Liquid medium can effectively improve
Bletilla striata (Thunb.) Reichb.f. is a perennial perennial herb of the genus Bletilla Reichb.f. of Orchidaceae. The roots are white and several are connected, hence the name Bletilla striata, alias Baiji, even grass and licorice root. Bletilla striata flowers are large in color and have high ornamental value. At the same time, Bletilla striata has thick fleshy lumpy pseudobulbs, which are one of the important Chinese medicinal materials, which have the functions of astringent hemostasis, swelling and muscle growth. With the continuous development of Bletilla striata, market demand has increased year after year, and prices have gradually risen, resulting in serious private mining and indiscriminate exploitation of wild resources, resulting in the exhaustion of wild resources of Bletilla striata. The existing resources of Bletilla striata have been seriously damaged and cannot meet market demand. Therefore, it has also been listed as one of the key protected wild
In the natural wild state, the mature Bletilla platensis seeds are powdery and very fine. After 15 weeks of flowering and pollination, the embryo rate of the seeds is only about 59%, and they rarely germinate into seedlings, so the natural germination rate of the seeds is extremely low, and the reproduction is difficult. At present, artificial breeding mostly uses plant division, that is, cutting the tubers into small pieces for planting, but the low reproduction coefficient, large consumption, and large-scale seed supply are still difficult to meet the needs of large-scale production. Adopting plant tissue culture technology can obtain a large number of Bletilla seedlings in a short time, which is a fast and effective propagation method. At present, it is mainly through sowing seeds on a solid medium with added hormones to germinate, regenerating sterile seedlings, or inducing original bulbs, multiplying and rooting for rapid propagation. This involves more than 4 to 5 kinds of media, the operation is cumbersome, and the cycle is long. The shortest seedling can be transplanted within 110 days, and the relative production cost is relatively high. For this purpose, the research group discussed a rapid propagation technology of Bletilla seedlings in vitro based on liquid medium.
This technique uses a liquid medium and uses liquid suspension to cultivate germinated seeds instead of the commonly usedSolid mediumSurface germination, which can effectively improve the germination rate of Bletilla striata seeds, shorten the seedling formation time of Bletilla striata, and quickly obtain Bletilla seedlings. The technology is described below.
In vitro culture of immature Bletilla platensis seeds
1.1 Seed surface sterilization and germination
After 15 weeks of flowering and pollination, the unopened Bletilla capsules (including seeds) were selected, and the surface was disinfected with 75% ethanol on the ultra-clean workbench of the Laboratory of Garden and Horticulture Branch of Chengdu University of Agricultural Science and Technology for 30 s, and rinsed with deionized water for 1 to 2 times. Carefully peel off the fruit with a scalpel and forceps, carefully shake off the seeds into a sterile tissue culture bottle, and add an appropriate amount of 5% sodium hypochlorite solution (add 1 to 2 drops of Tween-20 dropwise) to disinfect for 20 min. Filter the seeds with a sterilized 50-mesh cell sieve; then rinse and filter with deionized water, and repeat 4 to 6 times to rinse off the residual disinfectant on the surface. The seeds filtered on the cell sieve were rinsed into a triangular flask with MS + 1.0 mg/L TDZ + 30 g/L sucrose and pH 5.8 seed germination liquid medium, and cultured at 25 ° C, 100 r/min dark for 15-20 days. Usually after 5 days of culture, the seeds can germinate. At this time, they were transferred to a photoperiod environment of 16 h/8 h for light culture. Generally, the seeds developed into leaves after 15 days of inoculation.
1.2 Seeds grow into buds
After 15 days of inoculation, the germinated Bletilla platensis seeds were sifted off by 50 mesh cells, and the seed protobulbs were inoculated on MS + 0.2 mg/L 6-BA + 30 g/L sucrose + 6 g/L agar, pH 5.8 solid-induced bud medium. Seedlings were dispersed as much as possible, about 100 grains per dish, and cultured at 25 ° C, 16 h/8 h photoperiod. Generally, after 30 days of culture, when the buds grew into seedlings, about 2 cm and the root length was about 0.5 cm, rooting culture could be carried out.
1.3 Rooting culture
The seedlings of Bletilla platensis were inoculated on solid rooting medium with 1/2 MS + 0.1 mg/L NAA + 30 g/L sucrose + 2 g/L activated carbon + 6 g/L agar and pH 5.8. Each bottle was inoculated with 25-30 plants, and cultured at 25 ℃ and 16 h/8 h photoperiod environment to promote the growth of seedlings and roots, and improve the rooting rate of strong seedlings. After 30 days of rooting culture, the tissue-cultured seedlings grew into robust adult seedlings, and the morphology is shown in Figure 1.
1.4 Transplanting seedlings
When the roots of the Bletilla striata seedlings grow to 3-5 cm (usually 75-90 days after the seeds germinate), the seedlings can be refined and transplanted. Before planting, gradually open the bottle cap to adapt to the external environment for 3-5 days, and pay attention to maintaining the humidity in the bottle during this period. Wash the root agar with water to avoid damage to the roots and original bulbs; transplant the robust Bletilla striata tissue culture seedlings into the hole plate equipped with the seedling substrate, and choose the vegetable or flower seedling substrate. After planting, the substrate is watered and cultivated in the seedling refining shed. Pay attention to low-light moisturization in the early stage of the shed. The first week should be covered with a shading net to maintain low light and cover with a film to moisturize; after 15 days of transplanting, the seedlings of Bletilla striata will grow normally, and they can be planted after about 30 days of transplanting. After colonization, the field management is routine. The transplanting situation is shown in Figure 2. The key to the survival of Bletilla striata transplantation is the size and vitality of the original bulb. Generally, seedlings with original bulbs can be transplanted and survive, and the survival rate can reach more than 95%.
