Technology for shortening cotton breeding period and application

Abstract

The invention discloses a technology for shortening a cotton breeding cycle and application thereof, and the technology comprises the following steps: 1, accelerating budding and flowering under mixed light of blue light, green light, red light and far-red light; 2, performing GA3 treatment to promote germination of immature embryos; 3, using the created L-mAUH and mAUH cotton immature embryo culture medium to culture the 25-28-day-old cotton immature embryos into seedlings; fourthly, the first step, the second step, the third step and the fourth step are circularly carried out for multiple times, in the process, molecular markers are used for carrying out auxiliary selection on the good characters of the foreground, and meanwhile whole genome re-sequencing is used for carrying out background auxiliary selection. The method has the advantages that by means of efficient blue, green, red and far-red spectrum adjustment, GA3 treatment, immature embryo culture, molecular marker selection and whole genome re-sequencing, cotton growth and development can be remarkably accelerated, and the cotton breeding period is effectively shortened. According to the method, a conventional backcross breeding process can be effectively integrated, excellent characters can be quickly transformed, and an improved line with a high genetic background recovery rate can be obtained within 1.5 years.

Description

Technical Field

[0001] The invention belongs to the field of agricultural science and technology, and specifically relates to a technology for shortening the cotton breeding cycle and its application. Background Art

[0002] Cotton belongs to the Malvaceae family and the Gossypium genus. Cotton is an important multi-purpose economic crop and is widely planted around the world. Cotton is susceptible to adversities such as diseases and insects, which cause serious losses in its yield and quality. Variety improvement is the most economical and effective way to improve yield, quality, and resistance. Both traditional breeding techniques and biological breeding techniques play a very important role in cotton variety improvement. However, both traditional breeding and molecular breeding have the common problem of long breeding cycles, and distant hybridization takes even longer. Therefore, speeding up breeding and improving breeding efficiency have become one of the focuses of breeding research.

[0003] Light of different qualities or wavelengths has significantly different biological effects, including different effects on the morphological structure and chemical composition of plants, photosynthesis, and organ growth and development. The University of Queensland in Australia regulates light quality and photoperiod to accelerate the development of long-day plants such as wheat, barley, and rapeseed, achieving 4-6 generations of reproduction in one year. Researchers at the National Institute of Plant Breeding at the University of Hohenheim in Germany have promoted the rapid development of short-day crops such as soybeans and rice by adjusting light quality and photoperiod. Researchers at Huazhong Agricultural University have further shortened the growth cycle of two different rapeseed varieties by 12 days and 21 days respectively by regulating light quality and increasing far-red light. The cotton breeding team of Hebei Agricultural University has achieved rapid breeding technology of three generations of cotton in one year by using a greenhouse environment with controlled light and temperature, combined with cotton embryo culture. This study intends to further accelerate the development process of cotton and shorten the cotton breeding cycle by regulating light quality conditions.

[0004] Plant seeds often have the potential to develop into the next generation of plants before maturity, so the immature embryo culture technology can be used to shorten the plant growth cycle. For example, the immature embryos of wheat 12 days after flowering can achieve a germination rate of more than 95% through immature embryo culture; up to 97.99% of 30-day peanut immature embryos can develop into seedlings. The in vitro culture technology of cotton immature embryos uses tissue culture methods to improve the development conditions of immature embryos and provide the nutrients necessary for the development of immature embryos, thereby promoting their direct germination and development into healthy plants. This technology greatly saves the time required for cotton flowering to seed maturity, can effectively shorten the cotton generation cycle, and provide an important way to accelerate the breeding process. At present, the main in vitro culture media for cotton immature embryos are Mauney medium, BT medium, MS medium, CEM medium, etc. Although there are a variety of culture media and culture methods for in vitro culture of cotton immature embryos, there are still problems such as long immature embryo culture time and low seedling rate. Therefore, it is urgent to further improve the immature embryo culture medium and culture method to shorten the immature embryo culture time and improve the seedling rate, thereby accelerating cotton genetic improvement and promoting the rapid development of the cotton industry. Summary of the invention

[0005] The above technical purpose of the present invention is achieved through the following technical scheme: a technology and application for shortening the cotton breeding cycle.

