Culture medium composition and method for inducing plant regeneration through peony petioles

Abstract

The invention belongs to the field of plant tissue culture, and particularly relates to a culture medium composition and method for inducing plant regeneration through peony petioles, and the culture medium composition is characterized in that a callus induction tissue culture medium and an adventitious bud differentiation culture medium are MS culture mediums containing 0.5-1.5 mg / L TDZ and 0.05-0.25 mg / L NAA; the rooting induction culture medium is a 1 / 2 MS culture medium containing 2.0 to 4.0 mg / L of IBA, 0.3 to 0.7 mg / L of NAA and 0.4 to 0.6 g / L of TA. Petioles of peony sterile test-tube plantlets are used as explants; inoculating the explant into the callus induction culture medium for dedifferentiation culture to obtain a callus; inoculating the callus into the adventitious bud differentiation culture medium to induce bud differentiation to obtain adventitious buds; dividing the adventitious buds, and inoculating the adventitious buds into the rooting induction culture medium to induce rooting, so as to obtain regenerated seedlings. The culture medium is combined with a tissue culture method, petioles of peony seedlings are adopted for tissue culture, plant seedlings are obtained, the tissue culture seedling obtaining efficiency is high, the adventitious bud and adventitious root induction effect is good, and the transplanting survival rate is high.

Description

Technical Field

[0001] The invention belongs to the field of plant tissue culture, and particularly relates to a culture medium combination and a method for inducing plant regeneration through peony petioles. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Peony is a traditional famous flower in my country, known as "the most beautiful flower in the world" and "the king of flowers". In recent years, as the oil value, fresh cut flower value and health care function of peony have been gradually explored, peony has become an important economic plant that combines ornamental, medicinal and edible health care. Its status in the flower industry is gradually improving, and its market prospects are broad. Peony traditionally uses hybrid breeding, which has a long breeding cycle, low efficiency and slow reproduction speed, which seriously hinders the renewal of varieties and limits the high-quality development of the industry. Therefore, the lack of special varieties is an important problem facing the development of the peony industry.

[0004] Transgenic technology can improve breeding efficiency, and a stable and efficient plant regeneration system is the basis for using genetic engineering to breed new varieties. There have been many reports of successful cloning of peony genes. In theory, transgenic technology can be used to breed peony varieties that meet specific needs.

[0005] In the prior art, a perfect regeneration system for peony has not yet been established, making it impossible to apply transgenic technology to peony breeding. Domestic and foreign scholars have successively carried out research on the peony plant regeneration system using peony leaves, stem segments, cotyledons, receptacles, seeds, etc. as explants, but a perfect regeneration system has not yet been established. The main problems include the following aspects: (1) Severe browning of callus tissue and poor proliferation effect; (2) Low differentiation rate of adventitious buds; (3) Difficulty in inducing adventitious roots; (4) Transplantation does not survive. Therefore, it is urgent to establish a perfect peony plant regeneration system and then carry out genetic transformation research. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the object of the present invention is to provide a culture medium combination and method for inducing plant regeneration through peony petioles. The peony regeneration method provided by the present invention solves at least one of the above problems.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a culture medium combination for inducing plant regeneration through peony petioles, comprising a callus induction medium, an adventitious bud differentiation medium and a rooting induction medium;

[0008] The callus induction tissue culture medium and the adventitious bud differentiation culture medium are both MS culture medium containing 0.5-1.5 mg / L TDZ and 0.05-0.25 mg / L NAA;

[0009] The rooting induction medium is a 1 / 2MS medium containing 2.0-4.0 mg / L IBA, 0.3-0.7 mg / L NAA and 0.4-0.6 g / LTA.

[0010] In some embodiments, the root induction medium further comprises Ca 2+ , the preferred concentration is 0.003 mol / L.

[0011] In some embodiments, the invention further comprises an adventitious bud proliferation medium, wherein the adventitious bud proliferation medium contains Ca 2 + , 1.2-1.8 mg / L 6-BA and 0.03-0.07 mg / L NAA MS medium.

[0012] A second aspect of the present invention provides a method for inducing plant regeneration through peony petioles, comprising:

[0013] The petioles of sterile test tube seedlings of peony were used as explants;

[0014] Inoculating the explant into the above-mentioned callus induction medium for dedifferentiation culture to obtain callus;

[0015] Inoculating the callus tissue into the above adventitious bud differentiation medium to induce bud differentiation and obtain adventitious buds;

[0016] The adventitious buds were divided and inoculated into the above-mentioned root induction medium to induce rooting and obtain regenerated seedlings.

