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基于液体培养基的白芨种苗离体快繁技术白芨育苗技术

来源：花匠小妙招时间：2025-01-10 10:11

白芨[Bletilla striata（Thunb.）Reichb.f.]为兰科（Orchidaceae）白芨属（Bletilla Reichb.f.）多年生宿根草本植物，根呈白色，数个相连，故名白芨，别名白及、连及草、甘根。白芨花大色艳，具有较高的观赏价值；同时白芨生有肥厚的肉质块状假鳞茎，是重要的中药材之一，具有收敛止血、消肿生肌的功效。随着白芨的持续开发，市场需求连年增长，价格逐渐上涨，致使野生资源私挖滥采严重，导致白芨野生资源濒临枯竭，现有的白芨资源受到严重破坏，已无法满足市场需求，因而也被国家列为重点保护野生药用植物之一。在自然野生状态下，成熟的白芨种子呈粉末状，非常细小，开花授粉15周后的种子有胚率仅为59%左右，很少萌发成苗，因此其种子自然萌发率极低，繁殖困难。目前人工繁育多采用分株繁殖，即将块茎切成小块种植，但繁殖系数低、消耗量大、大面积供种依然较为困难，难以满足大规模生产的需求。采取植物组织培养技术，可在短时间内获得大量的白芨实生苗，是一种快速有效的繁殖方法，目前主要通过将种子撒播在添加激素的固体培养基上萌发，再生出无菌苗，或诱导原球茎、经增殖生根后进行快繁，这要涉及4～5种以上的培养基，操作较为繁琐，且周期较长，最短在110 d内方能成苗移栽，相对生产成本较高。为此课题组探讨了一种基于液体培养基的白芨种苗离体快繁技术，

该技术选用液体培养基，用液体悬浮培养萌发种子，而不是常用的固体培养基表面萌发，其能有效地提高白芨种子萌发率，缩短白芨成苗时间，快速获得白芨幼苗，下面将该技术介绍如下。

　　1 未成熟白芨种子离体培养

　　1.1 种子表面灭菌及萌发

　　选取开花授粉15周后、未开裂的白芨蒴果（含种子），在成都农业科技職业学院园林园艺分院实验室超净工作台上用75%乙醇进行表面消毒30 s，去离子水冲洗1～2次。用解剖刀及镊子仔细剥开果实，将种子小心地抖落到无菌组织培养瓶中，加适量5%的次氯酸钠溶液（滴加1～2滴吐温-20）消毒20 min。用灭菌的50目细胞筛过滤种子；再用去离子水冲洗、过滤，反复4～6次，冲去表面残留的消毒液。将细胞筛上过滤的种子以MS+1.0 mg/L TDZ+30 g/L蔗糖、pH 5.8的种子萌发液体培养基冲洗到三角瓶内，于25 ℃、100 r/min黑暗培养15-20 d。通常培养5 d后，种子即可萌发，此时转入16 h/8 h的光周期环境下光照培养，一般接种15 d后种子发育形成叶片。

　　1.2 种子生长成芽

　　将接种15 d、已萌发的白芨种子经50目细胞筛滤去液体，把种子原球茎接种于MS+0.2 mg/L 6-BA+30 g/L蔗糖+6 g/L琼脂、pH 5.8的固体诱导成芽培养基上，尽量分散幼苗，每皿约100粒，于25 ℃、16 h/8 h光周期培养。一般培养30 d后，当幼芽生长为幼苗，约2 cm左右、根长约0.5 cm左右时即可进行生根培养。

　　1.3 生根培养

　　将平皿中的白芨幼苗接种到1/2 MS+0.1 mg/L NAA+30 g/L蔗糖+2 g/L活性炭+6 g/L琼脂、pH 5.8的固体生根培养基上，每瓶接种25～30株，于25 ℃、16 h/8 h光周期环境里培养，促进幼苗和根的生长，提高壮苗生根率。生根培养30 d后，组培苗生长为健壮的成苗，形态见图1。

