基于液體培養(yǎng)基的白芨種苗離體快繁技術(shù) 白芨育苗技術(shù)
發(fā)布時間：
2022-12-27
作者：
白芨[Bletilla striata（Thunb.）Reichb.f.]為蘭科（Orchidaceae）白芨屬（Bletilla Reichb.f.）多年生宿根草本植物，根呈白色，數(shù)個相連，故名白芨，別名白及、連及草、甘根。白芨花大色艷，具有較高的觀賞價值；同時白芨生有肥厚的肉質(zhì)塊狀假鱗莖，是重要的中藥材之一，具有收斂止血、消腫生肌的功效。隨著白芨的持續(xù)開發(fā)，市場需求連年增長，價格逐漸上漲，致使野生資源私挖濫采嚴重，導致白芨野生資源瀕臨枯竭，現(xiàn)有的白芨資源受到嚴重破壞，已無法滿足市場需求，因而也被國家列為重點保護野生藥用植物之一。在自然野生狀態(tài)下，成熟的白芨種子呈粉末狀，非常細小，開花授粉15周后的種子有胚率僅為59%左右，很少萌發(fā)成苗，因此其種子自然萌發(fā)率極低，繁殖困難。目前人工繁育多采用分株繁殖，即將塊莖切成小塊種植，但繁殖系數(shù)低、消耗量大、大面積供種依然較為困難，難以滿足大規(guī)模生產(chǎn)的需求。采取植物組織培養(yǎng)技術(shù)，可在短時間內(nèi)獲得大量的白芨實生苗，是一種快速有效的繁殖方法，目前主要通過將種子撒播在添加激素的固體培養(yǎng)基上萌發(fā)，再生出無菌苗，或誘導原球莖、經(jīng)增殖生根后進行快繁，這要涉及4～5種以上的培養(yǎng)基，操作較為繁瑣，且周期較長，最短在110 d內(nèi)方能成苗移栽，相對生產(chǎn)成本較高。為此課題組探討了一種基于液體培養(yǎng)基的白芨種苗離體快繁技術(shù)，
該技術(shù)選用液體培養(yǎng)基，用液體懸浮培養(yǎng)萌發(fā)種子，而不是常用的固體培養(yǎng)基表面萌發(fā)，其能有效地提高白芨種子萌發(fā)率，縮短白芨成苗時間，快速獲得白芨幼苗，下面將該技術(shù)介紹如下。
1 未成熟白芨種子離體培養(yǎng)
1.1 種子表面滅菌及萌發(fā)
選取開花授粉15周后、未開裂的白芨蒴果（含種子），在成都農(nóng)業(yè)科技職業(yè)學院園林園藝分院實驗室超凈工作臺上用75%乙醇進行表面消毒30 s，去離子水沖洗1～2次。用解剖刀及鑷子仔細剝開果實，將種子小心地抖落到無菌組織培養(yǎng)瓶中，加適量5%的次氯酸鈉溶液（滴加1～2滴吐溫-20）消毒20 min。用滅菌的50目細胞篩過濾種子；再用去離子水沖洗、過濾，反復4～6次，沖去表面殘留的消毒液。將細胞篩上過濾的種子以MS+1.0 mg/L TDZ+30 g/L蔗糖、pH 5.8的種子萌發(fā)液體培養(yǎng)基沖洗到三角瓶內(nèi)，于25 ℃、100 r/min黑暗培養(yǎng)15-20 d。通常培養(yǎng)5 d后，種子即可萌發(fā)，此時轉(zhuǎn)入16 h/8 h的光周期環(huán)境下光照培養(yǎng)，一般接種15 d后種子發(fā)育形成葉片。
1.2 種子生長成芽
