CN112042529A

CN112042529A - Breeding method of tetraploid rice

Info

Publication number: CN112042529A
Application number: CN202011082936.5A
Authority: CN
Inventors: 曹国长; 舒园园; 王群
Original assignee: Wuhan Duobeiti Biotechnology Co ltd
Current assignee: Wuhan Duobeiti Biotechnology Co ltd
Priority date: 2020-10-12
Filing date: 2020-10-12
Publication date: 2020-12-08

Abstract

The invention provides a breeding method of tetraploid rice, which comprises the steps of selecting small and long fertile plants and small and long sterile plants from the hybridization of indica rice and japonica rice and the improved F2 generation segregation population; pollinating female flowers of the sterile plants by using the indica rice restorer line plants to obtain indica rice hybrid seeds with partial indica rice; pollinating female flowers of the sterile plants by using japonica rice restorer plants to obtain japonica-biased indica-japonica hybrid seeds; planting and selfing the indica-type and japonica-type hybrid seeds respectively to obtain hybrid F2 generation seeds; stripping rice and weighing and selecting seeds of the hybrid F2 generation to obtain diploid small and long rice seeds; doubling the doubled material of the diploid small long-grain rice seeds to obtain a tetraploid rice material; and breeding the tetraploid rice material to obtain tetraploid rice seeds. Compared with the traditional macroploid rice, the panicle number is increased by 40-60%, the effective panicle number per unit area is increased by 33.3-50.0%, the yield per unit area is increased by 20.0-25.0%, and the trait can be stably inherited.

Description

Breeding method of tetraploid rice

Technical Field

The invention belongs to the field of agricultural breeding science, and particularly relates to a breeding method of tetraploid rice.

Background

The rice native Asia tropical is one of three grain crops in the world and is also the most important grain crop in China. In China, rice comprises two subspecies of japonica rice and indica rice, the two subspecies show obvious difference in form and distribution, and the F1 generation after hybridization often shows pollen sterility with different degrees.

Currently, rice breeding in the world includes conventional breeding (systematic selection breeding), cross breeding, and molecular breeding. Natural plants originate from diploids; however, polyploidization is an important means of plant evolution in nature. Most of the rice plants are diploid, and several tetraploids have been mutated in wild rice. At present, tetraploid rice breeding units are not few in global scope, rice promoted varieties with thousand seed weight of more than 22-33 g are mostly selected as main materials for doubling, the rice promoted varieties belong to diploid materials with larger grains, the materials are used for doubling, the tetraploid varieties and lines which are doubled have thousand seed weight of more than 35.2-52.8 g, and the tetraploid rice varieties and lines are applied in production, so that the problems of poor rice quality, few grains per spike, few effective spikes, low yield per unit area and the like exist.

Disclosure of Invention

The invention provides a breeding method of tetraploid rice, which aims to solve the technical problems of poor rice quality, small number of grains per ear, small effective ears and low yield per unit area of tetraploid rice in the prior art.

The embodiment of the invention provides a breeding method of tetraploid rice, which comprises the following steps,

finding and selecting small and long fertile plants and small and long sterile plants in the hybrid of indica rice and japonica rice and the improved F2 generation segregation population of indica rice and japonica rice;

pollinating female flowers of the small long sterile plants with pollen of the indica rice restorer plants to obtain indica rice hybrid seeds with the weight of thousand seeds of 20-20.5 g;

pollinating the female flowers of the small long sterile plants with pollen of the japonica rice restorer plants to obtain the japonica-biased indica-japonica hybrid seeds with thousand seed weight of 17-17.5 g;

planting and selfing the partially indica-japonica hybrid seeds and the partially japonica indica-japonica hybrid seeds respectively to obtain hybrid F2 generation seeds;

peeling and weighing the hybrid F2 generation seeds to obtain double-bodied small-long rice seeds with thousand seed weight of 14-22 g;

doubling the doubled material of the diploid small-long-grain rice seeds to obtain a tetraploid rice material;

and breeding the tetraploid rice material to obtain the tetraploid rice variety line.

A method as claimed in claim 1, wherein the length to width ratio of both the partially indica and partially japonica hybrid seeds is > 3.

Further, the plant spike shape of the fertile plant is the same as that of the sterile plant.

Furthermore, the indica rice restorer line plant is an indica rice restorer line plant carrying a rice blast resistance gene, and the japonica rice restorer line plant is a japonica rice restorer line plant carrying a rice blast resistance gene.

