CN116210589A

CN116210589A - Method for hybridizing hemerocallis and six flowers and application of method in inducing callus of six flowers

Info

Publication number: CN116210589A
Application number: CN202310020166.9A
Authority: CN
Inventors: 程曼; 刘会君; 马志; 岳远志
Original assignee: Shandong Hezheng Ecological Agriculture Development Co ltd
Current assignee: Shandong Hezheng Ecological Agriculture Development Co ltd
Priority date: 2023-01-06
Filing date: 2023-01-06
Publication date: 2023-06-06

Abstract

The application discloses a method for hybridizing daylily with six flowers and application of the method in inducing six-flower callus, and belongs to the technical field of biology. In the application, the inventor utilizes an asymmetric fusion somatic cell hybridization technology to fuse hemerocalli and six-flower protoplasts by using a PEG fusion method, and then cultures to obtain embryogenic callus, compared with common leaf-induced callus, the method has higher induction success rate, not only can solve the problem of low induction rate of six-flower callus, but also can improve the fusion efficiency of the hemerocalli and two parents of six-flower, and breakthrough progress is made in the research of six-flower callus induction and differentiation.

Description

Method for hybridizing hemerocallis and six flowers and application of method in inducing callus of six flowers

Technical Field

The application discloses a method for hybridizing daylily with six flowers and application of the method in inducing six-flower callus, and belongs to the technical field of biology.

Background

The six flowers (Alstroemeria atrantiaca) are plants of genus six flowers of Amaryllidaceae, are rich in flower color, are butterfly-like in shape, are more elegant and gorgeous when blooming, are novel cut-flower materials, are in the Andies mountain region of south America where the six flowers originally produce, are in the shape of lily, and are locally called as 'Peruvian lily'. Introduction in 1754 to the United kingdom is not rapid until the 20 th century is used for ornamental cut flowers in Europe and America in the 50 th year, and the Netherlands are in the leading position in the aspects of breeding, propagation and production of new species of six flowers.

Six flowers are a great-potential ornamental flower with great genetic resource potential, but the six flowers are very difficult to form callus due to the characteristics of the six flowers, the research on the six flowers is still in the early stage at present, the research on tissue culture is only limited to regeneration seedlings induced by explants, and the research on the callus induction and differentiation of the six flowers in the prior art is little and has no breakthrough progress, so that a method capable of inducing the callus of the six flowers with high efficiency is needed, thereby fully utilizing the genetic resource of the six flowers and solving the problem that the callus is basically difficult to form by adopting six flower leaves as explants in the prior art.

Disclosure of Invention

The invention aims to provide a method for hybridizing hemerocalli with six flowers and application of the method in inducing the calli of the six flowers, which fuses the hemerocalli with the protoplasts of the six flowers by using a PEG fusion method, further cultures the hemerocalli to obtain embryogenic calli, solves the problem that the induction rate of inducing the calli of the six flowers by adopting common leaves is extremely low, improves the fusion efficiency of two parents of the hemerocalli and the six flowers, makes breakthrough progress in the research of the induction and differentiation of the calli of the six flowers, and provides a new idea for breeding new varieties of the hemerocalli.

According to one aspect of the present application, there is provided a method for hybridization of hemerocallis with six flowers, said hybridization method being somatic hybridization, said method comprising: the hemerocallis were fused with hexagons.

Alternatively, the fusion is performed in an asymmetric fusion mode.

Optionally, the method further comprises: the method comprises the steps of inactivating hemerocallis protoplast cytoplasm to obtain hemerocallis somatic cells; fragmenting chromosome of the hexaflower protoplast to obtain hexaflower somatic cells.

Optionally, the fusion method is one or more selected from polyethylene glycol fusion method, high-calcium fusion method, high-pH value fusion method, salt fusion method and electrofusion method; preferably, the fusion method is a polyethylene glycol fusion method.

