CN100426952C - Tissue culture seedling-growing method for bird's-net fern - Google Patents
Tissue culture seedling-growing method for bird's-net fern Download PDFInfo
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Abstract
The present invention discloses a tissue culture sprout raising method for nest fern. The method comprises the steps of aseptic material acquisition, primary culture, proliferation culture, rooting culture, sprout refining, etc.; aseptic materials are newly germinated bending spires of nest fern; a primary induction culture medium adopts improved MS+6-BA2.0 to 3.0 mg/L+NAA0.1 to 0.3 mg/L+AC1.0 g/L; a proliferation culture medium adopts MS+6-BA0.5 mg/L+AC0.5 g/L; a rooting culture medium adopts 1/2MS+NAA0.1 to 0.2 mg/L+IBA0.1 to 0.2 mg/L; and the sprout refining adopts an intelligent sprout raising system. The method has the following advantages of easy material acquisition, extremely easy aseptic material acquisition, low aberration rate and high propagation coefficient; the propagation times per month can achieve 5 to 6; the rooting rate of tissue culture sprouts can avhieve 100%; and the survival rate of sprout refining is as high as 95%. The tissue culture sprout raising method for nest fern has strong factory production capacity.
Description
Technical field
The present invention relates to field of plant tissue culture technique, specifically is that the group of new pteris fern plant is cultivated seedling-growing method.
Background technology
New pteris fern (Asplenium nidus) Aspleniaceae nest Cyclosorus, another name mountain Soviet Union flower, bird fern fern, mountain badderlocks, vicia unijuga, eagle wing belong to the perennial evergreen pteridophyte that grows nonparasitically upon another plant, and plant height can reach 1 meter～1.2 meters, root-like stock is short, the fibrous branch in top, curling.Leafage is born in short stem top, to radial arrangement all around, is nest like, and petiole is cylindrical, is about 5 centimetres, the wealthy needle-like of draping over one's shoulders of blade, and herbaceous stem, light green color, the two sides is smooth, and leaf is about 100 centimetres.New pteris fern strain shape is plentiful, and the leaf look pale yellowish green, likes shady and cool environment, and is more low temperature resistant, and maintenance management is comparatively extensive, and diseases and insect pests resistance is extremely strong, has very strong adaptability and high ornamental value, is the good sight leaf material of arranging the hall, meeting-place and the making gaily decorated basket.New pteris fern also is the widest fern of purposes in Taiwan, no matter vegetables, leaf material and potted plant its importance arranged all, this also is the main cause that brings up its commerial growing.New pteris fern tradition propagation method mainly adopts cutting maternal plant and two kinds of methods of spore sowing.(1) patterning method: new pteris fern does not have stolon, there is not indefinite bud yet, generally has only a terminal bud, growing point just can grow lateral bud when terminal bud is injured, so its cripetura stem rip cutting 1/2 or 1/4 is planted respectively, two, the sprout that makes new advances the president of the nearly sprout of wound place after three months is cut it with regard to separable again and goes out many plant.This method reproduction rate is very low, and injures maternal plant easily, so seldom adopt.(2) spore seeding method: the new pteris fern spore and is born in blade back, can change into brown when spore is ripe, and can gather sowing this moment.The blade that at first will have the spore maturation is sheared, and carefully spore is scraped with blade to collect, and is housed in dry shady and cool place, and the blade-section that maybe will have spore is cut into block and air-dry.Cleaning and more tiny medium such as thin letterwood bits, peat soil, charring rice husk or sphagna etc. are used in sowing, medium is contained in perspex box or the paddy rice seedling dish, with the spore uniform broadcasting on medium or the blade that will have a spore with the spore placement that faces up, cover lid or seal keeping high humility then with preservative film, and place the greenhouse bed that shades down or indoor bright place.Spore just can germinate and begin to form gametophyte in after planting about 10 days, began to grow sporophyll through 3 months, just can successively bigger plant be transplanted during to 6-8 month.Also long from the last seeding method cycle of spore as can be seen, it is very high to the more important thing is that it requires temperature and humidity, under the natural conditions of area, Zhejiang Province, spore seeding method spore germination rate is very low, be difficult to differentiation and seedling emergence, be not easy to obtain a large amount of seedlings, also be difficult to be produced employing.In addition, because the tiny and villous of new pteris fern spore, thereby the difficulty of drawing materials and sterilize, the production poor operability, and easily morph.