[0006] The present invention provides a technology for shortening the cotton breeding cycle, comprising:

[0007] 1. Accelerate budding and flowering under a mixed light of blue light, green light, red light and far-red light;

[0008] Second, GA3 treatment promotes the germination of young embryos;

[0009] 3. Using the L-mAUH and mAUH cotton embryo culture medium, 25-28-day-old cotton embryos were cultured into seedlings;

[0010] 4. The above steps 1 to 3 are repeated multiple times. During this process, molecular markers are used to assist in the selection of promising traits, while whole genome resequencing is used for background-assisted selection.

[0011] Furthermore, in step 1, a mixed light consisting of 450nm blue light, 530nm green light, 660nm red light and 730nm far-red light provided by an LED lamp is used as a light source.

[0012] Furthermore, the mixed light cycle is 12 hours of light and 12 hours of darkness, the day temperature is 30-32°C, the night temperature is 25-26°C, and the humidity is 35-45%.

[0013] Furthermore, the step 1 includes the following specific contents:

[0014] (1) Developmental period survey

[0015] The survey included the period of 1-9 true leaves per plant, the first fruiting branch node per plant, the first bud appearance period per plant, and the first flower flowering period per plant;

[0016] (2) Detection of expression levels of cotton flowering-related genes

[0017] From the first true leaf stage to the fifth true leaf stage, total RNA of the upper leaf was extracted, and three biological replicates were taken in each period. RNA was used as a template for reverse transcription into cDNA, and specific primers were designed based on the gene sequences of cotton flowering factors GhSOC1 and GhFT. The expression changes of GhSOC1 and GhFT genes were determined by qPCR technology.

[0018] (3) Observation of flower bud differentiation

[0019] From the first true leaf stage to the fifth true leaf stage, take 0.5-1 cm tissue from the top of the stem tip, peel off the leaf tissue and keep only the stem and stem tip meristem, and immediately put them into FAA fixative for fixation. After fixation, dehydrate, wax dip, embed, section, stain, and observe under a microscope.

[0020] Furthermore, the step three is specifically as follows: take young bolls 25-28 days after flowering, dry them in a culture room at a temperature of 28±2°C and a relative humidity of 40% to 50% for 3 days. In the sterile environment of a clean bench, soak them in 70% ethanol for 1 minute to sterilize the surface of the cotton bolls. Cut along the gap between the carpels of the cotton bolls with a scalpel, take out the young embryos, soak them in GA3 at a concentration of 60mmol / L for 4 hours, and then inoculate them in sterile L-mAUH culture medium, and culture them in the dark at 45r / min at 28°C; after 12 hours, transfer the young embryos to sterile mAUH culture medium and culture them in a light culture room.

[0021] Furthermore, the formula of the mAUH medium is: KNO3950mg / L, NH4NO3825mg / L, CaCl2·2H2O220mg / L, MgSO4·7H2O 185mg / L, KH2PO485mg / L, Na2-EDTA·2H2O 37.25mg / L, FeSO4·7H2O27.8mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, H3BO36.2mg / L, KI 0.83mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO 4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, inositol 100mg / L, niacin 1mg / L, thiamine hydrochloride 10mg / L, pyridoxine hydrochloride 1mg / L, indolebutyric acid 0.1mg / L, 6-benzylaminopurine 0.1mg / L, sucrose 20000mg / L, agar 3500mg / L.

[0022] The formula of the L-mAUH culture medium is: KNO3 475 mg / L, NH4NO3 412.5 mg / L, CaCl2·2H2O 110 mg / L, MgSO4·7H2O 92.5 mg / L, KH2PO4 42.5 mg / L, Na2-EDTA·2H2O 18.63 mg / L, FeSO4·7H2O 13.9 mg / L, MnSO4·4H2O 11.15 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO 4· 5H2O 0.025mg / L, CoCl2·6H2O0.025mg / L, inositol 100mg / L, niacin 1mg / L, thiamine hydrochloride 10mg / L, pyridoxine hydrochloride 1mg / L, indolebutyric acid 0.1mg / L, 6-benzylaminopurine 0.1mg / L, sucrose 20000mg / L.

