Tissue culture method for inducing cluster buds of paeonia delavayi embryo

Abstract

The invention provides a tissue culture method for inducing cluster buds of paeonia delavayi embryo, and relates to the technical field of tissue culture of paeonia delavayi. The tissue culture method mainly comprises the steps of explant acquisition, embryo germination culture, embryo cluster bud induction culture, cluster bud multiplication culture, cluster bud rooting culture, acclimatization and transplantation and the like. According to the method, the defects in the prior art are overcome, the embryos in fresh and mature Paeonia delavayi seeds in the current year are used as explants, a regeneration system of an embryo cluster bud induction way is preliminarily established through embryo cluster bud induction, proliferation and rooting culture medium research, and a foundation is laid for establishment of a Paeonia delavayi genetic transformation system.

Description

Technical Field

[0001] The invention relates to the technical field of Yunnan peony tissue culture, and in particular to a tissue culture method for inducing clustered buds of Yunnan peony seed embryos. Background Art

[0002] Yunnan peony (Paeonia delavayi Franch) is a national second-class protected plant distributed in the high-altitude and cold regions of southwest my country. It has rich and diverse flower colors and leaf shapes, and is of great ornamental value. It can be used in landscaping as specialized gardens, flower beds, flower mirrors and flower belts to create beautiful urban landscapes, which requires a large number of seedlings. Seedlings propagated by seeds have the disadvantages of long existence time, low efficiency, low germination rate, and large phenotypic differentiation, which cannot meet the requirements of large-scale production and breeding, and have become one of the constraints that have always plagued the promotion and use of Yunnan peony; and the double dormancy characteristics of the upper and lower hypocotyls of peony plants lead to their weak natural regeneration ability, which seriously restricts the increase of their population and the inheritance of excellent traits.

[0003] Plant tissue culture technology is the most important basic work for the genetic engineering breeding of new varieties. It is less restricted by time and space, can achieve the purpose of rapid expansion in a short time, and the excellent traits of the mother parent are also maintained with a small coefficient of variation, which can lay the foundation for peony breeding and industrial development. There are reports on the successful establishment of a complete regeneration system using embryos through callus induction or cluster bud induction for 'Fengdan' peony, peony, and large-flowered yellow peony. However, the research on the embryos of Yunnan peony only stays at the callus proliferation stage, and no differentiation of adventitious buds is observed. The tissue culture of petioles and stamens also only achieves the proliferation of callus. In addition, no related research on cluster bud induction has been found. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a tissue culture method for inducing clustered buds from seed embryos of Yunnan peony. The seed embryos in fresh and mature Yunnan peony seeds of the current year are used as explants. Through the research on seed embryo clustered bud induction, proliferation and rooting culture medium, a regeneration system for the seed embryo clustered bud induction pathway is preliminarily established, laying the foundation for the establishment of a Yunnan peony genetic transformation system.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0006] A tissue culture method for inducing clustered buds from Yunnan tree peony embryos, the culture method comprising the following steps:

[0007] S1. Obtaining explants: soaking the seeds of Paeonia dahliae in a gibberellin solution and then washing them, dissecting out the complete embryos and performing disinfection to obtain explants for later use;

[0008] S2. Germination culture of seed embryos: inoculate the above explants into germination medium, and culture in dark conditions to obtain embryo seedlings; and the formula of the germination medium is: MS+0.2mg / LGA3+0.5g / LPVP+7g / L agar+30g / L sucrose;

[0009] S3. Induction culture of clustered buds of seed embryos: inoculate the above embryo seedlings into the induction medium for clustered buds for culture, and induce multiple clustered buds; and the formula of the induction medium for clustered buds is: MS+0.5mg / L KT+0.5mg / L 6-BA+30g / L sucrose+7g / L agar+0.5g / LPVP+20mg / L ethyl isothiocyanate+30mg / L tea saponin;