Summary of the invention
The present invention aims to provide the group of a kind of new pteris fern plant and cultivates seedling-growing method, so that obtain a large amount of seedlings in a short time, realizes the factorial seedling growth of new pteris fern Plant Tissue Breeding.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: the group of this new pteris fern plant is cultivated seedling-growing method, it is characterized in that may further comprise the steps:
A, obtain sterilizable material
New pteris fern is placed the clean indoor maintenance of carrying out, avoids watering, treat that spire is sprouted after, the new crooked spire of sprouting of clip cleans, after the sterilization, the explant that is cut into small pieces;
B, cultivation
I, initial incubation; MS+6-BA2.0～3.0mg/L+NAA0.1～0.3mg/L+AC1.0g/L is initial inducing culture with improvement, explant is put on the inducing culture cultivated, wait to sprout sprout and become the seedling of growing thickly after carry out enrichment culture;
II, enrichment culture; With MS+6-BA0.5mg/L+AC0.5g/L is proliferated culture medium, behind the seedling cutting whole into sections of will growing thickly, is seeded on the proliferated culture medium and breeds, and again the seedling of growing thickly of new propagation is continued the cutting whole into sections inoculation, so continues, thereby obtains a large amount of fast propagating seedlings at short notice;
III, culture of rootage; With 1/2MS (macroelement reduces by half)+NAA0.1～0.2mg/L+IBA0.1～0.2mg/L is root media, fast propagating seedling is cut into to be inoculated on the root media behind single the seedling take root;
C, hardening
Fast propagating seedling after will taking root is transplanted to seedling medium, shade, be incubated, preserve moisture and ventilation condition under, cultivate into seedling.
Described improvement MS has replaced chlorion and has kept wherein the constant MS of nitrogen content with nitrate ion.Discover that the chlorion of higher concentration is unfavorable to the cultivation of new pteris fern plant, replace the back culture effect with nitrate ion and be clearly better.
Described improvement MS is preferably: contain NH
4NO
31410.5mg/L, KNO
31900mg/L, MgSO
47H
2O370mg/L, KH
2PO
4170mg/L, Ca (NO
3)
24H
2O 706.8mg/L, trace element, molysite and the same MS of organic principle consumption.
Described cleaning and the disinfectant program that obtains in the sterilizable material step is: use neutral detergent's soaking and washing, rinse well with running water flowing water then, under the superclean bench aseptic condition, earlier with 75% alcohol-pickled sterilization 30 seconds, aseptic water washing is 3 times then, put into 0.1% mercuric chloride solution again and sterilized 6～8 minutes, use aseptic water washing again 5 times, inhale the moisture that removes material surface with sterile gauze.
Described seedling medium is perlite and grit, and wherein the volume ratio of perlite and grit is 5: 1.
Described inducing culture, proliferated culture medium and root media have passed through sterilization treatment before use, and sterilising conditions is 121 ℃～126 ℃, 103kPa～137kPa, and three kinds of medium pH before sterilization are 5.9～6.0.
All be added with common edible sucrose 30g/L and intensity 900g/cm in described inducing culture and the proliferated culture medium
2Agar powder 5.0g/L is added with common edible sucrose 20g/L and intensity 900g/cm in the root media
2Agar powder 5.0g/L.
Described initial incubation and enrichment culture cultivation temperature are 25 ± 2 ℃, intensity of illumination 1500～2000lx; The culture of rootage temperature is 15 ℃～30 ℃, cultivates with natural daylight, and intensity of illumination is controlled at 1500～5000lx.
In the described hardening step, survive the back in hardening and apply fertilizer, to strengthen the plant growing way with the 1/4MS nutrient solution.
Adopted the seedling system of band smart electronics sensing equipment in the described hardening step, controlling seedling bed temperature and humidity more accurately, and this seedling system has blade face fertilising and sterilizing function automatically.
The present invention compared with prior art has following beneficial effect:
1, draws materials easily, very easily obtains sterilizable material, and keep breediness constant.
Bibliographical information was arranged in the past, adopt the mode of new pteris fern spore aseptic seeding to breed, but because the tiny and villous of new pteris fern spore causes the difficulty of drawing materials and sterilize, the production poor operability, and easily morph.And this method is to adopt the immature tender leaf of its New Development as explant, draws materials and sterilizes and all grasp than being easier to, and has very strong operability and repeatability, and can keep breediness seldom to make a variation.
