Cultivation method for upland cotton efficient genetic transformation receptor, and use

Abstract

Provided is a cultivation method for an upland cotton efficient genetic transformation receptor, comprising: using 'E cotton 22' (E22) as a female parent and using 'Yuzao No. 1' cotton (YZ-1) as a male parent to construct a genetic stable F9 recombinant inbred line, carrying out callus induction, differentiation and embryogenesis, and plant regenerative cultivation on the aseptic seedling hypocotyl of the genetic stable F9 recombinant inbred line, and screening for a highly regenerative material. The method comprises the following steps: using two different hormone combinations, i.e., IK(IBA+KT) and DK(2,4-D+KT), as a callus induction cultivation system to culture the aseptic seedling hypocotyl, carrying out cultivation to obtain an embryonic callus, carrying out statistical screening on the regeneration ability traits of a cotton to obtain the highly regenerative material, inoculating the embryonic callus into a differentiation culture medium, inoculating a seedling generated by differentiation into a rooting culture medium, and finally carrying out water cultivation to obtain a regenerative plant.

Description

Cultivation method and application of a high-efficiency genetic transformation receptor for upland cotton Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and in particular relates to a cultivation method and application of an efficient genetic transformation receptor for upland cotton. Background Art

[0002] Cotton is an important fiber and oil crop worldwide and plays a vital role in my country's economic development. Traditional hybrid breeding methods are no longer sufficient to meet the needs of cotton genetic improvement, and molecular breeding is a hot topic in cotton germplasm innovation. Somatic embryogenesis utilizes the totipotency of plant cells to recapitulate zygotic embryo morphogenesis. The application of modern molecular biotechnology is largely based on plant tissue culture techniques, making tissue culture technology crucial in the breeding of new cotton varieties. Steward and Reinert induced carrot phloem cells through suspension culture and obtained somatic embryos. Further culture yielded the first regenerated plant, ushering in a new era of exploration for plant regeneration. In 1912, Robins added different substances to the culture medium and used it to culture cotton root tips, obtaining stems and roots without greening. This was the first report on cotton tissue culture.

[0003] The development of cotton tissue culture systems started relatively late but has developed rapidly. Beasiey first induced callus in 1971, and Price and Smith (1979) successfully induced cotton somatic embryos using suspension culture. In 1983, Davidonis successfully obtained the first regenerated cotton plant after prolonged subculture. Zhang Xianlong proposed that cotton genotype is the primary factor influencing somatic embryogenesis and plant regeneration. A comparison of four cultivated varieties revealed significant differences in regeneration capacity between cultivars: Upland cotton > Asiatic cotton > Sea island cotton > African cotton. Variations also exist between different cotton varieties within the same cultivar. For example, within Upland cotton, the order is Kezi cotton > Daizi cotton > Sizi cotton. After more than a decade of research and exploration, cotton tissue culture systems have been gradually refined and subsequently entered a period of rapid development. Mapping and cloning genes associated with regeneration capacity, and using them to modify genotypes with high agronomic value but low regeneration capacity, is an effective means of achieving transgenic crop production.

[0004] Studies have shown that the upland cotton variety 'Yu Zao 1' (YZ-1) with chicken-foot leaves has strong regeneration capacity, a short transformation cycle, and a high seedling rate. However, YZ-1 has super-chicken-foot leaves and poor agronomic traits, making transgenic plants with it difficult to apply in production practices. Because cotton somatic embryogenesis and plant regeneration are highly dependent on the cotton genotype, based on the reported regeneration culture systems of different cotton varieties, we selected regeneration systems related to the parents to evaluate the regeneration capacity of constructed populations. We also attempted to locate loci associated with somatic embryogenesis and regeneration capacity. These loci can be targeted and introduced into varieties with excellent agronomic traits, broadening the selection of superior varieties as transgene recipients.

[0005] Summary of the Invention

[0006] One of the purposes of the present invention is to provide a method for cultivating high-efficiency genetic transformation receptors of upland cotton, comprising the following steps: material creation → high-regeneration material screening → high-generative transformation material screening, wherein the material creation comprises: hybridizing 'Emian 22' as the female parent and 'Yu Zao 1' as the male parent, and then selfing for multiple generations to construct a genetically stable F9 generation recombinant inbred line.

