CN1526278A - Tissue culture breeding method for clonic fibraurea stem - Google Patents
Tissue culture breeding method for clonic fibraurea stem Download PDFInfo
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Abstract
The tissue culture breeding process for clonic fibraurea stem includes the following steps: collecting flush explant of height 15-40 cm without wound and cracking; sterilizing the explant through first washing with washing powder and then sterilization with mercuric chloride in 0.1 % concentration for 1-2 min and thrice rinsing with bacteria-free water; soaking the explant with solution of cysteine or ascorbic acid to prevent browning; bushy bud introduction and secondary proliferation; and rooting culture with preferably seedling of 2 cm height. The said process with flush explant can propagate excellent seedling of clonic line and the present invention has wide application foreground.
Description
Affiliated technical field
The present invention relates to the herba fibraureae recisae breeding method, particularly herba fibraureae recisae clonal tissue culture propagation method.Belong to tropical forest particularly cultivation and the implantation technique field of herba fibraureae recisae.
Background technology
Herba fibraureae recisae (Daemonorops margaritae Beccari.) is that Palmae (Palmae) is economized rattan subfamily (Calamoideae) and economized the tool of rattan family (Calameae) Daemonorops (Daemonorops) liane of sting, climb, grow thickly, and is the torrid zone, south China, the distinctive accompanying plant of the regional forest of south subtropics.Rattan has good operational characteristic, is the good raw material of making furniture and handicraft.The tender tip of rattan is rich in the multiple nutritional components of needed by human body, and it is edible to do vegetables.Fruit can extract " kylin dragon's blood ".The seed quality is hard, is the traditional material of making " beads ".Therefore, herba fibraureae recisae is the precious forest plants of multipurpose with higher economic worth and DEVELOPMENT PROSPECT, also is the extensively staple rattan kind of cultivation of South China.At present, provinces and regions such as Hainan, Guangdong, Guangxi, Yunnan, Fujian commerial growing herba fibraureae recisae.
In the prior art, herba fibraureae recisae is mainly adopted seminal propagation, seedling growing process successfully be used to the to afforest cultivation of nursery stock.But there is following aspect shortcoming in seminal propagation: (1) is subjected to the influence in season, can only cultivate 1-2 season nursery stock in 1 year.Herba fibraureae recisae seed storage tolerance not under conventional room temperature condition has increased new difficulty to seminal propagation.(2) be 11～December the suitableeest seed collecting season of herba fibraureae recisae and since this in season the place of production humidity little, the easy dehydration of seed under the room temperature condition, thus lose germinating capacity rapidly.Even storage is six months under the optimum storaging condition of constant temperature (15 degree), constant humidity (the media water-bearing rate remains on 50%～65%), germination rate also is reduced to about 65%.(3) seminal propagation can only be bred conventional nursery stock, irreproducible clone nursery stock.Because herba fibraureae recisae is a cross-pollinatd plant, and the domestication degree is not high, so heterozygosis degree height of the herba fibraureae recisae in natural forest and the existing forest plantation, the nursery stock afforestation back of seminal propagation is big in the differentiation of aspects such as growth, sprout tillers, wood property, both be unfavorable for the raising of average yield, be unfavorable for also that the intensive management of rattan woods and rattan gathered and sell.
Last century, the nineties began, the begin one's study group culturation rapid propagating technology of herba fibraureae recisae of Tropical Foresty Inst., Chinese Academy of Foresty Sciences.But babassu tissue culture technology research difficulty is big, through the research of two five-year plans, also can only do the success of explant ability tissue-culturing rapid propagation with embryo, and do explant with no sexual organs such as terminal bud, coppice spout, root, leaves, then can not the tissue-culturing rapid propagation success.Thereby original rattan tissue culture technology only is confined to sapling multiplication, promptly by tissue culture industrial fast breeding nursery stock, but still irreproducible clone nursery stock.Compare with the seed growing nursery stock, the cost height of tissue-culturing rapid propagation, the market competitiveness is low, thereby the existing herba fibraureae recisae tissue culture technology of doing explant with embryo still do not have application prospect, can not produce economic benefit.Because forest genome heterozygosis degree height, the seedling forestation differentiation is big, and the road of clone breeding is normally walked in forest genetics, promptly selects select tree from breeding population, then by vegetative method reproduction improved variety.Existing can only be that the tissue culture technology of explant still can't satisfy the requirement that clone breeding and clone are promoted with the embryo.If do not solve the fast numerous technical problem of herba fibraureae recisae clonal tissue culture, will seriously restrict the pilot scale of the breeding of herba fibraureae recisae clone, breeding and the popularization of breeding.
