阅读说明：本技术 一种快速改良盐敏感水稻品种中花11提高耐盐性的方法 (Method for rapidly improving salt tolerance of flower 11 in salt-sensitive rice variety ) 是由 宗营杰 郭桂梅 张述伟 高润红 陈志伟 刘成洪 于 2020-05-06 设计创作，主要内容包括：本发明涉及植物生物技术领域,具体是一种快速改良盐敏感水稻品种中花11提高耐盐性的方法,利用<Sup>60</Sup>Coγ-射线辐照创造变异,采用小孢子培养单倍体技术快速纯合稳定,提出在小孢子诱导胚胎发生阶段采用800mg/L NaCl可达半数抑制筛选,获得DH株系在幼苗期施加8.7g/L NaCl胁迫处理,以幼苗的叶片茎秆失绿和卷曲程度为表型分级指标进行苗期耐盐性评价,以分级指标3.8以上筛选,最终获得苗期耐盐性显著提高的材料得率1％以上。本发明提供了快速改良盐敏感水稻品种的一套完整方法,建立了明确筛选与鉴定指标,提高了耐盐性改良效率；方法简单易观测,在规模化鉴定中具有可操作性。(The invention relates to the technical field of plant biology, in particular to a method for quickly improving salt tolerance of flower 11 in a salt-sensitive rice variety by utilizing 60 Co gamma-ray irradiation is used to create variation, microspore is used to culture haploid for fast purification and stabilization, 800 mg/L NaCl is used to half inhibition and screening in microspore inducing embryogenesis stage to obtain DH strain, 8.7 g/L NaCl stress treatment is applied in seedling stage, the green and curling degrees of seedling leaf and stem are used as the grading indexes to evaluate the salt tolerance of seedling stage, and grading index over 3.8 is used to screen out and obtain material with obviously raised salt tolerance in seedling stage in yield over 1%The complete method is adopted, clear screening and identification indexes are established, and the salt tolerance improvement efficiency is improved; the method is simple and easy to observe, and has operability in large-scale identification.)

1. A method for rapidly improving the salt tolerance of flower 11 in a salt-sensitive rice variety is characterized by comprising the following steps:

A) flower 11 dry seed adoption in rice variety⁶⁰Co gamma-ray irradiation with a dose of 300-400 GY;

B) irradiating seeds, sowing in a field, growing seedlings, transplanting, and managing normal water and fertilizer until heading;

C) after the young ear is processed for a week at low temperature, culturing free microspores, and carrying out salt stress by adopting 800 mg/L NaCl in the development process of microspore isolated embryos;

D) obtaining a regeneration plant, identifying ploidy by a flow cytometer, and selecting a doubled haploid;

E) breeding to obtain doubled haploid strain population;

F) adding 8.7 g/L NaCl into the doubled haploid strain Yoshida nutrient solution for water culture to stress and screen, carrying out phenotype grading evaluation according to the green loss and the crimp degree, identifying the doubled haploid strain with improved salt tolerance compared with the original parent, and obtaining the material with the grading index improved by about one time, namely the material with obviously improved salt tolerance.

2. The method for improving the salt tolerance of flower 11 of a rapid improved salt-sensitive rice variety as claimed in claim 1, wherein in the step C, the free microspore is cultured, young ears of the microspores in the mononuclear stage are taken, the freshness protection package is sealed and placed in a refrigerator at 4 ℃ for low-temperature treatment for one week, sodium hypochlorite solution is used for disinfection for 15min before inoculation, anthers are picked under the aseptic condition, a high-speed homogenizer is used for smashing, sieving is carried out, mannitol solution containing 60 g/L is used for heavy suspension, and the anthers are placed on improved N6 culture medium containing maltose 90 g/L, 2,4-D1.5 mg/L to induce the formation of embryogenic callus.

3. The method for improving salt tolerance of flower 11 of a rapid improved salt-sensitive rice variety as claimed in claim 1, wherein in step C, 800 mg/L NaCl stress treatment is used during microspore-induced embryogenic callus formation.

4. The method for improving the salt tolerance of flower 11 of the rice variety sensitive to the rapid improvement of the salt according to claim 1, wherein the embryogenic callus obtained in the step D is induced to obtain a regenerated plant, and the embryogenic callus obtained in the step C is obtained by stress-induced culture of microspores obtained in the step C for 21D and is transferred to an improved MS culture medium containing 30 g/L0.5.5 mg/L sucrose and 6-BA0.5mg/L sucrose to induce and form green seedlings.

