing the target carrier to prepare the agrobacterium infection solution; (c) Immersing the transformed explant of the step (a) into the agrobacterium infection solution of the step (b) for infection, and transferring the explant to a co-culture medium for co-culture after the infection is finished; (d) And (3) screening and regenerating, namely transferring the co-cultured explant into a culture medium for primary screening culture and secondary screening culture to obtain a transformed plant. In the step (a), the immature zygotic embryo is obtained by col