　　1.4 炼苗移栽

　　当白芨幼苗的根生长到3～5 cm时（一般在白芨种子萌发后75-90 d）即可炼苗移栽，栽前先逐渐揭开瓶盖适应3-5 d外界环境，期间注意保持瓶内湿度。移栽时用清水洗净根部琼脂，避免损伤根及原球茎；将健壮的白芨组培苗移栽到装好育苗基质的穴盘中，选蔬菜或花卉育苗基质即可，栽后基质浇透水，于炼苗棚中培养。棚中前期注意弱光保湿，第一周应覆盖遮阳网保持弱光，并覆盖薄膜保湿；移栽15 d后白芨成苗即生长正常，移栽30 d左右即可定植，定植后常规田间管理，移栽情况见图2。白芨移栽存活的关键是原球茎的大小及活力，一般有原球茎的幼苗均能移栽存活，存活率可达95%以上。

　　2 近成熟白芨種子离体培养

　　2.1 种子表面灭菌及萌发

　　将开花后20周以上、近成熟的白芨蒴果（未开裂，含种子）用纱布包好，自来水流水冲洗30 min，再在超净工作台上用75%乙醇进行表面消毒30 s，无菌水冲洗1～2次，用解剖刀及镊子小心剥开果实，将种子小心拨入无菌组培瓶中，加适量5%次氯酸钠溶液（滴加2～3滴吐温-20）消毒20 min。用灭菌的细胞筛（50目）过滤种子；再用去离子水冲洗、过滤，反复4～6次，冲去表面残留的消毒液。将细胞筛（50目）上过滤的种子，用MS+2.0 mg/L TDZ+30 g/L蔗糖、pH 5.8的种子萌发液体培养基冲洗到三角瓶内。25 ℃、100 r/min、黑暗培养15 d。

　　2.2 种子生长成芽

　　已萌发的种子经细胞筛（50目）滤去液体，将种子原球茎接种于MS+0.1 mg/L 6-BA+30 g/L蔗糖+6 g/L琼脂、pH 5.8的诱导成芽固体培养基上，尽量分散种子，每皿100粒。25 ℃、16 h/8 h光周期环境里培养20-30 d。

　　2.3 生根培养

　　将生成的幼苗及增殖的幼苗接种到1/2 MS+0.5 mg/L NAA+30 g/L蔗糖+0.5 g/L活性炭+6 g/L琼脂、pH 5.8的生根培养基里，每瓶25～30株，25 ℃、16 h/8 h光周期环境里培养30-40 d。

　　2.4 炼苗移栽

　　当白芨幼苗的根生长到3～5 cm长时，即可炼苗移栽，栽前先揭开瓶盖适应外界环境3-5 d。移栽时先用清水洗净根部琼脂，注意不要损伤根及原球茎结构；将健壮的白芨组培苗移栽到装有育苗基质的穴盘中，栽后浇透水，于炼苗棚中继续培养。管理上前期注意弱光保湿，第一周应加盖遮阳网保持弱光，并覆盖薄膜保湿；移栽15 d后白芨成苗即可生长正常，移栽30 d左右即可定植；定植后常规田间管理。

　　3 成熟白芨种子离体培养

　　3.1 种子表面灭菌及萌发

　　将成熟的白芨蒴果（未开裂，含种子）用纱布包好，自来水流水冲洗30 min；在超净工作台上用75%乙醇进行表面消毒30 s，无菌水冲洗1～2次，用解剖刀及镊子小心剥开果实，将种子小心拨入无菌组培瓶中，加适量5%次氯酸钠溶液（滴加2～3滴吐温-20）消毒20 min。将细胞筛（50目）上过滤的种子用MS+0.5 mg/L TDZ+30 g/L蔗糖、pH 5.8的种子萌发液体培养基冲洗到三角瓶内。25 ℃、100 r/min黑暗培养15 d，萌发率达99%。