Summary of the invention
The technical issues that need to address of the present invention, purpose promptly of the present invention is in order to overcome the shortcoming that seasonal effect is big, percentage of seedgermination is low, reproduction speed is slow that is subjected to of prior art seminal propagation method existence, herba fibraureae recisae clonal tissue culture propagation method to be provided.
Technical problem of the present invention can solve by taking following measure: herba fibraureae recisae clonal tissue culture propagation method is characterized in:
1) coppice spout explant collection prevents shattering of the sprout wound and the bud heart in the process of gathering the coppice spout explant, and to make the height of coppice spout explant be 15-40cm;
2) sterilization of explant is adopted sterilization twice, at first, with washing powder explant is cleaned up, peel off 1-2 layer leaf sheath, with 75% alcohol disinfecting 15-20 second, 0.1% mercuric chloride sterilization 8-10 minute, rinsed with sterile water 1 time 5 minutes, second step: peel off unnecessary leaf sheath and be trimmed to the size that is suitable for cultivating, promptly coppice spout explant 3-4cm is long, terminal bud explant 5-6cm is long, again with 0.1% mercuric chloride sterilization 1-2 minute, rinsed with sterile water 3 times was respectively 5 minutes, 5 minutes, 10 minutes;
3) prevent brown stain, adopt cysteine or ascorbic acid solution bubble to wash aforementioned coppice spout explant, or adopt cysteine or ascorbic acid mixed solution bubble to wash aforementioned coppice spout explant, can suppress the brown stain of bud explant effectively, the concentration of described cysteine and ascorbic acid is respectively 2mg/l and 10mg/l;
4) the clump bud is induced and shoot proliferation, and the clump bud induces the culture medium prescription with shoot proliferation to be: KNO3, NH4NO3, CaCl2, MgSO4 and KH2PO4, and its proportioning is respectively: 1.5MS, 0.5MS, 1.0MS, 1.0MS, 2.5MS; Clump bud induction duration 35～40 days, the cycle of successive transfer culture is 45～50 days;
5) culture of rootage, height is fit to culture of rootage the most greater than the bud seedling of 2 cm, root media is: 1.0mg/L NAA+2.0mg/L IBA+0.33MS macroelement+20.g/L sucrose solution+1.0MS trace element+1.0MS vitamin and amino acid, the time of culture of rootage needs 50～60 days, after the bud seedling of not taking root removed outer leaf sheath, carry out the culture of rootage second time.
In order to solve technical problem of the present invention better, also can adopt following scheme
Transplant through carrying out the field after the culture of rootage again, will the height greater than 4cm, root length greater than 4cm, Nursery stock transplanting with 2 above leaves is to the field, the medium that is fit to the transplanting of herba fibraureae recisae tissue cultivating seedling has fine sand, peat soil and yellow soil, also the three can be mixed in 1: 1: 2 ratio, as the transplanting medium of herba fibraureae recisae tissue cultivating seedling.
Transplant medium with 1/1000 disinfecting solution of potassium permanganate, transplant and spray in turn in each week of back with 1/800 tpn and carbendazim.
Fertilizing method is that composite fertilizer's solution of 1/1000 sprays weekly once, transplants the back 1～7 day, and 95% shading screen shades, 8～30 days, 85% shading screen shaded, 31～60 days, 70% shading screen shades, and adopts 50% shading screen to shade subsequently, manages outplanting afforestation always.