[0007] Furthermore, the high regeneration material screening includes: performing callus induction, embryo differentiation and plant regeneration culture on the hypocotyls of sterile seedlings of the genetically stable F9 generation recombinant inbred line to screen out high regeneration materials.

[0008] Further, the steps include:

[0009] (1) Callus induction: using two sets of callus induction culture systems, IBA+KT and / or 2,4-D+KT, to culture the hypocotyls of the sterile seedlings;

[0010] (2) Differentiation of embryos: culturing embryonic callus to obtain embryonic callus, statistically screening cotton regeneration ability traits, and obtaining the highly regenerative material;

[0011] (3) The embryonic callus is inoculated into a differentiation medium and a rooting medium in sequence, and hydroponically cultured to obtain regenerated plants.

[0012] Furthermore, the cotton regeneration ability traits include: callus induction rate, callus subculture reproduction capacity, callus embryo emergence time, and callus embryo emergence rate.

[0013] Furthermore, the calculation method of the cotton regeneration ability trait is:

[0014] Callus induction rate: number of callus blocks / total number of induced callus blocks × 100%;

[0015] Subculture propagation capacity of callus tissue: callus weight after 25 days of subculture - callus weight at 0 days of subculture;

[0016] Callus embryogenesis time: the earliest time when embryogenic callus appears in a single family;

[0017] Embryo emergence rate of callus tissue: the number of blocks with embryonic callus / the total number of subculture blocks × 100%.

[0018] Furthermore, families with callus induction rate higher than 98%, callus subculture reproduction capacity higher than 4.2 g, callus embryo generation time no higher than 116 d, and callus embryo generation rate higher than 82.4% were screened to obtain the high regeneration material.

[0019] Furthermore, the screening of the highly genetically transformed materials includes: using the Agrobacterium-mediated cotton genetic transformation system on the highly regenerative materials, using fluorescent protein as a reporter gene, obtaining transgenic plants, and screening to obtain transgenic families with a genetic transformation efficiency of not less than 80%.

[0020] Furthermore, the screening steps for highly genetically transformed materials are as follows:

[0021] (1) Selection of culture method: For lines that can be regenerated under both systems, the IBA+KT culture system is used; for lines that can only be regenerated under the IK system or the DK system, the IK or DK system is used.

[0022] (2) Selection of culture media, including sterile seedling germination medium, Agrobacterium activation medium, co-culture medium, callus induction medium, somatic embryo differentiation medium, and plant rooting medium;

[0023] (3) Analysis of genetic transformation efficiency: For each family's transgenic plants, the expression of fluorescent protein in the plants was observed using a stereo fluorescence microscope, and the number of transgenic positive plants was counted to analyze the genetic transformation efficiency.

[0024] Furthermore, the step also includes: examining the agronomic traits of the high genetic transformation materials, and ultimately screening out regenerated materials with excellent agronomic traits and high regeneration ability and genetic transformation efficiency.

[0025] The present invention also aims to provide a method for cultivating a high-efficiency genetic transformation receptor for upland cotton and its application in cultivating a high-efficiency genetic transformation receptor for upland cotton.

[0026] The present invention also aims to provide a method for cultivating a high-efficiency genetic transformation receptor for upland cotton, and obtain the high-efficiency genetic transformation receptor.

[0027] The present invention aims to provide a method for cultivating and applying high-efficiency genetic transformation receptors for upland cotton. The method employs a hybridization method using Hubei Province's high-yield broadleaf variety 'Emian 22' (E22) as the female parent and the highly regenerative chicken-foot-leaf variety 'Yu Zao 1' (YZ-1) as the male parent. Multiple generations of self-pollination are then performed to construct a genetically stable F9-generation recombinant inbred line. The regeneration capacity of the population is assessed using a culture system using two different hormone combinations, IK (IBA+KT) and DK (2,4-D+KT). The genetic transformation efficiency of the selected high-regeneration broadleaf cotton is then assessed to obtain cotton receptors with high genetic transformation efficiency. Further, through an examination of agronomic traits, cotton receptors with excellent agronomic traits and high genetic transformation efficiency can be prepared. This method broadens the range of regenerative upland cotton genotypes, provides excellent receptor materials for cotton genetic transformation, and enriches the currently mainstream cotton transgenic receptor materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a technical route of the present invention.

[0029] FIG2 is a flow chart of the regeneration capacity evaluation under the IK and DK systems of the present invention.