[0017] In some embodiments, the adventitious buds generated by induced differentiation are inoculated into the above-mentioned adventitious bud proliferation medium for proliferation culture, and then induced to root.

[0018] In some embodiments, the adventitious buds are divided and inoculated into the above-mentioned rooting induction medium, first treated in darkness, and then cultured under light conditions; preferably, the dark treatment time is 5-9 days, and more preferably 7 days.

[0019] In some embodiments, the regenerated seedlings are induced to produce dormant buds, and then the dormant buds are released from dormancy and hardened and transplanted.

[0020] In some embodiments, the regenerated seedlings are inoculated into a dormant bud induction medium for culturing to produce dormant buds, and the dormant bud induction medium comprises 0-2.0 mg / L IBA, 0-0.1 mg / L NAA and 50-200 mL / L coconut water.

[0021] In some embodiments, the regenerated seedlings producing dormant buds are cultured in the dark at 2-8° C., and transplanted after the dormancy of the buds is released.

[0022] In some embodiments, the dormancy-released regenerated seedlings are transplanted, and Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus megaterium is added to the transplanting medium.

[0023] In some embodiments, the transplanting medium includes peat and perlite.

[0024] The beneficial effects of the present invention are:

[0025] 1. The culture medium combination of the present invention is applied to the tissue culture regeneration system of the petiole of the sterile seedling of peony. By combining the culture medium of the present invention with the tissue culture method, the petiole of the peony seedling is used for tissue culture to obtain plant seedlings, and the efficiency of obtaining tissue culture seedlings is high, and the callus, adventitious bud and adventitious root induction effects are good, and the transplant survival rate is high. The callus induction medium can increase the callus induction ratio of the explant and improve the quality of the callus, the adventitious bud differentiation medium can increase the differentiation rate of the adventitious buds, and the rooting induction medium further increases the rooting rate of the tissue culture plant, greatly improving the adventitious bud differentiation rate and rooting rate.

[0026] 2. The invention can relieve the dormancy of the terminal buds after the test tube seedlings have taken root, and can make the root system develop well, so that the plant can grow normally after transplanting, and establish a perfect plant regeneration system.

[0027] 3. The present invention uses the petioles of peony seedlings for tissue culture to obtain plant seedlings, and the effect of inducing callus tissue is better than other parts, the callus induction ratio of explants is high, and the quality of callus tissue is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a physical picture of different links of the peony regeneration technology of the present invention. DETAILED DESCRIPTION

[0029] In view of the fact that most of the prior art peonies can only induce callus tissue through explants when conducting plant tissue culture, but fail to differentiate adventitious buds, even if a few induce adventitious buds and obtain rooted plants, there are still problems such as low rooting rate and transplant failure, so the regeneration system is very imperfect. The present invention proposes a culture medium combination and method for inducing plant regeneration through peony petioles, which establishes a complete callus tissue regeneration system and lays a foundation for peony molecular biology research and the application of transgenic technology.

[0030] Now, the English abbreviations of the hormones involved in the present invention are explained, TDZ (thidiazuron, N-phenyl-N-1,2,3-thiadiazol-5-urea), NAA (naphthaleneacetic acid, 2-(1-naphthyl)acetic acid), 6-BA (6-benzylaminopurine), IBA (3-indolebutyric acid), TA (triacontanol), 2,4-D (2,4-dichlorophenoxyacetic acid), and ZT (zeatin, 6-trans-4-hydroxy-3-methyl-but-2-enylaminopurine).

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0032] It should be noted that the materials and raw materials used in the following examples are all commercially available.

[0033] Example

[0034] The present invention takes the specific peony variety 'Hongxia Lanman' as an example, and specifically introduces the culture medium combination and method for inducing plant regeneration through peony petioles as follows. It should be noted that other peony varieties can also achieve the same or similar effects.