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 min; preferably, the concentration of polyethylene glycol in the polyethylene glycol fusion method is 40%, and the treatment time of the polyethylene glycol is 20min.

Optionally, in the polyethylene glycol fusion method, the polyethylene glycol inducer contains KH 0.01g/100ml ₂ PO ₄ 、0.147g/100ml CaC1 ₂ ·2H ₂ O, 1.822g/100ml mannitol and 40g/100ml polyethylene glycol.

Optionally, the method for fragmenting the chromosome of the hexafloras protoplasts to obtain hexafloras cells is selected from one or more of radiation, restriction endonuclease treatment, spindle toxin treatment, and chromosome shrinking agent treatment; preferably, a radiation method is adopted, and the radiation is one or more selected from X-rays, gamma-rays and ultraviolet rays; preferably, the radiation is ultraviolet; preferably, the ultraviolet radiation treatment time is 3-4 min.

Optionally, the method for obtaining hemerocallis from the cytoplasma inactivation of hemerocallis is selected from one or more of iodoacetic acid treatment, iodoethanamine treatment and rhodamine 6G treatment; preferably selected from the iodoethanamine treatment.

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

Optionally, the ratio of the number of the hemerocallis to the number of the hexagons is 1:1.

alternatively, after the hemerocalli and the hexagons are fused, the hybrid somatic cells are subjected to constant temperature dark culture at 25 ℃ in KM8P liquid medium to induce callus.

According to another aspect of the present application, the present application discloses the use of a method of hybridization of any one of the above-mentioned hemerocalli with hexafloras, for inducing hexafloras calli.

Benefits of the present application include, but are not limited to:

1. according to the method, the Hemerocallis is adopted to carry out somatic hybridization with the Hemerocallis, the Hemerocallis callus is successfully induced, the problem that the Hemerocallis leaves are adopted as explants to basically form the callus is solved, a novel solution is provided for inducing the Hemerocallis callus, and the scheme for obtaining the Hemerocallis callus disclosed by the application has the advantages of high inducing success rate and good callus inducing effect.

2. According to the method, the hemerocallis and the hemerocallis are adopted for somatic cell hybridization, ultraviolet radiation is carried out on protoplasts of the hemerocallis, so that chromosomes in cell nuclei are fragmented, and after the chromosome is fused with the hemerocallis protoplasts, part of genetic materials of the hemerocallis are combined with the hemerocallis, so that the genetic resources of the hemerocallis are fully utilized, and a new thought is provided for breeding new varieties of the hemerocallis.

3. According to the method, the method of performing somatic cell hybridization on the hemerocallis and the six flowers is adopted, and the passivation conditions of the donor protoplast and the acceptor protoplast and the induction fusion conditions are optimized by controlling, so that the fusion efficiency of the hemerocallis and the six parents is greatly improved, and the fusion rate is 8.1% at most.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:

FIG. 1 is a schematic diagram of a technical route for hybridization of hemerocallis and six flowers according to the embodiment of the present application;

fig. 2 is a protoplast and fusion map extracted from six flowers and hemerocallis, provided in the examples of the present application, wherein: a is a six-flower protoplast; b is a hemerocallis fulva protoplast; c is a fusion map; d is a fused partial enlargement (BAR: 1 mm);

FIG. 3 is a comparative drawing of fused embryogenic callus and conventional leaf-induced callus provided in the examples herein, wherein: A. panel B shows embryogenic callus obtained by culturing fused protoplasts of Hemerocallis fulva; C. panel D shows six-flower normal leaf and petiole induced calli (panel A BAR:1mm, panel B BAR:1dmm, panel C, D BAR:1 cm), respectively.

Detailed Description

The present application is described in detail below with reference to examples, but the present application is not limited to these examples.

Unless otherwise indicated, all starting materials and reagents in the examples herein were purchased commercially.

PEG is an English abbreviation of polyethylene glycol, IOA is an English abbreviation of iodoethanamine, and UV is an English abbreviation of ultraviolet.