[0030] Figure 3 illustrates the culture process for the IK and DK culture systems of the present invention. Figures (a) and (b) illustrate the regeneration process for the IK and DK systems, respectively, where A shows a cut hypocotyl segment cultured on callus induction medium, B shows the induced non-embryonic callus, C shows the induced embryonic callus, D shows the differentiated embryoid, E shows the differentiated rooted seedling, and F shows the hydroponic regenerated plant.

[0031] Figure 4 shows the frequency distribution histograms of CSCs, CETs, and CREs in callus tissue. (a) and (b) show the frequency distributions of CSCs, CETs, and CREs in callus tissue under the IK and DK systems, respectively, and in callus tissue from a lineage capable of regeneration under both the IK and DK systems. A represents CSCs, B represents CETs, and C represents CREs.

[0032] Figure 5 is an overview of regenerated plants from high regeneration families.

[0033] Figure 6 illustrates the genetic transformation process of a high-regeneration broadleaf cotton family. A sterile seedling culture of a high-regeneration broadleaf cotton family was performed using an Agrobacterium-mediated cotton genetic transformation system, using RFP as a reporter gene. A shows the cultured sterile seedlings; B shows a hypocotyl segment co-cultured with RFP-transformed Agrobacterium; C shows callus induction on screening medium; D shows induced non-embryogenic callus; E shows differentiated embryonic callus and embryoids; F shows differentiated regenerated plants; and G shows hydroponic transgenic T0 plants.

[0034] Figure 7 shows the RFP efficiency of high-regeneration broadleaf family transformation. Transgenic T0 plants were positively identified, and RFP expression in transgenic leaves was observed using a fluorescence microscope. Images (A, B, C, and D) are taken under white light, while images (E, F, G, and H) are taken under fluorescence. A and E show embryonic calli during culture, B and F show embryoids during culture, C and G show leaves of transgenic plants, and D and H show leaves of transgenic plants. The left image shows a positive transgenic plant, and the right image shows a negative transgenic plant. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1 Material Creation and Evaluation of Regeneration Capacity

[0037] The high-yielding broadleaf variety 'Emian 22' (E22) from Hubei Province was used as the female parent and the chicken-foot-leaf variety 'Yuzao 1' (YZ-1) with high regeneration ability was used as the male parent. After multiple generations of self-pollination, a genetically stable F9 generation recombinant inbred line YE was constructed.

[0038] The hypocotyls of seeds from the YE family and its parents, E22 and YZ-1, were subjected to callus induction, embryo differentiation, and plant regeneration culture to screen for high regeneration materials. The steps are as follows:

[0039] (1) Sterile seedling culture.

[0040] Select seeds with full grains and normal development, peel off the cotton seed coat, soak them in 10% mercuric chloride solution for 10 minutes, then rinse with sterile water for more than 3 times, inoculate them on sterile seedling culture medium, and after the seeds turn white, insert the roots into the culture medium for seedling support, seal the cover and place them in a dark incubator at 28℃ for 2-3 days.

[0041] (2) Acquisition of recycled materials.

[0042] Take the sterile seedling hypocotyls and cut them into 0.5cm-0.8cm small segments and inoculate them on two callus induction media (IK and DK). The flow chart of regeneration ability evaluation under IK and DK systems is shown in Figure 2, where the IK system includes: MS medium + 1mg / L IBA + 0.2mg / L KT; the DK system includes: MS medium + 1mg / L2,4-D + 0.2mg / L KT. Each family cultured 3 bottles, and each bottle initially placed 10 hypocotyl segments, weighed the weight at 0d and recorded. All cultures were placed at 28±2℃, light intensity (cold light source 135umolm -2 s - 1 ) and cultured under conditions of 14 h of light per day. After every 25-30 days, subculture was performed using their respective systems until embryonic callus appeared, and then transferred to differentiation medium (MS macroelements 50 ml, trace elements 10 ml, iron salts 10 ml, inositol 10 ml, potassium nitrate 50 ml, L-Gly 1 ml, B5 organic matter 1 ml, IBA 1 ml, KT 0.3 ml, glutamine 1 g, asparagine 0.5 g, glucose 30 g, Phytagel 2.6 g, pH 6.1-6.2, ddH2O to 1 liter) until somatic embryos and seedlings were cultured; the seedlings were transferred to rooting medium (MS macroelements 25 ml, trace elements 5 ml, iron salts 5 ml, inositol 10 ml, L-Gly 1 ml, B5 organic matter 1 ml, glucose 15 g, Phytagel 2.6 g, pH 5.90-5.9, ddH2O to 1 liter) until regenerated plants were obtained. The regenerated plants were hydroponically cultivated and hardened, then transplanted into soil pots in the greenhouse and the regenerated seedlings finally obtained from each family were counted.