　　3.2 种子生长成芽

　　已萌发的种子经细胞筛（50目）滤去液体，将种子原球茎接种于MS+0.5 mg/L 6-BA+30 g/L蔗糖+6 g/L琼脂、pH 5.8的诱导成芽固体培养基上，每皿100粒。25 ℃、16 h/8 h光周期环境里培养20-30 d。

　　3.3 生根培养

　　将长大的幼苗及增殖的幼苗接种到1/2 MS+1.0 mg/L NAA+30 g/L蔗糖+1.0 g/L活性炭+6 g/L琼脂、pH 5.8的生根培养基上，每瓶25～30株，25 ℃、16 h/8 h光周期环境里培养30-40 d。

　　3.4 炼苗移栽

　　当培养出的白芨幼苗根生长到3～5 cm长时，炼苗移栽，栽前先揭开瓶盖适应自然条件3-5 d。移栽先时用自来水洗净根部附着的琼脂，小心不要损伤根及原球茎结构；将健壮的白芨组培苗移栽到装有基质的育苗穴盘中，栽后浇透水，于炼苗棚中放置整齐，继续炼苗培养。管理上前期注意弱光保湿，第一周应加盖遮阳网保持弱光，并覆盖薄膜保湿；移栽15 d后白芨成苗即可生长正常，移栽30 d左右即可定植；定植后常规田间管理。

　　4 小结

　　采用近成熟的白芨种子进行液体悬浮培养，能显著提高种子萌发率，可达99%，并加快原球茎的生成速度（15 d左右）。且该技术操作简便，成本低廉，每瓶液体培养基可萌发数万粒种子，且种子萌发整齐，出苗一致，污染率低，结合简化的固体培养基培养，大大缩短了白芨组织培养周期，最快能在75～90 d内获得大量健壮的无菌苗用于移栽，实现了白芨组培苗的高效快速生产，并有效简化了操作程序，降低了生产成本，便于规模化生产。

作者：熊丙全廖相建郑雪莲

日水培养基qdrishui.cn

The Bletilla striata [Thunb.) reichb. f.] is a perennial herb of the Orchidaceae family. The bletilla striata bletilla striata is large in color and has high ornamental value. At the same time, bletilla striata, one of the important Chinese medicinal materials, has the function of astringency and detumescence. With continuous development of bletilla, continuously growing market demand, the price is rising, the wild resources and wasteful mining, private cause bletilla wild resources, was badly damaged by the existing resources of bletilla and have been unable to meet the market demand, and therefore one is listed as national key protected wild medicinal plants. Under natural wild state, mature bletilla seed powder, very small, the seeds of 15 weeks after pollination with embryo rate is only about 59%, germination to seedling rarely, so its seed germination rate is extremely low, natural reproductive problems. At present, a lot of artificial breeding using plant breeding, the tuber cut into small pieces, but the coefficient of low consumption, large, large area is being supported kind still more difficult, difficult to meet the needs of mass production. Plant tissue culture technology, may be achieved in a short time, a large number of bletilla west, is a rapid and effective breeding method, mainly through the seed sown on the solid medium added hormones, regenerate no vaccine, or after induction of protocorm and proliferation by rooting for rapid propagation, this involves more than 4 ~ 5 kinds of medium, operation is more tedious, and cycle is long, the shortest in the 110 d can into seedling transplanting, relatively high production cost. For this research based on liquid medium is discussed bletilla seedlings in vitro rapid propagation technology, the technology choose liquid medium, with liquid suspension culture germination of seeds, rather than the commonly used the surface of the solid medium, it can effectively improve bletilla seed germination rate, shortening the time of bletilla sprout, quickly get bletilla seedlings, the following will introduce the technology are as follows.