Furthermore, the chalkiness degree of the seeds used by the indica rice restorer line plant is less than or equal to 8 percent, the amylose is 14.0-24.0 percent, the whole polished rice rate of the seeds with the grain length of more than 6.5mm is more than 44 percent, the whole polished rice rate of the seeds with the grain length of 5.6-6.5 mm is more than 46 percent, and the whole polished rice rate of the seeds with the grain length of less than 5.6mm is more than 48 percent.

Further, the air conditioner is provided with a fan,

the rice stripping and weighing selection of the hybrid F2 generation seeds comprises,

pollinating female flowers of the small long sterile plants with pollen of the small long fertile plants to obtain artificial backcross BC1 generation seeds with thousand seed weight of 15-15.5 g and length-width ratio of more than 3;

planting the artificial backcross BC1 generation seeds to obtain backcross BC2 generation seeds;

selfing the small and long fertile plants to obtain selfing S1 seeds with thousand seed weight of 16.5-17.0 g and length-width ratio greater than 3;

planting and selfing the selfed S1 generation seeds to obtain selfed S2 generation seeds;

the backcross BC2 generation seeds, the selfed S2 generation seeds, and the hybrid F2 generation seeds were stripped of rice and weight selected.

Further, the doubling material is the diploid small and long rice seeds or young ears which are in the 3 rd to 4 th stage of young ear differentiation after planting.

Further, the doubling of the doubling material of the diploid small-long-grain rice seeds comprises:

inducing the double material of the diploid small and long rice seeds by using a culture medium to obtain callus;

doubling the callus with colchicine;

the doubled callus was differentiated and grown.

Furthermore, the breeding time is 3-5 generations.

Furthermore, in each generation of the breeding tetraploid material, the height of the plant is 100-110cm, the number of seeds per ear is 140-150, the setting percentage is about 80%, the thousand seed weight is 24-35g, and the effective ear per hectare is more than or equal to 262.5 ten thousand.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the invention provides a breeding method of tetraploid rice, which is characterized in that found small long-grain fertile plants and small long-grain sterile line plants are adopted to improve indica rice restorer lines and japonica rice restorer lines to form varieties, strains and materials with small grain shapes and long grain shapes, the thousand grain weight is generally between 14 and 22 grams, and more excellent diploid varieties, strains and materials are screened from the materials to be used as the doubling materials of the tetraploid, and the materials are doubled into the tetraploid and then bred, so that high-quality small long-grain tetraploid rice seeds with the thousand grain weight of 24 to 35 grams can be obtained in 5 generations at the fastest speed. After the high-quality small-long-grain tetraploid rice seeds bred by the invention are planted, compared with the traditional large-grain tetraploid (the thousand grain weight is more than 50g), the thousand grain weight is reduced by 30.0-50.0%, the ear number is increased by 40-60%, the unit area effective ear number is increased by 33.3-50.0%, the unit area yield is increased by 20.0-25.0%, the yield three-element coordination is better, the product of the three elements is maximum, the obtained yield is highest, and the characters can be stably inherited.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a process diagram of a method for breeding tetraploid rice according to an embodiment of the present invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

the embodiment of the invention provides a breeding method of tetraploid rice, figure 1 is a process step diagram of the breeding method of tetraploid rice of the embodiment of the invention, and the method comprises the following steps,

s1, finding and selecting small and long fertile plants and small and long sterile plants in the hybrid of indica rice and japonica rice and the improved F2 segregating population of indica rice and japonica rice;

s2, pollinating the female flowers of the small long sterile plants with pollen of the indica rice restorer line plants to obtain partially indica japonica hybrid seeds with thousand seed weight of 20-20.5 g;

s3, planting and selfing the partially indica-type indica-japonica hybrid seeds and the partially japonica indica-japonica hybrid seeds respectively to obtain hybrid F2 generation seeds;

s4, peeling and weighing the hybrid F2 generation seeds to obtain double-body small-long rice seeds with thousand seed weight of 14-22 g;

s5, doubling the doubled material of the diploid small-long-grain rice seeds to obtain a tetraploid rice material;

s6, breeding the tetraploid rice material to obtain the tetraploid rice variety.