2, growth coefficient is better, and month propagation reaches 5～6 times, can produce a large amount of seedlings at short notice, and produces and not to be subject to seasonal restrictions, and forms scale easily, has very strong batch production prospect of production.Calculate to obtain 30 aseptic explants, in 1 year, can obtain the unified seedling of 5,000 ten thousand strain proterties at least, have very strong batch production production capacity.
3, present technique is when guaranteeing the tissue cultivating seedling quality, taked Plant Tissue Breeding to simplify procedures as far as possible, produced relatively, saved a large amount of electric energy and greatly reduced production cost with the traditional group training, increase economic benefit, had very big value for applications.As: whole process of production adopts running water to replace distilled water, common edible sucrose replaces chemical pure sucrose, the tissue cultivating seedling of taking root adopts the pocket type natural daylight to cultivate (promptly the tissue cultivating seedling that will take root is put into group training Seedling bag, carry out natural daylight then and cultivate in the PC of cleaning plate greenhouse).
4, adopt the seedling system of independent development, the temperature and humidity in control seedbed that can be more accurate creates a good external environment for the new pteris fern tissue cultivating seedling, has improved new pteris fern training tissue culture seedling survival rate, and survival rate can reach more than 95%.This seedling system is by the smart electronics sensing equipment, during as summer high temperature, when surpassing the temperature upper limit of growing seedlings required or air humidity be lower than and grow seedlings when requiring, the seedbed all can the auto spraying cooling, can stop spraying in case satisfy the condition seedbed spraying system of setting.Because the seedbed adopts good especially perlite of air capacity of soils and grit (volume ratio 5: 1) as seedling medium, when keeping the seedbed moistening, can not produce the ponding problem.Seedling bed temperature also can satisfy the required temp. and humidity of growing seedlings fully by the intelligent management of the instrument of growing seedlings in the winter time.The seedling system of our exploitation has also added blade face fertilising and disinfection system automatically, at set intervals promptly, seedling system just can evenly be sprayed at us on the blade face of tissue cultivating seedling by the seedbed spraying system according to nutrient solution or bactericidal liquid that demand of plant growth disposed.By good environment control measure, well guarantee tissue cultivating seedling from heterotrophism to the process that autotrophy transforms, healthy growth still, and can develop into normal plant in the short period of time reaches our fast seedling growing requirement.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment one
1, medium
Initial inducing culture: improvement MS+6-BA2.0mg/L+NAA0.1mg/L+AC1.0g/L.Wherein, improvement MS contains NH
4NO
31410.5mg/L, KNO
31900mg/L, MgSO
47H
2O 370mg/L, KH
2PO
4170mg/L, Ca (NO
3)
24H
2O 706.8mg/L, trace element, molysite, the same MS of organic principle consumption.
Proliferated culture medium: MS+6-BA0.5mg/L+AC0.5g/L.
Root media: 1/2MS (macroelement reduces by half)+NAA0.2mg/L+IBA0.1mg/L.
(121 ℃-126 ℃, 103kPa～137kPa) the preceding PH of sterilization is between 5.9～6.0 to above-mentioned medium HTHP, adds common edible sucrose 30g/L and intensity 900g/cm respectively in initial inducing culture and the proliferated culture medium
2Agar powder 5.0g/L adds common edible sucrose 20g/L and intensity 900g/cm in the root media
2Agar powder 5.0g/L.
2, culture environment condition
Initial incubation and enrichment culture cultivation temperature are 25 ± 2 ℃, illumination 1500～2000lx.New pteris fern tissue cultivating seedling culture of rootage temperature is 15 ℃～30 ℃, cultivates with natural daylight, and intensity of illumination is controlled at 1500～5000lx.
3, the acquisition of sterilizable material
Earlier new pteris fern is put into clean indoorly before getting explant, maintenance one month is avoided watering.After one month,, use neutral detergent's soaking and washing again, rinse well with running water flowing water then the new crooked spire surgical scissors clip of sprouting of new pteris fern.Under the superclean bench aseptic condition, with 75% alcohol-pickled sterilization 30 seconds, aseptic water washing was 3 times then earlier, put into 0.1% mercuric chloride solution sterilization 6-8 minute again, use aseptic water washing again 5 times, inhale the moisture that removes material surface, be cut into the fritter about 1mm with sterile gauze.
4, induce differentiation situation (initial incubation)
Fritter put on the inducing culture cultivate.Incision begins to expand after 10 days, and the fritter surface begins to produce some green corpuscula bulboideas after 30 days.The fritter that has is brownization a bit, takes out to cut brownization and partly transfer on the new inducing culture again under aseptic condition.Along with the progressively increase of green corpuscula bulboidea, there is the part corpuscula bulboidea to begin to sprout after 50 days and sprouts, become the seedling of growing thickly then, show to induce and break up successfully.