5. The method for rapidly improving the salt tolerance of flower 11 in the salt-sensitive rice variety according to claim 1, wherein the method for identifying the ploidy of the regenerated plant in the step D comprises the steps of taking leaves of the regenerated plant, cutting the leaves, dissociating cell nuclei by using L B-01 buffer solution, treating the cell nuclei by using PI/RNase dye solution, taking filtrate, identifying the ploidy by using a flow cytometer, and identifying the doubled haploid by selecting a seedling of a seed as a position of a control peak.

6. The method of claim 1, wherein the step E: and (3) carrying out water culture and hardening on the doubled haploid plant obtained by microspore culture, packing and moisturizing the seedling when the seedling grows to about 10cm after a new root grows out, sending the seedling to a southeast Hainan breeding base, transplanting the seedling to a field in the same year, and harvesting single seeds in the second year to form a doubled haploid plant group.

7. The method of claim 1, wherein the evaluation criteria for the phenotypic grading in step F are:

according to the green degree and the curling degree of the leaves and the stems of the seedlings, 5 grades are divided, the 5 grades have the best salt tolerance, and the 1 grade has the worst property;

level 1: the whole seedling has no green part and is basically determined to die;

and 2, stage: heart leaves and stems are too green and curled seriously, and other leaves are withered and yellow and die;

and 3, level: the heart leaves and the stems are partly green, a small part of leaves are still green, and most of leaves are withered and yellow and die;

4, level: the tip of each leaf turns yellow and curls, the leaves and the stems at the middle and lower parts are still kept green, and part of the leaves are curled;

and 5, stage: only the tip of each leaf turns yellow and curls, the leaves and stems of the rest parts still keep green, and the heart leaves and half of the inverted two leaves can be seen from the unfolded parts;

by adopting the identification method, the doubled haploid strain with the salt tolerance improved compared with the original parent is identified by taking the grading evaluation index which is improved by one time compared with the original parent as a screening standard.

Technical Field

The invention relates to the technical field of plant biology, in particular to a method for quickly improving salt tolerance of flower 11 in a salt-sensitive rice variety.

Background

Salt stress is one of the main environmental factors for limiting plant growth and development and agricultural production, and soil salinization seriously restricts sustainable development of agriculture and grain production, and becomes one of the soil problems facing the world. Therefore, the method has important significance for improving the salt tolerance of crops and breeding the crop varieties with strong salt tolerance, and promoting the agricultural development. Rice is one of the most important food crops, and more than half of the world population uses rice as staple food. Billions of acres of saline-alkali soil in China are the land with no gross land, wherein nearly two hundred million acres of saline-alkali resistant 'sea rice' and the like can be planted in the saline-alkali resistant 'sea rice' field, and the saline-alkali resistant 'sea rice' field can be popularized and planted, and plays a strategic guarantee role in food safety in China. The medium flower 11 is a good japonica rice variety bred by applying a anther culture technology in China, is an important genetic transformation material in rice research, and belongs to a salt sensitive type. At present, some researches report the salt stress related phenotype and transgenic researches carried out in the variety (the influence of the stress of NaCl on the root growth and development of flower 11 seedlings in the rice variety [ J ]. seeds, 2012,31(7): 7-12; dawn, juyu, guohua, and the like; preliminary identification of the salt-resistant function of rice OsRab7 and construction of an expression vector thereof [ J ]. Wuhan botanic research, 2008,26(1):1-6.), but reports for rapidly improving the salt resistance by adopting a conventional breeding method are not seen.

Chinese patent document CN102106259A discloses a method for improving salt tolerance of cereal crops, which comprises respectively applying NaCl stress screening in the differentiation culture processes of dissociated microspores, embryoid induction and regeneration plants, carrying out in-vitro treatment on the microspores of the crops by using NaCl, respectively applying NaCl stress screening in the induction culture period and the plant differentiation period, eliminating layer by layer, obtaining regenerated plants with roots, transplanting to a greenhouse, and harvesting according to the plants after maturation. And (3) placing the harvested seeds on a stress culture medium added with NaCl for germination, and screening out a doubled haploid strain with strong salt tolerance. The method has the defects that the microspore stress screening method takes barley (dry land crops) as an implementation object, and the method conditions are not suitable for rice (paddy field crops); continuous NaCl salt stress screening in multiple culture processes is adopted, and the procedure is complicated; the effectiveness screening of half inhibitory concentration is lacked, and the effectiveness is unclear.