The aforementioned the 4th) step clump bud induces the culture medium prescription with shoot proliferation to increase hormone, and the hormone composition is: BA 4.0mg/L+IBA 0.25mg/L+2,4-D 0.75mg/L.
The aforementioned the 5th) 2～4 bud cuttings bud height of seedling degree and size is approaching in the culture of rootage are same budlet clump, are transferred to and carry out culture of rootage on the root media, can cultivate clump bud seedling after the transplanting.
The present invention has following outstanding effect:
Compare with original seed seedling-raising, this method has the advantage that sapling multiplication speed is fast, seedling production is not subject to seasonal restrictions, and is that the tissue culture technology of explant is compared with the embryo with what delivered, and this tissue culture method has the advantage that can breed choiceness.Original tissue culture technology is done explant with embryo, the hereditary basis of propagating materials can't be controlled fully, the nursery stock of breeding is compared with the nursery stock of seminal propagation does not have quality differential, but the cost of group training breeding nursery stock is more high than seed growing nursery stock, be difficult on market, compete, thereby the application prospect of original tissue culture technology with kind of embryo explants is little with the nursery stock of seed growing.This method is done explant with coppice spout, can breed the choiceness through seed selection and mensuration, production be the nursery stock of breeding, have the powerful market competitiveness, thereby present technique has broad application prospects, can produce huge economic benefit.
Embodiment
Embodiment 1:
The coppice spout explant is repeatedly gathered with the high forest farm, golden pheasant hole of wanting in Tropical Forest research institute group training chamber in Guangzhou dragon's cave-stalactite cave institute, use way of the present invention, with saw coppice spout is sawed from maternal plant, the size of coppice spout explant is the 15-40cm height, adopt twice sterilization step of the present invention, the sterilization success rate reaches as high as 59.1%, and average success rate is 34.9%--38.4%.Concrete grammar, step are as follows:
1) coppice spout explant collection, the key of gathering the coppice spout explant is to prevent shattering of the sprout wound and the bud heart.If sprout has wound, earth enters sprout inside easily, the success of can't sterilizing.After the bud heart shattered, the wound was rapid brown stain, easily causes the death of the bud heart.Thereby, can not coppice spout be dug from maternal plant with hoe, can not cut with cutter, must coppice spout be sawed from maternal plant with saw.Coppice spout is among the soil, must avoid gathering in 1-3 days behind rainy day and rain as far as possible.Otherwise pollution rate obviously rises.
The size of suitable coppice spout explant is the 15-40cm height.Coppice spout is too small, and explant sterilization back is easily dead, and does not form enough secondary coppice spout original hases, occurs the high growth of simple bud that continues after the cultured in vitro easily but does not sprout the phenomenon of secondary coppice spout.Coppice spout is excessive, and behind the especially super 50cm, coppice spout has begun to put forth, and stem apex is not at base portion.Keep stem apex, then explant is excessive, and the Nutrient Absorption supply is not gone up, and stem apex can't be grown, and the part brown stain of exposing outside medium is serious, finally causes the death of whole bud.Do not keep stem apex, explant also can lack stem apex and death.Explant collection season, growth and the inducing of bud of clump to sterilization success rate, bud all had remarkable influence.The explant that each month from February to August gathers shows: at the beginning of February-3 month and the explant of gathering September, because of antecedent soil moisture, sterilize successfully easily, 4-8 month rainwater is more, and temperature is higher, and the explant of gathering behind the rain is difficult to sterilize successfully.Do not grow for a long time at medium but 2-3 month gather explant, be difficult to the induced bundle bud.The explant that 4-8 month gathers is sprouted easily, and the secondary tiller bud of its base portion is also easily sprouted, and easily breeds, and wherein, best with the explant that the busy season of growing in 6-8 month gathers, the rate of increase is the highest.
