A tissue culture and rapid propagation method of pteryxia hispidula

Abstract

The present application relates to a kind of Pterocephalus heterophyllus tissue culture rapid propagation method, belong to plant tissue culture technical field, will be sterile Pterocephalus heterophyllus stem segment into the length containing 1 pair of opposite leaves, place in cluster bud induction medium, cluster bud is divided into single bud, each single bud contains 1 pair of opposite leaves, repeat bud induction. Single bud is no longer repeated induction is subcultured to rooting medium, and obtains rooted seedling. When rooted seedling grows to 3-4cm, can be transplanted from tissue culture bottle. The whole process is short, and propagation coefficient is high, can produce a large number of tissue culture seedlings in a short time, and it has important significance to the protection and utilization of Pterocephalus heterophyllus resources.

Description

TECHNICAL FIELD

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

[0002] Dracocephalum heterophyllum is a traditional Tibetan medicine with a long history of use and high medicinal value. According to the Crystal Bead Materia Medica, Dracocephalum heterophyllum has the effects of treating oral diseases, tooth diseases and clearing liver heat. The Chinese Materia Medica Dictionary records that it has the effect of clearing liver and is mainly used for the treatment of icteric hepatitis, gum swelling and pain due to liver fire rising, and oral ulcer. Modern medical research shows that it has the effects of protecting liver, protecting myocardial injury, resisting hypoxia and reducing blood pressure. It can be seen that the resource has good application and development prospects. At present, it mainly grows in habitats with an altitude of 2000-4700 m, but the resource distribution is scattered and discontinuous, and the plant is small and the resource amount is small. At the same time, excessive excavation also seriously destroys the surface vegetation, aggravates grassland degradation and soil erosion. In view of the contradiction between the protection of ecological environment and biological diversity and the development of biological resource industry and people's livelihood, the artificial rapid propagation technology of Dracocephalum heterophyllum is established and optimized, which lays a technical and resource foundation for the resource breeding and wild cultivation of Dracocephalum heterophyllum. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a tissue culture and rapid propagation method of Dracocephalum heterophyllum.

[0004] The present application is realized by the following technical scheme:

[0005] A tissue culture and rapid propagation method of Dracocephalum heterophyllum, which comprises the following steps:

[0006] In the first step, the sterile Dracocephalum heterophyllum stem segments are divided into lengths containing 1-2 pairs of opposite leaves and placed on a cluster bud induction medium. The culture conditions are 25-28 O C, 2200-3200 Lx, and placed in a 16h / 8h photoperiod culture condition;

[0007] The cluster bud induction medium is I: MS + 30g / L sucrose, 6-BA 0.2-0.5mg / L, KT 1-2mg / L + agar 7.5-7.8g / L, pH 5.8-6.0;

[0008] Or II: MS + 30g / L sucrose, 6-BA 0.2-0.3mg / L, TDZ 0.1-0.3mg / L + agar 7.5-7.8g / L, pH 5.8-6.0;

[0009] The second step is to elongate the adventitious buds: when the single bud of the cluster buds is 1-2 cm long, the single bud is cut off and placed on a bud elongation medium; the culture condition is 25-28 O C, 2200-3200 Lx, and the culture condition is a light period of 16h / 8h; the bud elongation medium is 1 / 2MS+15g / L sucrose+agar 7.5-7.8g / L, pH 5.8-6.0;

[0010] The third step is to root the adventitious buds: when the single bud is 3-6 cm long, the single bud is moved to a rooting medium; the composition and final concentration of the rooting medium are 1 / 2MS+15g / L sucrose+0.2-0.5mg / L NAA+0.5-1mg / L IBA+agar 7.5-7.8g / L, pH 5.8-6.0;

[0011] Further, the method further comprises a transplanting step: the obtained rooting seedlings are taken out when the length is 8-15 cm, the medium is washed off, and the seedlings are transplanted.

[0012] Further, the method further comprises a repeated propagation culture: the stem segment after cutting off the adventitious buds is divided into a length containing 1-2 pairs of opposite leaves, and is placed on the cluster bud induction medium again to induce cluster buds repeatedly.

[0013] Further, the explant is derived from seed culture; the seed disinfection condition is to use 75% alcohol to disinfect for 30s, 10% sodium hypochlorite to disinfect for 10min, and sterile water to wash for 3-6 times until there is no smell of sodium hypochlorite; the seed is soaked with 100mg / L gibberellin (GA3) for one night, and then is placed on a seed germination medium to germinate; the seed germination medium is 1 / 2MS+15g / L sucrose+agar 7.5-7.8g / L, pH 5.8-6.0.

