Tissue culture and rapid propagation method for flemingia philippinensis

Abstract

The invention relates to the technical field of plant tissue culture, and discloses a tissue culture and rapid propagation method of flemingia philippinensis. Comprising the following steps: accelerating germination and disinfecting seeds of flemingia philippinensis to obtain sterile test-tube plantlets; placing the sterile test-tube plantlet in a differential medium for multiplication culture, and inducing and differentiating adventitious clumpy buds to obtain a subculture seedling; the subculture seedlings are placed in a rooting culture medium for rooting culture, and rooting seedlings are obtained; and transplanting the rooted seedlings into matrix soil for seedling hardening culture. The tissue culture and rapid propagation method of the flemingia philippinensis has the advantages of convenience in material taking, low pollution rate, high multiplication multiple, high rooting rate and high seedling hardening survival rate, more high-quality sterile flemingia philippinensis plants can be obtained in a short time, and the requirements of large-scale production are met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture and reproduction, and particularly relates to a tissue culture and rapid propagation method of F. scandens. BACKGROUND

[0002] F. scandens belongs to a medicinal plant of F. scandens of perennial leguminous vines. It is used for expelling wind and removing dampness, removing blood stasis and detoxification, treating rheumatic arthralgia, chronic nephritis, contusion and injury, carbuncle and laryngeal papilloma. At present, due to the reasons such as overexploitation and scattered distribution of growth areas, the yield of wild F. scandens is getting less and less, the market demand is increasing year by year, and the price is rising year by year. The wild resources of F. scandens are close to exhaustion.

[0003] A tissue culture and propagation method of F. mimosica is disclosed in Chinese patent application publication No. CN105309317A. The method can maintain the original variety of F. mimosica through induction and propagation of adventitious buds of F. mimosica, and can propagate a large number of plants with consistent traits in a short period of time to meet the production needs and provide F. mimosica with excellent traits for factory cultivation. However, in the actual cultivation process, there are problems such as high pollution rate in the cultivation process, and low value-added multiple, rooting rate and seedling survival rate. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a tissue culture and rapid propagation method of F. scandens, which solves the above technical problems in the prior art.

[0005] The purpose of the present application can be achieved by the following technical solutions:

[0006] A tissue culture and rapid propagation method of F. scandens, comprising the following steps:

[0007] S1, catalysis and disinfection of seed raw materials: taking mature seeds of F. scandens as materials, soaking in a constant temperature water bath, and then obtaining mature seed raw materials after cleaning and disinfection;

[0008] S2, obtaining of sterile test tube seedlings: inoculating the disinfected mature seed raw materials into culture medium A to induce germination and obtain sterile test tube seedlings;

[0009] S3, subculture and proliferation of buds: inoculating the sterile test tube seedlings with stem segments into proliferation culture medium B to obtain subculture and proliferation of buds;

[0010] S4, rooting culture of cluster seedlings: cutting the subculture and proliferation seedlings into single buds, inoculating into culture medium C for culture to obtain root seedlings;

[0011] S5, the root seedling is transplanted: the root system seedling of the climbing mile-a-minute plant with 6-8cm of plant height and 3-6cm of root length is selected to carry out the seedling training in the room, and then is transplanted to the substrate soil and is cultured in the greenhouse.

[0012] Further, in S1, the constant-temperature water bath environment is 55 DEG C for 8-28h.

[0013] Further, in S1, the cleaning and disinfection method is as follows: first, washing with sterile water for 3-5 times, then disinfecting with 75% alcohol (75% by volume) for 0.5-1min, washing with sterile water again for 3-5 times, and then disinfecting with 2-8% sodium hypochlorite for 3-8min.

[0014] Further, in S2, the culture medium A is MS-based medium, with 7g / L agar and 30g / L sucrose, and the PH value is adjusted to 6.0.

[0015] Further, in S2, the culture environment is as follows: culture temperature is 26 DEG C, light intensity is 3800Lux, light time is 12h / d, and culture time is 45d.

[0016] Further, in S3, the culture medium B is MS-based medium, with 7g / L agar, 30g / L sucrose, 0.4-1.5mg / L 6-benzyladenine and 0.1-0.2mg / L naphthalene acetic acid, and the PH value is adjusted to 6.0.

[0017] Further, in S4, the culture environment is as follows: temperature is 26 DEG C, light intensity is 3800Lux, light time is 12h / d, and culture time is 25d.

[0018] Further, in S4, the culture medium C is 1 / 2MS-based medium, with 7g / L agar, 20g / L sucrose, 0.3-1.0mg / L vitamin B1, 0.3-1.0mg / L indole butyric acid and 0.3-1.0mg / L naphthalene acetic acid, and the PH value is adjusted to 6.0.