Culture of immature bletilla striata seeds in vitro

1.1 seed surface sterilization and germination

Select 15 weeks after pollination, did not crack bletilla capsule (seed), agricultural science and technology vocational college in chengdu garden branch laboratory ultraclean workbench use 75% ethanol for surface sterilization 30 s, deionized water flushing 1 ~ 2 times. Peel the fruit carefully with a scalpel and tweezers, carefully shake the seeds into a sterile tissue culture bottle, and apply an appropriate amount of 5% sodium hypochlorite solution (add 1 ~ 2 drops of twin-20) for 20 minutes. Seeds were filtered by sterilized 50 mesh cell sieves. Rinse and filter with deionized water 4 ~ 6 times to remove the remaining disinfectant from the surface. The seeds of the cells on the screen filter to MS + 1.0 mg/L TDZ + 30 g/L of cane sugar, pH 5.8 seed germination liquid medium flush to the triangle in the bottle, at 25 ℃, and 100 r/min dark train 15-20 d. Normally, seeds can germinate after 5 days of culture. At this time, seeds can be incubated under the light cycle of 16 h/8 h. Generally, seeds develop into leaves after 15 days of inoculation.

1.2 seeds grow into buds

'll vaccination 15 d, the seed germination of bletilla after 50 mesh sieve cells filtered liquid, put the seeds protocorm vaccination in MS + 0.2 mg/L 6 - BA + 30 g/L sucrose + 6 g/L AGAR, pH 5.8 solid induced into buds, scattered seedlings as far as possible, around 100 grains per dish, at 25 ℃, 16 h / 8 h light cycle training. Generally, after 30 days of culture, root culture can be carried out when the buds grow into seedlings, about 2 cm in length and about 0.5 cm in length.

1.3 rooting culture

Bletilla seedling inoculation of AGAR to 1/2 MS + 0.1 mg/L NAA + 30 g/L sucrose + 2 g/L activated carbon + 6 g/L on AGAR, pH 5.8 solid roots, bottle to vaccinate 25 ~ 30 strains at 25 ℃, 16 h / 8 h light cycle in the environment, promote the growth of seedlings and roots, raising seedling root rate. After 30 days of root culture, tissue culture seedlings grew into robust seedlings, as shown in figure 1.

1.4 transplanting the refined seedlings

When bletilla seedling root growth to 3 ~ 5 cm (generally 75-90 - d) after bletilla seed germination to seedling transplanting, plant gradually uncovered the cap fit before 3-5 d external environment, pay attention to during maintain humidity in the bottle. Rinse root AGAR with clean water during transplanting to avoid damaging root and bulb. Transplant the vigorous bletilla striata tissue culture seedlings into the hole dish with the seedling substrate, and select the vegetable or flower seedling substrate. After planting, the substrate can be watered and cultured in the seedling mixing shed. In the early stage of the shed attention to weak light moisture, the first week should cover sunshade to maintain weak light, and cover film moisturizing; After transplanting for 15 days, the seedlings of bletilla striata grew normally. After transplanting for about 30 days, the seedlings could be planted. The key to the survival of bletilla striata is the size and vitality of the original bulb. Generally, the seedlings with the original bulb can be transplanted and survived, and the survival rate can reach more than 95%.

Culture of hyacinth bletilla striata seeds in vitro

2.1 seed surface sterilization and germination

Will more than 20 weeks after flowering, nearly mature bletilla capsule (not cracking, including seed) wrapped in gauze, tap water rinse water for 30 min, then on the super net work 30 s with 75% ethanol for surface sterilization, sterile water rinse 1-2 times, with a scalpel and tweezers carefully peel away the fruit, the seed carefully dialed sterile seed in a bottle, add right amount 5% sodium hypochlorite solution (add 2 ~ 3 drops twain - 20) disinfection 20 min. Seeds were filtered by sterilized cell sieves (50 meshes); Rinse and filter with deionized water 4 ~ 6 times to remove the remaining disinfectant from the surface. The seeds filtered on the cell sieve (50 mesh) were rinsed into the triangular flask with MS+ 2.0mg /L TDZ+ 30g /L sucrose and pH 5.8. 25 ℃, 100 r/min, dark culture 15 d.