In view of the problem that six flowers are difficult to induce callus in the prior art, the quality of the induced callus is poor and the induction efficiency is low. The inventor has found a method for inducing six-flower calli by cultivating the filial generation of the hemerocalli and the six-flower protoplast through the asymmetric fusion of the protoplasts, namely the method for inducing the six-flower calli by adopting the asymmetric fusion culture method of the protoplasts through long-term and deep research. Through extensive research and experiments, the inventor overcomes the technical difficulty that the hemerocalli of the six flowers and the protoplast asymmetric fusion filial generation of the six flowers are difficult to obtain in the prior art through optimizing the hybridization conditions, not only successfully obtains the callus of the six flowers with high induction efficiency and high quality of the callus, but also cultivates the hemerocalli and the protoplast filial generation of the six flowers for the first time, excavates and uses genetic resources of the six flowers, and has important significance for improving the properties of the hemerocallis and breeding.

Plant cell hybridization is a plant breeding technology developed by combining a cell fusion technology and a plant tissue culture technology according to totipotency of plant cells. The plant somatic cell hybridization firstly removes the cell wall of the plant somatic cell to prepare complete viable protoplast, then fuses the heterologous protoplast by stimulation to form hybrid cells with biological activity, and further cultures the hybrid cells into hybrid plants by tissue culture and carries out selection and breeding of plants with excellent properties. The plant somatic cell hybridization technology does not need to undergo a sexual process, and hybrids can be manufactured only through somatic cell fusion, and protoplast fusion can be classified into symmetrical fusion, asymmetrical fusion and subprotoplast fusion according to different fusion degrees. The hybridization technology can break reproductive isolation among species, can overcome the condition that plants are not compatible with sexual hybridization in flowering phase, and creates an effective way for expanding genetic variation, updating germplasm resources and improving crop quality.

In the present application, protoplasts refer to cells from which cell walls have been removed, and since plant cells have a strong cell wall, protoplasts without cell walls are obtained before somatic hybridization.

In this application, asymmetric fusion is also referred to as asymmetric hybridization of cells, and refers to a technique of fusing two cells that cannot normally mate to produce hybrid offspring, and the procedure of protoplast fusion generally includes the steps of: removing cell walls of two cells belonging to the same or different species to form protoplasts, then using a fusion promoter to move proteins in certain areas on the two cell membranes, fusing the exposed phospholipid areas, and proliferating the hybrid somatic cells to form complete fused somatic cells.

Although protoplast asymmetric fusion has been successfully applied to the cultivation of some hybrid plants, only a small portion of plant species can successfully obtain hybrid progeny by protoplast fusion, which is largely dependent on the characteristics of the plant itself, the species, the size of the protoplasts, and the influence of many complications such as artificial manipulation, manipulation conditions, and the like, and in particular, it is more difficult for some flower varieties to cultivate hybrid progeny thereof by protoplast fusion. In the present application, the inventors have succeeded in inducing callus by hybridizing hemerocalli with hexagons, so that genetic material in hemerocalli can be combined with hexagons and the cellular activity of hexagons can be greatly improved.

In view of the above, it is necessary to provide a method for hybridizing daylily with hexagons, which can solve the problem of low callus induction rate of hexagons and can improve the fusion efficiency of two parents of daylily and hexagons. In order to achieve the above purpose, the technical scheme adopted by the invention is a method for hybridizing hemerocallis and hexagons, as shown in fig. 1, comprising the following steps:

(1) Preparation of protoplasts: extraction of plant protoplasts was performed using a protoplast preparation kit (model RTU 4052) from midwife biotechnology limited. Respectively weighing 0.5g of aseptic seedling tender leaves of hemerocallis and 0.5g of aseptic seedling tender leaves of six flowers, respectively cutting the seedling tender leaves into thin strips (finer and better), respectively adding the two parents into conical flasks containing 5ml of instant enzymolysis liquid, fully soaking, carrying out enzymolysis for 3 hours under the condition of avoiding light and 60rpm, and obtaining six-flower protoplasts and hemerocallis protoplasts.