[0014] Further, the sterile P. tibeticum stem segment is derived from a P. tibeticum plant; the disinfection method of the plant is to use 75% alcohol to disinfect for 30s, 10% sodium hypochlorite to disinfect for 5min, and 0.5% jie'er solution to disinfect for 5min; the plant is washed with sterile water for 3-5 times, and the water is absorbed with filter paper.

[0015] The present application has the following beneficial effects compared with the prior art:

[0016] The present application selects a pair of opposite leaves as the explant, and the budding rate of the single bud is improved.

[0017] In the elongation stage, the adventitious buds can be directly rooted on the elongation medium, and the work of rooting in the later stage can be greatly reduced. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The sterile seedling of P. tibeticum grown from the seed;

[0019] Figure 2 The heterophyllous leafy stem segments of P. heterophylla induce axillary bud sprouting to form clumps;

[0020] Figure 3 The clumps are divided and the sub-clumps are cultured on elongation medium;

[0021] Figure 4 The clumps are elongated and rooted on elongation medium;

[0022] Figure 5 The elongated clumps are rooted on rooting medium;

[0023] Figure 6 The P. heterophylla tissue culture seedlings are transplanted into soil. DETAILED DESCRIPTION

[0024] The technical solutions of the present application are further described below by way of examples, but the scope of protection of the present application is not limited in any form by the examples.

[0025] Example 1

[0026] 1) Take the seeds of P. heterophylla, divide them into two parts, one part is soaked with 100 mg / L gibberellin for 24 h, and the other part is not soaked with gibberellin, and the germination rate is calculated. After 24 h of soaking with 100 mg / L gibberellin, it is observed that the seeds of P. heterophylla without gibberellin treatment have not germinated, while the seeds soaked with gibberellin have germinated by 71.43%. After 4 days of observation, it is found that the germination rate of the seeds of P. heterophylla without gibberellin treatment reaches 14.29%, while the germination rate of the seeds treated with gibberellin reaches 85.71%. It can be seen that gibberellin treatment can make the seeds of P. heterophylla germinate earlier and increase the germination rate of the seeds of P. heterophylla.

[0027] 2) The seeds soaked with gibberellin are first sterilized with 75% alcohol for 30 s, then sterilized with 10% sodium hypochlorite for 10 min, washed with sterile water for 3-6 times until there is no smell of sodium hypochlorite, and then the water is absorbed with filter paper. Place on 1 / 2MS + 15 g / L sucrose, agar 7.5-7.8 g / L, pH 5.8-6.0 medium, 16 h / 8 h light cycle, light intensity 2200-3200 Lx culture conditions.

[0028] 3) When the aseptic seedlings grow to 3-4 cm, they are respectively subcultured into the above-mentioned medium, and when the aseptic seedlings grow to 5-10 cm, the healthy seedlings are selected for use, as shown in Figure 1

[0029] ​4) Select the healthy branches, cut them into lengths containing 1 or 2 pairs of opposite leaves, and place them on the cluster bud induction medium. Morphologically, the upper end is upward, and the lower end is inserted into the cluster bud induction medium, which can be any of the following:

[0030] Cluster bud induction medium I: MS + 30 g / L sucrose, 6-BA 0.2-0.5 mg / L, KT 1-2 mg / L + agar 7.5-7.8 g / L, pH 5.8-6.0.

[0031] Cluster bud induction medium II: MS + 30 g / L sucrose, 6-BA 0.2-0.3 mg / L, TDZ 0.1-0.3 mg / L + agar 7.5-7.8 g / L, pH 5.8-6.0.

[0032] After 1 month, cluster buds are produced at the axillary buds of the stem segments, as shown in Figure 2 .

[0033] The stem segments containing 6 pairs of opposite leaves were counted, and each was cut into 1 pair of opposite leaf stem segments, i.e. 6 segments, and 2 pairs of opposite leaf stem segments, i.e. 3 segments, and the number of buds induced was counted.

[0034] The results showed that the number of adventitious buds obtained by cutting 1 stem segment containing 6 pairs of opposite leaves into 1 pair of opposite leaf stem segments, i.e. 6 segments, was 28; and the number of buds obtained by cutting it into 2 pairs of opposite leaf stem segments, i.e. 3 segments, was 18. Therefore, to obtain a higher propagation coefficient, cutting the stem segments into only 1 pair of opposite leaves has the highest propagation efficiency.

[0035] At the same time, the number of cluster buds obtained by cutting the stem segments containing 1 pair of opposite leaves was counted. The number of cluster buds obtained by cluster bud induction medium I was 4-5, and the number of cluster buds obtained by cluster bud induction medium II was 4-5.

