Tissue culture method of medicinal plant divaricate saposhnikovia root

Abstract

The invention discloses a tissue culture method of a medicinal plant divaricate saposhnikovia root. The method comprises the following steps: taking a stem section with buds as an explant, adding 30g / L of sucrose, 8g / L of agar and 0.5 mg / L of 6-BA into an MS (Murashige and Skoog) culture medium as an induction culture medium, and ensuring that the pH value is A rooting culture medium is formed by adding 30 g / L of sucrose, 8 g / L of agar and 0.5 mg / L of IBA into an MS culture medium, and the pH value is 5.8. By using the method, the axillary bud induction rate is 100%, the multiplication coefficient reaches 2.29, and the average stem height is 1.1 cm; the rooting rate reaches 91%, the rooting coefficient reaches 17.25, and the formed roots are dense and thick and are suitable for transplanting. The method not only accelerates the breeding speed of the divaricate saposhnikovia root, but also keeps excellent characteristics of parents, is of great significance to rapid breeding and genetic improvement, and lays a technical foundation for commercial production.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture method of the medicinal plant fangfeng. Background Art

[0005] At present, the research on Radix Saposhnikoviae Radix by researchers is still limited, mainly focusing on pharmacognosy identification, chemical composition analysis and pharmacological activity, including the components such as lactones, glycosides, alkaloids, terpenes in Radix Saposhnikoviae Radix, and the application of Radix Saposhnikoviae Radix in the field of traditional Chinese medicine. After inquiry, no systematic research on the tissue culture of Radix Saposhnikoviae Radix has been found.

[0006] Sexual reproduction may not be conducive to the stability of genetically excellent traits due to its large genetic variability, and its reproduction cycle is relatively long. In contrast, plant tissue culture technology can efficiently cultivate new plants in a short period of time by culturing plant tissues, organs or embryos in vitro under laboratory conditions. This method not only speeds up the reproduction speed, but also maintains the excellent characteristics of the parents, which is of great significance for the rapid reproduction and genetic improvement of plants.

[0007] The history of plant tissue culture technology can be traced back more than a hundred years ago, when Gottlieb Haberlandt successfully created the first callus culture in the early 20th century. In 1940, with the continuous advancement of technology, plant cell suspension culture technology was further developed. This technology developed rapidly in the 1950s and was mainly used to produce valuable plant secondary metabolites, such as paclitaxel, shikonin, artemisinin, ginsenosides and wormwood alkaloids.

[0008] At present, rapid reproduction using tissue culture technology is an effective and important path. Tissue culture technology has the advantages of low cost, short growth cycle, fast reproduction speed, high efficiency, and controllable environmental conditions. It can reduce errors caused by seasons, geographical environment, etc., and obtain a large number of sterile seedlings in a short time.

[0009] The flowering period of Radix Saposhnikoviae is from August to September, and the fruiting period is from September to November. Under natural conditions, it takes about 7 months for a seed to germinate and grow into a complete plant. The natural growth cycle takes a long time, and the economic cost of the cultivation process is high. There is no relevant literature report on the tissue culture of Radix Saposhnikoviae. Summary of the invention

[0010] In view of the above-mentioned deficiencies in the prior art and in order to improve the efficiency of the seedling process of Saposhnikovia divaricata, the present invention is committed to constructing a set of efficient tissue culture systems for Saposhnikovia divaricata and providing a tissue culture method for Saposhnikovia divaricata, which can significantly reduce the time required for the natural growth cycle of Saposhnikovia divaricata and reduce the economic cost of its cultivation process.

[0011] A tissue culture method of fangfeng of the present invention comprises the following steps:

[0012] S1. Take the stems of sterile seedlings of Saposhnikovia divaricata, cut the stem segments with buds as explants, inoculate them into induction medium, and induce the growth of axillary buds; the induction medium is MS medium supplemented with 30g / L sucrose, 8g / L agar and 0.5mg / L 6-BA, pH5.8;

[0013] S2. Cut a single axillary bud and inoculate it into a rooting medium for cultivation to induce rooting and obtain regenerated plants of Saposhnikovia divaricata; the rooting medium is MS medium supplemented with 30 g / L sucrose, 8 g / L agar and 0.5 mg / L IBA, pH 5.8.

