CN-122004130-A - Strawberry anther culture regenerated plant optimization method based on multi-stage screening

CN122004130ACN 122004130 ACN122004130 ACN 122004130ACN-122004130-A

Abstract

The invention relates to the field of plant biotechnology breeding, and discloses a multi-stage screening-based strawberry anther culture regeneration plant optimization method which comprises the steps of constructing a regeneration first-generation regeneration plant group through in-vitro induction culture, detecting ploidy of a single plant by utilizing a flow cytometry, removing ploidy abnormal plants according to ploidy detection results, calculating genetic similarity coefficients of ploidy stable plants and parents by utilizing simple repeated sequence marks, removing genetic variation plants to reserve a major line group, determining agronomic, physiological and fruit quality indexes after the major line group is planted in a field, establishing a stage evaluation model by integrating ploidy, genetic coefficients and multidimensional character indexes, and dividing the plants into different stages to establish a major line. The invention improves the screening efficiency and accuracy of the regenerated plants cultured by the strawberry anther by constructing a three-level linkage screening system of cells, molecules and phenotypes, and realizes objective quantification of establishment of a major line.

Inventors

LUO JINGJING
TIAN RUNYU
WANG QINGJIE
GUO JINGRU
PENG FUTIAN

Assignees

山东农业大学

Dates

Publication Date: 20260512
Application Date: 20260226

Claims (10)

