Paeonia lactiflora germplasm innovation method

Abstract

The invention discloses a germplasm innovation method for paeonia lactiflora, and belongs to the technical field of polyploid breeding. The germplasm innovation method for the Chinese herbaceous peony comprises the step of carrying out polyploid induction on an explant of the Chinese herbaceous peony, and comprises the following steps: inoculating a disinfected underground bud into a germination culture medium containing a polyploid inducer on an ultra-clean workbench, culturing for one week, and then transferring into a germination culture medium without the polyploid inducer. According to the germplasm innovation method for the paeonia lactiflora, polyploidy induction is carried out on underground buds of the paeonia lactiflora, and polyploidy plants are successfully cultivated; the method for inducing the polyploidy is carried out based on underground buds of the Chinese herbaceous peony, the propagation mode of division propagation of the Chinese herbaceous peony is met, and the polyploidy induction method is simple and easy to implement.

Description

Technical Field

[0001] The invention relates to the field of polyploid breeding, and in particular to a method for germplasm innovation of peony. Background Art

[0002] Peony (scientific name: Paeonia lactiflora Pall.) is a perennial herbaceous plant of the genus Paeonia in the family Paeoniaceae. It is also known as wild peony, white peony, and peony flower. The roots are thick and the branches are dark brown; the stems are hairless, and the leaflets are narrowly ovate, elliptical or lanceolate; the flowers are located at the top of the stem and in the axils of the leaves; the flowering period is May-June; the fruiting period is August.

[0003] Peony likes to grow on hillsides and under forests. Peony likes a sunny environment, suitable for high terrain, good drainage, deep soil, loose and fertile sandy soil, and the soil pH suitable for growth is neutral or slightly acidic.

[0004] The root of peony can be used for medicinal purposes, called "white peony", which has the effects of analgesia, antispasmodic, blood stasis removal and menstruation regulation; the oil content of peony seeds is about 25%, which can be used for soap making and paint; the flowers of peony can be made into fresh cut flowers and sold to generate income.

[0005] Generally speaking, compared with ordinary diploid plants, polyploid plants have excellent morphological characteristics such as tall plants, thick stems, thick leaves, pure flower color, large flowers with extended flowering period, enhanced growth potential and stress resistance.

[0006] Colchicine-induced polyploidy is a common plant ploidy breeding technology. It is an in vitro induction method that combines tissue culture technology with chemical mutagenesis. It has become a common chromosome doubling method due to its advantages such as high induction rate, time and labor saving, and easy control of conditions.

[0007] Therefore, polyploid medicinal peony can be induced. The roots of polyploid peony are thick and strong, which can increase the yield of white peony and thus increase the economic value of peony.

[0008] A method for efficiently inducing polyploid pollen to cultivate polyploid peony (application number: 202011061629.9) provides a method for efficiently inducing polyploid pollen to cultivate polyploid peony, comprising the following steps: 1) determining the induction period; 2) preparing colchicine solution; 3) injecting colchicine solution; 4) collecting pollen; 5) identifying polyploid pollen; 6) hybridizing and pollinating; 7) collecting and sowing seeds; and 8) identifying polyploid offspring.

[0009] However, the invention is mainly aimed at ornamental peonies. The plant propagation method of medicinal peonies is usually division, and medicinal peonies need to be processed by bud picking so that all nutrients can be supplied to the roots. Therefore, the method of inducing polyploid pollen to cultivate polyploid peonies in the invention is not suitable for the cultivation of medicinal peonies. Summary of the invention

[0010] The purpose of the present invention is to provide a method for the germplasm innovation of peony, to induce polyploid medicinal peony, to increase the yield of white peony, and to increase the economic value of peony.

[0011] To achieve the above object, the present invention provides a method for the germplasm innovation of peony, comprising inducing polyploidy of peony explants, wherein the polyploidy induction comprises the following steps:

[0012] The sterilized underground buds were inoculated into a germination medium containing a polyploidy inducer on a clean bench, and after one week of cultivation, they were transferred into a germination medium without a polyploidy inducer.

[0013] Preferably, the polyploidy inducing agent is 0.2% colchicine.

[0014] Preferably, the polyploidy inducing agent is a mixture of 0.1% to 0.2% colchicine and 1% or 2% dimethyl sulfoxide.

[0015] Preferably, the polyploidy inducing agent is a mixture of 0.2% colchicine and 2% dimethyl sulfoxide.