[0014] Preferably, the sterile seedlings of Radix Saposhnikoviae are obtained by the following method: selecting complete and full-seeded Radix Saposhnikoviae seeds, rinsing, heating in a 60°C water bath for 5 minutes, sterilizing, and then inoculating into 1 / 2MS basal culture medium, and culturing to obtain sterile seedlings; the 1 / 2MS basal culture medium is 1 / 2MS culture medium supplemented with 30g / L sucrose and 8g / L agar, pH 5.8; the 1 / 2MS culture medium is a culture medium in which the concentrations of all components of the MS culture medium are halved.

[0015] More preferably, the rinsing is performed with tap water for 10 minutes, the disinfection is performed by soaking in a 75% by volume ethanol aqueous solution for 3 times for 15 seconds, then soaking in a 5% by mass NaClO solution for 5 minutes, rinsing with sterile water for 5 times and then absorbing excess water on the seeds with sterile filter paper.

[0016] Preferably, the stem segments with buds are obtained by the following method: taking the stems of sterile schefflera seedlings, removing the leaves and petioles on the stems, and then cutting the stems into small segments of 1 cm in length and each small segment containing an axillary bud, thereby obtaining the stem segments with buds.

[0017] Preferably, the axillary bud is a single bud with a bud length of more than 1.5 cm.

[0018] Preferably, the culture conditions are: a light cycle of 12 hours, a dark cycle of 12 hours, a light intensity of 2000lx-2500lx, a relative humidity of 80%, and a temperature of 22°C.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Rapid propagation by tissue culture can not only maintain the excellent characteristics of the parents, but also has important significance for the rapid propagation and genetic improvement of plants. Tissue culture technology has the advantages of low cost, short growth cycle, fast propagation speed, high efficiency, and controllable environmental conditions. It can reduce the errors caused by seasons and geographical environments. The method of the present invention can obtain a large number of sterile seedlings of Fangfeng in a short time.

[0021] Compared with the natural growth method, the tissue culture method of the present invention can significantly reduce the time required for the natural growth cycle of the Radix Saposhnikoviae and reduce the economic cost of the cultivation process. It provides a new approach and theoretical support for the application and in-depth research of the Radix Saposhnikoviae and lays a technical foundation for the commercial production of the Radix Saposhnikoviae. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure shows the effect of different concentrations of 6-BA on the induction rate of axillary buds; (the significant marks in the figure, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, the same below).

[0023] Figure 2 The effect of different concentrations of 6-BA on the proliferation coefficient of axillary buds.

[0024] Figure 3 The effect of different concentrations of 6-BA on the average stem height of axillary buds.

[0025] Figure 4 This is the effect of different concentrations of 6-BA on axillary bud induction.

[0026] Figure 5 This is the growth status of axillary buds at 30 days.

[0027] Figure 6 The effect of different concentrations of IBA on the rooting rate of axillary buds.

[0028] Figure 7 This is the effect of different concentrations of IBA on the rooting coefficient of axillary buds.

[0029] Figure 8 The effect of different concentrations of IBA on the average root length of axillary buds.

[0030] Fig. 9 This is the effect of different concentrations of IBA on rooting induction of axillary buds. DETAILED DESCRIPTION

[0031] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention. The reagents and instruments used are shown in Tables 1 and 2:

[0032] Table 1 Main reagents

[0033]

[0034] Table 2 Main instruments

[0035]

[0036]

[0037] Example 1

[0038] 1. Preparation of MS basal medium and culture conditions

[0039] In the MS medium, add 30g / L sucrose and 8g / L agar, and then adjust the pH to 5.80 with sodium hydroxide. Sterilize for 20 minutes at 121°C and 101MPa. After sterilization, pour the medium into the tissue culture bottle and wait for it to cool and solidify before use, thus preparing a solid MS basal medium.

[0040] In this embodiment, the culture conditions are set as follows: a light cycle of 12 hours, a dark cycle of 12 hours, a light intensity maintained at 2000 lx to 2500 lx, a relative humidity maintained at 80%, and a temperature controlled at 22°C.

[0041] 2. Clean and disinfect the seeds

[0042] Collect the seeds of Radix Angelicae Pubescentis, store them in a centrifuge tube filled with color-changing silica gel, and store them in a refrigerated environment at 4℃~8℃ for later use. Select the complete and plump seeds of Radix Angelicae Pubescentis, rinse them with tap water for 10min to clean them; transfer the rinsed seeds to a 1.5mL sterile centrifuge tube, add an appropriate amount of sterile water, and heat them in a 60℃ water bath for 5min to wake up the seeds; remove the sterile water in the centrifuge tube, soak them in a 75% ethanol aqueous solution for 3 times for 15s, and then soak them in a 5% NaClO solution for 5min for disinfection. Transfer the centrifuge tube to the clean bench, rinse them with sterile water 5 times to clean the seeds, and invert the seeds on sterile filter paper to absorb excess water for later use.

