Buckwheat interspecific hybridization offspring creation method

Abstract

The invention discloses a method for creating offspring of buckwheat interspecific hybridization. According to the invention, the immature embryo can be successfully obtained through artificial hybridization by taking wild buckwheat as a female parent and tartary buckwheat as a male parent; in order to solve the problems that the induction rate of an immature embryo on a callus induction culture medium is low and the differentiation rate is low when the callus is subjected to differentiation culture in a differentiation culture medium, the callus induction culture medium and the differentiation culture medium are optimized and screened; the obtained callus induction culture medium and the obtained differentiation culture medium remarkably improve the callus induction rate and the differentiation rate during differentiation culture, regenerated plants are obtained at high frequency, colchicine doubling is performed on the regenerated plants on the basis, and a doubled interspecific hybridization new germplasm is obtained by combining morphological and cytological identification. The method is applied to breeding of high-yield stress-resistant, high-flavone, perennial and other breakthrough new varieties or serves as a hybrid bridge parent, and the excellent characters of stress resistance, high flavone, perennial and the like of wild buckwheat are introduced into buckwheat cultivation.

Description

Technical Field

[0001] This invention relates to a method for creating hybrid offspring of crops, and more particularly to a method for creating offspring of interspecific hybridization in the genus *Fagopyrum*, belonging to the field of creating offspring through interspecific hybridization in the genus *Fagopyrum*. Background Technology

[0002] Buckwheat belongs to the genus *Fagopyrum* of the family Polygonaceae. Fagopyrum Wild buckwheat, a dicotyledonous plant, is characterized by high flavonoid content and strong resistance to adverse conditions, making it an important genetic material for breeding and improving cultivated buckwheat. Golden buckwheat, a close wild relative of cultivated bitter buckwheat, is characterized by rapid growth, high flavonoid content, large grains, and strong resistance to adverse conditions, effectively improving upon the shortcomings of cultivated bitter buckwheat.

[0003] Currently, there are no successful precedents for interspecific hybridization of the genus *Fagopyrum*. The main reason is that it is difficult to successfully rescue the embryos after hybridization, and the conditions for inducing, culturing, differentiating, and regenerating callus tissue of the embryos have not yet been understood, resulting in very low callus induction and differentiation rates, which are difficult to meet the needs of production and scientific research. Summary of the Invention

[0004] The main purpose of this invention is to address the current situation of narrow genetic base and lack of groundbreaking new germplasm in tartary buckwheat by developing a new method for creating hybrid offspring, thereby introducing the superior genes of wild closely related species into cultivated tartary buckwheat, laying the foundation for the development of groundbreaking new germplasm and new varieties. To achieve the above objectives, the main technical solutions adopted by the present invention include: A method for creating offspring from interspecific hybridization of the genus *Fagopyrum*, comprising: (1) Using golden buckwheat as the female parent and bitter buckwheat as the male parent for pollination and hybridization; (2) The immature embryos obtained after hybridization were cultured in an immature embryo callus induction medium to obtain callus tissue; (3) The obtained callus tissue was transferred to a differentiation medium for differentiation culture to obtain seedlings; (4) The obtained seedlings were transferred to a subculture medium for seedling subculture. (5) Transfer the seedlings after subculture to rooting medium for rooting culture; (6) After the rooted seedlings have been hardened off, they are transferred to soil for cultivation and then subjected to chromosome doubling treatment to obtain buckwheat interspecific hybrid germplasm with doubled chromosomes.

[0005] To overcome the technical obstacle of difficulty in forming immature embryos through interspecific hybridization of buckwheat, this invention utilizes the chloroplast genome information of bitter buckwheat to construct a phylogenetic tree for screening. Ultimately, diploid golden buckwheat, which is closely related to bitter buckwheat, was selected as the female parent and bitter buckwheat as the male parent for pollination and hybridization, and immature embryos of interspecific hybridization were successfully obtained.

[0006] In a preferred embodiment of the present invention, the maternal parent *Fagopyrum esculentum* or the paternal parent *Fagopyrum esculentum* is diploid; more preferably, diploid *Fagopyrum esculentum* with a long style is used as the maternal parent.

[0007] This invention reveals that embryos obtained through interspecific hybridization of *Fagopyrum esculentum* (female parent) and *Fagopyrum tartare* (male parent) are difficult to induce callus formation on conventional callus induction media, or the callus induction rate is low, failing to meet the needs of production or research. To address this issue, this invention optimizes and screens the callus induction media for embryos, ultimately finding that the following callus induction media significantly improves the callus induction rate: B5 basal medium + 1.5-3.0 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar. Specifically, when the 2,4-D content in this induction medium is 2.0 mg / L, the average callus induction rate of embryos reaches 60.62%, significantly higher than that of embryos with other 2,4-D contents.