5, the seedling and propagating (enrichment culture) of growing thickly
It is one clump that the seedling of will growing thickly cuts into 2～3, is seeded in respectively on the proliferated culture medium, and each Xiao Cong seedling of the left and right sides was just bred to about 10～12 in about 35 days.And all growing way is vigorous for the seedling of growing thickly, and color is emerald green, and part is also sprouted a spot of from giving birth to seedling.The seedling of growing thickly of new propagation is continued the cutting whole into sections inoculation, so go down, can obtain a large amount of fast propagating seedlings at short notice.
6, culture of rootage and hardening
The seedling of growing thickly is cut into single seedling to be inoculated on the root media then.Cultivate 25 days left and right sides new pteris ferns and sprout a lot of radiculas, rooting rate can reach 100%, and the elongation of also growing up rapidly of tissue cultivating seedling blade.Treat that root system grows to about 2cm and can transplant.The used seedling medium of hardening is perlite and grit, and wherein the volume ratio of perlite and grit is 5: 1.Transplant the back management, in 1 month after transplanting, most important work is will carry out to shade, and this aspects of works of ventilating is preserved moisture in insulation.We find out and utilize intelligent temperature control intermittent spraying seedling system, and the training tissue culture seedling survival rate of producing is reached more than 95%.To in time apply fertilizer after hardening survives, can strengthen the plant growing way, treat to go out the garden after 30 days with the 1/4MS nutrient solution.
Embodiment two
Present embodiment is except that used medium, and other ways are identical with embodiment one.
Initial inducing culture: improvement MS+6-BA3.0mg/L+NAA0.2mg/L+AC1.0g/L.Wherein, improvement MS contains NH
4NO
31410.5mg/L, KNO
31900mg/L, MgSO
47H
2O 370mg/L, KH
2PO
4170mg/L, Ca (NO
3)
24H2O 706.8mg/L, trace element, molysite, the same MS of organic principle consumption.
Proliferated culture medium: MS+6-BA0.5mg/L+AC0.5g/L.
Root media: 1/2MS (macroelement reduces by half)+NAA0.1mg/L+IBA0.2mg/L.
Embodiment three
Present embodiment is except that used medium, and other ways are identical with embodiment one.
Initial inducing culture: improvement MS+6-BA2.5mg/L+NAA0.3mg/L+AC1.0g/L.Wherein, improvement MS contains NH
4NO
31410.5mg/L, KNO
31900mg/L, MgSO
47H
2O 370mg/L, KH
2PO
4170mg/L, Ca (NO
3)
24H
2O 706.8mg/L, trace element, molysite, the same MS of organic principle consumption.
Proliferated culture medium: MS+6-BA0.5mg/L+AC0.5g/L.
Root media: 1/2MS (macroelement reduces by half)+NAA0.2mg/L+IBA0.1mg/L.
Claims (7)
1, the group of a kind of new pteris fern plant is cultivated seedling-growing method, it is characterized in that may further comprise the steps:
A, obtain sterilizable material
New pteris fern is placed the clean indoor maintenance of carrying out, avoids watering, treat that spire is sprouted after, the new crooked spire of sprouting of clip cleans, after the sterilization, the explant that is cut into small pieces;
B, cultivation
I, initial incubation; MS+6-BA2.0～3.0mg/l+NAA0.1～0.3mg/L+AC1.0g/L is initial inducing culture with improvement, explant is put on the inducing culture cultivated, wait to sprout sprout and become the seedling of growing thickly after carry out enrichment culture; Described improvement MS contains NH
4NO
31410.5mg/L, KNO
31900mg/L, MgSO
47H
2O 370mg/L, KH
2PO
4170mg/L, Ca (NO
3)
24H
2O 706.8mg/L, trace element, molysite and the same MS of organic principle consumption;
II, enrichment culture; With MS+6-BA0.5mg/L+AC0.5g/L is proliferated culture medium, behind the seedling cutting whole into sections of will growing thickly, is seeded on the proliferated culture medium and breeds, and again the seedling of growing thickly of new propagation is continued the cutting whole into sections inoculation, so continues, thereby obtains a large amount of fast propagating seedlings at short notice;
III, culture of rootage; With 1/2MS+NAA0.1～0.2mg/L+IBA0.1～0.2mg/L is root media, fast propagating seedling is cut into to be inoculated on the root media behind single the seedling take root; Described 1/2MS refers to the MS that macroelement reduces by half;
C, hardening
Fast propagating seedling after will taking root is transplanted to seedling medium, shade, be incubated, preserve moisture and ventilation condition under, cultivate into seedling.
