A method for hybridizing daylily and alstroemeria and application thereof in inducing alstroemeria callus

Abstract

The present application discloses a method for hybridizing daylily and Alstroemeria and its application in inducing Alstroemeria callus, belonging to the field of biotechnology. In the present application, the inventor uses asymmetric fusion somatic cell hybridization technology to fuse daylily and Alstroemeria protoplasts using PEG fusion method, and then culture to obtain embryonic callus. Compared with ordinary leaf induced callus, the induction success rate is higher. This method can not only solve the problem of low induction rate of Alstroemeria callus, but also improve the fusion efficiency of the two parents of daylily and Alstroemeria, and has made a breakthrough in the research of Alstroemeria callus induction and differentiation.

Description

Technical Field

[0001] The application discloses a method for hybridizing daylilies and Alstroemeria and an application in inducing Alstroemeria callus, belonging to the field of biotechnology. Background Art

[0002] Alstroemeria atrantiaca is a plant of the genus Alstroemeria in the Amaryllis family. It has rich colors and butterfly-like flowers. When in full bloom, it is more elegant and gorgeous. It is a novel cut flower material. Alstroemeria atrantiaca is native to the Andes Mountains in South America. Because its flower shape resembles a lily, it is called "Peruvian lily" locally. It was introduced to the UK in 1754. It did not begin to develop rapidly until it was used for cut flower viewing in Europe and the United States in the 1950s. The Netherlands is a world leader in the selection, breeding and production of new varieties of Alstroemeria.

[0003] Alstroemeria is an ornamental flower with great potential in genetic resources. However, due to the characteristics of Alstroemeria itself, it is very difficult to form callus tissue. Currently, research on Alstroemeria is still in its early stages, and research in tissue culture is limited to regenerated seedlings induced by explants. There are few studies on callus induction and differentiation of Alstroemeria in the prior art, and there has been no breakthrough progress. Therefore, a method that can induce Alstroemeria callus tissue with high efficiency is urgently needed, so as to make full use of the genetic resources of Alstroemeria and solve the problem that it is basically difficult to form callus tissue using Alstroemeria leaves as explants in the prior art. Summary of the invention

[0004] The purpose of the present application is to provide a method for hybridizing daylily and Alstroemeria and its application in inducing Alstroemeria callus tissue, wherein the protoplasts of daylily and Alstroemeria are fused using the PEG fusion method, and then the embryonic callus tissue is obtained by culture, thereby solving the problem of extremely low induction rate of Alstroemeria callus induction using ordinary leaves, and improving the fusion efficiency of the two parents of daylily and Alstroemeria, achieving a breakthrough in the research on Alstroemeria callus induction and differentiation, and providing new ideas for the breeding of new daylily varieties.

[0005] According to one aspect of the present application, the present application provides a method for hybridizing daylilies and Alstroemeria, wherein the hybridization method is somatic cell hybridization, and the method comprises: fusing daylily somatic cells with Alstroemeria somatic cells.

[0006] Optionally, the fusion adopts an asymmetric fusion method.

[0007] Optionally, the method further comprises: inactivating the cytoplasm of Hemerocallis protoplasts to obtain Hemerocallis somatic cells; and fragmenting the chromosomes of Alstroemeria protoplasts to obtain Alstroemeria somatic cells.

[0008] Optionally, the fusion method is selected from one or more of polyethylene glycol fusion method, high calcium fusion method, high pH fusion method, salt fusion method, and electric fusion method; preferably, the fusion method is polyethylene glycol fusion method.

[0009] Optionally, the concentration of polyethylene glycol in the polyethylene glycol fusion method is 35-45%, and the treatment time of the polyethylene glycol is 18-22 minutes; preferably, the concentration of polyethylene glycol in the polyethylene glycol fusion method is 40%, and the treatment time of the polyethylene glycol is 20 minutes.