[0019] Further, in S5, 32-hole plug is used for sealing and covering, and the seedling training is carried out in the room at room temperature of 25 DEG C; after 10d of seedling training, the seedling is transplanted to the substrate soil large pot and continues to grow in the greenhouse.

[0020] Further, in S5, the substrate soil comprises the following components: peat, coconut chaff and perlite in a ratio of 5:3:1.

[0021] The beneficial effects of the application are as follows:

[0022] 1、The present application disinfects by 75% alcohol and 2-8% sodium hypochlorite, has higher disinfection rate, and compared with using mercury, reduces the toxicity rate of seeds, advances the germination time of seeds, and the germination rate can reach more than 98%.

[0023] 2、The present application uses tissue culture technology, has convenient material taking, low pollution rate, high multiplication, high rooting rate and high seedling survival rate, can prevent plant virus damage, and can obtain more high-quality sterile F. cavaleriei plants in a short time, improve the overall yield, and meet the current market demand.

[0024] 3、The present application adds different proportions of cytokinin and auxin in three different stages of sterile test tube seedlings, subculture of buds, and rooting of cluster seedlings, the proliferation coefficient can reach 7.5, the rooting rate is as high as 100%, and the survival rate after transplanting reaches 100%, effectively solving the problems of shortage of F. cavaleriei medicinal material resources and large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0026] Figure 1 is the growth condition of the seed after disinfection in example 1 of the present application embodiment;

[0027] Figure 2 is the growth condition of the seed culture for 45d in example 2 of the present application embodiment;

[0028] Figure 3 is the plant growth condition of the stem section with buds induced to adventitious buds for 55d in different concentrations of 6-BA medium in example 3 of the present application embodiment.

[0029] Figure 4 is the browning condition of the medium of the stem section with buds induced to adventitious buds for 55d in different concentrations of 6-BA medium in example 3 of the present application embodiment;

[0030] Figure 5 is the plant growth condition of the stem section with buds induced to adventitious buds for 30d in different concentrations of 6-BA medium in example 3 of the present application embodiment.

[0031] Figure 6 is the root growth condition of the rooting medium under the action of different hormones for 25d in example 4 of the present application.

[0032] Figure 7 is the plant growth condition after seedling in the present application embodiment.

[0033] Figure 8 is the overall flowchart of the embodiment of the present application. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0035] As shown in Figure 8 The present application provides a tissue culture and rapid propagation method of F. scandens, comprising the following steps:

[0036] S1, catalysis and disinfection of seed raw materials: taking mature seeds of F. scandens as materials, soaking in a constant temperature water bath, then cleaning and disinfecting to obtain mature seed raw materials;

[0037] S2, obtaining of sterile test tube seedlings: inoculating the disinfected mature seed raw materials into culture medium A to induce germination and obtain sterile test tube seedlings;

[0038] S3, subculture and cluster bud proliferation culture: inoculating the sterile test tube seedlings with buds into proliferation culture medium B to obtain subculture cluster buds;

[0039] S4, cluster seedling rooting culture: cutting the subculture cluster seedlings into single buds and inoculating into culture medium C for culture to obtain root seedlings;

[0040] S5, rooting seedling hardening and transplanting: screening F. scandens plant root seedlings with a plant height of 6-8 cm and a root length of 3-6 cm for hardening in a room, and then transplanting into substrate soil for culture in a greenhouse.

[0041] Embodiment 1

[0042] Catalysis of seed raw materials: taking high-quality mature seeds of F. scandens as materials, washing 3-5 times with ultrapure water, placing in a constant temperature water bath, setting the temperature to 55°C, and soaking for 8-28h. After soaking, washing 3-5 times with ultrapure water, and then disinfecting in an ultraclean bench.

[0043] As shown in Table 1 below, the comparison of germination breaking rate under different soaking times.

[0044] Table 1

[0045] Serial number Temperature of constant temperature water bath Soaking time Crushing rate 1 55℃ 8h 1％ 2 55℃ 12h 1.5％ 3 55℃ 16h 2％ 4 55℃ 20h 8.7％ 5 55℃ 24h 15.7％ 6 55℃ 28h 27.2％

[0046] That is, in the present application, the highest breaking rate of mature seeds is achieved at a soaking time of 24 hours, and thus soaking the seeds at this time can shorten the germination period of the seeds.