The embodiment of the invention adopts the small long-grain sterile plant found in the hybridization of the indica rice and the japonica rice and the improved diploid progeny of F2 generation as an improved material, pollinates the sterile plant by using the existing indica rice restorer and japonica rice restorer respectively to obtain two hybrid seeds of partially indica type japonica hybrid and partially japonica type indica type japonica hybrid, and then selects and breeds high-quality small long-grain seeds from the separate generation, so that the original indica rice restorer and japonica rice restorer are actually improved, and the heterosis among the selected small long-grain sterile line, the indica rice restorer and the japonica rice restorer is also tested. The rice seeds with the two-fold small and long grains obtained by rice peeling and weighing selection have excellent properties, and then are doubled into tetraploids as raw materials, so that the breeding speed of the tetraploid rice can be improved, the breeding process is accelerated, the tetraploid rice can be connected with production, and meanwhile, the application range of the tetraploid rice can be expanded. By adopting the hybridization improvement method provided by the invention, the small and long-grain tetraploid rice variety line with thousand-grain weight of 24-35g can be obtained in 5 generations at the fastest speed.

The regeneration selection of the sterile plants can verify that the initially selected small long-grain sterile plants are two-line sterile line plants, the two-line sterile lines can be hybridized with any variety line material, the filial generation of the hybridization of any variety line material and the two-line sterile line can be hybrid of the two-line sterile line, and any variety line material is a restorer line, so that the combined application of strong superiority and high yield can be selected. Thus, not only the sterile line with strong superiority can be bred, but also the restoring line with strong superiority can be bred, and the new hybrid rice combination and new variety with strong superiority can be bred.

The characters of hybrid progeny between subspecies (indica-japonica cross) of cultivated rice are difficult to stabilize, the main reason is that indica rice has more than 9000 genes than japonica rice, and the problems of sterility, low fruiting rate and wild separation occur because the genes of indica rice and japonica rice are not matched with each other. The small long-grain sterile line plant selected by the invention is a two-line sterile line plant, the hybrid progeny between subspecies of cultivated rice (indica-japonica cross) is hybridized with the two-line sterile line, and when the hybrid progeny is separated into a new two-line sterile line, the genes of the hybrid progeny can be basically stabilized, so that the problems of sterility and low fruiting rate of the progeny can not occur, and the method is one of effective methods for rapidly and stably separating the indica-japonica cross progeny wild.

The excellent filial generation with stable gene is doubled to double the original single chromosome from male parent and female parent into double chromosomes, which are paired and stable.

The rice stripping selection standard in the embodiment of the invention is as follows: the method selects the tetraploid varieties with excellent rice quality (the rice reaches the standard of the national third-grade high-quality rice), the grains have good glume closing performance, the awn length is less than 0.5 cm, the cross section is less than 3.5 mm, the long-grain ultra-high-quality rice and the special rice are taken as the main materials, the high-quality small-grain tetraploid can be seeds formed by a sterile line, can also be seeds formed by a restorer line plant, can also be seeds formed by planting hybrid seeds, and the doubled materials of the seeds have popularization and application prospects when used for breeding the tetraploid varieties or materials.

Wherein the indica-type indica-japonica hybrid seeds and the japonica-type indica-japonica hybrid seeds are selected from a combination with good combining ability and strong advantages, namely a combination with high yield of the hybrid F2 generation.

As an implementation manner of the embodiment of the present invention, the aspect ratio of the partially indica-japonica hybrid seed to the partially japonica indica-japonica hybrid seed is both > 3.

As an implementation of the embodiments of the present invention, the plant ears of the fertile plant and the sterile plant are the same.

Compared with fertile plants, sterile plants have the advantages of phimosis, lack of auxin, short plant height, slightly small leaves, close panicle type and slightly small panicle type. Wherein, the plant panicle shape means that the grain traits are the same or similar and the leaf types are similar.

As an embodiment of the present invention, the indica rice restorer line plant is an indica rice restorer line plant carrying a rice blast resistance gene, and the japonica rice restorer line plant is a japonica rice restorer line plant carrying a rice blast resistance gene.

