CN114190268A

CN114190268A - Ploidy cyclic breeding method for polyploid rice

Info

Publication number: CN114190268A
Application number: CN202111225675.2A
Authority: CN
Inventors: 蔡得田; 宋兆建; 张献华; 陈阅军; 王维; 何玉池; 刘育华
Original assignee: Wuhan Polyploid Biotechnology Co ltd; Hubei University
Current assignee: Wuhan Polyploid Biotechnology Co ltd; Hubei University
Priority date: 2021-10-21
Filing date: 2021-10-21
Publication date: 2022-03-18

Abstract

The invention discloses a ploidy cycling breeding method of polyploid rice, which is based on that the genome of the rice is multiplied and increased to generate polyploid or haploid, and based on evolution and genetics, the ploidy of 4x → 2x → 1x is changed from high to low by continuously utilizing glume-removing young flower haploid culture to form diploid and haploid; and then, carrying out in-vitro culture and doubling on the young ear from the secondary branch stem primordium to the formation stage of the stamen and stamen continuously twice by utilizing somatic mitosis to realize the low-to-high alternation of 1x → 2x → 4x ploidy, and quickly breeding the homozygous tetraploid rice strain from the obtained plant through agronomic characteristic investigation, ploidy level detection, rice quality detection and resistance molecular marker auxiliary selection. The homozygous tetraploid rice line formed by ploidy cycle breeding by the method has the characteristics of obvious homozygous breeding time, high yield, high quality and multiple disease resistance. Promote the rapid breeding of new species of polyploid rice as soon as possible, and also provide valuable resource materials and breeding technology for the research of plant polyploids.

Description

Ploidy cyclic breeding method for polyploid rice

Technical Field

The invention relates to a ploidy cycle breeding method of polyploid rice, belonging to the technical field of new crop variety breeding in modern agriculture.

Background

Polyploidization is a main mode of plant evolution in nature, main grain, cotton and oil crops, wheat, cotton and rape are polyploids, and the yield is increased by times in the process of the polyploidy cultivars evolving through diploid wild species. This natural phenomenon implies a huge application prospect for polyploids. Therefore, a new strategy of 'breeding super rice by using double advantages of distant hybridization and polyploidy' was proposed in Chuattian, Yuanyongping and Luxingui 2001 (crop science, 2001). And a polyploid meiosis stability gene high-maturing strain (PMeS) is bred by adopting a wide distant hybridization and compound crossing method, the bottleneck problem of low maturing rate of polyploid rice breeding is solved (Chuaden and the like, China science C edition: life science, 2007), five technical systems are created, over 20 years are spent, 5000 polyploid rice materials are constructed, 100 polyploid excellent strains are bred, and 10 excellent strains show the advantages of polyploid rice such as thick stems, thick leaves, large panicle grains, strong stress resistance and the like. However, in the early stage, theoretical research is mainly used, breeding is assisted, so that the stable strain bred in the first batch has the advantages of excellent characteristics of high maturing rate of over 75 percent and thousand grain weight of 32-60 g (which is multiplied by the thousand grain weight of 18-30 g of a diploid), and strong adverse resistance, but the rice blast resistance is not strong, and the popularization and application of a new variety are influenced. In polyploid rice breeding, the number of carried genes is multiplied due to the multiplied increase of genome, so that the time for complete homozygous stabilization of a plurality of genes is long.

Tetraploid-based rice has a multiplied genome compared to that of diploid, contains a multiplied number of genes, and has 4 relative trait genes at each gene locus. From genetic analysis, 4 alleles relate to the whole homozygous process after pairing, exchanging and separating, which is much more complicated than that of diploids, and the obtained homozygous stable variety takes 16-20 generations. Therefore, the breeding mode of the polyploid rice needs to be further improved, the breeding range of the polyploid rice needs to be enlarged, the polyploid rice is applied to production practice as soon as possible, so that the grain yield is greatly improved, the world grain crisis is relieved, new thinking and new technology for breeding are urgently needed, and the problems of long homozygous breeding time, poor disease resistance and difficult popularization and application of the conventional polyploid rice are solved.