2) sterilization of explant by the characteristics that the multilayer leaf sheath wraps up, is adopted sterilization at bud twice.At first, explant is cleaned up (the coppice spout explant must clean up), peel off 1-2 layer leaf sheath with washing powder, with 75% alcohol disinfecting 15-20 second, 0.1% mercuric chloride sterilization 8-10 minute, rinsed with sterile water 1 time 5 minutes, second step: peel off unnecessary leaf sheath and be trimmed to size (the coppice spout explant 3-4cm length that is suitable for cultivating, terminal bud explant 5-6cm is long), with 0.1% mercuric chloride sterilization 1-2 minute, rinsed with sterile water 3 times was respectively 5 minutes again, 5 minutes, 10 minutes.The sterilization success rate is greater than 40%.
3) prevent brown stain, palm rattan coppice spout explant sterilization back brown stain is very serious, and it is dead rapidly not add the processing meeting.Find through series of studies, adopt methods such as dark place reason, additional activity carbon to be difficult to produce a desired effect, use polyvinylpyrrolidone then can cause explant to produce serious white secretion, adopt cysteine and ascorbic acid can suppress the brown stain of bud explant effectively, the two is in conjunction with better effects if.Be suitable for preventing that the cysteine of the anti-brown stain of rattan class bud explant and the concentration of ascorbic acid are respectively 2mg/l and 10mg/l.
4) the clump bud is induced and shoot proliferation, and the clump bud induces the culture medium prescription with shoot proliferation to be: KNO3, NH4NO3, CaCl2, MgSO4 and KH2PO4 are respectively: 1.5MS; 0.5MS; 1.0MS; 1.0MS; 2.5MS.Hormone is: BA 4.0mg/L+IBA 0.25mg/L+2,4-D 0.75mg/L.The cycle of successive transfer culture is 45～50 days, and the propagation multiple is 3～4 times.In addition, inoculation is induced the clump bud of coppice spout explant with cultural method also certain influence.After the explant sterilization, base portion 0.2-0.5cm is dead because of killing and wounding with brown stain, and dead part has a strong impact on the absorption of nutrition, bud must be tilted or keep flat, make it fully to contact medium, can significantly promote the growth of bud, promote the sprouting of secondary tiller bud, thereby promote propagation.
5) take root prescription and culture of rootage, height is fit to culture of rootage the most greater than the bud seedling of 2cm, and root media is 1.0mg/L NAA+2.0mg/L IBA+0.33MS macroelement+20.g/L sucrose solution+1.0MS trace element+1.0MS vitamin and amino acid.The time of culture of rootage needs 50～60 days, and rooting rate can reach more than 85%.The bud seedling of not taking root carries out the culture of rootage second time after removing outer leaf sheath, can take root.
If want to cultivate clump bud seedling, 2～4 bud cuttings that can bud height of seedling degree and size is approaching are transferred to and carry out culture of rootage on the root media same budlet clump, can cultivate clump bud seedling after the transplanting.
6) transplant in the field, and height is greater than 4cm, and root length has the most suitable transplanting of nursery stock of 2 above leaves greater than 4cm.The medium that is fit to the transplanting of herba fibraureae recisae tissue cultivating seedling has fine sand, peat soil and yellow soil, also the three can be mixed in 1: 1: 2 ratio, as the transplanting medium of herba fibraureae recisae tissue cultivating seedling.Transplant medium with 1/1000 disinfecting solution of potassium permanganate, transplant and spray in turn with 1/800 tpn and carbendazim in each week of back, the bacterium that prevents to mix grows.Fertilizing method is that composite fertilizer's solution of 1/1000 sprays once weekly.Transplanted the back 1～7 day, 95% shading screen shades, and 8～30 days, 85% shading screen shaded, and 31～60 days, 70% shading screen shaded, and adopted 50% shading screen to shade subsequently, manages outplanting afforestation always.Transplanting success is greater than 90%.