[0047] Seed sterilization: After the seed germination treatment, the seed is sterilized in a clean bench. The steps are as follows: first, rinse with sterile water for 3-5 times, second, sterilize with 75% alcohol for 30s-1min, third, rinse with sterile water for 3-5 times, fourth, sterilize with 2-8% sodium hypochlorite for 3-8min, and finally, rinse with sterile water for 3-5 times. After the water is absorbed, the seed is inoculated in MS medium A. After 15 days, the contamination rate is counted (as shown in Table 2). Figure 1 Compared with the mercury method in the prior art, a certain volatile toxicity is contained, which affects the safety of the user.

[0048] As shown in Table 2, the results of the seed treated by different sterilization methods are shown. Contamination rate = number of contaminated seeds ÷ number of transferred seeds; Germination rate = number of germinated plants ÷ (number of inoculated seeds - number of contaminated seeds)

[0049] Table 2

[0050]

[0051]

[0052] As shown in Table 1, the longer the treatment time of high-concentration NaClO, the higher the toxicity of the seed, and the seed will not germinate subsequently. Therefore, the efficiency is optimal when the sterilization time of 75% alcohol is 1min and the sterilization time of 4% NaClO is 5min.

[0053] Example 2

[0054] Obtaining of sterile test-tube seedlings: the seeds of the seed germination and sterilization treatment in example 1, No. 4 (Table 2) are inoculated in MS medium A to induce germination and obtain sterile test-tube seedlings. The culture environment is as follows: temperature is 26℃, light intensity is 3800Lux, light time is 12h / d. After 15 days of culture, the seeds start to germinate successively. After 45 days of culture, the seed germination rate reaches more than 98%, and the sterile test-tube seedlings with a height of 10cm are obtained. At this time, as shown in Table 3, the growth of the sterile test-tube seedlings of the climbing mile-a-minute weed is as shown in Table 4. Figure 2 Figure 2

[0055] Example 3

[0056] ​​Subculture of bud clusters: The sterile test-tube seedlings obtained in Example 2 were cut into 3cm stem segments with buds using surgical scissors and inoculated into proliferation medium B to obtain a large number of bud clusters. Five single buds were inoculated into each bottle of medium B, with 20 bottles inoculated for each medium B. The culture was maintained at 26℃, light intensity of 3800 Lux, and a photoperiod of 12h / d for 45 days to obtain sterile test-tube seedlings with a height of 6–10cm. Medium B consisted of MS and B5 as basal media, supplemented with 7g / L agar, 30g / L sucrose, 0.4–1.5mg / L 6-benzyladenine (6-BA), and 0.1–0.2mg / L naphthaleneacetic acid, with a pH of 6.0.

[0057] Table 3 below shows the plant growth coefficients after 55 days of induction of adventitious buds from stem segments with buds in different concentrations of 6-BA medium (e.g., ...). Figure 3 (As shown).

[0058] Table 3

[0059]

[0060] Among them, the parameters of culture medium B, serial number 7, were optimal, with a proliferation coefficient of 7.5. At this point, the higher the 6-BA content, the deeper the browning of culture medium B, the looser the callus tissue in the roots, and the more it affected the plant's absorption of nutrients. As the growth cycle lengthened, the plants began to show signs of drying out and became thin.

[0061] The utilization rate is low during rooting or redifferentiation. The differentiation coefficient decreases sharply after the 6-BA content exceeds 0.5 mg / L. In different types of plant tissue culture, the selection of the cytokinin to auxin ratio is crucial. This is a common characteristic of most growth regulators: they promote differentiation at appropriate concentrations, but inhibit it beyond these concentrations.

[0062] Table 3 shows the sequence number of the adventitious bud induction scheme 1 ( Figure 3 a) Plant proliferation after 55 days ( Figure 3 b) and Scheme 7 Figure 3 c) Proliferation and growth at different stages.

[0063] like Figure 4 As shown in Table 3, the browning of culture medium B was observed 55 days after the induction of adventitious shoots.

[0064] Example 4

[0065] Using different basal culture media B, the same hormones were added for proliferation culture. Table 4 below shows the plant proliferation coefficient of stem segments with buds after inducing adventitious buds for 30 days in different basal culture media B.

[0066] After 30 days of culture, the proliferation coefficient of one seed in different culture medium B was counted. The counting method of proliferation coefficient was: proliferation coefficient = the number of rooted transplanted plants ÷ the number of seeds.

[0067] Table 4

[0068]

[0069] As shown in Table 4, B5 medium is not suitable for differentiation experiment of Flemingia miocarpa, and the plantlets almost do not differentiate, the height is not high, and the leaves do not start to grow in 30 days. The difference is obviously great compared with Flemingia miocarpa cultured in MS medium. The plantlet proliferation after 30 days of induction of adventitious buds is shown in Table 6. Figure 5

[0070] Example 5

[0071] Rooting culture of cluster seedlings: the subculture cluster buds obtained in Example 4, MS + 0.4 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7 g / L agar, were cut into single buds and inoculated into rooting medium C for culture, and the culture environment was: temperature 26°C, light intensity 3800 Lux, light time 12 h / d, culture time 25 d, and then the healthy root seedlings were obtained.