The invention adopts indica rice restoring line plant carrying blast-resistant rice gene and japonica rice restoring line plant carrying rice blast-resistant gene as improved objects, can breed tetraploid rice seeds with high quality, small and long grains and added rice blast-resistant gene, after the tetraploid rice seeds are planted, the stems of the tetraploid rice seeds become thin, the flexibility and the lodging resistance become strong, the hardness of the leaves and the stems is reduced, the root system becomes long, the quantity becomes large, the absorption, storage, transportation and metabolism speed of water, inorganic matters and organic matters are accelerated, and the efficiency is improved. The plant type elasticity is moderate, the leaf type, the leaf size, the leaf hardness degree and the leaf color are moderate, the plant type elasticity is moderate, the growth is luxuriant, the tillering force is strong, the ear forming rate is high, the ear type is moderate, the branch and the stalk are short, the grain attachment density is moderate, the late-stage maturity is good, the leaves are yellow and synchronous, and the organic matters in the stem leaves and the glumes are fully transferred to the grains. The grains are thin and long, and the quality of the rice is excellent. Compared with the large-grain tetraploid rice with thousand kernel weight of more than 50g, the effective panicle number per hectare is 240-262.5 ten thousand, after the tetraploid rice seeds bred by the method are planted, the thousand kernel weight is reduced by 30.0-50.0%, the panicle number is increased by 40-60%, the effective panicle number per unit area is increased by 33.3-50.0%, the yield per unit area is increased by 20.0-25.0%, the yield is more harmonious, the product of the three factors is the largest, and the obtained yield is the highest. The small-grain tetraploid can be connected with production, can be directly applied to production and can be comparable to diploid varieties, the thousand grain weight of grains of diploid rice harvested in field production is more than 22-33 g, the thousand grain weight of newly bred small-grain tetraploid is 24-35g, and the small-grain tetraploid is similar to the diploid in field production in the thousand grain weight level. In terms of nutrients, the amino acid and protein contents of tetraploid rice are generally higher than those of diploid rice.

As an implementation manner of the embodiment of the invention, the chalkiness degree of the seeds used by the indica type rice restorer line plant is less than or equal to 8 percent, the amylose is 14.0-24.0 percent, the whole polished rice rate of the seeds with the grain length of more than 6.5mm is more than 44 percent, the whole polished rice rate of the seeds with the grain length of 5.6-6.5 mm is more than 46 percent, and the whole polished rice rate of the seeds with the grain length of less than 5.6mm is more than 48 percent.

As an implementation of an embodiment of the present invention,

As an implementation manner of the embodiment of the present invention, the doubling material is the diploid small and long-grained rice seeds or young ears of the diploid small and long-grained rice seeds which are in the 3 rd to 4 th stage of young ear differentiation after being planted.

As an implementation of the embodiment of the present invention, the doubling of the material of the diploid small and long rice seeds comprises:

doubling the callus with colchicine;

the doubled callus was differentiated and grown.

The method can cultivate strong test-tube seedlings by using the callus after being multiplied, the test-tube seedlings are transplanted to a field after seedling exercising, tetraploid seedlings can be identified manually when 4 or 5 tillers exist in the seedlings, the seedlings are not identified really, one tillered stem leaf can be taken and sent to a laboratory ploidy detector for ploidy detection, the tetraploid seedlings are identified to be managed intensively, multiple tillers, multiple spikes and multiple seeds are strived for, the propagation coefficient is enlarged, and more materials are provided for systematic breeding.

As an implementation manner of the embodiment of the invention, the breeding time is 3-5 generations. Namely whether the newly confirmed tetraploid accords with a breeding target or not, 3-5 generations of systematic breeding are needed, and materials, strains and varieties close to the breeding target are selected step by step.

The character of the filial generation of the cultivated rice subspecies (indica-japonica cross) is difficult to be stabilized, and the transformation into the sterile line is one of the stabilizing methods. Doubling the number of the steps is a second stabilizing method, and the method is a method which is more stable, faster and better in effect. The doubling is that each single chromosome originally from a parent and a maternal parent is doubled into double chromosomes, double pairing is realized, each chromosome is self-stabilized, and the tetraploid is stabilized. Certainly, the method can breed small-length tetraploids, and also can carry out parthenogenesis under the background of the tetraploids to obtain stable filial generation of the interspecific (indica-japonica cross) haploid between cultivated rice subspecies, namely a new line created by filial generation of the interspecific (indica-japonica cross) between cultivated rice subspecies, and can breed a new variety of diploid carrying excellent characters of a parent and a female parent of the interspecific (indica-japonica cross) filial generation of cultivated rice with strong advantages and high yield by moderate systematic breeding. Namely, whether the newly confirmed tetraploid meets the breeding target or not, 3-5 generations of breeding are needed, and materials, strains and varieties close to the breeding target are selected step by step.