Disclosure of Invention

The invention aims to provide a ploidy cycle breeding method for polyploid rice, which is used for solving the difficult points that the complete homozygous stability of a plurality of genes and long time are required because the number of loaded genes is multiplied due to the multiplied increase of genome in polyploid rice breeding.

The invention discloses a method for cultivating rice by utilizing a haploid of a glume-removing young flower, which is based on evolution and genetics, and realizes the change of the ploidy of 4x → 2x → 1x from high to low by continuously and secondarily utilizing the haploid of the glume-removing young flower to form a diploid and a haploid; and then carrying out in-vitro culture and doubling on two continuous chromosome groups of young ears from a secondary branch stem primordium to a female stamen forming stage in mitosis of the somatic cell by utilizing mitosis of the somatic cell, so as to realize the replacement of 1x → 2x → 4x ploidy from low to high, and quickly breeding the homozygous tetraploid rice strain from the obtained plant through agronomic characteristic investigation, ploidy level detection, rice quality detection and resistance molecular marker auxiliary selection.

The ploidy cycle breeding method of the polyploid rice comprises the following stages:

a. polyploid indica rice and japonica rice with various characteristics are adopted as parents; the parent strain contains excellent characteristics of high fruit set (PMeS), large seed, excellent rice quality, high yield, stress resistance, disease resistance and the like;

b. mutually hybridizing the polyploid parents of indica rice and japonica rice with excellent characteristics to obtain a tetraploid indica/japonica subspecies hybrid (4 x);

c. adopting a young glume-removed flower which contains anther and ovary together at the mature embryo sac stage of the interspecific hybrid of the tetraploid indica-japonica subspecies to carry out haploid culture I;

d. in the haploid culture I, a special induced callus culture medium of a plant growth regulator MCPA (2-methyl-4-chlorophenoxyacetic acid) and amide is applied, and haploid culture processes such as callus induction, differentiation seedling emergence, strong root transplantation and the like are carried out; the culture medium for inducing the callus comprises: n6+ 0.02% -0.05% MCPA +0.5 mg/L-1.2 mg/L NAA (naphthylacetic acid) +5 mg/L-10 mg/L asparagine +5 mg/L-10 mg/L glutamine + 5% -7% sucrose (no agar); aseptically transferring the young glume-removed flowers into an induction culture medium for suspension culture, inducing the ovary to expand until callus is contained (about 40-50 days) and anthers crack and callus is contained, transferring the ovary callus and the anthers callus into a differentiation culture medium (MS +0.3 mg/L-1.5 mg/L KT +0.5 mg/L-1.5 mg/L NAA +5 mg/L-10 mg/L asparagine +5 mg/L-10 mg/L glutamine + 3% -5% sucrose + 0.75% agar) for culture and differentiation seedling emergence; performing rooting and seedling strengthening culture in 1/2MS, 0.2 mg/L-0.3 mg/L NAA, 100mg/L lactoprotein hydrolysate, 0.01-0.03% active carbon and 0.8% agar culture medium;

e. through agronomic feature investigation and chromosome ploidy detection by a flow cytometry, and DNA molecular markers such as rice blast resistance, bacterial blight resistance and the like are used for detection, so that different single plants (2x) close to a breeding target are obtained;

f. growing the selected single plant (2x) in a planting field to the mature period of an embryo sac, adopting a young flower haploid for removing glumes to culture II, and then culturing according to the same method of c and d to obtain different 1x plants, thereby realizing the replacement process of the ploidy of a chromosome set from 4x → 2x → 1x from high to low;

