A rapid propagation method of Platycodon grandiflorum tissue culture

Abstract

The invention discloses a method for rapid propagation of Platycodon grandiflorum by tissue culture, which belongs to the technical field of rapid propagation of plants. The method selects stem segments as explants, provides a callus induction culture medium suitable for Platycodon grandiflorum, and combines a proliferation culture medium with a rooting culture medium. During seedling hardening and transplantation, the cover is not opened to ensure environmental humidity. The transplantation matrix adopts nutrient soil: vermiculite: perlite = 1:1:1. The method greatly improves the propagation coefficient of Platycodon grandiflorum, shortens the propagation time, optimizes the rapid propagation technology of Platycodon grandiflorum by tissue culture in the prior art, significantly saves the cultivation time cost, and is suitable for large-scale rapid tissue culture seedlings of Platycodon grandiflorum.

Description

Technical Field

[0001] The invention relates to a plant tissue culture rapid propagation technology, in particular to a platycodon tissue culture rapid propagation method. Background Art

[0002] Platycodon grandiflorum Platycodon grandiflorus ) is also known as Baoaohua, Baiyao, stalk grass, bitter platycodon, dolaji, bell flower, etc. It is a perennial herb. It not only has outstanding medicinal and edible value, but also has strong ornamental value due to its upright and beautiful plant shape, bright color and strong resistance to adversity. It is deeply loved and concerned by people. Common propagation methods of Platycodon include: seed propagation, cutting propagation, tissue culture, etc.

[0003] Tissue culture technology has attracted extensive attention and research from scholars at home and abroad because it can culture Platycodon grandiflorum in vitro without being affected by external environmental conditions, produce enough young shoots in a short period of time, thereby accelerating the growth and reproduction process of Platycodon grandiflorum and meeting market demand.

[0004] The existing technology for Platycodon grandiflorum tissue culture research is relatively complete, but there are still areas that need improvement, especially in the proliferation coefficient, rooting quality and quantity. Summary of the invention

[0005] The present invention aims to optimize the tissue culture rapid propagation technology of Platycodon grandiflorum, and obtains the optimal combination by selecting suitable explants, selecting suitable hormone types and concentrations for different stages, significantly improving the proliferation coefficient of Platycodon grandiflorum tissue culture, optimizing the rooting culture effect, and optimizing the efficiency of Platycodon grandiflorum tissue culture rapid propagation as a whole, thereby providing technical support for the high-efficiency production of Platycodon grandiflorum. The scheme of the present invention is as follows:

[0006] In order to achieve the above object, the present invention provides a method for rapid propagation of Platycodon grandiflorum through tissue culture, comprising the following steps to provide the following technical solution:

[0007] S1. Prepare the following culture medium;

[0008] MS basal medium: MS basal medium adopts MS formula, and MS basal medium also contains: carrageenan 7g / L, sucrose 30g / L, pH adjusted to 5.8-6.0, sterilized at 121°C for 20min for use;

[0009] Callus induction medium: Add 6-BA 3.0 mg / L and NAA 1.0 mg / L to MS basal medium, sterilize at 121°C for 20 min for later use;

[0010] Proliferation medium: Add 0.5 mg / L 6-BA and 0.05 mg / L NAA to MS basal medium, sterilize at 121°C for 20 min and set aside;

[0011] Rooting medium: Use 1 / 2MS basal medium and add IBA 2.0ml / L, sterilize at 121℃ for 20min for later use;

[0012] S2. Perform callus induction culture: select stem segments as explants, inoculate the stem segments with leaf nodes into the callus induction culture medium (MS+6-BA 3.0mg / L+NAA 1.0mg / L), place in a tissue culture room at room temperature of 25°C, illuminate for 12 hours, and cultivate with a light intensity of 1000 1x to form callus blocks and bud clusters;

[0013] S3, performing proliferation culture: selecting the bud cluster obtained in step S2 and inoculating it into the proliferation medium (MS+6-BA 0.5 mg / L+NAA 0.05 mg / L) to cultivate seedlings;

[0014] S4, performing rooting culture: after the seedlings cultured in step S3 grow to 2-3 cm, inoculate them into the rooting medium (1 / 2MS+IBA 2.0 mg / L) for rooting cultivation;

[0015] S5, tissue culture seedling hardening and transplanting: after the seedlings in step S4 grow roots, move the seedlings to a substrate, which is a mixture of nutrient soil: vermiculite: perlite in a ratio of 1:1:1. Ensure environmental humidity and perform normal management.