[0043] 3. Obtaining Sterile Seedlings

[0044] Use tweezers to inoculate the sterilized seeds into 1 / 2MS basal medium (i.e., 1 / 2MS medium + 30 g / L sucrose + 8 g / L agar, pH 5.8; wherein 1 / 2MS medium is a medium in which the concentrations of all components of MS medium are halved), culture for 10 days, and obtain sterile seedlings.

[0045] 4. Induction of axillary buds by different concentrations of 6-BA

[0046] Preliminary experiments have shown that callus and axillary buds can be directly induced from sterile seedlings of Saposhnikovia divaricata. Axillary buds are not only easy to form, but also differentiate quickly, starting to differentiate in about two weeks. A single explant can induce multiple small buds to grow, quickly obtaining a large number of differentiated axillary buds. Compared with inducing callus through leaf wounds, the method of using stem segments with buds significantly reduces the time required to obtain regenerated plants and greatly reduces the cost of cultivation. Therefore, stem segments with buds were used to induce axillary bud growth in subsequent experiments.

[0047] On an ultra-clean workbench, select sterile seedlings with good and consistent growth conditions, cut a section of stem with scissors, remove the leaves and petioles on it, and then cut the stem into small sections of about 1 cm in length, and make sure that each small section contains an axillary bud, thereby obtaining a stem section with buds.

[0048] Different concentrations of 6-BA were added to the MS basal medium as an induction medium, and the cut stem segments with buds were inoculated into the induction medium to induce the growth of axillary buds.

[0049] As shown in Table 3, 6 different concentration gradients were set, with group B1 as the control group and groups B2 to B6 as the treatment groups. Ten culture bottles were inoculated under each concentration condition, and 3 stem segments with buds were placed in each bottle. The whole experiment was repeated 3 times. After 15 days of cultivation, the growth of axillary buds was observed, and the axillary bud induction rate, axillary bud proliferation coefficient and average stem height were calculated.

[0050] Table 3 Different concentrations of 6-BA

[0051]

[0052] 5. Induction of rooting of axillary buds by different concentrations of IBA

[0053] Different concentrations of IBA were added to MS basal medium as rooting medium to evaluate its effect on the rooting effect of axillary buds. Samples were selected from axillary buds that had been induced for 30 days, had uniform growth, and had a bud length of approximately 1.5 cm. They were cut with a scalpel and vertically implanted into the rooting medium.

[0054] As shown in Table 4, the experiment was divided into 5 groups, with group C1 as the control group and groups C2 to C5 as the treatment groups. Each group was cultured in 10 bottles, with 3 single buds placed in each bottle, and the experiment was repeated 3 times. After 20 days of culture, the rooting was observed, and the rooting rate, rooting coefficient and average root length were calculated.

[0055] Table 4 Different concentrations of IBA

[0056]

[0057] 6. Statistical indicators

[0058] The experimental data obtained were sorted and analyzed by Excel program, and TTEST test was performed (P < 0.05 was statistically significant). The calculation method of each evaluation standard is as follows:

[0059] Axillary bud induction rate = (number of stem segments induced to produce axillary buds / number of inoculated stem segments) × 100%; axillary bud proliferation coefficient = (number of long axillary buds / number of inoculated stem segments) × 100%.

[0060] Rooting rate = (number of adventitious root seedlings / number of single buds grafted) × 100%; rooting coefficient = (number of roots / number of adventitious root seedlings) × 100%.

[0061] 7. Calculation results of different concentrations of 6-BA on axillary bud induction

[0062] The results showed that different concentrations of 6-BA medium could induce axillary buds from stem segments with buds. Figure 1 As shown in the figure, the axillary bud induction rates of 1.0 mg / L 6-BA in group B3 and 1.5 mg / L 6-BA in group B4 were statistically different from those of the control group B1 (P < 0.05), and the axillary bud induction rates were significantly lower; Figure 2 As shown, the proliferation coefficient (P<0.01 or P<0.05) and average stem height (P<0.001) of the treatment groups with concentration ratios of group B2 and group B4 were statistically different from those of the control group B1.

[0063] In all groups with different concentrations, although each group could induce axillary buds, there were large differences in the induction rates. Figure 1 As shown in Figure 2, when the 6-BA concentration is 0 mg / L and 0.5 mg / L, it is more conducive to the induction of axillary buds, and the highest induction rate is 100%. Figure 2 As shown in the figure, when 6-BA was 0.5 mg / L (group B2), the proliferation coefficient was the highest, which was 2.29. Figure 3 As shown, when 6-BA was 1.5 mg / L (B4 group) and 0.5 mg / L (B2 group), the average stem height was the highest, which was 1.2 cm and 1.1 cm, respectively.