[0010] Optionally, in the polyethylene glycol fusion method, the polyethylene glycol inducer used contains 0.01 g / 100 ml KH2PO4, 0.147 g / 100 ml CaC12·2H2O, 1.822 g / 100 ml mannitol and 40 g / 100 ml polyethylene glycol.

[0011] Optionally, the method for fragmenting the chromosomes of Alstroemeria protoplasts to obtain Alstroemeria somatic cells is selected from one or more of ray radiation method, restriction endonuclease treatment method, spindle toxin treatment method and chromosome shrinkage agent treatment method; preferably, ray radiation method is adopted, and the ray is selected from one or more of x-ray, gamma-ray and ultraviolet light; preferably, the ray is ultraviolet light; preferably, the ultraviolet radiation treatment time is 3 to 4 minutes.

[0012] Optionally, the method for inactivating the cytoplasm of Hemerocallis bioplast to obtain Hemerocallis somatic cells is selected from one or more of iodoacetic acid treatment, iodoacetamide treatment and rhodamine 6G treatment; preferably, it is selected from iodoacetamide treatment.

[0013] Optionally, the concentration of the iodoacetamide solution in the iodoacetamide treatment method is 4-6 mmol / L, the pH is 5.8-6.0, and the iodoacetamide treatment time is 13-17 min. Preferably, the concentration of the iodoacetamide solution is 5 mmol / L.

[0014] Optionally, the ratio of the number of the daylily somatic cells to the number of the alstroemeria somatic cells is 1:1.

[0015] Optionally, after fusing the Hemerocallis somatic cells with the Alstroemeria somatic cells, the hybrid somatic cells are cultured in a KM8P liquid culture medium at 25° C. in the dark to induce callus tissue.

[0016] According to another aspect of the present application, the present application discloses an application of any of the above-mentioned methods for hybridizing daylilies with Alstroemeria in inducing Alstroemeria callus.

[0017] The beneficial effects of this application include but are not limited to:

[0018] 1. The present application successfully induced callus tissue of Alstroemeria by using a method of somatic cell hybridization between Hemerocallis and Alstroemeria, thereby solving the problem in the prior art that it is basically difficult to form callus tissue using Alstroemeria leaves as explants, and providing a new solution for inducing callus tissue of Alstroemeria. In addition, the scheme for obtaining callus tissue of Alstroemeria disclosed in the present application has a high induction success rate and a good effect of inducing callus tissue.

[0019] 2. The present application adopts the method of somatic cell hybridization between daylilies and Alstroemeria, irradiates the protoplasts of Alstroemeria with ultraviolet rays, fragments the chromosomes in the cell nucleus, and after fusion with the daylily bioplast, part of the genetic material of Alstroemeria will be combined with daylily, thereby making full use of the genetic resources of Alstroemeria and providing new ideas for the breeding of new varieties of daylilies.

[0020] 3. The present application adopts the method of somatic cell hybridization between daylily and alstroemeria, optimizes the passivation conditions of donor protoplasts and recipient protoplasts, and optimizes the induced fusion conditions, thereby greatly improving the fusion efficiency of the two parents, daylily and alstroemeria, with a maximum fusion rate of 8.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0022] Figure 1 This is a schematic diagram of the technical route for hybridization of daylilies and alstroemeria provided in the examples of the present application;

[0023] Figure 2 The protoplasts extracted from Alstroemeria and Hemerocallis and the fusion diagram provided in the examples of the present application, wherein: A is the protoplasts of Alstroemeria; B is the protoplasts of Hemerocallis; C is the fusion diagram; D is the magnified diagram of the fusion part (BAR: 1 mm);

[0024] Figure 3 It is a comparison diagram of fused embryonic callus and traditional leaf induced callus provided in the examples of the present application, wherein: Figures A and B are embryonic callus obtained by culturing after fusion of Alstroemeria and Hemerocallis bioplasts; Figures C and D are callus induced by ordinary leaves and petioles of Alstroemeria, respectively (BAR: 1 mm in Figure A, BAR: 1 dmm in Figure B, BAR: 1 cm in Figures C and D). DETAILED DESCRIPTION

[0025] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0026] Unless otherwise specified, the raw materials and reagents in the examples of this application were purchased through commercial channels.