2 In vitro culture of near-mature Bletilla striata
2.1 Seed surface sterilization and germination
More than 20 weeks after flowering, the nearly ripe Bletilla capsules (uncracked, containing seeds) were wrapped in gauze, rinsed with running water for 30 min, and then surface disinfected with 75% ethanol on a clean workbench for 30 s. Rinse with sterile water for 1 to 2 times, carefully peel off the fruit with a scalpel and forceps, carefully dial the seeds into a sterile tissue culture bottle, and add an appropriate amount of 5% sodium hypochlorite solution (add 2 to 3 drops of Tween-20 dropwise) to disinfect for 20 min. Filter the seeds with a sterilized cell sieve (50 mesh); then rinse and filter with deionized water, and repeat 4 to 6 times to rinse off the residual disinfectant on the surface. The seeds filtered on a cell sieve (50 mesh) were rinsed into a triangular flask with MS + 2.0 mg/L TDZ + 30 g/L sucrose, pH 5.8 seed germination liquid medium. 25 ℃, 100 r/min, dark for 15 d.
2.2 Seeds grow into buds
The germinated seeds were filtered out by cell sieve (50 mesh), and the seed protobulbs were inoculated on MS + 0.1 mg/L 6-BA + 30 g/L sucrose + 6 g/L agar, pH 5.8 to induce germination. The seeds were dispersed as much as possible, 100 grains per dish. Culture at 25 ℃, 16 h/8 h photoperiod environment for 20-30 days.
2.3 Rooting culture
The resulting seedlings and proliferating seedlings were inoculated into 1/2 MS + 0.5 mg/L NAA + 30 g/L sucrose + 0.5 g/L activated carbon + 6 g/L agar, pH 5.8 rooting medium, 25-30 plants per bottle, 25 ℃, 16 h/8 h photoperiod environment for 30-40 days.
2.4 Transplanting seedlings
When the roots of the Bletilla platensis seedlings grow to 3-5 cm long, the seedlings can be transplanted. Uncover the bottle cap to adapt to the external environment for 3-5 days before planting. When transplanting, wash the root agar with water first, and be careful not to damage the roots and original bulb structure; Transplant the robust Bletilla platensis tissue-cultured seedlings into a hole plate equipped with a seedling substrate, water them well after planting, and continue to cultivate in the seedling refining shed. Pay attention to low-light moisturization in the early stage of management. In the first week, a shading net should be covered to keep low light, and a film should be covered to moisturize; after 15 days of transplanting, Bletilla platensis seedlings can grow normally, and transplanting can be colonized in about 30 days; regular field management after
In vitro culture of mature Bletilla platensis seeds
3.1 Seed surface sterilization and germination
The ripe Bletilla platyphylla capsule (uncracked, containing seeds) was wrapped in gauze and rinsed with running water for 30 min; the surface was sterilized with 75% ethanol for 30 s on an ultra-clean workbench, and the sterilized water was rinsed 1-2 times. Carefully peel off the fruit with a scalpel and forceps, carefully dip the seeds into a sterile tissue culture bottle, and add an appropriate amount of 5% sodium hypochlorite solution (add 2-3 drops of Tween-20 dropwise) to sterilize for 20 min. The seeds filtered on a cell sieve (50 mesh) were rinsed into a triangular bottle with MS + 0.5 mg/L TDZ + 30 g/L sucrose, pH 5.8 seed germination liquid medium. The germination rate was 99% at 25 ℃ and 100 r/min for 15 d.
3.2 Seeds grow into buds
The germinated seeds were inoculated with MS + 0.5 mg/L 6-BA + 30 g/L sucrose + 6 g/L agar, pH 5.8, 100 grains per dish. Culture at 25 ℃, 16 h/8 h photoperiod environment for 20-30 days.
3.3 Rooting culture
Growing seedlings and proliferating seedlings were inoculated on 1/2 MS + 1.0 mg/L NAA + 30 g/L sucrose + 1.0 g/L activated carbon + 6 g/L agar, pH 5.8 rooting medium, 25-30 plants per bottle, cultured at 25 ℃, 16 h/8 h photoperiod environment for 30-40 days.
3.4 Transplanting seedlings
When the roots of the cultivated Bletilla platensis seedlings grow to 3-5 cm long, the seedlings should be transplanted, and the bottle cap should be opened to adapt to natural conditions for 3-5 days before planting. Wash the agar attached to the roots with tap water at the beginning of transplanting, be careful not to damage the roots and original bulb structure; Transplant the robust Bletilla platensis tissue-cultured seedlings into a seedling hole plate equipped with a substrate, water them well after planting, place them neatly in the seedling refining shed, and continue to cultivate seedlings. Pay attention to low-light moisturization in the early stage of management. In the first week, a sunshade net should be added to keep low-light and cover with a film to moisturize; 15 days after transplanting, Bletilla platensis seedlings can grow normally, and about 30 days after transplanting can be planted; regular field management after
4 Summary
The liquid suspension culture of near-mature Bletilla platensis seeds can significantly improve the seed germination rate, up to 99%, and accelerate the generation rate of protobulbs (about 15 days). And the technology is easy to operate and low cost, each bottle of liquid medium can germinate tens of thousands of seeds, and the seeds germinate neatly, emerge consistently, and the pollution rate is low. Combined with the simplified solid medium culture, the tissue culture cycle of Bletilla platensis is is greatly shortened, and a large number of robust sterile seedlings can be obtained for transplanting within 75-90 days at the fastest, which realizes the efficient and rapid production of Bletilla platensis tissue culture seedlings, and effectively simplifies the operation procedure, reduces the production cost, and facilitates large-scale production.
Authors: Xiong Bingquan, Liao Xiangjian, Zheng Xuelian