[0016] The environmental humidity is ensured in step S5 by adopting a seedling hardening method without opening the cover: the seedlings are directly moved onto a substrate, the substrate adopts nutrient soil, vermiculite and perlite with a mass ratio of 1:1:1, and covered with a plastic cover to ensure the environmental humidity, and the cover is gradually opened after 7 days for normal management.

[0017] Furthermore, a 1 cm long stem segment with two leaf nodes is used in step S2.

[0018] Furthermore, in step S3, the bud clusters are cut into small square pieces of 0.5 cm×0.5 cm×0.5 cm.

[0019] Furthermore, in step S4, a 2-3 cm proliferating seedling with 3-4 pairs of leaves is selected, and the lower incision is inserted into the rooting medium without touching the bottom of the bottle.

[0020] Furthermore, the environmental humidity is ensured in step S5 by adopting a seedling hardening method without opening the cover: the seedlings are directly moved onto a substrate, the substrate adopts nutrient soil, vermiculite and perlite in a mass ratio of 1:1:1, and covered with a plastic cover to ensure the environmental humidity, and the cover is gradually opened after 7 days for normal management.

[0021] Beneficial effects of the technical solution of the present invention:

[0022] The present invention obtains the optimal combination by configuring and screening the explants, the types and concentrations of culture medium hormones, thereby improving the propagation coefficient of Platycodon grandiflorum, optimizing the rooting culture effect, and improving the efficiency of Platycodon grandiflorum tissue culture rapid propagation as a whole. The specific conclusions based on the embodiments of the present invention are summarized as follows:

[0023] (1) In the callus formation stage, in combination with the induction medium MS+6-BA 3.0 mg / L+NAA 1.0 mg / L proposed in the present invention, the stem segments selected in the present invention were used as explants, and callus was formed as soon as 23-25 ​​days after inoculation (see Figure 3 A) or bud cluster (see Figure 3 B); while in the leaf explant group, it took at least 35 days to form callus or several small buds (see Figure 2 C).

[0024] Moreover, the induction rate of stem segments was almost 100%, while the highest induction rate of leaves was only 80%, see Table 2.

[0025] It can be seen that using stem segments as explants for callus induction not only saves 10 to 12 days compared to using leaves as explants, but the induction rate is also at least 20% higher than that of leaves.

[0026] (2) In the proliferation culture stage, in combination with the proliferation medium MS+6-BA 0.5mg / L+NAA0.05mg / L proposed in the present invention, the bud cluster group selected by the present invention can reach a height of 5cm after 29 days (see Figure 4 B); while the callus group showed no significant changes at 29 days (see Figure 4 A); The proliferation coefficient of the bud cluster group reached a maximum of 77.

[0027] (3) In the rooting culture stage, the present invention proposes the best rooting medium 1 / 2MS + IBA 2.0mg / L for rooting time, rooting amount and root strength (see Figure 5 B), survival rate of seedlings after transplanting (see Figure 6 C), factors such as cost are optimized.

[0028] (4) Based on technical improvements at different stages, the seedlings were finally transplanted directly without opening the cover, with a survival rate of 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The diagram is a diagram of the process of forming callus tissue after the stem segment is inoculated into the callus induction medium in the embodiment of the present invention, wherein A: the stem segment is inserted into the callus induction medium for 0 days, B: the stem segment is inserted into the callus induction medium for 15 days, and C: the stem segment is inserted into the callus induction medium for 23 days;

[0030] Figure 2The diagram is a process diagram of callus formation after leaves are inoculated into callus induction medium in the embodiment of the present invention, wherein A: leaves are inoculated into callus induction medium for 0 days, B: leaves are inoculated into callus induction medium for 15 days, and C: leaves are inoculated into callus induction medium for 35 days;