(2) Passivation of protoplasts: suspending the hemerocallis obtained in the step (1) in 5mmol/L IOA solution at room temperature for 20min, spreading the hexaflorigens in 3cm small culture dish at room temperature, irradiating with UV for 1min, centrifuging and washing the two protoplasts after IOA and UV treatment with CPW solution respectively twice, wherein each centrifugation speed is 800rpm, centrifugation time is 5min, and culturing in dark condition until density is 5×10 ⁵ Day lily acceptor protoplasts and six flower donor protoplasts were obtained per ml.

(3) Fusion of protoplasts: mixing the acceptor protoplast and the donor protoplast obtained in the step (2) according to the volume ratio of 1:1 to obtain a parent suspension, carefully sucking a PEG inducer (PEG concentration of 40%) by a pipette, slowly dripping the PEG inducer along small droplets around, standing at room temperature for 20min for induction, washing twice by using 1ml of high-calcium high-pH washing liquid and 1ml of CPW solution, washing twice by using KM8P Medium (Kao & Michakuk Medium), finally adding KM8P fresh Medium for shallow culture, and performing dark culture at 25 ℃ until regenerated embryogenic callus particles grow out.

The solution ratios used in this application are shown in table 1 below:

TABLE 1 solution formulation

Example 1

Preparation of protoplasts

1) And (5) enzymolysis. Using a protoplast preparation kit (model RTU 4052) produced by Zhongkeshitai Biotechnology Co., ltd.), cutting well-grown and tender leaves of Hemerocallis and Hemerocallis into fine strips (finer and finer), adding 0.5g of the leaves into a conical flask, adding 5ml of an instant enzymolysis solution, soaking thoroughly, and carrying out enzymolysis for 3 hours under the conditions of 60rpm and 25 ℃. The green color of the enzyme solution indicates that protoplasts have been isolated, and the leaf protoplasts are microscopically green and spherical (as shown in A, B of FIG. 2);

2) And (5) rinsing. After enzymolysis, an equal volume of 1x rinse solution was added, and in this example, 5ml of the enzymolysis system was used, so that 5ml of 1x rinse solution was added and mixed gently;

3) And (5) filtering. Filtering the solution obtained in the step 2) by using a screen or a piece of mirror paper, removing undigested leaves, washing the enzymolysis vessel and undigested leaves with 5-10ml of 1x rinsing solution 1-2 times by using a suction tube, and collecting all the liquid into a 10ml centrifuge tube;

4) And (5) collecting for the first time. Centrifuging the filtrate at 1200rpm at normal temperature for 5min, and removing the supernatant;

5) And (5) collecting for the second time. Adding 5ml of 1x rinsing solution, re-suspending the protoplast by using a suction pipe, centrifuging at 1200rpm for 5min at normal temperature, and collecting the protoplast;

6) And (5) re-suspending. Carefully remove the supernatant without encountering protoplast precipitation, add to the self-contained heavy suspension in the kit, i.e. 1ml solution III, i.e. protoplast solution.

Passivation of protoplasts

1) IOA treatment (recipient treatment) of hemerocalli protoplasts: the CPW solution is used for preparing iodoacetamide solution with the concentration of 5mmol/L and the pH of 5.8-6.0. Dissolving, sterilizing, and storing at-20deg.C; the protoplast is resuspended by CPW solution, 1ml protoplast is sucked, iodoacetamide is added, the operation is carried out on ice, and the protoplast is gently shaken; standing at room temperature for 10min, 15min, and 20min (the results are shown in Table 2); centrifuging at 1200rpm after standing is finished, and performing 2min; the supernatant was discarded, washed 2 times with CPW solution, and resuspended with 1ml of CPW solution.