[0043] (3) Identification of callus-related traits.

[0044] During callus induction, subculture was performed every 25 days using the respective system, and the callus induction rate (CIF) was calculated. The proliferation weight of each family was weighed at the first subculture (25 days), and the average was calculated to calculate the callus subculture fecundity (CSC). As the callus grew rapidly, the number of callus blocks was randomly reduced during the subculture process. During the subculture process, the callus embryo emergence time (CET) and the number of callus embryo blocks and bottles were counted to calculate the callus embryo emergence rate (CRE). The specific calculation method is as follows:

[0045] Callus induction rate (CIF): number of callus blocks / total number of induced callus blocks × 100%;

[0046] Callus subculture capacity (CSC): callus weight after 25 days of subculture minus callus weight at 0 days of subculture;

[0047] Callus embryogenesis time (CET): the earliest time when embryogenic callus appears in a single family;

[0048] Callus embryo emergence rate (CRE): the number of embryonic calli / the total number of subcultured calli × 100%;

[0049] The results are shown in Figure 4 , which shows that the callus induction rate of all families was close to 100%, with no difference between the two systems. In terms of key regeneration indicators such as callus subculture fecundity, earliest embryo emergence time and embryo emergence rate, there were certain differences between the IK (IBA+KT) and DK (2,4-D+KT) systems. As shown in Figure 4(b), for the CSC trait, the DK system was 2.1g-7.4g, with an average of 4.8g, and the IK system was 2.8g-5.7g, with an average of 4.2g; for the CET trait, the DK system was 75d-154d, and the IK system was 67d-116d; for the CRE trait, the DK system was 33.3%-100%, with an average of 86.3%, and the IK system was 25%-100%, with an average of 82.4%. Based on the statistical results of the above three traits, the DK system has a faster callus proliferation, and the IK system has an earlier embryonic time than the DK system. This shows that excessive callus proliferation does not accelerate the appearance of embryonic callus. In terms of the choice of culture system for the YE family, the IK system is more suitable.

[0050] The high-regeneration material was obtained by screening plants with a callus induction rate higher than 98%, close to 100%, a callus subculture reproduction capacity higher than 4.2 g, a callus embryo generation time no longer than 116 days, and a callus embryo generation rate higher than 82.4%. An overview of the high-regeneration plants is shown in FIG5 .

[0051] Example 2 Identification of genetic transformation efficiency of high regenerated broadleaf cotton.

[0052] The genetic transformation efficiency of the obtained high-regeneration broadleaf cotton family was analyzed. Using red fluorescent protein (RFP) as a reporter gene, transgenic plants were obtained using the Agrobacterium-mediated cotton genetic transformation system. The process is shown in Figure 6. The specific steps are as follows:

[0053] (1) Selection of culture system. For lines that can regenerate under both systems, the IBA+KT (IK) culture system is used; for lines that can only regenerate under the IK system or the DK system, the IK or DK system is used.

[0054] (2) Obtaining transgenic plants.

[0055] Select seeds with full grains and normal development, peel off the cotton seed coat, soak them in 0.1% mercuric chloride solution for 10 minutes, rinse with sterile water for more than 3 times, and inoculate them on sterile seedling culture medium. After the seeds turn white, insert the roots into the culture medium for seedling support, seal the culture medium and culture it in a dark incubator at 28℃ for 2-3 days; cut the hypocotyls of the sterile seedlings into 0.5cm-0.8cm small segments and inoculate them on callus induction medium. Culture 6 bottles for each family. All cultures are placed at 28±2℃ and the light intensity (cold light source 135umolm -2 s -1 ) and cultured under conditions of 14 hours of light per day. Subculture was performed every 25-30 days using their respective systems until embryonic callus appeared. Callus was then transferred to differentiation medium until somatic embryos and seedlings were cultured. The seedlings were transferred to rooting medium and cultured until transgenic plants were obtained. The obtained transgenic plants were hydroponically cultivated and hardened, then transplanted into soil pots in the greenhouse. The number of transgenic plants obtained from each family was counted.