[0072] The rooting medium C used was: 1 / 2MS as basic medium, 7 g / L agar, 20 g / L sucrose, 0.3-1.0 mg / L vitamin B1, 0.3-1.0 mg / L indolebutyric acid, 0.3-1.0 mg / L naphthalene acetic acid, and the pH value was 6.0.

[0073] The rooting medium C used is shown in Table 5:

[0074] Table 5

[0075]

[0076]

[0077] After 25 days of rooting culture, the rooting of single buds in different rooting medium C was counted, and the results are shown in Table 6. The root growth speed of rooting medium C No. 2 is the slowest, and the roots of the plantlets are easy to break in the washing stage before hardening-off due to hormone problem; the root growth speed of rooting medium C No. 5 is fast, the root length reaches 3-5 cm, and the time of growing fibrous roots is advanced. The rooting of plantlets after 25 days of rooting culture shown in Table 5 is shown in Table 7. Figure 6 Figure 6

[0078] ​​​Rooted seedling acclimatization and transplanting: when the height of the robust F. scandens plants obtained in Example 4 is 6-8 cm and the root length is 3-6 cm, the robust plants are selected, washed with ultrapure water to remove the culture medium on the surface of the plants, and acclimatized in a 32-hole plug tray under closed cover in a room with a room temperature of 25°C. After 10 days of acclimatization, the plants are transplanted into a large pot of substrate soil (peat: coconut coir: perlite = 5:3:1), and continue to grow in a greenhouse. The growth of the plants after acclimatization is shown in Table 2. Figure 7

[0079] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application.​

Claims

1. A method for rapid propagation of *Millettia dielsiana* via tissue culture, characterized in that, Includes the following steps: S1. Catalysis and disinfection of seed raw materials: Take mature seeds of the vine-like *Millettia dielsiana* as raw materials, soak them in a constant temperature water bath, and then clean and disinfect them to obtain mature seed raw materials. S2. Obtaining sterile test-tube seedlings: Sterilized mature seed material is inoculated into culture medium A to induce germination and obtain sterile test-tube seedlings; S3. Subculture of bud clusters: Sterile test-tube seedlings with buds are inoculated into proliferation medium B to obtain subculture of bud clusters. S4. Rooting culture of clump seedlings: Take subcultured clump seedlings, cut them into single buds, inoculate them into culture medium C for culture, and obtain rooted seedlings; S5. Hardening off and transplanting of rooted seedlings: Selected root seedlings are hardened off indoors and then transplanted into substrate soil for cultivation in a greenhouse.

2. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In S1, the constant temperature water bath environment is: soaking at 55℃ for 8 to 28 hours.

3. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In S1, the cleaning and disinfection method is as follows: first, rinse with sterile water 3 to 5 times, then disinfect with 75% alcohol for 0.5 to 1 minute, rinse with sterile water again 3 to 5 times, and then disinfect with 2 to 8% sodium hypochlorite for 3 to 8 minutes.

4. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In S2, culture medium A is based on MS medium, with 7 g / L agar and 30 g / L sucrose added, and the pH of culture medium A is adjusted to 6.

0.

5. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In S2, the culture environment is as follows: culture temperature 26℃, light intensity 3800 Lux, light duration 12h / d, and culture time 45d.

6. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In S3, culture medium B is based on MS medium, with the addition of 7 g / L agar, 30 g / L sucrose, 0.4–1.5 mg / L 6-benzyladenine, and 0.1–0.2 mg / L naphthaleneacetic acid, and the pH of culture medium B is adjusted to 6.

0.

7. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In S4, the culture environment is: temperature 26℃, light intensity 3800 Lux, light duration 12h / d, culture for 25d.

8. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In S4, culture medium C is based on 1 / 2 MS medium, with the addition of 7 g / L agar, 20 g / L sucrose, 0.3–1.0 mg / L vitamin B1, 0.3–1.0 mg / L indolebutyric acid, and 0.3–1.0 mg / L naphthaleneacetic acid, and the pH of culture medium C is adjusted to 6.

0.

9. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In the S5, seedlings are covered with 32-cell seedling trays and hardened off indoors at a temperature of 25°C. After 10 days of hardening off, the seedlings are transplanted into large pots with substrate soil and continue to grow in a greenhouse.

10. The method for rapid propagation of *Millettia dielsiana* via tissue culture according to claim 1, characterized in that, In S5, the substrate soil used consists of the following components: peat: coconut coir: perlite = 5:3:1.

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