[0027] The PEG described in this application is the English abbreviation of polyethylene glycol, IOA is the English abbreviation of iodoacetamide, and UV is the English abbreviation of ultraviolet light.

[0028] In view of the problem that it is difficult to induce callus tissue of Alstroemeria in the prior art, the induced callus tissue is of poor quality and has low induction efficiency. After long-term and in-depth research, the inventor has found a method for cultivating asymmetric fusion hybrid offspring of daylily and Alstroemeria protoplasts, that is, a method for inducing Alstroemeria callus tissue by using asymmetric fusion culture of protoplasts. Through extensive research and experiments, the inventor has overcome the technical difficulties in the prior art that it is difficult to obtain asymmetric fusion hybrid offspring of protoplasts of daylily and Alstroemeria by optimizing hybridization conditions. Not only has the callus tissue of Alstroemeria been successfully obtained, and the induction efficiency is high, the callus tissue is high in quality, and the protoplast hybrid offspring of daylily and Alstroemeria are cultivated for the first time, and the genetic resources of Alstroemeria are excavated and used, which is also of great significance for the trait improvement and breeding of daylily.

[0029] Plant somatic cell hybridization is a plant breeding technology developed by combining cell fusion technology and plant tissue culture technology based on the totipotency of plant cells. Plant somatic cell hybridization first removes the cell wall of plant somatic cells to prepare complete and viable protoplasts, and then stimulates the fusion of heterologous protoplasts to form hybrid cells with biological activity, and then tissue culture is used to form hybrid plants and select and breed plants with excellent traits. Plant somatic cell hybridization technology does not require a sexual process, but only requires the fusion of somatic cells to produce hybrids. Depending on the degree of fusion, protoplast fusion can be divided into symmetrical fusion, asymmetrical fusion and sub-protoplast fusion. This hybridization technology can break the reproductive isolation between species, and at the same time overcome the incompatibility between flowering and sexual hybridization, and create an effective way to expand genetic variation, update germplasm resources and improve crop quality.

[0030] In the present application, protoplasts refer to cells without cell walls. Since plant cells have strong cell walls, protoplasts without cell walls must be obtained before somatic cell hybridization.

[0031] In the present application, asymmetric fusion is also called asymmetric cell hybridization, which refers to the technology of fusing two cells that cannot mate normally to produce hybrid offspring. The procedure for protoplast fusion generally includes the following steps: removing the cell walls of two cells belonging to the same or different species to form protoplasts, and then using a fusion promoter to move proteins in certain areas of the two cell membranes. After the exposed phospholipid areas are fused, the hybrid somatic cells proliferate and develop cell walls to form complete fused somatic cells.

[0032] Although the asymmetric protoplast fusion method has been successfully applied to the cultivation of some hybrid plants, only a small number of plant species can successfully obtain hybrid offspring through the protoplast fusion method, which mainly depends on the characteristics, species, protoplast size of the plant itself, and the influence of many complex factors such as artificial operation methods and operation conditions. In particular, some flower varieties are more difficult to cultivate their hybrid offspring through the protoplast hybridization method. In the present application, the inventors carried out somatic cell hybridization of daylilies and alstroemeria, so that the genetic material in daylilies can be combined with alstroemeria, and the cell activity of alstroemeria can be greatly improved, and callus tissue is successfully induced.