[0035] 1. Callus Induction

[0036] 1.1 Effects of different parts of test tube seedlings on callus induction

[0037] Preparation of callus induction medium: The callus induction medium is MS medium supplemented with hormones, 30 g / L sugar and 6 g / L agar as shown in Table 1;

[0038] The petioles and leaves of sterile test tube seedlings of peony were used as explants, and the petioles and leaves were cut into small sections of about 0.5 cm on a clean bench;

[0039] The petioles and leaf segments were inoculated into different callus induction mediums as shown in Table 1 for culture. The culture conditions were as follows: temperature (23±1)°C, light intensity 2000 lx, light hours 12 h·d-1, and induction time 35 days. The callus induction culture effect was observed after 35 days.

[0040] As shown in Table 1, callus tissue was induced by leaves and petioles of test tube seedlings. The induction rate of petioles was better than that of leaves under different hormone combinations. In addition, when inducing with leaves of test tube seedlings, the leaves were severely browned as a whole, and the induced callus blocks were very small. The induction effect of petioles was better than that of leaves, and the best effect was achieved under the hormone combinations of ZT+NAA and TDZ+NAA.

[0041] Table 1 Effects of different parts of test tube seedlings on callus induction

[0042]

[0043] 1.2 Different hormone combinations to induce callus

[0044] Preparation of callus induction medium: The callus induction medium is MS medium supplemented with hormones as shown in Table 2, 30 g / L sugar and 6 g / L agar;

[0045] The petioles of sterile test tube seedlings of peony were used as explants, and the petioles were cut into small segments of about 0.5 cm on a clean bench;

[0046] The petiole segments were inoculated into different callus induction medium as shown in Table 2 for culture. The culture conditions were: temperature (23±1)℃, light intensity 2000lx, and light duration 12h·d. -1 The induction time was 35 days. The callus induction effect was observed after 35 days.

[0047] The results in Table 2 show that the callus color and volume of Group 4 are better than those of Groups 1-3. The best combination of culture medium hormones for petiole callus induction is TDZ and NAA. The callus induction rate is 84.6%. The induced callus is tender green in color and has a larger tissue size.

[0048] Table 2 Effects of different hormone combinations on callus induction

[0049]

[0050] 1.3 Different concentrations of hormones to induce callus

[0051] Preparation of callus induction medium: The callus induction medium is MS medium supplemented with hormones as shown in Table 3, 30 g / L sugar and 6 g / L agar;

[0052] The treated petiole segments were inoculated into different callus induction medium as shown in Table 3 for culture. The culture conditions were the same as above. The callus induction culture effect was observed after 35 days.

[0053] The results in Table 3 showed that the callus induction effects obtained in groups 4-7 were better, among which group 4 had the best effect and group 7 had the worst effect. The best hormone concentration was TDZ 1.0 mg / L+NAA 0.1 mg / L.

[0054] Table 3 Effects of different hormone concentrations on callus induction

[0055]

[0056]

[0057] 2. Adventitious bud differentiation

[0058] 2.1 Different hormone combinations induce bud differentiation

[0059] Prepare adventitious bud differentiation medium: The differentiation medium is MS medium supplemented with hormones as shown in Table 4, 30 g / L sugar and 6 g / L agar;

[0060] The above callus tissue was cut into two sections and inoculated into the differentiation medium shown in Table 4 for culture to induce bud differentiation. Culture conditions: temperature (23±1)℃, light intensity 2000lx, light hours 12h·d -1 The culture time is 40 days, and the effect is observed after 40 days.

[0061] As shown in Table 4, the number of induced budding callus and the bud differentiation induction rate of Group 3 were significantly better than those of Groups 1-2. The optimal culture medium for bud differentiation was MS + TDZ 1.0 mg / L + NAA 0.1 mg / L + sugar 30 g / L + agar 6 g / L, and the bud differentiation rate was 51%.

[0062] Table 4 Effects of different hormone concentration combinations on bud differentiation

[0063]

[0064] 2.2 Different hormone concentrations induce bud differentiation

[0065] Prepare adventitious bud differentiation medium: The differentiation medium is MS medium supplemented with hormones as shown in Table 5, 30 g / L sugar and 6 g / L agar;

[0066] The callus tissue was cut into two sections and inoculated into the differentiation medium shown in Table 5 to induce bud differentiation. The culture conditions were the same as above, and the effect was observed after 40 days.

[0067] As shown in Table 5, groups 3-5 were significantly better than group 6, and group 3 had the best effect.