1. A preferred method for culturing regenerated plants based on multi-stage screening of strawberry anthers, comprising the steps of: constructing a regenerated first-generation regenerated plant population through in-vitro induction culture; detecting ploidy of each individual plant in the regenerated first-generation regenerated plant population by utilizing a flow cytometry, obtaining ploidy detection results, removing ploidy abnormal plants according to the ploidy detection results and retaining ploidy stable plants; Extracting genome DNA of the ploidy stable plants, amplifying by using simple repeated sequence markers, calculating genetic similarity coefficients between the ploidy stable plants and donor parents, eliminating genetic variation plants according to the genetic similarity coefficients, and retaining a major population with consistent genetic background; planting the major line group in a field environment, and measuring agronomic character indexes, physiological indexes and fruit quality indexes; And establishing a grading evaluation model by integrating the ploidy detection result, the genetic similarity coefficient, the agronomic character index, the physiological index and the fruit quality index, and dividing single plants in the major line group into different grades to establish a major line.
2. The method for optimizing regenerated plants in strawberry anther culture based on multi-stage screening according to claim 1, wherein the step of detecting ploidy of each individual plant in the regenerated first-generation regenerated plant population by flow cytometry, and rejecting ploidy abnormal plants and retaining ploidy stable plants according to the ploidy detection result comprises: introducing a reference substance with known ploidy as an internal reference standard; detecting the DNA fluorescence intensity of each single plant leaf tissue and the internal reference standard in the regenerated first-generation regenerated plant population by utilizing the flow cytometry, and respectively obtaining the DNA fluorescence intensity peak value of the leaf tissue and the DNA fluorescence intensity peak value of the internal reference standard; Calculating the ratio of the DNA fluorescence intensity peak value of the leaf tissue to the DNA fluorescence intensity peak value of the internal reference standard to obtain a relative peak value ratio as the ploidy detection result; determining an individual plant with the relative peak ratio which is displayed by the ploidy detection result and accords with a first preset characteristic ratio or a second preset characteristic ratio as the ploidy stable plant and reserving the plant; And determining the single plant of which the ploidy detection result shows that the relative peak ratio deviates from the first preset characteristic ratio and deviates from the second preset characteristic ratio as the ploidy abnormal plant, and removing.
3. A preferred method of regenerating plants for strawberry anther culture based on multi-stage screening according to claim 1, characterized in that the step of eliminating genetically variant plants and preserving a major population of consistent genetic background according to said genetic similarity coefficients comprises: setting a preset genetic similarity coefficient screening threshold; Rejecting the single plant with the genetic similarity coefficient lower than the preset genetic similarity coefficient screening threshold value as the genetic variation plant; And classifying the residual single plants by using cluster analysis, and selecting the single plants clustered in the same main branch with the donor parent to form a major line group with consistent genetic background.
4. A method for regenerating a plant by strawberry anther culture based on a multistage screening according to claim 3, wherein in the step of establishing a priority line by dividing individual plants in the priority line group into different priority levels, a preset high genetic stability threshold is set, and the judgment criteria of the class I highly stable plants in the priority evaluation model comprises: The ploidy detection result shows that the chromosome number is stable to eight ploidy, the DNA fluorescence intensity distribution is concentrated, and a single peak is presented; The genetic similarity coefficient is greater than the preset high genetic stability threshold; The agronomic trait index and the donor parent are not significantly different; Setting the chromosome number, the genetic similarity coefficient and the agronomic character index of the plant as I grade judging conditions, and establishing the plant meeting the I grade judging conditions as a directly utilizable major material.
5. The method for preferentially culturing regenerated plants from strawberry anthers based on multi-stage screening according to claim 4, wherein the decision criteria of the class II substantially stable plants in the hierarchical evaluation model comprises: The ploidy detection result shows that the chromosome number is mainly eight ploidy; The genetic similarity coefficient is between the preset genetic similarity coefficient screening threshold value and the preset high genetic stability threshold value; The phenotype is slightly variable in non-critical traits; Setting the chromosome number, the genetic similarity coefficient and the phenotype as II grade judging conditions, and taking the plants meeting the II grade judging conditions as secondary materials for retention and observation.
6. A preferred method of regenerating plants for strawberry anther culture based on a multi-stage sieve according to claim 3, characterized in that the decision criteria for class III low-stable plants in the hierarchical evaluation model comprise: the ploidy detection result shows that the chromosome number is disordered; The genetic similarity coefficient is smaller than the preset genetic similarity coefficient screening threshold value; Phenotypes are manifested by stunted plant growth, leaf curl and fruit deformity; Setting the judgment standard of at least one of the chromosome number, the genetic similarity coefficient or the phenotype as a class III judgment condition, and eliminating the plants meeting the class III judgment condition.
7. A preferred method of regenerating plants for strawberry anther culture based on multi-stage screening according to claim 1, wherein in the step of calculating a genetic similarity coefficient between each of said ploidy-stable plants and the donor parent, said genetic similarity coefficient calculating logic comprises: Multiplying the number of the stripes shared by the ploidy stable plant to be detected and the donor parent by two to obtain a molecular value, multiplying the number of the stripes shared by the ploidy stable plant to be detected and the donor parent by two, adding the number of the stripes unique to the ploidy stable plant to be detected and the number of the stripes unique to the donor parent to obtain a denominator value, and calculating the ratio of the molecular value to the denominator value to obtain the genetic similarity coefficient.
8. The method for optimizing regenerated plants in strawberry anther culture based on multi-stage screening according to claim 1, wherein in the step of determining agronomic performance index, physiological index and fruit quality index, the agronomic performance index comprises plant height, stem thickness, leaf area, biomass and root morphology; the physiological index comprises chlorophyll content, net photosynthetic rate, stomatal conductance and root system activity; The fruit quality index comprises single fruit quality, soluble solids content, titratable acid content, soluble sugar content, soluble protein content and vitamin C content.
9. A preferred method for regenerating plants in strawberry anther culture based on multi-stage screening according to claim 8, characterized in that the root activity measurement process comprises: and obtaining the root system of the plant planted in the field environment of the major line group as a sample, reducing phenyl tetrazolium in the sample into formazan by using dehydrogenase, measuring the generation amount of the formazan, and calculating the generation amount of the formazan in the unit root weight of unit time by combining the fresh weight of the root system and the reaction time of the sample as the root system activity.
10. A preferred method of regenerating plants for strawberry anther culture based on a multi-stage screen according to claim 1, characterized in that in said step of constructing a population of regenerating first generation regenerated plants by in vitro induction culture, said in vitro induction culture comprises: selecting a bud which grows to a mononuclear borderline as an explant; Inoculating anthers in the flower buds into a callus induction culture medium for dark culture induction to form callus; transferring the callus into a differentiation medium, and inducing differentiation of adventitious buds under illumination conditions; And transferring the adventitious buds into a rooting culture medium for culturing and constructing the regenerated first-generation regenerated plant population.