將接種15 d、已萌發(fā)的白芨種子經(jīng)50目細胞篩濾去液體，把種子原球莖接種于MS+0.2 mg/L 6-BA+30 g/L蔗糖+6 g/L瓊脂、pH 5.8的固體誘導成芽培養(yǎng)基上，盡量分散幼苗，每皿約100粒，于25 ℃、16 h/8 h光周期培養(yǎng)。一般培養(yǎng)30 d后，當幼芽生長為幼苗，約2 cm左右、根長約0.5 cm左右時即可進行生根培養(yǎng)。
1.3 生根培養(yǎng)
將平皿中的白芨幼苗接種到1/2 MS+0.1 mg/L NAA+30 g/L蔗糖+2 g/L活性炭+6 g/L瓊脂、pH 5.8的固體生根培養(yǎng)基上，每瓶接種25～30株，于25 ℃、16 h/8 h光周期環(huán)境里培養(yǎng)，促進幼苗和根的生長，提高壯苗生根率。生根培養(yǎng)30 d后，組培苗生長為健壯的成苗，形態(tài)見圖1。
1.4 煉苗移栽
當白芨幼苗的根生長到3～5 cm時（一般在白芨種子萌發(fā)后75-90 d）即可煉苗移栽，栽前先逐漸揭開瓶蓋適應(yīng)3-5 d外界環(huán)境，期間注意保持瓶內(nèi)濕度。移栽時用清水洗凈根部瓊脂，避免損傷根及原球莖；將健壯的白芨組培苗移栽到裝好育苗基質(zhì)的穴盤中，選蔬菜或花卉育苗基質(zhì)即可，栽后基質(zhì)澆透水，于煉苗棚中培養(yǎng)。棚中前期注意弱光保濕，第一周應(yīng)覆蓋遮陽網(wǎng)保持弱光，并覆蓋薄膜保濕；移栽15 d后白芨成苗即生長正常，移栽30 d左右即可定植，定植后常規(guī)田間管理，移栽情況見圖2。白芨移栽存活的關(guān)鍵是原球莖的大小及活力，一般有原球莖的幼苗均能移栽存活，存活率可達95%以上。
2 近成熟白芨種子離體培養(yǎng)
2.1 種子表面滅菌及萌發(fā)
將開花后20周以上、近成熟的白芨蒴果（未開裂，含種子）用紗布包好，自來水流水沖洗30 min，再在超凈工作臺上用75%乙醇進行表面消毒30 s，無菌水沖洗1～2次，用解剖刀及鑷子小心剝開果實，將種子小心撥入無菌組培瓶中，加適量5%次氯酸鈉溶液（滴加2～3滴吐溫-20）消毒20 min。用滅菌的細胞篩（50目）過濾種子；再用去離子水沖洗、過濾，反復4～6次，沖去表面殘留的消毒液。將細胞篩（50目）上過濾的種子，用MS+2.0 mg/L TDZ+30 g/L蔗糖、pH 5.8的種子萌發(fā)液體培養(yǎng)基沖洗到三角瓶內(nèi)。25 ℃、100 r/min、黑暗培養(yǎng)15 d。
2.2 種子生長成芽
已萌發(fā)的種子經(jīng)細胞篩（50目）濾去液體，將種子原球莖接種于MS+0.1 mg/L 6-BA+30 g/L蔗糖+6 g/L瓊脂、pH 5.8的誘導成芽固體培養(yǎng)基上，盡量分散種子，每皿100粒。25 ℃、16 h/8 h光周期環(huán)境里培養(yǎng)20-30 d。
2.3 生根培養(yǎng)
將生成的幼苗及增殖的幼苗接種到1/2 MS+0.5 mg/L NAA+30 g/L蔗糖+0.5 g/L活性炭+6 g/L瓊脂、pH 5.8的生根培養(yǎng)基里，每瓶25～30株，25 ℃、16 h/8 h光周期環(huán)境里培養(yǎng)30-40 d。
2.4 煉苗移栽