As an implementation manner of the embodiment of the invention, in each generation of the breeding tetraploid material, the height of the plant is 100-110cm, the number of seeds per ear is 140-150, the setting percentage is about 80%, the thousand seed weight is 24-35g, and the effective ear per hectare is 240-262.5 ten thousand.

In each generation of the tetraploid breeding material, the comprehensive characters of plants are slightly thin, the flexibility and the lodging resistance are enhanced, the hardness of leaf and stem is reduced, the root system is lengthened, the quantity is increased, the absorption, storage, transportation and metabolism speed of water, inorganic matters and organic matters is accelerated, and the efficiency is improved. The plant type elasticity is moderate, the leaf type, the leaf size, the leaf hardness degree and the leaf color are moderate, the plant type elasticity is moderate, the growth is luxuriant, the tillering force is strong, the ear forming rate is high, the ear type is moderate, the branch and the stalk are short, the grain attachment density is moderate, the late-stage maturity is good, the yellow and yellow spikes of the leaves are synchronous, and the organic matters in the stem leaves and the glumes are fully transferred to the grains. The grains are thin and long, and the quality of the rice is excellent. The breeding of the tetraploid material of each generation also comprises the following standards: the total growth period and other characters, the total growth period is 3-5 days longer than that of the early-maturing control Huanghua and 1-3 days shorter than that of the late-maturing control Fengliou No. four; in addition, the grain length is more than or equal to 6.5mm (except for japonica rice and indica-japonica cross grain), the maximum cross section width is less than or equal to 3.5 mm, the length of the miscanthus is less than or equal to 0.5 cm, the quality of rice reaches the standard of over 3-grade high-quality rice, and particularly, in the breeding process, the plant type, the stem leaf hardness, the stem thickness, the tillering force, the growth potential, the late maturing phase, the yield and the quality need to be comprehensively considered.

The stem becomes thin, the flexibility and the lodging resistance become strong, the hardness of the leaf stem is reduced, the root system becomes long, the quantity becomes large, the absorption, storage, transportation and metabolism speed of water, inorganic matters and organic matters are accelerated, the efficiency is improved, the plant type tightness is moderate, the leaf type, the leaf size, the leaf hardness degree and the leaf color are moderate, the plant type tightness is moderate, the growth is luxuriant, the tillering capability is strong, the ear forming rate is high, the ear type is moderate, the branches and the stalks are short, the grain attachment density is moderate, the late maturing phase is good, the yellow and yellow spikes of the leaves are synchronous, and the organic matters in the stem leaf glumes are fully transferred to grains. The grains are thin and long, and the quality of the rice is excellent.

The following will specifically describe a method for breeding tetraploid rice of the present invention with reference to specific examples.

Example 1

In 2016, 8 months and 20 days, in the Xiangyang base, a small-grain sterile plant is found in an F2 generation segregation population among subspecies of rice cultivated in a seed selection garden (indica-japonica cross), and a small-grain fertile plant similar to the leaf spike type of the plant is found. Then, pollinating the female flowers of the sterile plants by the male flowers of the fertile plants to obtain backcross BC1 generation seeds; simultaneously, a good indica rice restoring line containing a rice blast resistance gene and a good japonica rice restoring line containing the rice blast resistance gene are selected to respectively pollinate female flowers of the sterile plants, and two hybrid F1 seeds of partially indica type indica rice hybrid seeds and partially japonica type indica rice hybrid seeds are harvested after fertilization and maturity; and selfing the fertile plants to obtain selfed S1 seeds. Wherein, the thousand seed weight of the hybrid seeds obtained by pollinating the sterile plants by the indica rice restorer line containing the rice blast resistance gene is 20.5 g, the thousand seed weight of the hybrid seeds obtained by pollinating the sterile plants by the japonica rice restorer line containing the rice blast resistance gene is 17.2 g, the thousand seed weight of the backcross BC1 generation seeds obtained by pollinating the sterile plants by the fertile plants is 15.4 g, and the thousand seed weight of the inbred S1 generation seeds obtained by inbreeding the fertile plants is 16.9 g. The length-width ratio of the seeds of the four seeds is more than 3, and the rice quality is excellent.

The backcross BC1 generation, the selfing S1 generation, the partially indica type indica-japonica hybrid seeds and the partially japonica type indica-japonica hybrid seeds harvested in Xiangyang in 2016 at 9 months are planted and selfed in Hainan breeding base at 11 months bottom in 2016, after sowing for 11 months and 25 days, transplanting for 12 months and 24 days, and field management for 4 months from 12 months bottom to 2017 at 4 months bottom, the backcross BC2 generation seeds, the selfing S2 generation seeds and the hybridization F2 generation seeds are harvested at 4 months and 25-27 days in 2017.