[0008] In a preferred embodiment of the present invention, the conditions for inducing and culturing the callus tissue are preferably: dark culture at 24°C for 1 to 2 weeks.

[0009] The present invention further discovered that the differentiation rate of callus tissue obtained by differentiation culture in conventional differentiation media is very low. To solve this problem, the present invention optimized and screened the differentiation media. The results showed that a specific medium consisting of MS basal medium + 1.0-3.0 mg / L 6-BA + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel could significantly improve the differentiation rate of callus tissue. Among them, the use of MS basal medium + 2.0 mg / L 6-BA + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel could increase the differentiation rate of callus tissue to over 82.2%.

[0010] In a preferred embodiment of the present invention, the differentiation culture conditions are preferably: culture temperature 24℃, 16h / 8h (light / dark).

[0011] In a preferred embodiment of the present invention, the subculture medium in step (4) is MS basal medium; the subculture conditions are preferably: 24℃, 16h / 8h (light / dark).

[0012] In a preferred embodiment of the present invention, the rooting medium in step (5) is MS basal medium; the preferred conditions for the rooting culture are: 24℃, 16h / 8h (light / dark).

[0013] In a preferred embodiment of the present invention, the method for chromosome doubling in step (6) is preferably to use colchicine treatment for chromosome doubling: after the seedlings have fully adapted to the soil, prepare a 0.2% colchicine aqueous solution, dip a cotton ball in the solution and place it on the seedling growth point, once every morning, for 3 days.

[0014] The present invention provides a method for creating hybrid offspring of the genus *Fagopyrum*, using *Fagopyrum esculentum* as the female parent and *Fagopyrum truncatum* as the male parent to successfully obtain immature embryos through artificial hybridization. To address the issues of low induction rate of immature embryos on callus induction medium and low differentiation rate of callus tissue during differentiation culture, the present invention optimizes and screens the callus induction and differentiation media. The screened callus induction and differentiation media significantly improve the callus induction rate and differentiation rate during differentiation culture, resulting in high-frequency regeneration of plants. Based on this, the regenerated plants are subjected to colchicine doubling, and combined with morphological and cytological identification, new interspecific hybrid germplasm is obtained after doubling. The obtained new interspecific hybrid germplasm of the genus *Fagopyrum* can be directly used for the breeding of high-yield, stress-resistant, high-flavonoid, and perennial breakthrough varieties, or as a bridging parent for hybridization, introducing the special and superior traits of wild buckwheat, such as stress resistance, high flavonoid content, and perenniality, into cultivated buckwheat, promoting the genetic improvement of cultivated buckwheat and the breeding and application of breakthrough new varieties. Attached Figure Description

[0015] Figure 1 This is a phylogenetic tree of buckwheat constructed based on chloroplast genome data; orange represents golden buckwheat, purple represents golden buckwheat which is more closely related to bitter buckwheat, and green represents bitter buckwheat.

[0016] Figure 2 This diagram illustrates the morphological changes during the embryo rescue process of a hybrid offspring of golden buckwheat and bitter buckwheat.

[0017] Figure 3 The inflorescence characteristics of bitter buckwheat (left), interspecific hybrid offspring (middle), and golden buckwheat (right).

[0018] Figure 4 The images show the chromosome number and in situ hybridization results of the interspecific hybrid offspring; from left to right, they show the chromosome observation results of the hybrid offspring, the in situ hybridization results using the tartary buckwheat genome as a probe, the in situ hybridization results using the golden buckwheat genome as a probe, the confocal results of the two in situ hybridization results, and the arrows pointing to the tartary buckwheat-derived chromosomes in the hybrid offspring. Detailed Implementation

[0019] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, these experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0020] Experimental Example 1: Selection of a maternal parent closely related to buckwheat To overcome the technical obstacle of difficulty in forming immature embryos during interspecific hybridization of buckwheat, this invention utilizes the chloroplast genome information of bitter buckwheat to construct a phylogenetic tree (…). Figure 1 Through screening, diploid golden buckwheat with a long style, which is closely related to bitter buckwheat, was finally selected as the female parent and diploid bitter buckwheat as the male parent for pollination and hybridization. In the end, the interspecific hybrid embryos were successfully rescued. Figure 2 This diagram illustrates the morphological changes during the embryo rescue process of a hybrid offspring of golden buckwheat and bitter buckwheat.