[0010] S4. Proliferation culture of clustered buds: the embryo seedlings induced with multiple clustered buds are transferred to a proliferation medium for culture until the clustered buds proliferate more than 2 times; and the formula of the proliferation medium is: MS + 0.2mg / L GA3 + 0.5mg / L KT + 0.5mg / L 6-BA + 0.3g / L CH + 30g / L sucrose + 7g / L agar + 1g / L PVP + 15mg / L tocopherol acetate;

[0011] S5. Cluster bud rooting culture: The cluster buds obtained in the above step S4 are inoculated into the cluster bud rooting medium to obtain Yunnan peony seedlings; and the formula of the cluster bud rooting medium is improved 1 / 2MS+1mg / L IBA+0.2mg / L NAA+0.5mg / L IAA+7g / L agar+1g / L PVP+2g / LAC+15mg / L ethyl isothiocyanate+30mg / L tocopherol acetate; and the formula of the improved 1 / 2MS is: 720mg / LKNO3, 580mg / LNH4NO3, 106mg / LKH2PO4, 146mg / LMgSO4·7H2O, 255mg / L CaCl2·2H2O, 0.83mg / LKI, 6.2mg / L H3BO3, 22.3mg / LMnSO4·H2O, 8.6mg / LZnSO4·7H2O, 0.25mg / L Na2MoO4·2H2O, 0.025mg / L CuSO4·5H2O, 0.025mg / LCoCl2·6H2O, 27.6mg / LEDTA-Na2, 30.5mg / LFeSO4·7H2O, 100mg / L inositol, 3.5mg / L glycine, 0.1mg / L thiamine hydrochloride, 0.5mg / L pyridoxine hydrochloride;

[0012] S6. Seedling hardening and transplanting: Select the robust Yunnan tree peony seedlings grown in S4 above, move them out of the incubator for acclimatization for 2 days, remove the rooting medium of the root clusters, and transplant them into the growth medium for cultivation.

[0013] Preferably, in step S1, the Yunnan tree peony seeds are soaked in a gibberellin solution by soaking the Yunnan tree peony seeds in a 500 mg / L gibberellin solution for 48-50 hours.

[0014] Preferably, the Yunnan tree peony seeds are disinfected in step S1 by soaking them in a sodium hypochlorite solution with a mass concentration of 0.4%-0.5% for 5-6 minutes, and then rinsed with sterile water for 2-3 times.

[0015] Preferably, the embryo germination culture condition in step S2 is 25±2° C. in the dark.

[0016] Preferably, the temperature condition for the induction culture of clustered buds in step S3 is 15±1°C, and during the culture process, dark culture is first performed, and then the daily lighting time is controlled to 16h for supplementary light culture, and the dark culture time is 10d, and white LED light is used as the light source during the supplementary light culture.

[0017] Preferably, the temperature of the proliferation culture of the clustered buds in step S4 is 25±2° C., and the lighting time is controlled to 16 h / d using a white LED lamp.

[0018] Preferably, the temperature of the clustered bud rooting culture in step S5 is 20±1° C., and the white LED lamp is used to control the lighting time to 16 h / d.

[0019] Preferably, the standards for healthy growth of Paeonia dahliae seedlings in step S6 are seedling height>8cm, root length>5cm, and number of fibrous roots>3.

[0020] Preferably, the growth matrix in step S6 is any one of a matrix with a mass ratio of humus: laterite: perlite = 1:1:1 or a matrix with a mass ratio of humus: perlite = 1:0.5, and the growth matrix is ​​sterilized at 121° C. and high temperature and high pressure for 20 min the day before use and then naturally cooled before use.

[0021] Preferably, the conditions for transplanting to the growth medium for cultivation in step S6 are light cultivation at 28±2°C, controlling the light exposure for 16 hours per day, and covering with a transparent plastic cover 7 days before transplanting.