當白芨幼苗的根生長到3～5 cm長時，即可煉苗移栽，栽前先揭開瓶蓋適應(yīng)外界環(huán)境3-5 d。移栽時先用清水洗凈根部瓊脂，注意不要損傷根及原球莖結(jié)構(gòu)；將健壯的白芨組培苗移栽到裝有育苗基質(zhì)的穴盤中，栽后澆透水，于煉苗棚中繼續(xù)培養(yǎng)。管理上前期注意弱光保濕，第一周應(yīng)加蓋遮陽網(wǎng)保持弱光，并覆蓋薄膜保濕；移栽15 d后白芨成苗即可生長正常，移栽30 d左右即可定植；定植后常規(guī)田間管理。
3 成熟白芨種子離體培養(yǎng)
3.1 種子表面滅菌及萌發(fā)
將成熟的白芨蒴果（未開裂，含種子）用紗布包好，自來水流水沖洗30 min；在超凈工作臺上用75%乙醇進行表面消毒30 s，無菌水沖洗1～2次，用解剖刀及鑷子小心剝開果實，將種子小心撥入無菌組培瓶中，加適量5%次氯酸鈉溶液（滴加2～3滴吐溫-20）消毒20 min。將細胞篩（50目）上過濾的種子用MS+0.5 mg/L TDZ+30 g/L蔗糖、pH 5.8的種子萌發(fā)液體培養(yǎng)基沖洗到三角瓶內(nèi)。25 ℃、100 r/min黑暗培養(yǎng)15 d，萌發(fā)率達99%。
3.2 種子生長成芽
已萌發(fā)的種子經(jīng)細胞篩（50目）濾去液體，將種子原球莖接種于MS+0.5 mg/L 6-BA+30 g/L蔗糖+6 g/L瓊脂、pH 5.8的誘導成芽固體培養(yǎng)基上，每皿100粒。25 ℃、16 h/8 h光周期環(huán)境里培養(yǎng)20-30 d。
3.3 生根培養(yǎng)
將長大的幼苗及增殖的幼苗接種到1/2 MS+1.0 mg/L NAA+30 g/L蔗糖+1.0 g/L活性炭+6 g/L瓊脂、pH 5.8的生根培養(yǎng)基上，每瓶25～30株，25 ℃、16 h/8 h光周期環(huán)境里培養(yǎng)30-40 d。
3.4 煉苗移栽
當培養(yǎng)出的白芨幼苗根生長到3～5 cm長時，煉苗移栽，栽前先揭開瓶蓋適應(yīng)自然條件3-5 d。移栽先時用自來水洗凈根部附著的瓊脂，小心不要損傷根及原球莖結(jié)構(gòu)；將健壯的白芨組培苗移栽到裝有基質(zhì)的育苗穴盤中，栽后澆透水，于煉苗棚中放置整齊，繼續(xù)煉苗培養(yǎng)。管理上前期注意弱光保濕，第一周應(yīng)加蓋遮陽網(wǎng)保持弱光，并覆蓋薄膜保濕；移栽15 d后白芨成苗即可生長正常，移栽30 d左右即可定植；定植后常規(guī)田間管理。
4 小結(jié)
采用近成熟的白芨種子進行液體懸浮培養(yǎng)，能顯著提高種子萌發(fā)率，可達99%，并加快原球莖的生成速度（15 d左右）。且該技術(shù)操作簡便，成本低廉，每瓶液體培養(yǎng)基可萌發(fā)數(shù)萬粒種子，且種子萌發(fā)整齊，出苗一致，污染率低，結(jié)合簡化的固體培養(yǎng)基培養(yǎng)，大大縮短了白芨組織培養(yǎng)周期，最快能在75～90 d內(nèi)獲得大量健壯的無菌苗用于移栽，實現(xiàn)了白芨組培苗的高效快速生產(chǎn)，并有效簡化了操作程序，降低了生產(chǎn)成本，便于規(guī)?；a(chǎn)。
作者：熊丙全 廖相建 鄭雪蓮
日水培養(yǎng)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.