[0033] In view of the above, it is necessary to provide a method for somatic cell hybridization of daylily and Alstroemeria, which can not only solve the problem of low callus induction rate of Alstroemeria, but also improve the fusion efficiency of the two parents of daylily and Alstroemeria. To achieve the above purpose, the technical solution adopted by the present invention is a method for somatic cell hybridization of daylily and Alstroemeria, such as Figure 1 As shown, the following steps are included:

[0034] (1) Preparation of protoplasts: Use the protoplast preparation kit (model RTU4052) produced by Zhongke Ruitai Biotechnology Co., Ltd. to extract plant protoplasts. Weigh 0.5 g of sterile young leaves of Hemerocallis fulva seedlings and sterile young leaves of Alstroemeria philadelphica seedlings, cut the young leaves into thin strips (the thinner the better), add the two parents into a conical flask containing 5 ml of ready-to-use enzymatic solution, soak them fully, and enzymolyze them at 60 rpm in a dark environment at 25°C for 3 h to obtain Alstroemeria fulva protoplasts and Hemerocallis fulva protoplasts.

[0035] (2) Protoplast passivation: The Hemerocallis protoplasts obtained in step (1) were suspended in a 5 mmol / L IOA solution at room temperature for 20 min, and the Alstroemeria protoplasts were spread in a 3 cm small culture dish at room temperature and irradiated with UV for 1 min. The two types of protoplasts after IOA and UV treatment were centrifuged and washed twice using CPW solution, wherein each centrifugation speed was 800 rpm and the centrifugation time was 5 min. After washing, they were cultured in the dark until the density was 5×10 5 / ml, and obtained Hemerocallis recipient protoplasts and Alstroemeria donor protoplasts.

[0036] (3) Protoplast fusion: The recipient protoplasts obtained in step (2) and the donor protoplasts were mixed in a volume ratio of 1:1 to obtain a parent suspension. The PEG inducer (PEG concentration 40%) was carefully aspirated with a pipette and slowly dripped into the suspension in small droplets along the periphery. The suspension was allowed to stand at room temperature for 20 min for induction. The suspension was then washed twice with 1 ml of high calcium and high pH washing solution, washed twice with 1 ml of CPW solution, and washed once with KM8P medium (Kao & Michayluk Medium). Finally, fresh KM8P medium was added for shallow culture. The suspension was cultured in the dark at 25° C. until regenerated embryonic callus particles grew.

[0037] The solution ratios used in this application are shown in Table 1 below:

[0038] Table 1 Solution ratio

[0039]

[0040]

[0041] Example 1

[0042] Preparation of protoplasts

[0043] 1) Enzymatic hydrolysis. Use the protoplast preparation kit (model RTU4052) produced by Zhongke Ruitai Biotechnology Co., Ltd., take well-grown, young leaves of Alstroemeria and Hemerocallis, cut them into thin strips (the thinner the better), add 0.5g of leaves to a conical flask, then add 5ml of ready-to-use enzymatic hydrolysis solution, soak them fully, and perform enzymatic hydrolysis for 3h at 60rpm and 25℃ in a dark environment. The enzyme solution turns green, indicating that protoplasts have been separated. Under a microscope, the leaf protoplasts are green spherical (such as Figure 2 (shown in A and B);

[0044] 2) Rinsing. After enzymatic hydrolysis, add an equal volume of 1x rinsing solution. In this example, a 5 ml enzymatic hydrolysis system is used, so 5 ml of 1x rinsing solution is added and gently mixed;

[0045] 3) Filter. Use a sieve or lens paper to filter the solution obtained in step 2) to remove undigested leaves. Use a pipette to wash the enzymatic vessel and undigested leaves 1-2 times with 5-10 ml of 1x rinse solution, and collect all the liquid into a 10 ml centrifuge tube;

[0046] 4) First collection: Centrifuge the filtrate at 1200 rpm for 5 min at room temperature and remove the supernatant;

[0047] 5) Second collection: Add 5 ml of 1x rinsing solution, resuspend the protoplasts with a pipette, and centrifuge at 1200 rpm for 5 min at room temperature to collect the protoplasts;

[0048] 6) Resuspend. Carefully remove the supernatant without touching the protoplast precipitate, and add it to the resuspension solution provided in the kit, i.e. 1 ml of solution III, to obtain the protoplast solution.