[0022] The present invention provides a tissue culture method for inducing clustered buds from Yunnan tree peony embryos, which has the following advantages over the prior art:

[0023] The present invention can establish a regeneration system for the cluster bud induction pathway of seed peony embryos by sequentially culturing them with a cluster bud induction medium, a proliferation medium, and a cluster bud rooting medium, so as to obtain complete Yunnan peony plants, facilitate the breeding of Yunnan peony, ensure the healthy growth of the induced Yunnan peony root system, and improve the survival rate of subsequent transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of seed embryo seedlings cultured in different cluster bud induction mediums according to the embodiments of the present invention;

[0025] Figure 2 This is a schematic diagram of the rooting of clustered buds cultured on the rooting medium 1 according to an embodiment of the present invention;

[0026] Figure 3 This is a schematic diagram of the rooting of clustered buds cultured on the rooting medium 2 in the embodiment of the present invention;

[0027] Figure 4 This is a schematic diagram of the rooting of clustered buds cultured in the rooting medium 3 according to the embodiment of the present invention;

[0028] Figure 5 This is a schematic diagram of the rooting of clustered buds cultured in the rooting medium 4 according to the embodiment of the present invention;

[0029] Figure 6 This is a schematic diagram of the rooting of clustered buds cultured in the rooting medium 5 according to an embodiment of the present invention;

[0030] Figure 7 This is a schematic diagram of the rooting of clustered buds cultured in the rooting medium 6 according to an embodiment of the present invention;

[0031] Figure 8 Schematic diagram of the growth of seedlings 15 days after transplanting in an embodiment of the present invention, wherein a is the growth condition after transplanting into substrate 1; b is the growth condition after transplanting into substrate 2. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example:

[0034] Yunnan peony embryo tissue culture experiment:

[0035] 1. Test material selection:

[0036] Fresh Yunnan peony seeds were collected from the Yunnan Xiang Guo Se Yunnan Peony Germplasm Resource Garden (102°53′55″E, 24°45′38″N, 2,733m above sea level) of Yunnan Dian Xiang Guo Se Agricultural Technology Co., Ltd. in Chengjiang City, Yunnan Province. After collection, they were placed in a ventilated place indoors to dry in the shade and stored in a dark refrigerator at 4°C for later use;

[0037] 2. Selection of sterilization methods for embryos:

[0038] The Yunnan peony seeds soaked in 500 mg / L GA3 for 48 hours were transferred to tap water containing detergent to gently scrub the surface dirt and impurities, and then rinsed with running water for 30 minutes. The complete embryos were picked out on the laboratory bench using a scalpel and a dissecting needle and placed in a 2ml centrifuge tube. Then, they were disinfected in a clean bench using three different concentrations (0.3%, 0.4%, and 0.5%) of sodium hypochlorite (NaClO) for 4 minutes, 5 minutes, and 6 minutes, respectively. They were washed with sterile water 3 times and inoculated into 1 / 2MS medium containing 0.2 mg / L GA3. There were a total of 9 treatments, 20 embryos were inoculated in each treatment, repeated 3 times, and 60 embryos were inoculated in each treatment. The contamination rate of different treatments was counted 7 days after inoculation, and the browning rate and mortality rate of the embryos were counted 30 days later; the specific results are shown in Table 1:

[0039] Table 1

[0040]

[0041] Note: The data in the table are mean ± standard deviation.

[0042] That is, the best disinfection method is to select a NaClO concentration of 0.4% and a disinfection time of 5 minutes.

[0043] 3. Germination and culture of embryos:

[0044] The embryos treated by the above optimal disinfection method were inoculated into different germination cultures in Table 2 below, 30 embryos were inoculated into each basic culture medium, repeated 3 times, 90 embryos were inoculated into each culture medium, cultured in the dark at 25±2°C, and the germination was counted every 7 days. After 30 days of culture, the seedling rate, seedling height, root length, etc. of the embryos were counted to select the best germination basic culture medium;

[0045] Table 2

[0046] Germination medium formula Germination medium 1 <![CDATA[MS + 0.2 mg / L GA3 + 0.5 g / L PVP + 7 g / L agar + 30 g / L sucrose]]> Germination medium 2 <![CDATA[1 / 2MS + 0.2 mg / L GA3 + 0.5 g / L PVP + 7 g / L agar + 30 g / L sucrose]]> Germination medium 3 <![CDATA[WPM + 0.2 mg / L GA3 + 0.5 g / L PVP + 7 g / L agar + 30 g / L sucrose]]>

[0047] The results of 30-day culture are shown in Table 3 below (wherein germination rate (%) = (number of embryos with changes / total number of inoculated embryos) × 100%; seedling rate (%) = (number of embryos with elongated upper and lower hypocotyls / total number of inoculated embryos) × 100%):

[0048] Table 3

[0049]

[0050] That is, germination medium 1 is the best germination medium.