[0023] Furthermore, the culture conditions of the light culture room are: 12h light / 12h dark; the temperature under light conditions is 28±2°C, the light intensity is 1500-2500lux, and the relative humidity is 40%-50%; the temperature under dark conditions is 25±2°C, and the relative humidity is 40%-50%. The cotton embryo seedlings are counted after 6-8 days of culture, and the true leaf growth rate is investigated after 10-12 days of culture.

[0024] The invention provides an application of a technology for shortening the cotton breeding cycle: using IF1-1 containing the iaaM gene as a donor parent and high-yield and disease-resistant Jinongda No. 24 as a recipient parent for backcross breeding. The method is used to quickly obtain multiple BC4F3 generation Jinongda No. 24 improved lines containing the iaaM gene within 1.5 years, among which the background recovery rate of the Jinongda 24-i3 improved line reaches 97.36%, and the lint and fiber quality are significantly improved.

[0025] The present invention provides a technology for shortening the cotton breeding cycle and its application. Through efficient blue, green, red and far-red spectrum regulation, combined with GA3 treatment, young embryo culture, molecular marker selection and whole genome resequencing, the growth and development of cotton can be significantly accelerated, the cotton breeding cycle can be effectively shortened, and the invented multi-spectrum LED plant growth lamp can be used to promote cotton to bloom about 15-20 days in advance; through GA3 treatment and young embryo culture, the seedling rate of 25-28-day cotton young embryos can reach more than 90%, and the cotton growth period can be shortened by 20-23 days. The growth period of field cotton is usually 120-130 days. This method can shorten the cotton generation cycle to 71-85 days, achieve 5 generations a year locally, significantly reduce the breeding years, and provide technical support for improving the efficiency of new cotton variety selection. This method can be effectively integrated into the conventional backcross breeding process, quickly transfer excellent traits, and obtain improved lines with high genetic background recovery rates within 1.5 years. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The combined light source in step 1 of the present invention;

[0027] Among them, (a) is the red light spectrum; (b) is the far-red light spectrum; (c) is the blue-green combined spectrum (6500K); (d) is the blue-green combined spectrum (13000K).

[0028] Figure 2 Optimizing the spectrum of the LED lamp in step 1 of the present invention;

[0029] Figure 3 The LED lamp layout in step 1 of the present invention;

[0030] Figure 4 The cotton growth condition under the LED light optimized spectrum condition in step 1 of the present invention;

[0031] Figure 5 The expression levels of flowering key genes under optimized spectral conditions;

[0032] Figure 6 To optimize the starting time of flower bud differentiation under spectral conditions;

[0033] Figure 7 The invention is applied to the process of creating new materials;

[0034] Figure 8 The iaaM gene expression level and IAA content in the new material used in the present invention;

[0035] Fig. 9 A comparison diagram of the protrusions of the seed coat fibers of the present invention used in creating new materials;

[0036] Fig.10 This is the genetic background recovery situation used in the creation of new materials by the present invention. DETAILED DESCRIPTION

[0037] The following is combined with Figure 1-10 The present invention is further described in detail with the following embodiments:

[0038] (1) Cotton varieties tested

[0039] Agricultural University 601 and Ji Shi 929.

[0040] (2) Spectral conditions

[0041] According to the absorption spectra of plant chlorophyll a, chlorophyll b, and phytochrome and the effects of different lights on plant growth and development, the radiation spectrum of the light source is regulated. The mixed light composed of 450nm blue light, 530nm green light, 660nm red light, and 730nm far-red light provided by LED lamp is used as the light source. The spectrum is shown in the supplement. The photon flux density at 10cm under the lamp is 650μmol / m 2 ·s.