[0049] Inactivation of protoplasts

[0050] 1) IOA treatment of Hemerocallis protoplasts (acceptor treatment): Prepare iodoacetamide solution 5mmol / L with CPW solution, pH 5.8-6.0. Sterilize after dissolution and store at -20°C; resuspend protoplasts with CPW solution, take 1ml protoplasts, add iodoacetamide, operate on ice, and shake gently; let each treatment stand at room temperature for 10min, 15min, and 20min (the results are shown in Table 2); centrifuge at 1200rpm for 2min after standing; discard the supernatant, add CPW solution to wash twice, and then add 1ml CPW solution to resuspend.

[0051] Table 2 Effects of different IOA treatment times on phytoplasts of Hemerocallis fulva

[0052]

[0053] As shown in Table 2, the different IOA treatment times had significant differences in the passivation effect on the activity of the protoplasts of daylilies: after 0min, 10min, 15min, and 20min IOA treatment, the activity of the protoplasts of daylilies decreased by 68.5%, 82.3%, and 86.9% respectively compared with the control, indicating that the protoplasts of daylilies were very sensitive to IOA. With the increase of IOA treatment time, the degree of protoplast cell fragmentation increased; after 15min IOA treatment, about 93.5% of the protoplasts of daylilies were inactivated and the degree of cell fragmentation was not very high, indicating that the 15min IOA treatment can effectively passivate the protoplasts of daylilies.

[0054] 2) UV treatment of Alstroemeria protoplasts (donor treatment): Take an appropriate amount of 3-5 ml of protoplast suspension and spread it in a 3 cm small culture dish to form a thin layer, then place it directly below the UV lamp on the clean bench at a distance of 20 cm, and irradiate for 1 min, 3 min, and 6 min respectively (the results are shown in Table 3), and remove the cover during treatment. Use a pipette to transfer the irradiated protoplasts to a centrifuge tube, add CPW suspension and centrifuge, and the final concentration is consistent with the receptor.

[0055] Table 3 Effects of different UV treatment times on protoplasts of Alstroemeria

[0056]

[0057] As can be seen from Table 3, the passivation effect of UV treatment time at different concentrations on the vitality of Alstroemeria protoplasts is significantly different: after 1min, 3min, and 6min UV treatment, the vitality of Alstroemeria protoplasts decreased by 35.1%, 74.6%, and 86.4% respectively compared with the control. With the extension of UV irradiation time, the passivation effect of UV on the vitality of Alstroemeria protoplasts is enhanced, and the degree of cell fragmentation is deepened; among them, the degree of cell fragmentation of protoplasts treated with 3min UV irradiation begins to increase. If the treatment time is further extended, the passivation of protoplasts will be enhanced, and more protoplast cells will die and fragment. Therefore, UV irradiation for 3min can effectively passivate the protoplasts of Alstroemeria and reduce the degree of protoplast fragmentation.

[0058] Fusion of protoplasts

[0059] 1) The two purified protoplasts (UV-passivated donor and IOA-passivated acceptor) were suspended in CPW medium and the density was adjusted to the same concentration;

[0060] 2) Take equal amounts of the same protoplasts and mix them (v / v, 1:1). Use a pipette to aspirate 0.2 ml of the mixture and drop it on the bottom of a 3 cm culture dish. Let it stand for 5 min and observe until the protoplasts have attached to the bottom of the dish.

[0061] 3) Use a pipette to draw 0.6 ml of PEG inducer (PEG concentration 40%) and carefully drip it in small drops along the four sides to make the PEG contact with the protoplasts. Let it stand at room temperature for 0 min, 15 min, 20 min, and 25 min to induce (the results are shown in Table 4);

[0062] Table 4 Effects of different PEG treatment times on cell fusion

[0063]

[0064]

[0065] As can be seen from Table 4, with the increase of PEG treatment time, the heterologous fusion rate first increased and then decreased: when the PEG treatment time was 20 minutes, the fusion rate was the highest at 8.1%; but when the PEG treatment time was 25 minutes, the fusion product could not survive, a large number of protoplasts were broken and gradually died. Since PEG itself has certain toxicity to protoplasts, in order to reduce its side effects on the growth and development of fusion products, PEG treatment for 20 minutes was selected as the appropriate time for the fusion of Hemerocallis and Alstroemeria protoplasts. At this time, most protoplasts were one-to-one fused, the heterologous fusion rate was as high as 8.1%, and the degree of protoplast fragmentation was low.