[0051] 4. Induction of clustered buds from seed embryos:

[0052] The embryo seedlings with the same growth obtained by culturing with the above germination medium 1 for 20 days were inoculated into different cluster bud induction mediums in Table 4 below, 10 bottles were inoculated with each medium, 3 embryo seedlings were inoculated with each bottle, and 90 embryo seedlings were inoculated with each medium. The embryo seedlings were cultured in a dark environment at 15±1°C for 10 days and then transferred to a white LED light with a light-dark alternation of 16:8 for culture.

[0053] Table 4

[0054]

[0055] After 30 days of culture, the germination of embryos, epicotyl elongation and cluster bud induction in various culture media were observed and counted. The specific results are shown in Table 5 below (epicotyl elongation rate (%) = (length of epicotyl after culture - length of epicotyl before culture) / length of epicotyl before culture × 100%; cluster bud induction rate (%) = (number of embryos induced to cluster buds / total number of inoculated embryos) × 100%):

[0056] Table 5

[0057] Culture medium Germination rate (%) Epicotyl elongation rate (%) Cluster bud induction rate (%) Cluster shoot induction medium 1 78.89％ 63.72％ 46.67％ Cluster shoot induction medium 2 74.44％ 43.24％ 34.44％ Cluster shoot induction medium 3 77.78％ 47.21％ 42.22％ Cluster shoot induction medium 4 66.67％ 28.57％ 26.67％

[0058] That is, the induction medium 1 for clustered buds is the optimal induction medium for clustered buds, and the specific induction medium for clustered buds is as follows: Figure 1 As shown, the bud clusters induced by the induction medium 1 are as follows Figure 1 As shown in middle d, the number of clustered buds is greater than 5; the clustered buds induced by culturing in the induction medium 4 are as follows Figure 1 As shown in a, the number of clustered buds is 1-2; the clustered buds induced by the induction medium 3 are as follows Figure 1 As shown in b, the number of clustered buds is 1-2; the clustered buds induced by the induction medium 2 are as follows Figure 1 As shown in middle c, the number of clustered buds is 3-4.

[0059] 5. Proliferation culture of seed embryo cluster buds:

[0060] The above-mentioned cluster bud induction medium 1 was cultured for 30 days, and the seed embryo seedlings with consistent growth and epicotyl growth in single bud form were selected, and the cluster buds were inoculated into the proliferation medium in the following Table 6 for cluster bud proliferation culture. Each culture medium was inoculated with 20 seed embryo seedlings with epicotyl growth in single bud form, and repeated 3 times. The culture was carried out at 25±2°C under white LED light with 16:8 light and dark alternation. After 30 days, the proliferation rate and average proliferation coefficient of the cluster buds were calculated (proliferation rate (%) = (the number of seed embryos increased by the number of cluster buds / the total number of seed embryos inoculated) × 100, and the proliferation coefficient = the number of cluster buds increased / the number of cluster buds before proliferation culture);

[0061] Table 6

[0062]

[0063]

[0064] The culture results of 30 days are shown in Table 7:

[0065] Table 7

[0066] Culture medium Proliferation rate (%) Proliferation coefficient Proliferation Medium 1 75.79 3.14 Proliferation Medium 2 51.67 1.47 Proliferation Medium 3 55.00 1.50 Proliferation Medium 4 63.33 2.81

[0067] That is, proliferation medium 1 is the optimal proliferation medium.

[0068] 6. Cluster bud rooting culture:

[0069] The clustered buds cultured in the above proliferation medium 1 for 30 days and with the same growth momentum were selected and inoculated into the rooting medium with different formulations in Table 8 below. Thirty seedlings were inoculated into each medium and cultured under light at 20±1°C. After 30 days, the rooting rate and seedling growth status were counted.