[0031] Figure 3 Schematic diagram of the expansion of the base of the stem segment in the embodiment of the present invention, wherein A: the tubercle callus is formed after the expansion of the stem segment B: the bud cluster is directly formed after the expansion of the stem segment;

[0032] Figure 4 The results of different callus morphologies in the embodiments of the present invention after inoculation into the proliferation medium for 29 days, from left to right are 2-1, 2-2, 2-3, 2-4; A: 29 days after the callus tissue was inoculated into the proliferation medium, B: 29 days after the bud cluster was inoculated into the proliferation medium;

[0033] Figure 5 This is a comparison diagram of the rooting effects of different groups in the embodiments of the present invention, wherein A: the rooting situation of group 3-4 after 30 days of culture, B: the rooting situation of group 3-6 after 53 days of culture;

[0034] Figure 6 It is a comparison chart of different seedling hardening effects of different groups in the embodiments of the present invention, wherein A: the growth of the NAA group after 45 days of hardening with the cover opened, B: the growth of the NAA group after 45 days of hardening with the cover not opened, and C: the growth of the IBA group after 7 days of hardening with the cover not opened. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the examples and drawings.

[0036] The following experiment was conducted at the Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences. Self-cultivated Platycodon grandiflorum tissue culture seedlings were selected and the sterile seedlings were used as experimental materials. The experimental steps are as follows:

[0037] Step 1: Basic culture medium culture

[0038] The basic culture medium uses the MS formula, and the rooting culture medium uses 1 / 2MS culture medium and MS culture medium. The culture medium contains: 7g / L carrageenan, 30g / L sucrose, and the pH is adjusted to 5.8-6.0. On this basis, the subsequent culture medium is added with hormones such as 6-BA, NAA and IBA of different concentrations and ratios to form various combinations (see Table 1-Table 3 for details). The prepared culture medium is placed in a volumetric flask and sterilized at 121°C.

[0039] Step 2: Induced callus culture

[0040] Under sterile conditions, 1cm×1cm leaves and 1cm long stem segments with two leaf nodes were taken from sterile Platycodon grandiflorum seedlings and inoculated on callus induction medium. The callus medium hormones are shown in Table 1. 5 leaves / bottle, and 4 bottles were inoculated for each treatment; 5 stem segments / bottle, and 6 bottles were inoculated for each treatment. After inoculation, they were placed in a tissue culture room at 25℃, illuminated for 12 hours, and the light intensity was 1000lx.

[0041] The time of axillary bud emergence, the time and number of base swelling, the time and number of callus emergence were observed, and the differentiation rate and callus induction rate were calculated.

[0042] Step 3: Proliferation Culture

[0043] The leafless callus blocks and differentiated clustered bud blocks were cut into square blocks of 0.5 cm × 0.5 cm × 0.5 cm, respectively, and inoculated into the concentration culture medium shown in Table 2, 4 blocks / bottle, and 3 bottles were inoculated for each replicate.

[0044] Observe and compare the effects of different morphologies on the proliferation of Platycodon grandiflorum and calculate the corresponding proliferation coefficient (number of seedlings in the bottle after proliferation / number of inoculated seedlings).

[0045] Step 4: Rooting and cultivation

[0046] Cut tissue culture seedlings about 2.5 cm long with roughly the same growth status and strong growth, 3-4 pairs of leaves, and insert the lower incision into the rooting medium without touching the bottom of the bottle. Repeat 5 bottles for each treatment, observe and calculate the rooting rate, and compare the number of roots and root length.

[0047] Step 5: Hardening and transplanting of tissue culture seedlings

[0048] Select tissue culture seedlings with strong root growth, more than 3-5 roots of similar length, loosen the bottle stopper in the greenhouse, place for 0h, 24h, 48h, and 72h for seedling hardening, wash off the carrageenan on the roots of the seedlings, transplant them into nutrient soil: vermiculite: perlite = 1:1:1, and cover with plastic film for 7 days.

[0049] During these 7 days, you need to pay attention to shading and control the temperature at 20-30℃. After the seedlings grow new roots, gradually increase the light to improve the adaptability of the seedlings. After 7 days, open the cover and manage normally.