g. inducing the second branch stem primordium of the selected haploid (1x) plant to young ears of the stamens and pistils, and doubling the in vitro chromosome added with colchicine through callus tissue to obtain a homozygous diploid plant (2 x); the callus induction culture medium is as follows: n6+1.0 mg/L-2.5 mg/L2, 4-D +0.5 mg/L-1.5 mg/L NAA +100mg/L hydrolyzed milk protein + 0.5-0.6% sucrose + 0.8% agar, and the differentiation medium is MS +1.5 mg/L-2.5 mg/L KT +0.5 mg/L-1.5 mg/L NAA +0.1 mg/L-0.5 mg/L IAA +100mg/L hydrolyzed milk protein + 0.01-0.03% active carbon + 0.3% sucrose + 0.8% agar; the culture medium for inducing chromosome doubling of callus cells is: n6+1.5 mg/L-2.0 mg/L KT +0.5 mg/L-0.75 mg/L NAA +0.5mg/L IAA + 0.3% sucrose +50 mg/L-150 mg/L colchicine (no agar). Inducing the callus for 35-50 days, inducing chromosome doubling culture for 48-60 hours, after the treatment is finished, cleaning the callus by using an MS basic culture medium, then transferring the callus into an induced callus culture medium for recovery culture for 8-10 days, then transferring the callus into a differentiation culture medium until the callus is differentiated and emerges, and transferring the callus into a field for planting after strong root cultivation.

h. Carrying out agronomic trait observation, ploidy detection and resistance gene marker assisted selection on the plant, and carrying out in vitro culture and reduplication on the chromosome set by adopting the step g to obtain homozygous tetraploid (4 x); through agronomic character observation, ploidy detection, rice quality and disease resistance detection and disease resistance molecular marker auxiliary selection, the technical program of 1x → 2x → 4x of the chromosome group from low to high is completed; this is based on the principle that meiosis reduces chromosome composition by fold, whereas somatic mitosis increases chromosome composition by fold after treatment with colchicine. And (3) adopting haploid cultures I and II of the young flower without glume after continuous second meiosis, and breeding the 4x indica-japonica subspecies hybrid to form 2x and 1x haploid plants so as to complete the technical program and ploidy replacement of the 4x → 2x → 1x chromosome group from high to low. In addition, the young ear culture in-vitro chromosome group is doubled to form a homozygous diploid (2x), then the young ear culture in-vitro chromosome group is doubled to form a homozygous tetraploid (4x), the low-to-high technical program and ploidy replacement of the chromosome group from 1x → 2x → 4x are completed, and the two are fused together to form the ploidy circular breeding method of the polyploid rice.

The invention relates to a ploidy cycle breeding method of polyploid rice. The homozygous tetraploid rice line can be rapidly bred by the ploidy replacement of 4x → 2x → 1x and 1x → 2x → 4 x; and the homozygous diploid rice line can be rapidly bred according to the ploidy alternation of 4x → 2x → 1x and 1x → 2 x.

The invention has obvious advantages, and the homozygous tetraploid rice line is rapidly bred by utilizing a polyploid rice ploidy circulating breeding method. The homozygous tetraploid rice line formed by ploidy cycle breeding has the characteristics of obvious homozygous breeding time, high yield, high quality and multiple disease resistance.

The main innovation points of the invention are as follows: (1) according to the characteristics of biological meiosis and mitosis, the chromosome set from high to low of 4x → 2x → 1x is replaced by two continuous young flower haploid cultures, and the chromosome is doubled in vitro by two continuous colchicine treatments in mitosis of the cell, so that a plant of 1x → 2x → 4x is rapidly formed; (2) the MCPA special plant growth regulating substance is adopted to improve the callus induction efficiency; (3) the young glume-removing flowers comprise anthers and ovaries, the beneficial aggregation effect in tissue culture is exerted, the induction rate is improved, and the most easily-appearing genotype influence in tissue culture is avoided. (4) The invention can open a new approach for breeding tetraploid and diploid rice, greatly shorten the breeding period, promote the breeding of new varieties at an early stage, gradually apply to production practice, and provide valuable resource materials and breeding methods for plant polyploid research.