[0056] (3) Analysis of transformation efficiency and investigation of main agronomic traits of heritable transformation families.

[0057] For each family's transgenic plants, the expression of red fluorescent protein in the plants was observed using a stereo fluorescence microscope, and the number of transgenic positive plants was counted to analyze the genetic transformation efficiency.

[0058] Genetic transformation efficiency = transgenic positive individual plants / total number of transgenic individual plants*100%.

[0059] The graph for evaluating the efficiency of transformation of RFP is shown in FIG7 .

[0060] The transgenic broadleaf family YE3 with a genetic transformation efficiency of no less than 80% was screened and its genetic transformation efficiency was 82.9%.

[0061] That is, the method of the present invention can ultimately screen and obtain cotton recipients with high genetic transformation efficiency, wherein the highest genetic transformation efficiency can reach 82.9%.

[0062] Further investigation was conducted on the main agronomic traits of the broadleaf families with higher genetic transformation efficiency as well as the maternal parent E22 and the paternal parent YZ-1, including fiber length, fiber strength, fiber fineness, fiber elongation, fiber uniformity and lint percentage, as shown in Table 1.

[0063] Table 1. Agronomic traits of transformable broadleaf cotton families and their parents

[0064] Results showed that the resulting material significantly outperformed the regenerable parent in key fiber-related agronomic traits, particularly fiber strength, fiber elongation, and lint percentage. In summary, the method described herein, combined with analysis of genetic transformation efficiency and examination of key agronomic traits, ultimately yielded transgenic recipient material with high transformation efficiency and excellent agronomic traits.

[0065] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for cultivating an efficient genetic transformation receptor of upland cotton, characterized in that: The following steps are involved: Material creation→high regeneration material screening→high genetic transformation material screening, wherein the material creation includes: after hybridization with 'Emian 22' as the female parent and 'Yu Zao 1' as the male parent, multiple generations of self-pollination are performed to construct a genetically stable F9 generation recombinant inbred line.

2. The method according to claim 1, characterized in that The high regeneration material screening comprises: performing callus induction, embryo differentiation and plant regeneration culture on the hypocotyls of sterile seedlings of the genetically stable F9 generation recombinant inbred line to screen out the high regeneration material.

3. The method according to claim 2, characterized in that the steps include: (1) Callus induction: using two sets of callus induction culture systems, IBA+KT and / or 2,4-D+KT, to culture the hypocotyls of the sterile seedlings; (2) Differentiating embryos: culturing embryonic callus, statistically screening cotton regeneration ability traits, and obtaining the highly regenerative material; (3) The embryonic callus is inoculated into a differentiation medium, the differentiated seedlings are inoculated into a rooting medium, and finally hydroponically cultured to obtain regenerated plants.

4. The method according to claim 3, characterized in that The cotton regeneration ability traits include: callus induction rate, callus subculture reproduction capacity, callus embryo emergence time, and callus embryo emergence rate.

5. The method according to claim 4, characterized in that The calculation method of the cotton regeneration ability trait is: Callus induction rate: number of callus blocks / total number of induced callus blocks × 100%; Subculture reproduction capacity of callus tissue: callus weight after 25 days of subculture - callus weight at 0 days of subculture; Callus embryogenesis time: the earliest time when embryonic callus appears in a single family; Embryo emergence rate of callus tissue: the number of blocks with embryonic callus / the total number of subculture blocks × 100%.

6. The method according to claim 4, characterized in that The families with callus induction rate higher than 98%, callus subculture reproduction capacity higher than 4.2g, callus embryo generation time no higher than 116d, and callus embryo generation rate higher than 82.4% were screened to obtain the high regeneration material.

7. The method according to claim 1, characterized in that The screening of the high genetic transformation material includes: using the cotton genetic transformation system mediated by Agrobacterium to obtain transgenic plants with fluorescent protein as a reporter gene, and screening to obtain transgenic families with a genetic transformation efficiency of not less than 80%.

8. The method according to claim 1, characterized in that The step also includes: investigating the agronomic traits of the high genetic transformation material.

9. Use of the method according to any one of claims 1 to 8 in cultivating recipient materials for efficient genetic transformation of upland cotton.

10. The upland cotton efficient genetic transformation receptor obtained by the method according to claim 1.

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