Description

Strawberry anther culture regenerated plant optimization method based on multi-stage screening Technical Field The invention relates to the field of plant biotechnology breeding, in particular to a strawberry anther culture regeneration plant optimization method based on a multi-stage screening. Background Strawberry anther culture technology is an important way for germplasm creation and variety improvement, but in the isolated culture process, the regenerated plant generally has the problem of high chromosome fold variation rate. The prior art generally lacks an effective link for systematically screening ploidy of a regenerated population in a seedling stage, and undetected regenerated plants are often transplanted directly to a field for full-period planting and management, so that a large number of ploidy confusion or ineffective individuals of aneuploidy occupy land resources and production management cost. Meanwhile, in the process of screening the major lines aiming at regenerated plants, the traditional method mainly relies on direct observation of agricultural characters in the field. However, the expression of plant phenotype is easy to be interfered by environmental factors such as soil fertility, climate fluctuation and the like, and it is difficult to accurately distinguish environmental decoration changes from substantial genetic variation simply by phenotype observation, so that hysteresis exists in variation identification, and it is difficult to ensure that the genetic background of a selected major line is truly consistent. In addition, the existing evaluation system is often focused on single character investigation, and lacks a comprehensive quantitative evaluation standard capable of fusing cytological characteristics, molecular genetic data and field phenotype expression. The method makes it difficult for breeders to objectively and accurately judge the application value of plants when facing complex regeneration groups, and results in greater blindness and randomness in the screening process of good strains. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a strawberry anther culture regeneration plant optimization method based on a multi-stage screening, which solves the problems of high field screening workload, high blindness and poor genetic stability of a major line caused by the fact that regenerated plants are easy to have ploidy confusion and unexpected genetic variation and lack of quantitative grading evaluation standards in the existing strawberry anther culture regeneration technology. The invention aims at realizing the technical scheme that the method for optimizing the regenerated plant by strawberry anther culture based on the multi-stage screening is realized. The method establishes a set of multidimensional evaluation system integrating cytology detection, molecular marker analysis and phenotype identification, and aims at eliminating variant individuals from a regenerated population layer by layer and establishing a priority level. The specific technical scheme is as follows: The method comprises the steps of obtaining a first generation regeneration plant group through in-vitro induction culture, carrying out ploidy screening on single plants of the group by utilizing a flow cytometry, directly removing ploidy abnormal plants according to ploidy detection results of cell nucleus DNA content, only preserving ploidy stable plants to enter the next round of screening, extracting genome DNA of ploidy stable plants, amplifying by utilizing a simple repeated sequence marking technology, quantifying genetic variation degree by calculating genetic similarity coefficients between each plant and donor parents, removing the genetic variation plants according to the genetic variation degree, preserving a major line group with a highly consistent genetic background, after finishing the screening in a laboratory, fixing the major line group in a field environment, collecting multidimensional data of agronomic characters, physiological indexes, fruit quality and the like, and finally, dividing the regeneration plants into different grades by establishing a grading evaluation model, comprehensively considering ploidy states, genetic similarity and field expression data, thereby establishing a target major line. As a preferable scheme of the invention, in the in vitro induction culture stage, a bud which develops to a mononuclear approach period is selected as an explant. The anther in the flower bud is inoculated to a callus induction culture medium for dark culture, pollen cells are promoted to dedifferentiate to form callus, the callus is induced to dedifferentiate into adventitious buds by utilizing a differentiation culture medium under the illumination condition, and finally, a regenerated population is obtained through rooting culture. The process is regulated and controlled by specific light temperature and culture medium conditions, a