[0050] Experimental results and analysis

[0051] 1. Effects of different hormone concentrations on the induction of Platycodon grandiflorum callus and axillary bud formation

[0052] like Figure 1 , Figure 3 The figure shows the process of callus formation after the stem segment is inoculated into the callus induction medium. The medium combination with different concentrations of hormones is added to the MS basal medium, and the leaves of the explant stem segment are cut off, leaving two leaf nodes (see Figure 1 A), axillary buds began to grow on the 7th day of culture, and the base of the stem segment swelled on the 15th day (see Figure 1 B), 20 days later, a tumor gradually formed at the bottom of the enlarged part of the stem segment (see Figure 1 C), the color changes from light green to white, and callus is formed on the stem segment as soon as 23-25 ​​days later (see Figure 3 A) or bud cluster (see Figure 3 B).

[0053] like Figure 2 The figure shows the process of callus formation after the leaves are inoculated into the callus induction medium. The leaves are cut into 1cm×1cm pieces and inoculated into the medium (see Figure 2 A), no significant changes in the first 15 days of culture (see Figure 2 B), it takes at least 35 days to form callus or several small buds (see Figure 2 C). Specific experimental groups and results are shown in Table 1.

[0054] Table 1 Effects of different hormone concentration combinations on callus and axillary bud formation in induction medium

[0055]

[0056] Table 1 shows the experimental grouping and results of step 2. It can be seen from Table 1 that the culture medium combination of adding different concentrations of hormones to the MS basal medium is conducive to the induction of callus tissue. The induction rate of stem segments is almost 100%, and the induction rate of leaves is slightly lower, at 80%.

[0057] Different concentrations of hormones had little effect on the induction rate of stem segments, only a certain difference in the time of callus appearance. A higher concentration of 6-BA could shorten the time for axillary buds to grow. Group 1-2 had a poor induction effect on leaves. As the concentration of 6-BA increased, the leaf induction rate was positively correlated. Within the experimental concentration range of NAA, there was no significant difference in the effect on the formation of new buds in Platycodon grandiflorum stem segments. The 1-3 combination had the highest differentiation rate, and the seedlings grew fast, the buds were emerald green, and the growth was good.

[0058] Therefore, the best induction culture medium was determined to be group 1-3 (MS+6-BA 3.0mg / L+NAA 1.0mg / L). Some stem segments swelled at the base and gradually formed yellow-green cakes. Callus tissue was not formed, and bud clusters were directly formed, differentiating into a large number of seedlings.

[0059] 2. Effects of different hormone concentrations and different callus morphologies on callus differentiation

[0060] like Figure 4 The results are shown in Figure 2 after 29 days of culture in proliferation medium using tissues of different morphologies:

[0061] A: 29 days after callus was inoculated into proliferation medium;

[0062] B: 29 days after the bud cluster was inoculated into the proliferation medium (from left to right: 2-1, 2-2, 2-3, 2-4);

[0063] Depend on Figure 4 It can be seen that the callus inoculated on the proliferation medium could not differentiate into small buds, and there was no obvious change in morphology after 29 days of culture (see Figure 4 A), while the height of the bud cluster group can reach 5cm after 29 days (see Figure 4 B).

[0064] The specific experimental groups and results are shown in Table 2.

[0065] Table 2 Effects of different concentrations of hormone combinations on callus differentiation

[0066]

[0067] Table 2 shows the experimental grouping and results of step 3. Figure 4 It can be seen that the callus blocks inoculated onto the proliferation medium could not differentiate into small buds; and the results of the bud cluster group showed that the proliferation was better in the group with a relatively high 6-BA mass concentration, while the proliferation was poor in the group with a relatively high NAA mass concentration.

[0068] This study also found that although the bud clusters can be proliferated in MS basal medium, vitrification will occur, while no vitrification phenomenon occurred in the groups with added hormones, so it is believed that the addition of hormones can reduce the vitrification phenomenon of bud clusters. Except for the number of seedlings, there is no significant difference in the development speed of bud clusters, the thickness of bud cluster stems, and the color of bud cluster leaves among the groups.