[0021] In this experiment, other wild buckwheat varieties were selected as the female parent and diploid bitter buckwheat as the male parent for pollination and hybridization, but none of them successfully obtained hybrid embryos.

[0022] Experiment Example 2: Optimization and Screening of Callus Induction Culture Medium for Hybrid Progeny of Fagopyrum cymosum and Fagopyrum dibotrys Embryos obtained from different hybridization combinations of three tartary buckwheat parents (K1, K2, K3) and three golden buckwheat parents (J1, J2, J3) were used to induce callus culture using the following five callus induction media: (1) B5 basal medium + 1.0 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar; (2) B5 basal medium + 1.5 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar; (3) B5 basal medium + 2.0 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar; (4) B5 basal medium + 2.5 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar; (5) B5 basal medium + 3.0 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar; The induction culture method was to incubate in the dark at 24℃ for 1 to 2 weeks.

[0023] The specific results of the induction culture experiment are shown in Table 1.

[0024] Table 1. Callus induction rate at different 2,4-D concentrations in callus culture medium for immature embryos.

[0025] As shown in Table 1, when the concentration of 2,4-D in the callus induction medium was 2.0 mg / L, the average callus induction rate was 60.62%, which was significantly better than the callus induction rate at other concentrations.

[0026] Experiment Example 3: Optimization and Screening Experiment of Differentiation Culture Medium for Callus Progeny of Fagopyrum esculentum and Fagopyrum dibotrys The callus tissue obtained in Experiment 2 was cultured and differentiated in the following eight differentiating media: (1) MS basal medium + 30 g / L sucrose + 3 g / L plant gel; (2) MS basal medium + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel; (3) MS basal medium + 1.0 mg / L 6-BA + 30 g / L sucrose + 3 g / L plant gel; (4) MS basal medium + 1.0 mg / L 6-BA + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel; (5) MS basal medium + 2.0 mg / L 6-BA + 30 g / L sucrose + 3 g / L plant gel; (6) MS basal medium + 2.0 mg / L 6-BA + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel; (7) MS basal medium + 3.0 mg / L 6-BA + 30 g / L sucrose + 3 g / L plant gel (8) MS basal medium + 3.0 mg / L 6-BA + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel; The differentiation culture was carried out at 24℃ for 16h / 8h (light / dark). The results of the differentiation culture experiments on the eight different differentiation media are shown in Table 2.

[0027] Table 2. Callus differentiation rate of 8 different differentiation culture media

[0028] As shown in Table 2, the differentiation rate of callus tissue using the differentiation medium of "MS basal medium + 2.0 mg / L 6-BA + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel" reached 82.2 ± 4.52%, which was significantly higher than the differentiation rate of other differentiation media.

[0029] Experimental Example 4: Creation of hybrid offspring of golden buckwheat and bitter buckwheat Interspecific hybridization and the acquisition of offspring 1.1 Hybridization methods Based on the phylogenetic tree constructed from chloroplast genomes, diploid *Fagopyrum tsao-ko*, which is closely related to tartary buckwheat, was screened out. Using its long style type as the female parent, the inflorescence was arranged, the open flowers were removed, and the inflorescence was bagged and isolated. The opening status of the flowers in the bag was observed after 3 to 5 days. After the flowers opened, pollen from tartary buckwheat was collected from the female parent and applied to the stigma of the diploid *Fagopyrum tsao-ko* from 11:00 to 13:00 every day. After completion, the inflorescence of the female parent was bagged and isolated again, and this process was repeated for 3 days.

[0030] 1.2 Embryo rescue and callus induction Ten days after hybridization, the ovary was removed, disinfected with 70% alcohol for 2 minutes, then with 10% sterile calcium hypochlorite for 20 minutes, and rinsed 5-6 times with sterile water for aseptic treatment. The ovary was then opened using a stereomicroscope to observe the formation of the embryonic stage. The formed embryonic stage was gently removed and placed in callus induction medium (B5 basal medium + 2.0 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar) for callus induction culture. Callus formation was observed after 1-2 weeks of incubation at 24°C in the dark. After 3-4 weeks, a white pre-embryonic cell complex appeared on the surface of the callus, which could continue to divide and had stable regeneration capacity.