However, no report is found about a method for rapidly improving the salt tolerance of flower 11 in a salt-sensitive rice variety by adopting a conventional breeding means.

Disclosure of Invention

The invention aims to provide a method for quickly improving salt tolerance of a salt-sensitive rice model variety medium flower 11, and the salt-sensitive rice model variety medium flower 11 is quickly screened to obtain a homozygous stable seed material with improved salt tolerance by applying proper NaCl salt stress on microspore in-vitro embryonic development and regeneration doubled haploid strains after seed irradiation mutagenesis.

Compared with the method for improving the salt tolerance of the cereal crops, disclosed by Chinese patent document CN102106259A, the method has the advantages that: aiming at the rice model variety 'Zhonghua 11', an improvement method is established, the object is clear, and the guidance is strong; the improvement method comprises the following 3 steps: in the induction process of seed irradiation mutagenesis-microspore embryo development, NaCl salt stress screening-regeneration doubling haploid strain seedling-stage NaCl salt stress identification is adopted, so that the steps are simpler; the screening concentration of NaCl is determined according to the half inhibition ratio of microspore to start embryo development for the first time, and the effectiveness is high; and (4) providing seedling stage identification grading indexes according to the green degree and curling degree of the leaves and the stems of the seedlings, and performing the operation strongly.

In order to achieve the above object, the present invention provides a method for rapidly improving the salt tolerance of flower 11 in a salt-sensitive rice variety, comprising the steps of:

A) flower 11 dry seed adoption in rice variety⁶⁰Co gamma-ray irradiation with a dose of 300-400GY (according to the experimental observation, the germination rate is influenced by more than 400, the mutagenesis effect is not obvious when the germination rate is lower than 300, and 350GY is preferred, and the irradiation effect is better);

B) irradiating seeds, sowing in a field, growing seedlings, transplanting, and managing normal water and fertilizer until heading;

C) after the young ear is processed for a week at low temperature, culturing the free microspore, and adopting 800 mg/L NaCl salt stress in the development process of the microspore isolated embryo, so that an ideal screening effect (half inhibition concentration) can be obtained, the quantity proportion of 4d microspore expansion starting embryo development and the 21d induced embryogenic callus yield are reduced to about half of the control (no NaCl stress is added);

according to the literature, the salt tolerance of rice is poorer than that of barley, but when 400 mg/L NaCl stress screening is applied (the concentration range determined by the earlier patent is 50-500 mg/L), although the inhibition effect on microspore initiation embryo development is obviously inhibited, the inhibition effect on embryogenic callus induction is not obviously different, and when 800 mg/L NaCl stress screening is applied, the two indexes are close to about half of the control, so that the ideal effect is achieved.

D) Obtaining a regeneration plant, identifying ploidy by a flow cytometer, and selecting a doubled haploid (diploid); the lowest yield is more than 50 percent;

E) breeding to obtain a doubled haploid strain (DH strain) population;

F) and (3) carrying out water culture of the doubled haploid strain (DH strain) Yoshida nutrient solution, adding 8.7 g/L NaCl for stress screening, carrying out phenotype grading evaluation according to the green loss and curling degrees, identifying the doubled haploid strain (DH strain) with the salt tolerance improved by being compared with the original parent, and obtaining the material with the evaluation grading index improved by about one time, namely the material with the salt tolerance obviously improved.

And further, in the step C, free microspore culture, namely, taking young ears of microspores in a mononuclear period, sealing a freshness protection bag, placing the freshness protection bag in a refrigerator for low-temperature treatment for one week at 4 ℃, disinfecting the young ears by using a sodium hypochlorite solution for 15min before inoculation, picking anthers under an aseptic condition, crushing the anthers by using a high-speed homogenizer, sieving the anthers, re-suspending the anthers by using a mannitol solution containing 60 g/L, and placing the anthers on an improved N6 culture medium containing 90 g/L, 2, 4-D1.5mg/L to induce and generate embryogenic callus.

Further, in the step C, during microspore induced embryogenic callus formation, 800 mg/L NaCl stress treatment is adopted.

And further, in the step D, the embryogenic callus is induced to obtain a regeneration plant, namely, the embryogenic callus obtained by inducing and culturing microspore obtained by stress screening in the step C for 21D is transferred to an improved MS culture medium containing 30 g/L0.5.5 mg/L sucrose and 6-BA0.5 mg/L sucrose to induce and form green seedlings.