[0042] (3) Cotton plant cultivation

[0043] Mature and healthy seeds of Nongda 601 and Jishi 929 were delinted, soaked at 28°C for 8-12 hours, and germinated at 25-28°C for 18-24 hours. When the roots grew to 0.5-1.0 cm, the seeds were sown in seedling pots filled with nutrient soil, with 4-5 germinated seeds per pot, and then placed in a culture room with controlled light, temperature and humidity for cotton seedling culture. The culture conditions were: the light conditions were the same as the spectrum conditions in (2) above, the photoperiod was 12 hours of light / 12 hours of darkness, the day temperature was 30-32°C, the night temperature was 25-26°C, and the humidity was 35-45%.

[0044] (4) Developmental period survey

[0045] The investigations were conducted on the stage when individual plants have 1-9 true leaves, the first fruiting branch node of individual plants, the stage when the first bud of individual plants appears, and the stage when the first flower of individual plants blooms.

[0046] (5) Detection of expression levels of cotton flowering-related genes

[0047] From the first true leaf stage to the fifth true leaf stage, total RNA was extracted from the upper leaf, and three biological replicates were taken in each period. RNA was used as a template for reverse transcription into cDNA, and specific primers were designed based on the cotton flowering factor GhSOC1 and GhFT gene sequences. qPCR technology was used to determine the changes in the expression of GhSOC1 and GhFT genes.

[0048] (6) Observation of flower bud differentiation

[0049] From the first true leaf stage to the fifth true leaf stage, take 0.5-1 cm tissue from the top of the stem tip, peel off the leaf tissue and retain only the stem and stem tip meristem, and immediately put them into FAA fixative for fixation. After fixation, dehydrate, wax dip, embed, section, stain, and observe under a microscope. The specific steps refer to Wu Hongping's method.

[0050] (7) The cotton immature embryos were cultured into seedlings using the developed L-mAUH and mAUH cotton immature embryo culture media.

[0051] Take young bolls 25-28 days after flowering and dry them in a culture room at a temperature of 28±2℃ and a relative humidity of 40% to 50% for 3 days. Under the sterile environment of a clean bench, soak them in 70% ethanol for 1 minute to sterilize the surface of the cotton bolls. Cut along the gap between the carpels of the cotton bolls with a scalpel, take out the young embryos, soak them in GA3 with a concentration of 60mmol / L for 4 hours, and then inoculate them in sterile L-mAUH medium (Table 1), and culture them in the dark at 45r / min at 28℃; after 12 hours, transfer the young embryos to sterile mAUH medium (Table 2) and culture them in a light culture room. The culture conditions in the light culture room are: 12h light / 12h dark; under light conditions, the temperature is 28±2℃, the light intensity is 1500-2500lux, and the relative humidity is 40% to 50%; under dark conditions, the temperature is 25±2℃ and the relative humidity is 40%-50%. The cotton immature embryos were counted for seedling formation on day 6-8 of culture, and the true leaf growth rate was investigated on day 10-12 of culture. Table 1 below shows the formula of mAUH medium and L-mAUH medium.

[0052] Table 1

[0053]

[0054]

[0055] 3 Results and analysis

[0056] (1) Created multi-spectral conditions to induce the expression of key genes for flowering

[0057] The results of fluorescence quantitative PCR showed that under the optimized spectral conditions, the expression levels of GhSOC1 and GhFT genes were significantly higher than those in the control group, and the peak expression time was advanced by 3 days ( Figure 5 ). This indicates that the spectral conditions optimized in this study play an important regulatory role in the expression of key flowering genes.

[0058] (2) The multi-spectral conditions created promote the early start of flower bud differentiation

[0059] Through microscopic observation of stem tip differentiation tissue sections, it was found that the cotton plants grown under optimized spectral conditions had flower bud differentiation earlier, starting at the second true leaf stage, while the control plants started to differentiate at the third to fourth true leaf stage, 3 to 6 days earlier than the control ( Figure 6 ). This indicates that the optimized spectrum plays an important regulatory role in the initiation of cotton flower bud differentiation.