The hybrid F2 generation seeds are seeds of plants with strong dominance of the indica-type indica-japonica hybrid F1 generation hybrid rice and the indica-type japonica hybrid F1 generation hybrid rice, the yield is increased by more than 15% compared with the traditional indica-japonica hybrid F1 generation (diploid rice), and the characters are stable. Backcrossed BC2 generation seeds were seeds harvested from the self-pollination of 1 fertile plant found in plants planted with backcrossed BC1 generation seeds. The inbred S1 seed is seed obtained by self-pollination of plants planted with the hybrid S1 seed.

The amount of the backcross BC2 seed, the inbred S2 seed and the cross F2 seed harvested in the above is 300 plant seeds.

And at the end of 4 months in 2017, after the seeds of the 300 plants are harvested, airing and sending back to Wuhan.

In 5 months in 2017, 300 plant seeds in total are harvested from Hainan in Wuhan base (backcross BC2 generation seeds, inbred S2 generation seeds and hybrid F2 generation seeds), rice peeling and weighing selection are carried out on individual plants, rice peeling is an important link of rice breeding, poor-quality seeds are eliminated by rice peeling, excellent-quality seeds are kept, the relationship between the size and thickness of grains and the quality of rice can be found in the period, and the rice with better compactness can be better only with small grains and higher quality; weighing is to select small particle materials; after stripping and weighing, 30 parts of high-quality small and long seeds with thousand seed weight of 14-22g are screened out from 300 parts of single seeds and used as doubling materials.

30 parts of seeds of good quality small and long plants are frozen to break dormancy (after-ripening effect), and the treatment time is 15 days. The seed after dormancy breaking has good permeability, strong water absorption and high germination rate.

And in 2017, in 5 months, sending the first doubling material, namely 30 parts of high-quality small long-grain plant seeds to a laboratory for seed doubling.

The doubling method comprises the following steps: the diploid rice seeds are sterilized aseptically, then are placed into 1/2N6 culture medium, and are cultivated in a thermostat at 27 +/-2 ℃ for about one month without illumination, and callus is induced. Transferring the callus into 0.05% colchicine inducer solution when the callus grows to soybean size, and performing double treatment at 23 deg.C or lower under dark condition for 48 hr. After the doubling treatment, the plant needs to be recovered for about half a month, the plant is transferred to a 1/2N6 culture medium, the plant is cultured in a thermostat with the temperature of 27 +/-2 ℃ without light to induce root buds, the root buds are induced after 30-50 days, and finally, robust plants with complete root systems and stem leaves are differentiated. Generally, the seedlings have more than 3 complete leaves, and can be transplanted to a field after the seedlings are trained for about 5 days. The doubling process takes about 4 and a half months.

The doubling start time is: generally, the expected lodging time of the field plantlet is taken as the standard, and the doubling procedure is started after about 4 months.

Doubling 30 parts of tetraploid doubling material seeds in a laboratory for about 4 half months, wherein only 21 parts of the tetraploid doubling material seeds are doubled to form seedlings, and the part of the doubled seedling material accounts for 70% of 30 parts of the doubling material; when the 21 doubled seedlings are sown in 2017, 11 and 25 days in Hainan, the seedlings are planted in a Hainan breeding base together, after the cultivation for about one month, when the plants grow to 6-8 leaves, the planted plants are detected by a ploidy determinator and are identified by manual visual inspection, tetraploid rice plants are determined in 12 materials in total, and the doubling success rate is 40%.

The 12 tetraploid plants are systematically selected from the Wuhan Huang waver big pond base in the positive season of 2018, the Wuhan big pond base in the spring south China in the winter of 2018 to 2019, the Wuhan big pond base in the winter of 2019 and the Wuhan Nannan China base in the spring of 2020 in the winter to 2020, and a new high-quality small-long-grain rice blast-resistant tetraploid strain with the thousand seed weight of more than 100 and the weight of 24-35g is cultivated. The rice variety test has remarkable performance of individual strains, has participated in the rice variety test, has good performance and is hopeful to be obtained.