TABLE 2 Effect of different IOA treatment times on Hemerocallis protoplasts

As can be seen from table 2, the difference in passivation effect of different IOA treatment times on hemerocallis protoplast viability was significant: after the IOA treatment for 0min, 10min, 15min and 20min, the activity of the hemerocallis is respectively reduced by 68.5 percent, 82.3 percent and 86.9 percent compared with the control, which shows that the hemerocallis protoplast is very sensitive to the IOA. With the increase of IOA treatment time, the degree of disruption of protoplast cells tends to increase; about 93.5% of hemerocallis protoplasts are inactivated after 15min IOA treatment and the degree of cell disruption is not very high, indicating that 15min IOA treatment can effectively inactivate hemerocallis protoplasts.

2) UV treatment of hexaflower protoplasts (donor treatment): spreading proper amount of protoplast suspension 3-5ml in a small culture dish of 3cm to form a thin layer, placing under ultraviolet lamp tube on an ultra-clean workbench, and respectively radiating for 1min, 3min and 6min (the result is shown in Table 3) at a distance of 20 cm. The irradiated protoplasts were transferred to centrifuge tubes with pipettes, respectively, and the final concentration was consistent with the recipient by centrifugation with addition of CPW suspension.

TABLE 3 influence of different UV treatment times on Liuzhua protoplasts

As can be seen from table 3, the passivation effect of UV treatment time at different concentrations on the protoplast viability of hexaflorae was significantly different: after UV treatment for 1min, 3min and 6min, the activity of the hexagons protoplast is respectively reduced by 35.1%,74.6% and 86.4% compared with that of a control, and along with the extension of the UV irradiation time, the passivation effect of UV on the hexagons protoplast activity is enhanced, and the cell disruption degree is deepened; the cell disruption degree of the protoplast is increased after the protoplast is treated by UV irradiation for 3min, for example, the passivation of the protoplast is enhanced by prolonging the treatment time, and more cell death and disruption of the protoplast occur, so that the protoplast with six flowers can be effectively passivated by UV irradiation for 3min, and the disruption degree of the protoplast is lower.

Fusion of protoplasts

1) Suspending the purified two protoplasts (the UV ray passivation donor and the IOA passivation acceptor) in CPW culture medium to the same density;

2) Mixing equal amount of protoplast (v/v, 1:1), sucking out 0.2ml of the mixed solution with a suction pipe, dripping the mixed solution to the bottom of a 3cm culture dish, standing for 5min, and observing until the protoplast is attached to the bottom of the culture dish;

3) Sucking up PEG inducer 0.6ml (PEG concentration 40%) with a pipette, carefully dripping along small droplets around, contacting PEG with protoplast, standing at room temperature for 0min, 15min,20min, and 25min respectively for induction (results are shown in Table 4);

TABLE 4 effects of different PEG treatment times on cell fusion

As can be seen from table 4, the heterologous fusion rate changed from rising to falling with increasing PEG treatment time: wherein the fusion rate is 8.1% at maximum when the PEG treatment time is 20 min; however, when the PEG treatment time was 25min, the fusion product could not survive, and a large amount of protoplasts were broken and gradually dead. Because PEG itself has certain toxicity to protoplast, in order to reduce the side effect of PEG on the growth and development of fusion products, PEG treatment is selected for 20min as the proper time for fusion of hemerocallis and hexaflorus protoplast, at the moment, most protoplasts are fused one to one, the heterologous fusion rate is higher than 8.1%, and the breaking degree of the protoplasts is lower.

4) Then slowly adding 1ml of high-calcium high-pH washing liquid at the edge of the liquid drop;

5) After 10min, adding 1ml of high-calcium high-pH washing liquid, sucking a small amount of washing liquid until the glass slide is observed under a microscope, and recovering the protoplast into a sphere;

6) Standing at room temperature for 1h, slowly sucking out PEG and high-calcium high-pH solution, and slowly adding 1ml of CPW fresh washing liquid from one side;

7) After 5min, washing for the second time, wherein the method is the same until the PEG and the high-calcium high-pH solution are cleaned;

8) And (3) washing once by using a KM8P culture medium, and finally adding a KM8P fresh culture medium for shallow culture, and performing dark culture at 25 ℃, wherein the fresh culture medium is added every 7 days or so, so as to gradually reduce the mannitol concentration (the photo of fusion of hemerocallis and hexaflorida protoplasts in the invention is shown as C and D in figure 2).