[0060] (3) The multi-spectral conditions created promote the early appearance and flowering of thunder

[0061] The results of the developmental period survey showed that the optimized spectrum group significantly accelerated both vegetative growth and reproductive growth compared with the control group. Specifically, the second true leaf of Agricultural University 601 was 5.0-8.0 days earlier, 6.8 days earlier on average; the fourth true leaf was 5.3-9.3 days earlier, 7.2 days earlier on average; the sixth true leaf was 5.7-10.7 days earlier, 8.3 days earlier on average; the first bud was 4.5-9.5 days earlier, 6.5 days earlier on average; the first flower was 6.5-11.5 days earlier, 9.3 days earlier on average. The specific performance of Ji Shi 929 is that the second true leaf appears 6.7-9.7 days earlier, 7.9 days earlier on average; the fourth true leaf appears 6.7-10.7 days earlier, 9.2 days earlier on average; the sixth true leaf appears 7.7-13.7 days earlier, 10.9 days earlier on average; the first bud appearance is 8.3-12.3 days earlier, 9.7 days earlier on average; the first flower blooms 8.0-13.0 days earlier, 11.0 days earlier on average.

[0062] (4) The invented immature embryo culture method effectively shortens the growth period of cotton

[0063] This study created a shaking culture method for L-mAUH medium. After 12 hours of shaking culture with L-mAUH medium, the cotyledons began to stretch and the roots extended to about 0.5-1cm. After the immature embryos were transferred to the created mAUH medium for culture for 6 days, the seedling rate reached 94.4%, and the seedlings began to grow true leaves after 8 days of culture. The results of the study showed that the in vitro culture of immature embryos using L-mAUH and mAUH medium can effectively promote the germination of cotton immature embryos and greatly improve the seedling rate of immature embryos. This culture technology effectively solves the problems of long culture time and low seedling rate of cotton immature embryos, and shortens the growth period of cotton by about 22 days, providing an important way to shorten the cotton generation cycle and speed up breeding.

[0064] (5) Rapid creation of new germplasm with an average of 5 generations per year

[0065] Backcross breeding was carried out with IF1-1 containing the iaaM gene as the donor parent and the high-yield and disease-resistant JNU 24 as the recipient parent. This method was used to quickly obtain multiple BC4F3 generation JNU 24 improved lines containing the iaaM gene within 1.5 years, among which the background recovery rate of JNU 24-i3 improved line reached 97.36%, and the lint and fiber quality were significantly improved. Figure 7-10 , Figure 7 The invention is applied to the process of creating new materials; Figure 8 The iaaM gene expression level and IAA content in the new material used in the present invention; Fig. 9 This is a comparison diagram of the protrusions of the seed coat fibers of the new material created by the present invention, which shows that the protrusions of the seed coat fibers of the new material created are increased; Fig.10 This is the genetic background recovery situation used in the creation of new materials by the present invention.

[0066] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A technology for shortening the cotton breeding cycle, characterized in that: The following steps are involved:

1. Accelerate budding and flowering under a mixed light of blue light, green light, red light and far-red light; Second, GA3 treatment promotes the germination of young embryos; 3. Using the L-mAUH and mAUH cotton embryo culture medium, 25-28-day-old cotton embryos were cultured into seedlings; 4. The above steps 1 to 3 are repeated multiple times, and in this process, molecular markers are used to assist in the selection of promising traits, while whole genome resequencing is used for background-assisted selection.

2. A technology for shortening the cotton breeding cycle according to claim 1, characterized in that: The step 1 uses a mixed light consisting of 450nm blue light, 530nm green light, 660nm red light and 730nm far-red light provided by an LED lamp as a light source.

3. A technology for shortening the cotton breeding cycle according to claim 2, characterized in that: The mixed light cycle of step 1 is 12 hours of light and 12 hours of darkness, with a day temperature of 30-32°C, a night temperature of 25-26°C, and a humidity of 35-45%.