[0016] Preferably, the germination medium comprises: 1 / 2MS+6-BA 1.0 mg / L+IAA 0.2 mg / L.

[0017] On the other hand, the present invention provides an application of a method for germplasm innovation of peony, wherein the above-mentioned method for germplasm innovation is applied to polyploid breeding of peony.

[0018] Therefore, the present invention provides a method for the innovation of peony germplasm, and successfully cultivates polyploid plants by inducing polyploidy in the underground buds of peony; the method for inducing polyploidy is based on the underground buds of peony, which is in line with the reproduction method of peony division reproduction, and the polyploidy induction method of the present invention is simple and easy to implement.

[0019] The technical solution of the present invention is further described in detail below through embodiments. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention clearer, the embodiments of the present invention are further described in detail in conjunction with the embodiments below. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of this application.

[0021] It should be noted that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or server that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units that are not explicitly listed or inherent to these processes, methods, products or devices.

[0022] Embodiment 1

[0023] A method for the germplasm innovation of peony, comprising selecting plump underground buds of normally grown white peony plants planted in a Bozhou Chinese medicinal material planting base, and doubling them after disinfection, comprising the following steps:

[0024] The immersion method used different concentrations of sterile colchicine aqueous solution (0.05%, 0.1%, 0.2%) to soak the underground buds for 8h, 12h, and 16h respectively, and then rinsed them with sterile water on an ultra-clean workbench for 3 to 5 times, each time for 3 minutes. After absorbing the water with sterile filter paper, they were inoculated into the culture medium.

[0025] (2) Add 7 g / L agar and 30 g / L sucrose to the culture medium, and adjust the pH to 5.8-6.0. The culture temperature is 23±2°C, the light exposure time is 14 h / d, and the light intensity is 2000-3000 lx.

[0026] The underground buds without colchicine treatment were used as the control treatment. Thirty underground buds were treated each time.

[0027] Fresh and tender leaves of mutant plants were collected, and the relative content of DNA in the cell nucleus was determined using an Epics Ahra flow cytometer from the United States. The results were analyzed using PartecDPAC software, and the ploidy of the sample to be tested was determined based on the results. The results are shown in Table 1:

[0028] Table 1 Effect of colchicine immersion method on polyploid induction of underground buds

[0029]

[0030]

[0031] As can be seen from Table 1, with the increase of colchicine concentration, the mortality rate of underground buds gradually increased and was proportional to the treatment time. Among them, the mortality rate of underground buds soaked in 0.2% colchicine for 16 hours was the highest, at 76.67%, while the mortality rate of underground buds soaked in 0.05% colchicine for 8 hours was the lowest, at 23.67%.

[0032] After the explants treated by the immersion method were transferred to the start-up culture medium, they grew slowly compared with the control group. This phenomenon was aggravated with the increase of concentration and treatment time. Some plants were obviously poisoned. With the increase of concentration and treatment time, the explants turned brown seriously and gradually wilted and died. The reason for the browning may be that colchicine has a toxic effect on the explants after being immersed in them, causing the phenolic substances in the plant to be oxidized and converted into quinone substances, which in turn caused the plant to turn brown.

[0033] Flow cytometry ploidy test results: no plants with variable ploidy. This may be because the immersion method is more toxic to the explants, and the colchicine residue in the plant body affects its normal growth.

[0034] Embodiment 2

[0035] A method for the germplasm innovation of peony, comprising selecting plump underground buds of normally grown white peony plants planted in a Bozhou Chinese medicinal material planting base, and doubling them after disinfection, comprising the following steps:

[0036] In the mixed culture method, the sterilized underground buds were inoculated into germination medium supplemented with different concentrations of colchicine (0.05%, 0.1%, 0.2%) on a clean bench, and after one week of culture, they were transferred to germination medium without colchicine.

[0037] The germination medium is 1 / 2MS+6-BA 1.0mg / L+IAA 0.2mg / L.

[0038] The underground buds without colchicine treatment were used as the control treatment. Thirty underground buds were treated each time.