[0064] Observation found that Figure 4 As shown in Figure 1, when the axillary buds begin to germinate, a small amount of callus tissue will appear at the incision. The small amount of callus tissue that appears at the incision can further differentiate and develop into buds. After 30 days of cultivation, Figure 5 As shown, a cluster-like structure consisting of 3 to 5 small buds can be observed in the leaf axil. These buds are usually thin and grow slowly.

[0065] From this experiment, we can see that 6-BA has little effect on the induction rate. As the concentration of 6-BA increases, it does not promote the increase of the induction rate, but it can effectively increase the proliferation coefficient and promote the proliferation of axillary buds. Therefore, group B2 (MS basal medium + 0.5 mg / L 6-BA) was used as the best induction medium to obtain the best axillary bud induction effect.

[0066] 8. Calculation results of different concentrations of IBA on rooting induction of axillary buds

[0067] like Figure 6 As shown in the figure, the rooting rate of axillary buds under 0.3mg / L IBA and 0.5mg / L IBA conditions was as high as 91%, with no statistical difference. Figure 7 As shown in the figure, the rooting coefficient was the highest (P < 0.01) when the MS basal medium was supplemented with 0.5 mg / L IBA, which was 17.25. Figure 8 As shown in the figure, the average root length decreased with the increase of concentration, and there was a statistical difference (P < 0.01 or P < 0.001). In the MS basal medium without IBA (i.e., the IBA concentration was 0 mg / L), the average root length reached the maximum, with an average value of 3.29 cm. However, it was found during the experiment that the more developed the root system, the more conducive it was for the plant to capture the nutrients it needed. At a concentration of 0.5 mg / L IBA, the roots formed were dense and thick, as shown in the figure. Fig. 9 The results of comprehensive analysis showed that the rooting medium with the best rooting effect was MS basal medium + 0.5 mg / L IBA.

Claims

1. A tissue culture method of fangfeng, characterized in that: The following steps are involved: S1. Take the stems of sterile seedlings of Saposhnikovia divaricata, cut the stem segments with buds as explants, inoculate them into induction medium, and induce the growth of axillary buds; the induction medium is MS medium supplemented with 30g / L sucrose, 8g / L agar and 0.5mg / L 6-BA, pH5.8; S2. Cut a single axillary bud and inoculate it into a rooting medium for cultivation to induce rooting and obtain regenerated plants of Saposhnikovia divaricata; the rooting medium is MS medium supplemented with 30 g / L sucrose, 8 g / L agar and 0.5 mg / L IBA, pH 5.

8.

2. The tissue culture method of siler according to claim 1, characterized in that: The sterile seedlings of Radix Saposhnikoviae are obtained by the following method: complete and full-seed Radix Saposhnikoviae seeds are selected, washed, heated in a water bath at 60°C for 5 minutes, sterilized, and then inoculated into 1 / 2MS basic culture medium, and cultured to obtain sterile seedlings; the 1 / 2MS basic culture medium is 1 / 2MS culture medium supplemented with 30g / L sucrose and 8g / L agar, pH 5.8; the 1 / 2MS culture medium is a culture medium in which the concentrations of all components of the MS culture medium are halved.

3. The tissue culture method of fangfeng according to claim 2, characterized in that: The washing is to wash with tap water for 10 minutes, the disinfection is to soak the seeds in a 75% ethanol aqueous solution for 3 times for 15 seconds, then soak them in a 5% NaClO solution for 5 minutes, wash them with sterile water for 5 times, and then use sterile filter paper to absorb excess water on the seeds.

4. The tissue culture method of siler according to claim 1, characterized in that: The stem segments with buds are obtained by the following method: taking the stems of sterile fangfeng seedlings, removing the leaves and petioles on the stems, and then cutting the stems into small segments with a length of 1 cm and each small segment containing an axillary bud, thereby obtaining the stem segments with buds.

5. The tissue culture method of siler according to claim 1, characterized in that: The axillary bud single bud is an axillary bud single bud with a bud length of more than 1.5 cm.

6. The tissue culture method of fangfeng according to claim 1 or 2, wherein the culture conditions are: light cycle 12h, dark cycle 12h, light intensity 2000lx-2500lx, relative humidity 80%, and temperature 22°C.

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