Drawings

FIG. 1 ploidy cycle method for breeding new variety "duo super 69" pedigree

Detailed Description

The present invention will be further described below by breeding polyploid rice with the polyploid rice gene 69.

a. Polyploid indica rice and japonica rice with various important characteristics are selected as parents. The indica rice line DT66 tetraploid with PMeS (polyploid meiosis stability) high-maturing gene is taken as a female parent, and 106 BxPSR 120 tetraploid japonica type material with rice blast resistance and PMeS gene is taken as a male parent;

b. and (3) carrying out parent hybridization of PMeS, non-PMeS, high-quality, high-yield, disease-resistant and other characters on the polyploid parents to obtain a tetraploid indica/japonica subspecies hybrid. Hybridizing tetraploid japonica rice, DT66 with rice blast resistance and PMeS with tetraploid 106B/PSR120 indica japonica rice with PMeS gene, rice blast resistance and bacterial leaf blight resistance to obtain tetraploid compound cross indica japonica subspecies hybrid;

c. and (3) carrying out haploid culture I by using the young glume-removed flowers in the mature embryo sac stage of the tetraploid interspecies hybrids. The method comprises the steps of adopting the young spikes which are in the mature embryo sac stage in the tetraploid interspecific hybrid booting ear, have the young flower glumes reaching the size of normal chaffs, have light green tips and light yellow and white glumes, sterilizing, using sharp tweezers to remove the two glumes of the young flowers aseptically, keeping complete anthers, filaments and ovaries, planting on a receptacle, and inoculating on a callus induction culture medium.

d. The haploid culture process of callus induction, differentiation seedling emergence and strong root transplantation is carried out by applying a special callus induction culture medium. Suspension culturing glume-removing young flowers in a N6+ 0.02-0.05% MCPA (2-methyl-4-chlorophenoxyacetic acid) + 0.5-1.2 mg/L NAA (naphthylacetic acid) +5 mg/L-10 mg/L asparagine +5 mg/L-10 mg/L glutamine + 5-7% sucrose culture medium, inducing ovary to expand, containing callus and dehiscent anther containing callus, transferring the embryo sac callus and anther callus into a differentiation culture medium (MS +0.3 mg/L-1.5 mg/L KT +0.5 mg/L-1.5 mg/L NAA +5 mg/L-10 mg/L asparagine +5 mg/L-10 mg/L glutamine + 3% sucrose + 0.75% agar) to emerge; the seedlings are rooted and strengthened in 1/2MS +0.2 mg/L-0.3 mg/L NAA +100mg/L lactoprotein hydrolysate + 0.01% -0.03% active carbon + 0.8% agar and cultured to the field for transplanting the seedlings.

e. Agronomic characteristic observation, flow cytometry ploidy determination and molecular marker-assisted breeding of disease-resistant genes. The agronomic characteristic observation focuses on investigating spike number, seed setting rate, thousand grain weight and rice quality; ploidy measurements were performed using young leaves and detected by flow cytometry. Plants (2x) close to the breeding target are selected by molecular markers of rice blast resistance genes Pi1, Pi2 and Pi9, and bacterial leaf blight resistance gene Xa 23.

f. And (4) completing the haploid culture II of the young glume-removed flowers, and realizing the replacement of the ploidy of the chromosome set from 2x → 1x and the ploidy of 4x → 2x → 1x from high to low. And (3) growing the selected single plant in the field to the mature period of an embryo sac, and adopting a glume-removing young flower haploid culture II (the process is like the glume-removing young flower haploid culture I) to obtain different haploid (1x) plants, thereby completing the technical approach of changing the ploidy of a chromosome group from 4x to 2x to 1x in tetraploid rice breeding from high to low.

g. The young ear is used for chromosome set in vitro culture doubling I to obtain a homozygous diploid plant (2 x). The second branch stem primordium of the selected haploid (1x) plant to the young ear of the stamen forming stage is subjected to chromosome set in vitro culture doubling I to obtain homozygous different diploid plants (2 x).

h. And (3) carrying out chromosome set in-vitro culture II on the selected diploid plant by the step g to obtain homozygous tetraploid (4x), finishing 1x → 2x → 4x of the chromosome set from low to high, and carrying out agronomic character observation, ploidy detection and resistance molecule marker auxiliary selection to obtain a plant meeting the breeding target.