[0069] Therefore, the proliferation coefficient was used as the basis for selecting the best culture medium, and the best proliferation culture medium was determined to be group 2-3 (MS+6-BA 0.5mg / L+NAA0.05mg / L).

[0070] 3. Effects of different concentrations and types of hormones on the rooting of Platycodon grandiflorum

[0071] like Figure 5 As shown in the figure, it is a comparison of rooting effects of different groups, A is the rooting situation of group 3-4 after 30 days of cultivation, and B is the rooting situation of group 3-6 after 53 days of cultivation. The specific groups and results are shown in Table 3.

[0072] Table 3 Effects of different concentrations and types of hormones on the rooting of Platycodon grandiflorum

[0073]

[0074] As shown in Table 3, it is the experimental grouping and results of step 4. It can be seen from Table 3 that NAA and IBA both promote the rooting of Platycodon grandiflorum, and the rooting rate and average number of roots increase with increasing concentration.

[0075] The NAA concentration is low, the number of roots formed is small, about 5-8 roots, the root length is long, the lateral root growth is small, and the root shape is thin and fragile (see Figure 5 A). With high IBA concentration, the number of roots formed is significantly increased, about 30-44 roots, the rooting rate is 100%, the root shape is short and thick, and it is easy to transplant (see Figure 5 B). It was also observed that Platycodon grandiflorum also took root in the basic culture medium, but the speed was slower than that in the hormone-added group, and the number of roots was small and thin. In the group with the same IBA addition, the rooting rate of the 1 / 2MS group and the MS group was 100%, and the number of roots increased with the increase of IBA concentration, and the number of roots could reach between 27 and 42. The root shape was the same as that of the MS group. Although the number of roots decreased, it still met the transplanting requirements. In subsequent experiments, it was found that the 1 / 2MS seedlings were more vigorous after transplanting.

[0076] In summary, the best rooting medium is 1 / 2MS+IBA 2.0mg / L.

[0077] 4. Hardening and transplanting

[0078] According to the experimental method, the cap of the culture medium bottle of the seedlings with 5-8 strong roots in the NAA group was gradually opened, and the sterile seedlings were allowed to adapt for 7 days. The agar at the roots of the seedlings was washed off and transplanted into the matrix. The matrix was made of nutrient soil: vermiculite: perlite = 1:1:1, and covered with plastic film. The seedlings were placed in the greenhouse for training. It was found that the seedlings wilted and grew poorly (see Figure 6 A), the survival rate was only 31.8% after 45 days.

[0079] The experimental method was subsequently adjusted to hardening the seedlings without opening the cover. The seedlings were directly moved to the substrate and covered with a plastic cover to ensure the environmental humidity. After 7 days, the cover was gradually opened. The growth of the seedlings was significantly improved compared with the hardening group (see Figure 6 B), the survival rate after 45 days was 54.6%.

[0080] The IBA group was subsequently transplanted without opening the cover, and the survival rate of the seedlings was 95% (see Figure 6 C) Subsequent observations revealed that groups 3-6 grew faster than other groups, and the leaves of the seedlings were dark green, which was helpful in confirming the best rooting medium group.

[0081] To sum up, when hardening and transplanting seedlings, it is best to use the method of not opening the cover, and the environmental humidity needs to be ensured. The matrix should be nutrient soil: vermiculite: perlite = 1:1:1.

[0082] 5. Experimental Conclusion

[0083] In the experiment, leaves were used as explants, but the induction rate was low, the buds formed were small, the induction speed was slow, and the subsequent buds grew slowly, so they were considered unsuitable as explants.

[0084] Using stem segments as explants, the induction rate is almost 100%, each segment can produce at least 2 buds, the growth rate is fast, and the growth state is good. Therefore, stem segments are selected as explants.

[0085] Different hormone concentration combinations can effectively improve the induction, proliferation and rooting efficiency of Platycodon grandiflorum, so as to achieve the purpose of shortening the growth cycle of Platycodon grandiflorum. The best hormone combination and its concentration in the present invention are as follows: the best induction medium is (MS+6-BA3.0mg / L+NAA 1.0mg / L), the best proliferation medium is (MS+6-BA 0.5mg / L+NAA 0.05mg / L), and the best rooting medium is 1 / 2MS+IBA 2.0mg / L.