[0031] 1.3 Callus differentiation and seedling hardening Callus tissue was selected and placed on MS medium (containing 2.0 mg / L 6-BA, 1.0 mg / L Kinetin, 30 g / L sucrose, and 3 g / L plant gel) for differentiation culture at 24°C for 16 h / 8 h (light / dark). After about 3 weeks, the callus tissue expanded and turned red, and embryoids with buds grew on the surface. The developed embryoids or buds were transferred to MS medium for further culture, and seedlings were formed after about 3 weeks.

[0032] Seedlings were rooted on MS basal medium. Once a strong root system had developed, the seedlings were transferred to a regular greenhouse for 1-2 weeks to adapt, and then transplanted into soil for further growth.

[0033] 1.4 Chromosome doubling treatment Once the seedlings can grow normally in the soil, prepare a 0.2% colchicine solution. Dip a cotton ball in the solution and place it on the seedling growth point once every morning at 8:00 AM for 3 days.

[0034] Identification of offspring from interspecific hybridization 2.1 Morphological identification Morphological observations were conducted at different stages of seedling growth, and the results showed that the hybrid offspring were intermediate between the two parents in terms of inflorescence morphology, leaf size, and flower size. Figure 3 ).

[0035] 2.2 Cytological identification Chromosome observation of the shoot tip meristems of the hybrid offspring revealed that the offspring had 32 chromosomes, twice the number of chromosomes in the paternal parent (bitter buckwheat) and maternal parent (golden buckwheat) (16 chromosomes each). In situ hybridization analysis confirmed that the hybrid offspring contained a complete set of golden buckwheat chromosomes and a complete set of bitter buckwheat chromosomes, indicating an allotetraploid genome with a chromosome configuration of 2n=4x=32. Figure 4 ).

Claims

1. A method for creating a progeny of an interspecific Fagopyrum cross, characterized by, The application comprises the following steps: (1) pollinating hybridization is carried out by taking Fagopyrum dibotrys as female parent and Fagopyrum tataricum as male parent; (2) the obtained young embryo is induced and cultured in a young embryo callus induction medium to obtain callus; the young embryo callus induction medium is B5 basic medium + 1.5-3.0 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar; (3) the obtained callus is transferred into a differentiation medium to carry out differentiation culture and obtain seedlings; the differentiation medium is MS basic medium + 1.0-3.0 mg / L 6-BA + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel; (4) the obtained seedlings are transferred into a subculture medium to carry out subculture of the seedlings; (5) the subcultured seedlings are transferred into a rooting medium to carry out rooting culture; (6) the rooted seedlings are transferred into soil after seedling training, then are cultivated, and then are subjected to chromosome doubling treatment to obtain doubled buckwheat interspecific hybrid germplasm.

2. The offspring creation method according to claim 1, characterized in that, The female parent Fagopyrum dibotrys or the male parent Fagopyrum tataricum is diploid.

3. The offspring creation method according to claim 1, characterized in that, The young embryo callus induction medium is B5 basic medium + 2.0 mg / L 2,4-D + 0.5 mg / L NAA + 0.5 mg / L IAA + 0.2 mg / L Kinetin + 2.0 mg / L casein hydrolysate + 25 g / L sucrose + 0.8% agar.

4. The offspring creation method according to claim 1, characterized in that, The induction culture condition in step (2) is 24 DEG C dark culture for 1-2 weeks.

5. The offspring creation method according to claim 1, characterized in that, The differentiation medium is MS basic medium + 2.0 mg / L 6-BA + 1.0 mg / L Kinetin + 30 g / L sucrose + 3 g / L plant gel.

6. The progeny creation method of claim 1, wherein, The differentiation culture condition in step (3) is 24 DEG C, 16 h light / 8 h dark.

7. The progeny creation method of claim 1, wherein, The subculture medium in step (4) is MS basic medium; and the subculture condition is 24 DEG C, 16 h light / 8 h dark.

8. The progeny creation method of claim 1, wherein, The rooting medium in step (5) is MS basic medium; and the rooting culture condition is 24 DEG C, 16 h light / 8 h dark.

9. The progeny creation method of claim 1, wherein, The method for carrying out chromosome doubling treatment in step (6) is adopting colchicine treatment to carry out chromosome doubling.

10. The progeny creation method of claim 9, wherein, The method for adopting colchicine treatment to carry out chromosome doubling comprises the following steps: after the seedlings completely adapt to the soil, 0.2% colchicine aqueous solution is prepared, the colchicine aqueous solution is dipped and placed on the growth point of the seedlings, once a day in the morning, and the operation is repeated for 3 days.

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