[0068] Table 5 Effects of different hormone concentrations on bud differentiation

[0069]

[0070] 3. Adventitious bud proliferation

[0071] Preparation of adventitious bud proliferation medium: The bud proliferation medium is MS medium supplemented with hormones as shown in Table 6, 150 mL / L coconut milk, 30 g / L sugar and 6 g / L agar;

[0072] The differentiated adventitious buds were cut and inoculated into the bud proliferation medium shown in Table 6 for proliferation culture. The culture conditions were the same as above and the culture time was 45 days. The effect was observed after 45 days.

[0073] As shown in Table 6, different hormone concentration combinations have different effects on bud proliferation, with proliferation coefficients ranging from 1.52 to 2.36. Groups 1 to 3 have better effects than group 4, among which group 2 has the best bud proliferation effect. The best medium formula for bud proliferation is: MS+Ca 2+ 0.003mol / L+6-BA1.5mg / L+NAA 0.05mg / L+coconut juice 150mL / L+sugar 30g / L+agar 6g / L, the proliferation coefficient reached 2.36, and the buds grew vigorously.

[0074] Table 6 Effects of different hormone concentration combinations on bud proliferation

[0075]

[0076]

[0077] 4. Rooting Induction

[0078] 4.1 Root induction with different hormone concentrations

[0079] Preparation of root induction medium: The root induction medium was supplemented with hormones, Ca 2+ , 1 / 2MS medium with 40 g / L sugar and 6 g / L agar;

[0080] The above-mentioned adventitious buds (i.e., rootless test tube seedlings) with a plant height of more than 2 cm were selected, cut into individual plants, and cultured in the rooting medium shown in Table 7 to induce adventitious roots. The culture conditions were the same as above, and the rooting situation was counted after 45 days of culture.

[0081] As shown in Table 7, groups 1-4 were better than group 5, among which group 4 had the best effect, with a rooting rate of 70%, a strong induced root system, and less callus at the base. It can be seen that the best medium formula for inducing adventitious roots is: 1 / 2MS+Ca 2+ 0.003mol / L+IBA3.0mg / L+NAA 0.5mg / L+TA0.5g / L+sugar 40g / L+agar 6g / L.

[0082] Table 7 Effects of different hormone concentration combinations on adventitious root induction

[0083]

[0084] 4.2 Effect of dark treatment time on adventitious root induction

[0085] Inoculate the rootless test tube seedlings

[0086] Ca 2+The effects of different dark treatment times on adventitious root induction were studied in 1 / 2MS medium containing 0.003mol / L+IBA 3.0mg / L+NAA 0.5mg / L+TA0.5g / L+sugar 40g / L+agar 6g / L. The dark treatment time is shown in Table 8. The culture was then placed under light conditions and the culture conditions were the same as above. The rooting situation was counted after 45 days of culture.

[0087] It can be seen from Table 8 that dark treatment at different times can promote the occurrence of adventitious roots. The best treatment time is 7 days, and the rooting rate reaches 90.8%.

[0088] Table 8 Effects of dark treatment at different times on adventitious root induction

[0089] Dark treatment time Rooting rate% Root status 3 77.8 The root system is strong and the base callus is small 5 82.0 The root system is strong and the base callus is small 7 90.8 The root system is strong and the base callus is small

[0090] 5. Dormant Bud Induction

[0091] Prepare dormant bud induction medium, which is 1 / 2MS medium with 40g / L sugar and 6g / L agar. The induction time is about 60 days.

[0092] The above tissue culture seedlings were transferred to dormant bud induction medium for culture. The culture conditions were: 15-20°C, light intensity 2000-3000lx, and light hours 10-12h·d. -1 The terminal buds are formed after about 60 days of cultivation. This is the prior art, see patent CN 113875596 A for details.

[0093] 6.Release from sleep mode

[0094] The test tube seedlings with dormant buds are placed in a dark culture at 4°C to break the dormancy of the buds. The culture time is 40-50 days. When the dormant buds begin to germinate, they are taken out and transplanted.

[0095] 7. Hardening and transplanting

[0096] After the dormancy of the terminal buds is released, the tissue culture seedlings are hardened to gradually adapt to the external environment and then transplanted:

[0097] During hardening, the tissue culture seedlings are hardened in a low to high gradient temperature environment (10 to 23° C.) for 8 to 12 days (the specific gradient design is, for example, a temperature increase of 1 to 3° C. per day, and each temperature gradient is cultured for 1 to 2 days);

[0098] When transplanting, the transplanting medium is a mixture of peat and perlite in a volume ratio of 3:1;

[0099] To ensure the survival rate, in the initial stage of transplanting (within 10 to 20 days after transplanting), the environmental conditions are controlled as follows: relative humidity above 90%, temperature controlled at 20 to 25°C, and the transplanting survival rate can reach more than 85%; 0.2-0.4g / strain of composite microbial agent (including Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus megaterium) can also be added. The composite agent is the Yangshao composite microbial agent purchased from Henan Yangshao Biochemical Engineering Co., Ltd.