Thus, 2x is formed by carrying out glume-removing young flower haploid culture on 4x indica-japonica subspecies hybrid, 1x is formed by carrying out young flower haploid culture on the hybrid again, 2x is formed by carrying out haploid doubling on an isolated chromosome group I by young ear culture to form a homozygous diploid, and 4x is formed by carrying out homozygous doubling on an isolated chromosome group II by young ear culture. The DTS69 has the excellent characteristics of tidiness, more ears, more seeds, high quality, stronger disease resistance and plump grains, and is named as 'more than 69' according to plant line selection and variety comparison. And a large amount of tetraploid and diploid rice new seed materials are created in the process of breeding the variety, and the method is applied to breeding and theoretical research and development of new varieties.

Claims

1. A polyploid rice ploidy circulation breeding method, set up the rice genome and increase and decrease by times and produce polyploid or haploid, on the basis of evolution and genetics, characterized by that through removing the young flower haploid culture of glume continuously and secondarily and realizing the ploidy of 4x → 2x → 1x is changed from high to low, form diploid and haploid; and then carrying out in-vitro culture and doubling on two continuous chromosome groups of young ears from a secondary branch stem primordium to a female stamen forming stage in mitosis of the somatic cell by utilizing mitosis of the somatic cell, realizing the replacement of 1x → 2x → 4x ploidy from low to high, and quickly breeding the homozygous tetraploid rice strain from the obtained plant through agronomic characteristic investigation, ploidy level detection, rice quality detection and resistance molecular marker auxiliary selection.

2. The ploidy cycle breeding method of polyploid rice as claimed in claim 1, characterized by the following concrete steps:

a. selecting polyploid indica rice and japonica rice with various excellent characteristics as parents;

b. carrying out interspecific hybridization on the polyploid parents of the indica rice and the japonica rice with excellent characteristics to obtain a tetraploid indica/japonica subspecies interspecific hybrid (4 x);

c. adopting a young glume-removing flower containing anthers and ovaries together at the mature embryo sac stage of the tetraploid interspecific hybrid to carry out haploid culture I;

d. in the haploid culture I, a special induced callus culture medium of plant growth regulators MCPA and amide is applied, and 2x thick and strong seedlings are grown through the haploid culture processes of callus induction, differentiation seedling emergence, strong root transplantation and the like;

e. through agronomic feature investigation and ploidy determination of a flow cytometry, different single plants (2x) close to a breeding target are obtained by using DNA molecular markers for resisting rice blast, bacterial blight and the like;

f. growing the selected single plant (2x) in a planting field to a mature period of an embryo sac, adopting a glume-removing young flower haploid culture II, and then culturing according to the same method of c and d to obtain different haploid (1x) plants, thereby realizing the process of replacing ploidy of a chromosome set from 4x to 2x to 1x from high to low;

g. carrying out chromosome set in-vitro culture doubling I on young ears from the second branch stem primordium to the formation stage of a stamen and a stamen of the selected haploid (1x) plant to obtain a homozygous diploid plant (2x), and transferring the diploid plant into a field for planting after root strengthening culture;

h. the plant after agronomic characteristic investigation, ploidy detection and resistance gene marker assisted selection adopts the step g to carry out the process of chromosome group in vitro culture doubling II to obtain homozygous tetraploid (4x), and the technical process of 1x → 2x → 4x of the chromosome group from low to high is completed; the process of 4x → 2x → 1x and 1x → 2x → 4x is totally completed, and the two ploidy alternation programs are integrated to form a ploidy circular breeding method of the polyploid rice;

i. after agronomic characteristics, ploidy level, rice quality detection and resistance molecular marker auxiliary selection of different plants, selecting excellent plants to enter a quality ratio and a region test to form a new tetraploid rice variety.

3. The ploidy cycle breeding method of polyploid rice as claimed in claim 1, wherein the homozygous tetraploid rice line is rapidly bred by the ploidy alternation of 4x → 2x → 1x and 1x → 2x → 4 x.

4. The ploidy cycle breeding method of polyploid rice as claimed in claim 1, wherein the homozygous diploid rice line is selected and bred rapidly in a manner of 4x → 2x → 1x and 1x → 2x ploidy alternation.