[0086] During the seedling hardening and transplanting stage, it is necessary to ensure humidity. The seedlings should be hardened without opening the lid. The substrate should be nutrient soil: vermiculite: perlite = 1:1:1.

[0087] According to the above experimental conclusions, the method for rapid propagation of Platycodon grandiflorum through tissue culture proposed in the present invention is better than the existing known methods.

[0088] In summary, the rapid propagation method of Platycodon grandiflorum tissue culture of the present invention is compared with the existing known methods. The combination of explants used in the present invention combined with hormones in various stages is significantly optimized in the callus induction stage (the induction rate reaches 100% while saving time), the proliferation culture stage (the proliferation coefficient of the bud cluster group is as high as 77) and the rooting culture stage (the rooting rate is 100%, and the root system is numerous and strong), which greatly improves the tissue culture efficiency of Platycodon grandiflorum; and the method provided by the present invention can ensure that the growth of the clustered buds is not affected and the growth rate is not significantly affected when the proliferation coefficient is as high as 77, which is a major breakthrough in this technology in the field.

[0089] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for rapid propagation of Platycodon grandiflorum through tissue culture, characterized in that: The following steps are involved: S1. Prepare the following culture medium: MS basal medium: It is composed of MS medium + carrageenan 7g / L + sucrose 30g / L, and the pH is adjusted to 5.8-6.

0. It is sterilized at 121℃ for 20min before use; Callus induction medium: It is composed of the MS basal medium + 6-BA 3.0 mg / L + NAA 1.0 mg / L, and is sterilized at 121°C for 20 minutes before use; Proliferation medium: It is composed of the MS basal medium + 6-BA 0.5mg / L + NAA 0.05mg / L, and is sterilized at 121°C for 20min before use; Rooting medium: It is composed of 1 / 2MS basal medium + IBA 2.0ml / L, sterilized at 121℃ for 20min before use; S2. Inducing callus culture: selecting stem segments as explants, inoculating the stem segments with leaf nodes into the inducing callus culture medium, placing them in a tissue culture room at room temperature of 25° C., irradiating them with light for 12 h at a light intensity of 1000 1x, and cultivating them to form callus blocks and bud clusters; S3, performing proliferation culture: selecting the bud cluster obtained in step S2, and inoculating it into the proliferation culture medium to cultivate seedlings; S4, performing rooting culture: when the seedlings cultured in step S3 grow to 2-3 cm, inoculate them into a rooting medium for rooting cultivation; S5, tissue culture seedling hardening and transplanting: after the seedlings in step S4 grow roots, move the seedlings to the substrate. The matrix is ​​a mixture of nutrient soil, vermiculite and perlite in a ratio of 1:1:

1. Ensure the environmental humidity and carry out normal management.

2. The method for rapid propagation of Platycodon grandiflorum through tissue culture according to claim 1, wherein: The stem segment with leaf nodes in step S2 refers to a 1 cm long stem segment with two leaf nodes.

3. The method for rapid propagation of Platycodon grandiflorum through tissue culture according to claim 1, wherein: The bud clusters in step S3 are cut into small square pieces of 0.5 cm×0.5 cm×0.5 cm.

4. The method for rapid propagation of Platycodon grandiflorum through tissue culture according to claim 1, wherein: In step S4, a 2-3 cm long seedling with 3-4 pairs of leaves is selected for proliferation culture, and the lower incision is inserted into the rooting medium without touching the bottom of the bottle.

5. The method for rapid propagation of Platycodon grandiflorum through tissue culture according to claim 1, wherein: The environmental humidity is ensured in step S5 by adopting a seedling hardening method without opening the cover: the seedlings are directly moved onto a substrate, the substrate adopts nutrient soil, vermiculite and perlite with a mass ratio of 1:1:1, and covered with a plastic cover to ensure the environmental humidity, and the cover is gradually opened after 7 days for normal management.

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