Through 3 years of half-7 generations of multi-round hybridization, test crossing and backcross improvement, at present, in double-bodied small and long rice seeds with the thousand-grain weight of 14-22g, the high-quality small and long rice-blast-resistant gene-carrying two-line sterile line accounts for more than 80% of the total amount of all sterile lines, the rice-blast-resistant gene-carrying restoring line accounts for more than 90% of all restoring lines, and the rice-blast-resistant gene-carrying japonica rice accounts for 60% of all japonica rice. These large quantities of material have the potential to be doubled up with small particle tetraploids.

Example 2

20 plant seeds were selected from 270 plant seeds remaining after 30 plant seeds were selected in example 1 at the beginning of 5 months in 2017, and were treated by freezing to break seed dormancy (after-ripening effect) for 15 days. The seed after dormancy breaking has good permeability, strong water absorption and high germination rate.

And (5), in 2017, in 5, 16, soaking seeds of 20 parts of plant seeds breaking dormancy, accelerating germination and planting in a field, in about 7, 20 days, and taking young ears when the young ears of the planted plants grow to 3-4 stages to obtain a second doubling material.

And 7, month, 20-30 in 2017, and 3 batches of the second doubled material are sent to a laboratory for young ear doubling to obtain the tetraploid material.

The doubling method comprises the following steps: and (3) carrying out sterile disinfection on the young ear, then putting the young ear into 1/2N6 culture medium, and carrying out non-illumination cultivation in a thermostat at 27 +/-2 ℃ for about one month to induce the callus. Transferring the callus into 0.05% colchicine inducer solution when the callus grows to soybean size, and performing double treatment at 23 deg.C or lower under dark condition for 48 hr. After the doubling treatment, the plant needs to be recovered for about half a month, the plant is transferred to a 1/2N6 culture medium, the plant is cultured in a thermostat with the temperature of 27 +/-2 ℃ without light to induce root buds, the root buds are induced after 30-50 days, and finally, robust plants with complete root systems and stem leaves are differentiated. Generally, the seedlings have more than 3 complete leaves, and can be transplanted to a field after the seedlings are trained for about 5 days. The doubling process takes about 4 and a half months.

The doubling start time is: and taking the expected transplanting time of the field plantlets as a standard, reversing for about 4 and a half months, and entering a doubling procedure.

12 months and 24 days in 2017, 12 doubled seedlings obtained by doubling 20 parts of doubling materials are planted together in a field in a southern Hainan breeding base, about 1 month of the parasitic transplanting is achieved, and when the seedlings grow to 6-9 leaves, tetraploid individuals can be recognized by human eyes.

Through artificial identification and ploidy detector detection, tetraploid plants with 7 parts of materials successfully doubled are identified, and high-quality small-long-grain rice blast-resistant tetraploid new lines with thousand seed weight of 24-35g are cultivated by systematic selection in the same way from the positive season of 2018 to the large blue and wave pond base of Wuhan in the first year, from the winter of 2018 to the south propagation base of 2019 in the spring, from the large blue and wave pond base of Wuhan in the first year and from the winter of 2019 to the south propagation base of 2020 in the spring. There are also individual lines that perform prominently.

Example 3

In the south Hainan propagation base at the bottom of 11 months in 2016, after the backcross BC1 generation seeds in example 1 are planted, sterile plants separated in 3 months in 2017 are backcrossed again, sterile plants separated in 16 months in 5 months in 2018 are planted in Wuhan Datan, sterile plants are separated again, ears of the sterile plants are cut and regenerated in 24 days in 8 months in the current year, sterile seeds are harvested from the sterile regenerated plants at the bottom of 9 months, and therefore it can be determined that a small-sized sterile plant found in F2 generation segregating population among rice subspecies (indica-japonica cross) cultivated in a Xiangyang selection garden in 8 months in 2016 is a two-line sterile material, and the hybrid seed prepared in the later year is a true two-line hybrid seed. The hybrid seeds prepared by the high-quality small-grain two-line sterile line in each season have strong advantages, the seeds are long in children, and can be selectively doubled, hybrid F2 generations are planted, new excellent sterile lines and restoring lines are selected from the segregating population of the hybrid F2 generations, and diploid rice resources are enriched.

The thousand seed weight of the new high-quality small-long-grain rice blast-resistant tetraploid line bred in the examples 1 and 2 is more than 24-35g, the new high-quality small-long-grain rice blast-resistant tetraploid line belongs to the high-quality small-long-grain tetraploid type, the thousand seed weight is reduced by about 15 g compared with that of the conventional macroploid, the bred new tetraploid line is basically equivalent to that of diploid rice applied to production (between 22-33 g), and the thousand seed weight, the number of grains per spike and the number of effective spikes per unit area are all similar to that of diploid rice.