At 3～11 monthly portable herba fibraureae recisae tissue cultivating seedling, wherein be the best seasonal migration of herba fibraureae recisae tissue cultivating seedling March～June, and temperature, humidity all be fit to the further cultivation of nursery stock, reaches afforestation height and requirement.In 7-9 month, temperature is too high, and transplanting success is lower slightly, and by the intensity fertilising, nursery stock still can reach the afforestation height.10～November can transplanting survival, but seedling growth season mistake, spring next year, nursery stock still can't reach the afforestation height.
In the present embodiment: BA is the 6-benzylaminopurine, and IBA is an indolebutyric acid, and NAA is a methyl, 2, and 4-D is a 2,4 dichlorophenoxyacetic acid.
Macroelement is made of KNO3, NH4NO3, CaCl2, MgSO4 and KH2PO4, and trace element is made of H3BO3, MnSO44H2O, ZnSO44H2O, KI, Na2MoO42H2O, Na2EDTA, Fe2SO47H2O, CuSO45H2O, CoCl26H2O.
Embodiment 2:
The successful explant of sterilizing adopts anti-browning inducing culture of the present invention and proliferated culture medium, and just visible clump bud produced in 2-3 month, and the clump bud induction rate is about 10%, and after successive transfer culture 45-50 days/generation, the propagation multiple is 3～4 times.
Claims (6)
1, herba fibraureae recisae clonal tissue culture propagation method is characterized in that:
1) coppice spout explant collection prevents shattering of the sprout wound and the bud heart in the process of gathering the coppice spout explant, and to make the height of coppice spout explant be 15-40cm;
2) sterilization of explant is adopted sterilization twice, at first, with washing powder explant is cleaned up, peel off 1-2 layer leaf sheath, with 75% alcohol disinfecting 15-20 second, 0.1% mercuric chloride sterilization 8-10 minute, rinsed with sterile water 1 time 5 minutes, second step: peel off unnecessary leaf sheath and be trimmed to the size that is suitable for cultivating, promptly coppice spout explant 3-4cm is long, terminal bud explant 5-6cm is long, again with 0.1% mercuric chloride sterilization 1-2 minute, rinsed with sterile water 3 times was respectively 5 minutes, 5 minutes, 10 minutes;
3) prevent brown stain, adopt cysteine or ascorbic acid solution bubble to wash aforementioned coppice spout explant, or adopt cysteine or ascorbic acid mixed solution bubble to wash aforementioned coppice spout explant, can suppress the brown stain of bud explant effectively, the concentration of described cysteine and ascorbic acid is respectively 2mg/l and 10mg/l;
4) the clump bud is induced and shoot proliferation, and the clump bud induces the culture medium prescription with shoot proliferation to be: KNO3, NH4NO3, CaCl2, MgSO4 and KH2PO4, and its proportioning is respectively: 1.5MS, 0.5MS, 1.0MS, 1.0MS, 2.5MS; Clump bud induction duration 35～40 days, the cycle of successive transfer culture is 45～50 days;
5) culture of rootage, height is fit to culture of rootage the most greater than the bud seedling of 2cm, root media is 1.0mg/L NAA+2.0mg/L IBA+0.33MS macroelement+20.g/L sucrose solution+1.0MS trace element+1.0MS vitamin and amino acid, the time of culture of rootage needs 50～60 days, after the bud seedling of not taking root removed outer leaf sheath, carry out the culture of rootage second time.
2, herba fibraureae recisae clonal tissue culture propagation method according to claim 1, it is characterized in that: transplant through carrying out the field after the culture of rootage again, will the height greater than 4cm, root length greater than 4cm, Nursery stock transplanting with 2 above leaves is to the field, the medium that is fit to the transplanting of herba fibraureae recisae tissue cultivating seedling has fine sand, peat soil and yellow soil, also the three can be mixed in 1: 1: 2 ratio, as the transplanting medium of herba fibraureae recisae tissue cultivating seedling.
3, herba fibraureae recisae clonal tissue culture propagation method according to claim 2 is characterized in that: transplant medium with 1/1000 disinfecting solution of potassium permanganate, transplant and spray in turn with 1/800 tpn and carbendazim in each week of back.