2, the group of new pteris fern plant according to claim 1 is cultivated seedling-growing method, it is characterized in that described cleaning and the disinfectant program that obtains in the sterilizable material step is: use neutral detergent's soaking and washing, rinse well with running water flowing water then, under the superclean bench aseptic condition, earlier with 75% alcohol-pickled sterilization 30 seconds, aseptic water washing was 3 times then, put into 0.1% mercuric chloride solution again and sterilized 6～8 minutes, use aseptic water washing again 5 times, inhale the moisture that removes material surface with sterile gauze.
3, the group of new pteris fern plant according to claim 1 is cultivated seedling-growing method, it is characterized in that described seedling medium is perlite and grit, and wherein the volume ratio of perlite and grit is 5: 1.
4, the group of new pteris fern plant according to claim 1 is cultivated seedling-growing method, it is characterized in that described inducing culture, proliferated culture medium and root media have passed through sterilization treatment before use, sterilising conditions is 121 ℃～126 ℃, 103kPa～137kPa, and three kinds of medium pH before sterilization are 5.9～6.0.
5, the group of new pteris fern plant according to claim 1 is cultivated seedling-growing method, it is characterized in that all being added with in described inducing culture and the proliferated culture medium common edible sucrose 30g/L and intensity 900g/cm
2Agar powder 5.0g/L is added with common edible sucrose 20g/L and intensity 900g/cm in the root media
2Agar powder 5.0g/L.
6, the group of new pteris fern plant according to claim 1 is cultivated seedling-growing method, it is characterized in that described initial incubation and enrichment culture cultivation temperature are 25 ± 2 ℃, intensity of illumination 1500～2000lx; The culture of rootage temperature is 15 ℃～30 ℃, cultivates with natural daylight, and intensity of illumination is controlled at 150～5000lx.
7, the group of new pteris fern plant according to claim 1 is cultivated seedling-growing method, it is characterized in that in the described hardening step, survives the back in hardening and applies fertilizer with the 1/4MS nutrient solution, to strengthen the plant growing way.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101790962B (en) * | 2010-04-12 | 2012-09-05 | 东莞市生物技术研究所 | Production method of adiantum |
| CN102138522B (en) * | 2010-12-30 | 2012-08-22 | 江苏省农业科学院 | Method for regenerating plants under high frequency by inducing adventitious buds with leaves of davallia bullata |
| CN102715092B (en) * | 2012-07-06 | 2013-10-23 | 中国热带农业科学院热带作物品种资源研究所 | Method for rapidly reproducing new pteris fern seedlings by using prothallium reproduction approaches |
| CN103004606A (en) * | 2012-12-29 | 2013-04-03 | 吉林农业大学 | Germplasm test tube storage and ex-situ conservation method for East Asian phyllitis japonica |
| CN103098712B (en) * | 2013-02-07 | 2014-03-26 | 云南省农业科学院花卉研究所 | Davallia mariesii breeding method |
| CN103125390A (en) * | 2013-03-06 | 2013-06-05 | 中国科学院西双版纳热带植物园 | Tissue-culture rapid propagation method of platycerium |
| CN103444534B (en) * | 2013-09-05 | 2015-07-15 | 江苏省农业科学院 | Regeneration method for high-frequency small green ball plants by using induction of new pteris fern |
| CN103548692B (en) * | 2013-11-04 | 2016-04-06 | 珠海市园艺研究所 | A kind of tissue culture and rapid propagation method of ripple leaf new pteris fern seedling |
| CN104026018B (en) * | 2014-06-20 | 2016-08-17 | 南京工业大学大丰海洋产业研究院 | A kind of new pteris fern Fast-propagation tissue culture medium (TCM) of improvement |
| CN105557515B (en) * | 2015-12-14 | 2018-01-16 | 海南中科花海云商科技股份有限公司 | A kind of tissue culture and rapid propagation method of roundleaf new pteris fern |
| CN114027190B (en) * | 2021-11-08 | 2023-03-24 | 广州花卉研究中心 | Method for tissue culture propagation of pteridium aquilinum by using short stem slices |
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| 鸟巢蕨的组织培养. 秦廷豪等.植物生理学通讯，第40卷第3期. 2004 |
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