After the tetraploid rice bred by the invention is planted, thousand kernel weight is reduced by 30.0-50.0%, ear number is increased by 40-60%, unit area effective ear number is increased by 33.3-50.0%, unit area yield is increased by 20.0-25.0%, three elements of yield have better harmony, the product of the three elements is maximum, the obtained yield is highest, the tetraploid rice can be directly applied to production, and characters can be stably inherited.

The method provided by the embodiment of the invention is adopted to find and introduce high-quality small long grain and rice blast resistant variety materials, the existing widely-applied and successfully bred excellent diploid nonglutinous rice variety, strain and material are improved by finding and discovering the introduced high-quality small long grain sterile line, restorer line and rice blast resistant variety materials, so that the diploid nonglutinous rice variety, strain and material have excellent comprehensive properties, and the improved diploid small long grain is used as the breeding material for breeding tetraploids, and the tetraploid rice seeds have at least the following purposes:

(1) can be directly used for production;

(2) the sterile line and the restorer line can be selected from the bred tetraploid rice seeds, and the sterile line and the restorer line are prepared into hybrid seeds for production; heterosis is stronger than that of diploid.

(3) The bred tetraploid rice variety line can be improved into a traditional tetraploid rice variety with the thousand-grain weight of 24-35 g;

(4) the method is used as a new resource for cultivating diploid rice (by a haploid breeding method of parthenogenesis), compensates for the shortage of rice resource reduction, and delays the speed of rice resource exhaustion.

(5) The new created diploid sterile line and restorer line carrying excellent genes among the subspecies of the cultivated rice and resisting rice blast are utilized to carry out two-line hybrid seed production, and the yield increasing potential of the generated diploid hybrid is expected to break through 35 percent.

(6) After subsequent artificial hybridization and selfing, the sterile plant selected initially is proved to be a two-line sterile line, so that the progeny has the character of stable gene. The excellent filial generation with stable gene is doubled to double the original single chromosome from male parent and female parent into double chromosomes, which are paired and self-stabilized and have stable character.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method of breeding tetraploid rice, comprising,

selecting small and long fertile plants and small and long sterile plants from the indica rice and japonica rice hybrid and the improved F2 generation segregation population;

2. A method as claimed in claim 1, wherein the length to width ratio of both the partially indica and partially japonica hybrid seeds is > 3.

3. A method as claimed in claim 1, wherein said fertile plant and sterile plant have the same panicle shape.

4. A method as claimed in claim 1, wherein the indica rice restorer line plant is an indica rice restorer line plant carrying a rice blast resistance gene, and the japonica rice restorer line plant is a japonica rice restorer line plant carrying a rice blast resistance gene.

5. A breeding method of tetraploid rice as claimed in claim 1, wherein said indica restorer line plant uses seeds with chalkiness degree less than 8%, amylose is 14.0-24.0%, said indica restorer line plant uses seeds with whole rice rate > 44% with grain length > 6.5mm, seed whole rice rate > 46% with grain length > 5.6-6.5 mm, and seed whole rice rate > 48% with grain length <5.6 mm.

6. A breeding method of tetraploid rice as claimed in claim 1, wherein said crossing F2 generation seed is peeled and weight selected, comprising,

planting and selfing the artificial backcross BC1 generation seeds to obtain backcross BC2 generation seeds;

7. A breeding method of tetraploid rice as claimed in claim 1 or 6, wherein said doubling material is any one of the following: the diploid small and long rice seeds and the young ears which are just in the 3 rd to 4 th stage of young ear differentiation after the diploid small and long rice seeds are planted.

8. A breeding method of tetraploid rice as claimed in claim 1 or 6, wherein said doubling of doubling material of said diploid small-and long-grain rice seeds comprises:

doubling the callus with colchicine;

the doubled callus was differentiated and grown.

9. A method as claimed in claim 1, wherein the breeding time is 3-5 generations.

10. A breeding method of tetraploid rice as claimed in claim 1, wherein in each generation of tetraploid material, the height of the plant is 100-110cm, the number of seeds per ear is 140-150, the maturing rate is about 80%, the thousand seed weight is 24-35g, and the effective ear per hectare is not less than 262.5 ten thousand.