2.2 seeds grow into buds

Have seed germination of the sieve cells (50 eyes) filter the liquid, the seed protocorm vaccination in MS + 0.1 mg/L 6 - BA + 30 g/L sucrose + 6 g/L AGAR, pH 5.8 induced into bud on the solid medium, scattered seeds as far as possible, every dish 100 grains. 25 ℃, 16 h / 8 h environment light cycle 20-30 d.

2.3 root culture

Will generate seedlings and proliferation of seedling inoculation to 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 to take root in the culture medium, a bottle of 25 ~ 30 strains, 25 ℃, 16 h / 8 h environment light cycle 30-40 d.

2.4 transplanting the refined seedlings

When the root of bletilla striata seedlings grows to 3 ~ 5 cm long, the seedlings can be purified and transplanted. The root AGAR was washed with clean water before transplanting. The vigorous bletilla striata tissue culture seedlings were transplanted into the pit dish containing the seedling substrate, and then watered to continue the culture in the seedling mixing shed. In the early stage of management, attention should be paid to the weak light moisturizing, the first week should be covered with a shading net to maintain weak light, and cover the film moisturizing; The seedlings of bletilla striata can grow normally after 15 days of transplantation, and can be fixed after 30 days of transplantation. Routine field management after transplanting.

Culture of mature bletilla striata seeds in vitro

3.1 seed surface sterilization and germination

Wrap the mature capsule of bletilla striata (undehisced, containing seeds) in gauze and rinse with running water for 30 min. On the super net work 30 s with 75% ethanol for surface sterilization, sterile water rinse 1-2 times, with a scalpel and tweezers carefully peel away the fruit, the seed carefully dialed sterile seed in a bottle, add right amount 5% sodium hypochlorite solution (add 2 ~ 3 drops twain - 20) disinfection 20 min. The seeds filtered on the cell sieve (50 mesh) were rinsed into the triangular flask with MS+0.5 mg/L TDZ+30 g/L sucrose and pH 5.8. 25 ℃, and 100 r/min train 15 d, dark germination rate was 99%.

3.2 seeds grow into buds

The germinated seeds were filtered by cell sieve (50 meshes) to remove the liquid, and the seed proglobules were inoculated into MS+0.5 mg/L 6-ba +30 g/L sucrose +6 g/L agarose and pH 5.8 to induce buds on solid medium, 100 grains per dish. 25 ℃, 16 h / 8 h environment light cycle 20-30 d.

3.3 rooting culture

Will grow seedlings and the proliferation of seedling inoculation to 1/2 MS + 1.0 mg/L NAA + 30 g/L sucrose + 1.0 g/L activated carbon + 6 g/L on AGAR, pH 5.8 take root, a bottle of 25 ~ 30 strains, 25 ℃, 16 h / 8 h environment light cycle 30-40 d.

3.4 transplanting the refined seedlings

When the root of the cultured bletilla striata seedlings grows to 3 ~ 5 cm long, the refined seedlings are transplanted. Before transplanting, use tap water to clean the agar-agar attached to the root. Be careful not to damage the structure of the root and the bulb. Transplanting the vigorous bletilla striata tissue culture seedlings into the seedling hole dish containing the substrate, pouring water after planting, and placing them neatly in the seedling mixing shed, the seedling culture was continued. In the early stage of management, attention should be paid to the weak light moisturizing, the first week should be covered with a shading net to maintain weak light, and cover the film moisturizing; The seedlings of bletilla striata can grow normally after 15 days of transplantation, and can be fixed after 30 days of transplantation. Routine field management after transplanting.

4 summary

Liquid suspension culture of nearly mature bletilla striata seeds can significantly improve seed germination rate, up to 99%, and accelerate the growth rate of the original bulb (around 15d). And the technology is simple, low cost, a bottle of liquid medium can be tens of thousands of seed germination, and germination, emergence, low pollution rate, combining with simplified solid medium culture, greatly shorten the cycle bletilla tissue culture, the fastest can get a lot of within 75 ~ 75 d robust no vaccine for transplanting, implements the bletilla somaclone efficient production, fast and effectively simplify the operating procedure, reduces the production cost, convenient for mass production.