And D, further, the method for identifying the ploidy of the regenerated plant in the step D comprises the steps of taking a leaf of the regenerated plant, cutting the leaf, dissociating cell nucleus by using L B-01 buffer solution, treating the cell nucleus by using PI/RNase dye solution, taking filtrate, carrying out flow cytometry to identify the ploidy, and identifying the doubled haploid (diploid) by selecting a seed seedling as a peak position of a control (diploid).

Further, the step E: and (3) carrying out water culture and hardening on the doubled haploid plants obtained by microspore culture, packing and moisturizing when the seedlings grow to about 10cm after new roots grow out, sending the seedlings to a southeast Hainan breeding base, transplanting the seedlings to a field in the same year, and harvesting single seeds in the second year to form a doubled haploid strain (DH strain) group.

Further, in the step F, the table type grading evaluation criteria are:

according to the green degree and the curling degree of the leaves and the stems of the seedlings, 5 grades are divided, the 5 grades have the best salt tolerance, and the 1 grade has the worst property;

level 1: the whole seedling has no green part and is basically determined to die;

and 2, stage: heart leaves and stems are too green and curled seriously, and other leaves are withered and yellow and die;

and 3, level: the heart leaves and the stems are partly green, a small part of leaves are still green, and most of leaves are withered and yellow and die;

4, level: the tip of each leaf turns yellow and curls, the leaves and the stems at the middle and lower parts are still kept green, and part of the leaves are curled;

and 5, stage: only the tip of each leaf turns yellow and curls, the leaves and stems of the rest parts still keep green, and the heart leaves and half of the inverted two leaves can be seen from the unfolded parts;

by adopting the identification method, the DH strain with the salt tolerance improved than that of the original parent is identified by taking the grading evaluation index which is doubled compared with that of the original parent (the average grading value is 3.8) as the screening standard.

In a preferred embodiment of the present invention, the method for improving salt tolerance of flower 11 in a rapidly improved salt-sensitive rice variety comprises the following steps:

A)seed irradiation mutagenesis: taking dry seeds of flower 11 in rice variety, adopting⁶⁰Co gamma-ray irradiation with a dose of 350 GY;

B) sowing in a field and transplanting seedlings: irradiating rice seeds for field sowing, transplanting after seedling raising, and managing by conventional water and fertilizer until heading; 20kg of base fertilizer per mu, 10kg of urea for tillering fertilizer and additional application, and 15kg of compound fertilizer for jointing fertilizer and additional application;

C) culturing free microspore, namely placing young ears of microspores in the mononuclear stage in a refrigerator for low-temperature treatment for one week at 4 ℃, sterilizing for 15min by using a sodium hypochlorite solution before inoculation, picking anthers under aseptic condition, crushing by using a high-speed homogenizer, sieving, re-suspending by using a mannitol extract containing 60 g/L, and placing on a modified N6 culture medium containing maltose 90 g/L, 2,4-D1.5 mg/L to induce and generate embryogenic callus;

D) salt stress treatment in the process of embryo development, wherein in the process of microspore inducing embryogenic callus formation, 800 mg/L NaCl stress treatment is adopted;

E) inducing the embryogenic callus to regenerate plant, namely transferring the embryogenic callus obtained by microspore induced culture 21d to an improved MS culture medium containing 30 g/L0.5.5 mg/L of sucrose and 0.5 mg/L of 6-BA to induce and form green seedling;

F) the method comprises the following steps of (1) regenerated plant ploidy identification, namely obtaining a regenerated plant, taking leaves, cutting the leaves, dissociating cell nucleuses by using L B-01 buffer solution, treating the cut leaves by using PI/RNase staining solution, taking filtrate, and identifying ploidy by using a flow cytometer, selecting seed seedlings as the peak position of a control (diploid), identifying the doubled haploid (diploid), and estimating the spontaneous doubling frequency by a statistical result to be about 50%;

G) and (3) seed reproduction in Hainan: culturing the obtained doubled haploid plant by using microspores, water-culturing and hardening the seedling by using nutrient solution, packing and moisturizing the seedling when the seedling grows to about 10cm after a new root grows out, sending the seedling to a southeast Hainan breeding base, transplanting the seedling to a field in the same year, and harvesting single seeds in the second year to form a doubled haploid plant (DH plant) group;