Example 2

Culture of protoplasts

Adding KM8P liquid culture medium into the fused hybrid cells to adjust the density to 2X 10 ⁵ And (3) culturing protoplast per mL by taking KM8P as a basic culture medium and adopting a liquid culture medium method, sealing by a sealing film, and culturing in an incubator at a constant temperature of 25 ℃. Protoplast isolated from embryogenic callus is subjected to 6-7d protoplast double wall on KM8P medium, first division occurs about 14-15d, second division occurs about 20-25d, macroscopic small callus appears about 35-40d, and compared with the conventional explant induced callus such as leaf or petiole, the method has short induction time and high induction success rate (as shown in figure 3 and figure 3)Shown in table 5).

TABLE 5 comparison of traditional explant callus induction and cell fusion hybrid callus induction

The foregoing is merely exemplary of the present application, and the scope of the present application is not limited to the specific embodiments, but is defined by the claims of the present application. Various modifications and changes may be made to the present application by those skilled in the art. 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 hemerocallis with six flowers, wherein the hybridization method is somatic hybridization, the method comprising: the hemerocallis were fused with hexagons.

2. The method for hybridization of hemerocallis and hexagons according to claim 1, wherein said fusion is by means of asymmetric fusion.

3. The method of hybridization of hemerocallis with six flowers according to claim 2, further comprising: the method comprises the steps of inactivating hemerocallis protoplast cytoplasm to obtain hemerocallis somatic cells; fragmenting chromosome of the hexaflower protoplast to obtain hexaflower somatic cells.

4. The method for hybridization of hemerocallis and six flowers according to claim 1, wherein said fusion method is selected from one or more of polyethylene glycol fusion, high calcium fusion, high pH fusion, salt fusion, and electrofusion; preferably, the fusion method is a polyethylene glycol fusion method.

5. The method for hybridization of hemerocallis and six flowers according to claim 4, wherein 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 min;

preferably, the concentration of polyethylene glycol in the polyethylene glycol fusion method is 40%, and the treatment time of the polyethylene glycol is 20min.

6. The method for hybridization of hemerocallis with hexagons according to claim 3, wherein said method for fragmenting the protoplast chromosomes of hexagons to obtain hexagons is selected from one or more of radiation, restriction endonuclease treatment, spindle toxin treatment and chromosome constrictor treatment;

preferably, a radiation method is adopted, and the radiation is one or more selected from X-rays, gamma-rays and ultraviolet rays;

more preferably, the radiation is ultraviolet;

more preferably, the ultraviolet radiation treatment is performed for 3 to 4 minutes.

7. The method for hybridization of hemerocallis with six flowers according to claim 3, wherein said method for obtaining hemerocallis somatic cells by inactivation of hemerocallis protoplast is selected from one or more of iodoacetic acid treatment, iodoethanamine treatment and rhodamine 6G treatment;

preferably selected from the iodoethanamine treatment.

8. The method for hybridizing daylily with six flowers according to claim 7, wherein said iodoethanamine treatment method has a concentration of iodoacetamide solution of 4-6 mmol/L, a pH of 5.8-6.0, a time period of iodoacetamide treatment of 13-17 min,

preferably, the concentration of the iodoacetamide solution is 5mmol/L.

9. The method for hybridization of hemerocallis with six flowers according to claim 1, wherein the ratio of the number of hemerocallis somatic cells to the number of hemerocallis somatic cells is 1:1.

10. use of a method of hybridization of hemerocalli according to any one of claims 1 to 9 with hexaflorae for inducing hexaflorae calli.