[0036] 5) When the single buds of the cluster buds grow to 1-2 cm, they are cut off and placed on the bud elongation medium, which has the following composition and final concentration: 1 / 2MS + 15 g / L sucrose + agar 7.5-7.8 g / L, pH 5.8-6.0, as shown in Figure 3 .

[0037] After they grow to 4-6 cm, 20-30 days, about half of the cluster buds root on the elongation medium, as shown in Figure 4 .

[0038] 6) The single buds that have not rooted can be moved to the rooting medium to continue rooting, as shown in Figure 5 .

[0039] The single buds were placed on two different rooting media respectively, and the rooting efficiency was counted after 20 days.

[0040] Rooting medium I: 1 / 2MS + 15 g sucrose / L + 0.2-0.5 mg / L NAA + 0.5-1 mg / L IBA + agar 7.5-7.8 g / L, pH 5.8-6.0; rooting efficiency 85.32% ± 5.31%;

[0041] Rooting medium II: 1 / 2MS + 15 g sucrose / L + agar 7.5-7.8 g / L, pH 5.8-6.0, rooting efficiency 50% ± 2.41%.

[0042] Therefore, the single bud is transplanted on the rooting medium I to root faster.

[0043] 7) Transplanting: when the tissue culture seedling grows 5-8 true roots, it is removed from the tissue culture bottle, washed clean of the medium, and transplanted in nutrient soil, covered with plastic wrap, and kept moist. After 10-20 days, new bud points grow at the root stem connection of the heterophyllous chenopodium, small holes are punched in the plastic wrap, and after a week, the plastic wrap is completely removed, as shown in Fig. 5. Figure 6

[0044] 8) The stem segment after cutting the adventitious bud in step 5) is divided into a length containing 1-2 pairs of opposite leaves, and placed again on the cluster bud induction medium to repeat the induction of cluster buds. Rooting, transplanting, and increasing the utilization efficiency of the explants are performed again.​

Claims

1. A tissue culture rapid propagation method of Pteryxia hispidula, characterized in that, The method comprises the following steps: First step, the sterile P. lanceolata stem segments were divided into lengths containing 1 pair of opposite leaves, placed on a shoot cluster induction medium, and cultured under conditions of 25-28 O C, 2200-3200 Lx, under a 16 h / 8 h photoperiod culture condition; The said cluster bud induction medium is I: MS+30 g / L sucrose+0.2-0.5 mg / L 6-BA+1-2 mg / L KT+7.5-7.8 g / L agar, pH 5.8-6.0; Or II: MS+30 g / L sucrose+0.2-0.3 mg / L 6-BA+0.1-0.3 mg / L TDZ+7.5-7.8 g / L agar, pH 5.8-6.0; Second step, elongation of adventitious buds: when the single bud of the cluster buds grows to 1-2 cm, it is cut off and placed on bud elongation medium; the culture condition is 25-28 O C, 2200-3200 Lx, under the culture condition of 16 h / 8 h photoperiod; bud elongation medium: 1 / 2MS+15 g / L sucrose+agar 7.5-7.8 g / L, pH 5.8-6.0; The third step, rooting of adventitious buds: when the single bud grows to 3-6 cm, it is moved to the rooting medium, and the said rooting medium is: 1 / 2 MS+15 g / L sucrose+0.2-0.5 mg / L NAA+0.5-1 mg / L IBA+agar 7.5-7.8 g / L, pH 5.8-6.

0.

2. The tissue culture rapid propagation method of Pertya asperma according to claim 1, characterized in that, The said method further comprises the step of transplanting: the rooted seedling after rooting of adventitious buds is taken out when it grows to 8-15 cm, the medium is washed away, and then it is transplanted.

3. The method for tissue culture propagation of Pseudostellaria heterophylla according to claim 1, characterized in that, The said method further comprises the step of repeated proliferation culture: the stem segment after cutting off adventitious buds is divided into a length containing a pair of opposite leaves, and then it is placed on the cluster bud induction medium again to induce cluster buds repeatedly.

4. The tissue culture rapid propagation method of Pertya aspera according to claim 1, characterized in that, The said sterile P. heterophylla stem segment is derived from a P. heterophylla plant, and the disinfection method of the plant is: 75% alcohol is used for disinfection for 30 s, 10% sodium hypochlorite is used for disinfection for 5 min, 0.5% jie'er disinfectant solution is used for disinfection for 5 min, and then the plant is washed with sterile water for 3-5 times and dried with filter paper.

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