H) the Yoshida nutrient solution salt stress water culture screening comprises the steps of taking about 50 seeds of each DH strain, soaking the seeds for accelerating germination, transferring seedlings to nutrient solution water culture, adding 8.7 g/L NaCl for stress treatment, taking the leek green variety with strong salt tolerance as a control, and taking the growth phenotype of the seedlings as the graded evaluation of the salt tolerance of the seedling stage, wherein the specific standard is as follows:

according to the green degree and the curling degree of the leaves and the stems of the seedlings, 5 grades are divided, the 5 grades have the best salt tolerance, and the 1 grade has the worst property;

level 1: the whole seedling has no green part and is basically determined to die;

and 2, stage: heart leaves and stems are too green and curled seriously, and other leaves are withered and yellow and die;

and 3, level: the heart leaves and the stems are partly green, a small part of leaves are still green, and most of leaves are withered and yellow and die;

4, level: the tip of each leaf turns yellow and curls, the leaves and the stems at the middle and lower parts are still kept green, and part of the leaves are curled;

and 5, stage: only the tip of each leaf turns yellow and curls, the leaves and stems of the rest parts still keep green, and the heart leaves and half of the inverted two leaves can be seen from the unfolded parts;

by adopting the identification method, the DH strain with the salt tolerance improved than that of the original parent is identified by taking the average grading value 3.8 which is twice as high as that of the original parent (the average grading value of 11 rice seedlings in middle blossom) as the screening standard. And (3) providing a seedling stage identification grading index according to the green loss degree and the curling degree of the leaves and the stems of the seedlings, taking a material with the grading index improved by about one time as a seedling stage salt tolerance improvement identification standard, and screening to obtain the material with the proportion of more than 1%.

The invention provides a method for quickly improving the salt tolerance of flower 11 in a salt-sensitive rice variety, which utilizes⁶⁰Co gamma-ray irradiation is used for creating variation, microspore culture haploid technology is adopted for rapid homozygous stabilization, half inhibition screening is provided at the microspore induced embryogenesis stage by adopting 800 mg/L NaCl, a DH strain is obtained, 8.7 g/L NaCl stress treatment is applied at the seedling stage, the seedling stage salt tolerance evaluation is carried out by taking the greening and curling degrees of leaf and stem of seedlings as phenotype grading indexes, the grading index is selected to be more than 3.8 (2 times of the grading index of a control material), the material yield of the seedling stage salt tolerance is finally improved by more than 1 percent, the proportion of target characters obtained by conventional mutagenesis is ten thousandths or lower, the yield can reach one ten thousandths or lower through early stress screening, and the inventionThe yield is more than 1 percent, and is obviously improved compared with the conventional method.

The main improvement points of the invention are as follows: 1. the whole improvement process is completed by a six-step method; 2. the screening concentration of NaCl is determined according to two indexes of half inhibition proportion of microspore starting embryo development, embryogenic callus yield formed by microspore embryo development and the like for the first time, so that the effectiveness is better; 3. the identification grading index of the seedling stage is provided for the first time according to the green degree and the curling degree of the leaves and the stems of the seedlings, and the operation is strong; 4. the salt tolerance of the medium flowering plant at the 11 seedling stage improved by the method is high in proportion and stable in homozygosis.

Compared with the prior art, the invention has the advantages that:

1. the invention provides a complete method for quickly improving salt-sensitive rice varieties, and specific screening and identification indexes are established;

2. the invention adopts the variation generated by fast homozygous stable irradiation mutagenesis of the microspore culture haploid technology, and doubled haploid strains obtained within two years of south propagation and generation addition can be directly used for identification, thereby saving the time of 3-5 years for the selfing, homozygous and stable material;

3. NaCl salt stress treatment is adopted in two periods of microspore initiation embryo development, seedling stage and the like to carry out early salt tolerance screening and identification, so that the salt tolerance improvement efficiency is improved;

4. the salt tolerance screening and identifying method established by the invention is simple and easy to observe, and has operability in large-scale identification.

Drawings

FIG. 1 is a photograph showing microscopic observations of the development of embryos initiated by isolated microspores of rice flower 11 under salt stress at different concentrations (red arrows indicate expanded microspores) (CK control for A; 400 mg/L NaCl treatment for B; 800 mg/L NaCl; and 1200 mg/L NaCl treatment for C).

FIG. 2 quantitative proportion of floral 11 isolated microspores in rice that initiate embryonic development (expansion) under salt stress at different concentrations;

FIG. 3 shows embryogenic callus yields induced by floral 11 isolated microspores in rice under salt stress of different concentrations;

FIG. 4 shows an example of the evaluation of salt tolerance of 11 seedlings in rice (left-hand numbers indicate grading values).

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.