[0100] To prevent the occurrence of stem rot in tissue culture seedlings and promote the production of capillary roots. The data in Table 9 are the survival rates calculated two months after transplantation. From the results, it can be seen that adding 0.2-0.6g of microbial agent to each plant can effectively prevent the occurrence of stem base rot and significantly improve the survival rate of test tube seedlings, but the difference of adding 0.2-0.6g is not obvious. Considering the cost issue, 0.2-0.4g / plant is appropriate.

[0101] Table 9 Effects of different dosages of microbial agents on the survival rate of test tube seedlings transplanted

[0102]

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A culture medium combination for inducing plant regeneration through peony petioles, characterized in that: It includes callus induction medium, adventitious bud differentiation medium and rooting induction medium; The callus induction tissue culture medium and the adventitious bud differentiation culture medium are both MS culture medium containing 0.5-1.5 mg / L TDZ and 0.05-0.25 mg / L NAA; The root induction medium is a 1 / 2MS medium containing 2.0-4.0 mg / L IBA, 0.3-0.7 mg / L NAA and 0.4-0.6 g / L TA.

2. The culture medium combination for inducing plant regeneration through peony petioles according to claim 1, characterized in that: Also included is an adventitious bud proliferation medium, wherein the adventitious bud proliferation medium contains Ca 2+ , 1.2-1.8 mg / L 6-BA and 0.03-0.07 mg / L NAA MS medium.

3. The culture medium combination for inducing plant regeneration through peony petioles according to claim 1, characterized in that: The root induction medium also includes Ca 2+ , the preferred concentration is 0.003 mol / L.

4. A method for inducing plant regeneration through peony petioles, characterized in that: include: The petioles of sterile test tube seedlings of peony were used as explants; The explants are inoculated into a callus induction medium for dedifferentiation culture to obtain callus tissue; Inoculating the callus tissue into an adventitious bud differentiation medium to induce bud differentiation and obtain adventitious buds; The adventitious buds are divided and inoculated into a rooting induction medium to induce rooting and obtain regenerated seedlings; The callus induction tissue culture medium and adventitious bud differentiation culture medium are both MS culture medium containing 0.8-1.2 mg / L LTDZ and 0.08-0.12 mg / L NAA; The root induction medium is a 1 / 2MS medium containing 2.0-4.0 mg / L IBA, 0.3-0.7 mg / L NAA and 0.4-0.6 g / L TA.

5. The method for inducing plant regeneration through peony petioles as claimed in claim 4, characterized in that: After the adventitious buds were divided, they were inoculated into the rooting induction medium, first treated in the dark, and then cultured under light conditions.

6. The method for inducing plant regeneration through peony petioles as claimed in claim 4, characterized in that: The dark treatment time is 5-9 days, preferably 7 days.

7. The method for inducing plant regeneration through peony petioles as claimed in claim 4, characterized in that: The adventitious buds produced by induced differentiation are inoculated into the adventitious bud proliferation medium according to claim 2 or claim 4 for proliferation culture, and then induced to root.

8. The method for inducing plant regeneration through peony petioles as claimed in claim 4, characterized in that: The regenerated seedlings are induced to produce dormant buds, which are then released from dormancy and hardened and transplanted.

9. The method for inducing plant regeneration through peony petioles as claimed in claim 4, characterized in that: The regenerated seedlings are inoculated into a dormant bud induction medium for cultivation to produce dormant buds, wherein the dormant bud induction medium comprises 0-2.0 mg / L IBA, 0-0.1 mg / L NAA and 50-200 mL / L coconut juice; further, the regenerated seedlings producing dormant buds are placed in a dark culture at 2-8° C., and transplanted after the dormancy of the buds is released.

10. The method for inducing plant regeneration through peony petioles according to claim 4, characterized in that: The dormancy-released regenerated seedlings are transplanted, and Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus megaterium are added to the transplanting medium; further, the transplanting medium also includes peat and perlite.

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