[0066] 4) Then slowly add 1 ml of high calcium and high pH wash solution to the edge of the droplet;

[0067] 5) After 10 minutes, add 1 ml of high calcium and high pH washing solution, pipette a small amount onto a glass slide and observe under a microscope. The protoplasts will return to a spherical shape.

[0068] 6) Leave at room temperature for 1 hour, slowly aspirate the PEG and high calcium and high pH solutions, and then slowly add 1 ml of fresh CPW wash solution from one side;

[0069] 7) After 5 minutes, perform a second wash using the same method until the PEG and high calcium and high pH solution are cleaned up;

[0070] 8) Wash once more with KM8P medium, and finally add fresh KM8P medium for shallow culture, dark culture at 25°C, add fresh medium every 7 days or so, and gradually reduce the mannitol concentration (the photo of the fusion of the protoplasts of Hemerocallis and Alstroemeria of the present invention is shown in FIG. Figure 2 C and D).

[0071] Example 2

[0072] Protoplast culture

[0073] The fused hybrid cells were added into KM8P liquid medium and the density was adjusted to 2×10 5 / mL, KM8P was used as the basic culture medium, and the protoplasts were cultured in liquid culture medium. After sealing with sealing film, the protoplasts were cultured in a culture box at a constant temperature of 25℃ in the dark. The protoplasts separated from embryonic callus on KM8P culture medium were re-walled in about 6-7 days, the first division appeared in about 14-15 days, the second division appeared in 20-25 days, and small callus visible to the naked eye appeared in about 35-40 days. Compared with the traditional callus induction of explants such as leaves or petioles, the induction time is short and the induction success rate is high (such as Figure 3 and Table 5).

[0074] Table 5 Comparison of traditional explant callus induction and cell fusion hybrid callus induction

[0075]

[0076] The above is only the embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the technical ideas and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for hybridizing daylilies and alstroemeria, and its application in inducing alstroemeria callus, characterized in that: The method for hybridizing daylily and Alstroemeria is somatic cell hybridization, which includes: inactivating the cytoplasm of daylily protoplasts to obtain daylily somatic cells, fragmenting the chromosomes of Alstroemeria protoplasts to obtain Alstroemeria somatic cells, and then fusing the daylily somatic cells with the Alstroemeria somatic cells, wherein the fusion adopts an asymmetric fusion method; The method for fragmenting the protoplast chromosomes of Alstroemeria to obtain Alstroemeria somatic cells adopts an ultraviolet radiation method, and the ultraviolet radiation treatment time is 3 to 4 minutes; The method for inactivating the cytoplasm of Hemerocallis bioplast to obtain Hemerocallis somatic cells is selected from an iodoacetic acid treatment method, wherein the concentration of the iodoacetamide solution in the iodoacetamide treatment method is 4 to 6 mmol / L, the pH is 5.8 to 6.0, and the iodoacetamide treatment time is 13 to 17 minutes; The fusion method is a polyethylene glycol fusion method, in which the concentration of polyethylene glycol is 35-45%, and the treatment time of the polyethylene glycol is 18-22 minutes.

2. The use according to claim 1, characterized in that: The concentration of polyethylene glycol in the polyethylene glycol fusion method is 40%, and the treatment time of the polyethylene glycol is 20 minutes.

3. The use according to claim 1, characterized in that: The concentration of the iodoacetamide solution is 5 mmol / L.

4. The use according to claim 1, characterized in that: The number ratio of the daylily somatic cells to the alstroemeria somatic cells is 1:1.

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