[0039] Fresh and tender leaves of mutant plants were collected, and the relative content of DNA in the cell nucleus was determined using an Epics Ahra flow cytometer from the United States. The results were analyzed using PartecDPAC software, and the ploidy of the sample to be tested was determined based on the results. The results are shown in Table 2:

[0040] Table 2 Effect of colchicine mixed culture on polyploid induction of underground buds

[0041]

[0042] As can be seen from Table 2, the mortality rate of underground buds treated by mixed cultivation is relatively low. Among them, after 1 week of mixed cultivation with 0.2% colchicine, 2 polyploid plants were obtained after being cultured in a medium without colchicine. After flow cytometer detection, the DNA content of the plants was twice that of the control group (diploid). And the appearance of the plants was as follows: the leaves were obviously enlarged, the leaf edges were obviously curled, and the stems were thickened, so they were judged to be tetraploid plants.

[0043] Embodiment 3

[0044] A method for the germplasm innovation of peony, comprising selecting plump underground buds of normally grown white peony plants planted in a Bozhou Chinese medicinal material planting base, and then sterilizing and doubling them by mixed cultivation, comprising the following steps:

[0045] The sterilized underground buds were inoculated into a germination medium supplemented with different concentrations of colchicine and dimethyl sulfoxide (DMSO) on a clean bench, and after one week of culture, they were transferred to a germination medium without colchicine.

[0046] The germination medium is 1 / 2MS+6-BA 1.0mg / L+IAA 0.2mg / L.

[0047] The groups included: 0.1% colchicine + 1% dimethyl sulfoxide, 0.1% colchicine + 2% dimethyl sulfoxide, 0.2% colchicine + 1% dimethyl sulfoxide, 0.2% colchicine + 2% dimethyl sulfoxide.

[0048] Fresh and tender leaves of mutant plants were collected, and the relative content of DNA in the cell nucleus was determined using an Epics Ahra flow cytometer from the United States. The results were analyzed using PartecDPAC software, and the ploidy of the sample to be tested was determined based on the results. The results are shown in Table 3:

[0049] Table 3 Effect of colchicine + dimethyl sulfoxide mixed culture on polyploid induction of underground buds

[0050]

[0051] As can be seen from Table 2, compared with Example 2, the polyploid induction rate of underground buds increased after adding dimethyl sulfoxide. Among them, after being treated with 0.2% colchicine + 2% dimethyl sulfoxide mixed culture method for 1 week, 9 polyploid plants were obtained after being cultured in germination medium. After flow cytometer detection, the DNA content of the plants was twice that of the control group (diploid). And the appearance of the plants was as follows: the leaves were obviously enlarged, the leaf edges were obviously curled, and the stems were thickened, so they were judged to be tetraploid plants.

[0052] The increase in polyploidy after the addition of DMSO may be because DMSO increases the rate of colchicine penetration into underground buds, which has a certain effect on cell division and promotes the formation of polyploid plants.

[0053] The 9 polyploid plants obtained from the 0.2% colchicine + 2% dimethyl sulfoxide treatment group were transplanted into soil for cultivation, and 6 of them survived, demonstrating that the method of inducing polyploidy has certain feasibility.

[0054] Therefore, the present invention provides a method for the innovation of peony germplasm, and successfully cultivates polyploid plants by inducing polyploidy in the underground buds of peony; the method for inducing polyploidy is based on the underground buds of peony, which is in line with the reproduction method of peony division reproduction, and the polyploidy induction method of the present invention is simple and easy to implement.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for the germplasm innovation of peony, comprising inducing polyploidy of peony explants, characterized in that: Polyploid induction includes the following steps: The sterilized underground buds were inoculated into a germination medium containing a polyploidy inducer on a clean bench, and after one week of cultivation, they were transferred into a germination medium without a polyploidy inducer.

2. The method for germplasm innovation of peony according to claim 1, characterized in that: The polyploidy inducer was 0.2% colchicine.

3. The method for germplasm innovation of peony according to claim 1, characterized in that: The polyploidy inducing agent is a mixture of 0.1% to 0.2% colchicine and 1% or 2% dimethyl sulfoxide.

4. The method for germplasm innovation of peony according to claim 3, characterized in that: The polyploidy inducing agent is a mixture of 0.2% colchicine and 2% dimethyl sulfoxide.

5. The method for germplasm innovation of peony according to claim 1, characterized in that: The germination medium includes: 1 / 2MS+6-BA 1.0mg / L+IAA 0.2mg / L.

6. An application of a method for the germplasm innovation of peony, characterized in that: The method for germplasm innovation according to any one of claims 1 to 5 is applied to polyploid breeding of peony.

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