4. A technology for shortening the cotton breeding cycle according to claim 1, characterized in that: The step 1 includes the following specific contents: (1) Developmental period survey The survey included the period of 1-9 true leaves per plant, the first fruiting branch node per plant, the first bud appearance period per plant, and the first flower flowering period per plant; (2) Detection of expression levels of cotton flowering-related genes From the first true leaf stage to the fifth true leaf stage, total RNA of the upper leaf was extracted, and three biological replicates were taken in each period. RNA was used as a template for reverse transcription into cDNA, and specific primers were designed based on the gene sequences of cotton flowering factors GhSOC1 and GhFT. The expression changes of GhSOC1 and GhFT genes were determined by qPCR technology. (3) Observation of flower bud differentiation From the first true leaf stage to the fifth true leaf stage, take 0.5-1 cm tissue from the top of the stem tip, peel off the leaf tissue and keep only the stem and stem tip meristem, and immediately put them into FAA fixative for fixation. After fixation, dehydrate, wax dip, embed, section, stain, and observe under a microscope.

5. A technology for shortening the cotton breeding cycle according to claim 1, characterized in that: The step three is specifically as follows: taking young bolls 25-28 days after flowering, drying them in a culture room at a temperature of 28±2°C and a relative humidity of 40%-50% for 3 days, soaking them in 70% ethanol for 1 minute in a sterile environment of a clean bench to sterilize the surface of the bolls, cutting them open along the gaps between the carpels of the bolls with a scalpel, taking out young embryos, soaking them in GA3 with a concentration of 60 mmol / L for 4 hours, and then inoculating them in a sterile L-mAUH culture medium, and culturing them in the dark with shaking at 45 r / min at 28°C; After 12 hours, the young embryos were transferred to sterile mAUH medium and cultured in a light culture room.

6. A technology for shortening the cotton breeding cycle according to claim 1, characterized in that: The formula of the mAUH medium in step 3 is: KNO3 950 mg / L, NH4NO3 825 mg / L, CaCl2·2H2O 220 mg / L, MgSO4·7H2O 185 mg / L, KH2PO4 85 mg / L, Na2-EDTA·2H2O 37.25 mg / L, FeSO4·7H2O 27.8 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, H3BO3 6.2 mg / L, KI 0.83 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO 4· 5H2O0.025mg / L, CoCl2·6H2O 0.025mg / L, inositol 100mg / L, niacin 1mg / L, thiamine hydrochloride 10mg / L, pyridoxine hydrochloride 1mg / L, indolebutyric acid 0.1mg / L, 6-benzylaminopurine 0.1mg / L, sucrose 20000mg / L, agar 3500mg / L.

7. A technology for shortening the cotton breeding cycle according to claim 1, characterized in that: The formula of the L-mAUH medium in the step 3 is: KNO3475mg / L, NH4NO3412.5mg / L, CaCl2·2H2O 110mg / L, MgSO4·7H2O92.5mg / L, KH2PO442.5mg / L, Na2-EDTA·2H2O 18.63mg / L, FeSO4·7H2O 13.9mg / L, MnSO4·4H2O 11.15mg / L, ZnSO4·7H2O 8.6mg / L, H3BO36.2mg / L, KI 0.83mg / L, Na2MoO4·2H2O0.25mg / L, CuSO 4· 5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, inositol 100mg / L, niacin 1mg / L, thiamine hydrochloride 10mg / L, pyridoxine hydrochloride 1mg / L, indolebutyric acid 0.1mg / L, 6-benzylaminopurine 0.1mg / L, sucrose 20000mg / L.

8. A technology for shortening the cotton breeding cycle according to claim 1, characterized in that: The culture conditions in the light culture room in step 3 are as follows: 12 h light / 12 h dark; under light conditions, the temperature is 28±2°C, the light intensity is 1500-2500 lux, and the relative humidity is 40%-50%; under dark conditions, the temperature is 25±2°C, and the relative humidity is 40%-50%. The cotton immature embryo seedling situation is counted after 6-8 days of culture, and the true leaf growth rate is investigated after 10-12 days of culture.

9. Application of a technology to shorten the cotton breeding cycle: using IF1-1 containing the iaaM gene as the donor parent and the high-yield and disease-resistant Ji Nong Da No. 24 as the recipient parent, backcross breeding is carried out, and this method is used to quickly obtain multiple BC4F3 generation Ji Nong Da No. 24 improved lines containing the iaaM gene within 1.5 years.

Images