4, herba fibraureae recisae clonal tissue culture propagation method according to claim 2, it is characterized in that: fertilizing method is that composite fertilizer's solution of 1/1000 sprays weekly once, transplanted the back 1～7 day, 95% shading screen shades, and 8～30 days, 85% shading screen shaded, 31～60 days, 70% shading screen shades, and adopts 50% shading screen to shade subsequently, manages outplanting afforestation always.
5, herba fibraureae recisae clonal tissue culture propagation method according to claim 1 is characterized in that: the 4th) step clump bud induces the culture medium prescription with shoot proliferation to increase hormone, and the hormone composition is: BA4.0mg/L+IBA0.25mg/L+2,4-D0.75mg/L.
6, herba fibraureae recisae clonal tissue culture propagation method according to claim 1, it is characterized in that: the 5th) 2～4 bud cuttings bud height of seedling degree and size is approaching in the culture of rootage are same budlet clump, be transferred to and carry out culture of rootage on the root media, can cultivate clump bud seedling after the transplanting.
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| CNB031469051A CN100512629C (en) | 2003-09-23 | 2003-09-23 | Tissue culture breeding method for clonic fibraurea stem |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101156527B (en) * | 2007-09-07 | 2011-05-11 | 屏边苗族自治县生物资源开发创新办公室 | Asexual breeding method for fibraurea recisa pierre |
| CN102172142A (en) * | 2011-01-28 | 2011-09-07 | 云南大围山生物制药有限公司 | Method for cultivating fibraurea recisapierre by keeping same short |
| CN102805032A (en) * | 2012-08-20 | 2012-12-05 | 广西壮族自治区药用植物园 | Method for preventing daemonorops margaritae callus browning phenomena from occurring |
| CN102972277A (en) * | 2012-12-27 | 2013-03-20 | 中国林业科学研究院热带林业研究所 | Method for quickly raising clonal seedlings of teakwood by light matrix tray |
| CN105145358A (en) * | 2015-09-15 | 2015-12-16 | 广西壮族自治区药用植物园 | Tissue culture and rapid propagation method for common fibraurea stem |
-
2003
- 2003-09-23 CN CNB031469051A patent/CN100512629C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101156527B (en) * | 2007-09-07 | 2011-05-11 | 屏边苗族自治县生物资源开发创新办公室 | Asexual breeding method for fibraurea recisa pierre |
| CN102172142A (en) * | 2011-01-28 | 2011-09-07 | 云南大围山生物制药有限公司 | Method for cultivating fibraurea recisapierre by keeping same short |
| CN102172142B (en) * | 2011-01-28 | 2012-07-04 | 云南大围山生物制药有限公司 | Method for cultivating fibraurea recisapierre by keeping same short |
| CN102805032A (en) * | 2012-08-20 | 2012-12-05 | 广西壮族自治区药用植物园 | Method for preventing daemonorops margaritae callus browning phenomena from occurring |
| CN102805032B (en) * | 2012-08-20 | 2014-03-26 | 广西壮族自治区药用植物园 | Method for preventing daemonorops margaritae callus browning phenomena from occurring |
| CN102972277A (en) * | 2012-12-27 | 2013-03-20 | 中国林业科学研究院热带林业研究所 | Method for quickly raising clonal seedlings of teakwood by light matrix tray |
| CN102972277B (en) * | 2012-12-27 | 2013-12-04 | 中国林业科学研究院热带林业研究所 | Method for quickly raising clonal seedlings of teakwood by light matrix tray |
| CN105145358A (en) * | 2015-09-15 | 2015-12-16 | 广西壮族自治区药用植物园 | Tissue culture and rapid propagation method for common fibraurea stem |
| CN105145358B (en) * | 2015-09-15 | 2017-08-04 | 广西壮族自治区药用植物园 | A kind of herba fibraureae recisae tissue cultivation rapid breeding method |
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| Publication number | Publication date |
|---|---|
| CN100512629C (en) | 2009-07-15 |
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