[0070] Table 8

[0071]

[0072] The formula of the above improved 1 / 2MS is as follows:

[0073] 720mg / L KNO3, 580mg / L NH4NO3, 106mg / LKH2PO4, 146mg / LMgSO4·7H2O, 255mg / LCaCl2·2H2O, 0.83mg / L KI, 6.2mg / L H3BO3, 22.3mg / L MnSO4·H2O, 8.6mg / L ZnSO4·7H2O, 0.25mg / LNa2MoO4·2H2O, 0.025mg / L CuSO4·5H2O, 0.025mg / L CoCl2·6H2O, 27.6mg / LEDTA-Na2, 30.5mg / L FeSO4·7H2O, 100mg / L inositol, 3.5mg / L glycine, 0.1mg / L thiamine hydrochloride, 0.5mg / L pyridoxine hydrochloride.

[0074] The rooting rate and seedling growth status at 30 days are shown in Table 9 below (rooting rate (%) = (number of tissue culture seedlings with adventitious roots / total number of inoculated tissue culture seedlings) × 100%):

[0075] Table 9

[0076] Rooting medium Rooting rate (%) Root growth status Rooting medium 1 60.00％ The main root is 0.5 cm long, with 4-8 lateral roots, and the average lateral root length is 2 cm Rooting Medium 2 53.33％ The main root is 0.3 cm long, with 1-3 lateral roots, 0.3-1.2 cm in length Rooting Medium 3 46.67％ The main root is 0.2 cm long, with 1-2 lateral roots, 0.45-0.55 cm in length Rooting Medium 4 40.00％ The main root is 0.2 cm long, with 2-3 lateral roots, 0.2-0.5 cm in length Rooting Medium 5 33.33％ The main root is 0.2 cm long, with one lateral root, 0.1-0.2 cm long Rooting Medium 6 23.33％ The main root is 0.2 cm long and has no lateral roots.

[0077] That is, rooting medium 1 is the optimal rooting medium.

[0078] 7. Hardening and transplanting:

[0079] The seedlings cultured in rooting medium 1 with a height of >8 cm, a root length of >5 cm, and a number of fibrous roots >3 were hardened and transplanted. The matrix was configured as shown in Table 10 below:

[0080] Table 10

[0081] Matrix Formula (mass ratio) Matrix 1 Humus: laterite: perlite = 1:1:1 Matrix 2 Humus: Perlite = 1:0.5

[0082] The substrate was sterilized at 121℃ for 20min and cooled naturally the day before use. The seedlings were then removed from the incubator and acclimated for 2 days before being transplanted into the substrates of each group. 100 seedlings were transplanted into each group of substrates and watered thoroughly. The substrates were covered with transparent plastic covers for the first 7 days to prevent water loss. After 15 days of planting, the survival rate of each group was calculated, as shown in Table 11 below:

[0083] Table 11

[0084] Matrix Survival rate Matrix 1 97％ Matrix 2 98％

[0085] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tissue culture method for inducing clustered buds from seed embryos of Paeonia dahliae, characterized in that: The culture method comprises the following steps: S1. Obtaining explants: soaking the seeds of Paeonia dahliae in a gibberellin solution and then washing them, dissecting out the complete embryos and performing disinfection to obtain explants for later use; S2. Germination culture of seed embryos: inoculate the above explants into germination medium, and culture in dark conditions to obtain embryo seedlings; and the formula of the germination medium is: MS+0.2mg / LGA3+0.5g / LPVP+7g / L agar+30g / L sucrose; S3. Induction culture of clustered buds of seed embryos: inoculate the above embryo seedlings into the induction medium for clustered buds for culture, and induce multiple clustered buds; and the formula of the induction medium for clustered buds is: MS+0.5mg / L KT+0.5mg / L 6-BA+30g / L sucrose+7g / L agar+0.5g / LPVP+20mg / L ethyl isothiocyanate+30mg / L tea saponin; S4. Proliferation culture of clustered buds: the embryo seedlings induced with multiple clustered buds are transferred to a proliferation medium for culture until the clustered buds proliferate more than 2 times; and the formula of the proliferation medium is: MS + 0.2mg / L GA3 + 0.5mg / L KT + 0.5mg / L6-BA + 0.3g / L CH + 30g / L sucrose + 7g / L agar + 1g / L PVP + 15mg / L tocopherol acetate; S5. Cluster bud rooting culture: The cluster buds obtained in the above step S4 are inoculated into the cluster bud rooting medium to obtain Yunnan peony seedlings; and the formula of the cluster bud rooting medium is improved 1 / 2MS+1mg / L IBA+0.2mg / L NAA+0.5mg / L IAA+7g / L agar+1g / L PVP+2g / LAC+15mg / L ethyl isothiocyanate+30mg / L tocopherol acetate; and the formula of the improved 1 / 2MS is: 720mg / LKNO3, 580mg / LNH4NO3, 106mg / LKH2PO4, 146mg / LMgSO4·7H2O, 255mg / L CaCl2·2H2O, 0.83mg / LKI, 6.2mg / L H3BO3, 22.3mg / LMnSO4·H2O, 8.6mg / LZnSO4·7H2O, 0.25mg / L Na2MoO4·2H2O, 0.025mg / L CuSO4·5H2O, 0.025mg / LCoCl2·6H2O, 27.6mg / LEDTA-Na2, 30.5mg / LFeSO4·7H2O, 100mg / L inositol, 3.5mg / L glycine, 0.1mg / L thiamine hydrochloride, 0.5mg / L pyridoxine hydrochloride; S6. Hardening and transplanting of seedlings: Select the robust Yunnan tree peony seedlings grown in S4 above, move them out of the incubator for acclimatization for 2 days, remove the rooting medium of the root clusters, and transplant them into the growth medium for cultivation.

2. The tissue culture method according to claim 1, wherein: In the step S1, the Yunnan peony seeds are soaked in a gibberellin solution by soaking the Yunnan peony seeds in a 500 mg / L gibberellin solution for 48-50 hours.

3. The tissue culture method according to claim 1, wherein: In step S1, the Yunnan tree peony seeds are disinfected by soaking them in a sodium hypochlorite solution with a mass concentration of 0.4%-0.5% for 5-6 minutes, and then rinsing them with sterile water for 2-3 times.

4. The tissue culture method according to claim 1, characterized in that: The conditions for germination culture of the embryos in step S2 are dark culture at 25±2°C.

5. The tissue culture method according to claim 1, characterized in that: The temperature condition for the induction culture of clustered buds in step S3 is 15±1°C, and during the culture process, dark culture is first performed, and then the daily lighting time is controlled to 16h for supplementary light culture, and the dark culture time is 10d. White LED light is used as the light source during the supplementary light culture.

6. The tissue culture method according to claim 1, characterized in that: The temperature of the proliferation culture of the clustered buds in step S4 is 25±2° C., and the white LED lamp is used to control the lighting time to 16 h / d.

7. The tissue culture method according to claim 1, characterized in that: The temperature of the clustered bud rooting culture in step S5 is 20±1° C., and the white LED lamp is used to control the lighting time to 16 h / d.

8. The tissue culture method according to claim 1, characterized in that: The standards for healthy and strong Yunnan peony seedlings in step S6 are seedling height>8cm, root length>5cm, and number of fibrous roots>3.

9. The tissue culture method according to claim 1, characterized in that: In step S6, the growth substrate is any one of a substrate with a mass ratio of humus: laterite: perlite = 1:1:1 or a substrate with a mass ratio of humus: perlite = 1:0.5, and the growth substrate is sterilized at 121° C. and high temperature and high pressure for 20 minutes the day before use and then naturally cooled before use.

10. The tissue culture method according to claim 1, characterized in that: The step The conditions for transplanting to the growth medium for cultivation in step S6 are culturing at 28±2°C under light conditions. Control the light intensity to 16 hours per day and cover with transparent plastic cover 7 days before transplanting.

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