CN102227976B - Litsea cubeba pers asexual quick propagation and seedling breeding method - Google Patents
Litsea cubeba pers asexual quick propagation and seedling breeding method Download PDFInfo
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Abstract
The invention discloses a litsea cubeba pers asexual quick propagation and seedling breeding method. The method is a method which comprises the following steps that: rooting is performed outside a litsea cubeba pers tissue cultured sub-generation bud test tube; a litsea cubeba pers high-quality clonal tissue cultured sub-generation bud serves as a cutting shoot; seedling breeding is performed outside a litsea cubeba pers high-quality strain tissue cultured sub-generation bud test tube or through tissue cultured seedling young stem cutting seedling cultivation; MSC (MS+2,380mg/L of Ca(NO3) +38.2mg/L of H3BO)+ 1.6mg/L of BA+0.1mg/L of IAA+10mg/L of VC serves as a sub-generation multiplication culture medium; MSC (MS+2,380mg/L of Ca(NO3) +38.2mg/L of H3BO)+ 0.6mg/L of BA+0.3mg/L of IAA+15mg/L of VC serves as a sub-generation strong seedling culture medium; and yellow soil serves as a substrate; and a litsea cubeba pers sub-generation bud cutting seedling or a litsea cubeba pers tissue cultured young stem cutting seedling is cultured under the culture condition of irradiation of natural scattered light. The cutting survival rate of the sub-generation bud is 85 to 95 percent and the cutting survival rate of the young stem is 80 to 90 percent. The method has the advantages of high reproductive rate, large seedling culture amount, simple method, easiness in operation and low material consumption. The excellent genetic characteristics of a mother strain can be kept in the filial generation; and the problem on asexual seedling breeding of high-quality litsea cubeba pers is solved.
Description
Technical field
The invention belongs to plant Fast Asexual Propagation Technique field, relate to the asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree.
Background technology
(Litsea Cubeba (Lour.) has another name called cubeb litsen tree, Litsea pungens, crosses bee saage the fruit of a cubeb litsea tree; Be Lauraceae (Lauraceae) Litsea (Litsea Lam); Be precious fragrant oil plant dungarunga, the retortable extraction litsea cubeba oil of its flower, leaf, fruit and bark, the mellow fruit oil content is the highest.Contain citral, higher alcohol, organic acid in the litsea cubeba oil, citral content is the primary raw material of preparation essence, spices and synthetic vitamin A, E up to 70%, also can directly be used for Pesticidal and sterilizing, is the maximum natural plant essential oils of China's foreign exchange earning always.The fruit of a cubeb litsea tree mainly is distributed in China Zhejiang, Fujian, Hunan, Hubei, Jiangxi, Guangdong, Guangxi, Yunnan, Guizhou, Taiwan etc. and economizes (district); Also there is distribution in states such as India, Indonesia, Malaysia, mainly are grown in tailo, cutting blank, burned area, deserted mountain shrubbery and the sparse woods.The fruit of a cubeb litsea tree is a kind of with seeds ecological, the economical, societal benefits high unity, because of it has very high economic worth.At present; The fruit of a cubeb litsea tree major part of China still is in wild state, and the fruit of a cubeb litsea tree of processing is picked up from wild fruit more than 85%, and how to be main harvesting mode with destructive chopping at a tree; This not only heavy damage the ecological balance; Also the continuation utilization to fruit of a cubeb litsea tree resource causes extremely bad influence, causes resource exhaustion and output to descend, and makes litsea cubeba oil in that supply falls short of demand at present.Fruit of a cubeb litsea tree wide adaptability, barren-resistant, intercept water enclose soil nature can be good, can be used to administer the open forest land water and soil loss.Traditional cultivation is mainly used seminal propagation, and not only percentage of seedgermination is low, and merit heredity is also unstable, seriously affects the output and quality of litsea cubeba oil.Vegetative propagation is the stable effective method for culturing seedlings that keeps hereditary maternal merit, obtains the fruit of a cubeb litsea tree nursery stock of neat and consistent fast by the Plant Tissue Breeding method.Fruit of a cubeb litsea tree asexual multiplication seedling new technology; It is fruit of a cubeb litsea tree choiceness group training subculture bud with the fruit high yield; Study the outer cuttage root-taking bud technology of its bottle; And tissue cultivating seedling juvenile form branch cuttage raise seedling technique, success cultivate whole plant, intend to the improved variety of fruit of a cubeb litsea tree forest plantation, clone development science and technology support be provided.Take passages as follows about the vegetative prior art of the fruit of a cubeb litsea tree:
One, Ma Chongjian, Hu Jiakai, the tissue culture of fruit of a cubeb litsea tree different explants, forestry science and technology, the 30th volume, the 4th phase, 2005 years.Get then and to give birth to the wild plant of the fruit of a cubeb litsea tree healthy and strong, no damage by disease and insect, the semi-lignified branch of clip diameter 0.3～0.5cm, with tender, tender leaf and more sturdy stem as culture materials.With adopt material clean up earlier with running water, soak 10～15min, use 70% alcohol disinfecting 20min again, use 0.1%HgCl then, conventional sterilization 10～15min, aseptic water washing 4～5 times.Be cut into the segment of 3-4cnl to stem, bud and stem divide the dual mode inoculation: a part of traverse is seeded on the medium, another partly vertical medium that inserts.Leaf then full wafer is put into medium, does not cut.Each culture materials all is inoculated in the agar of additional 3.0% sugar of MS+6-BA 2.0mg/L+2.0rag/L 2.4-D medium and 0.7%), cultivation temperature is 25 ± 2 ℃, the illumination that bud is cultivated is 1500～2000Lx, light application time 12hid.Blade and stem place dark condition to carry out callus induction earlier.
The tissue culture of fruit of a cubeb litsea tree bud: bud produces a part of brown stain in inoculation after 3～10 days, some is penetrated in the medium, makes medium present brown in various degree.Generally inoculation about 5 days, bud began to sprout; Inoculate after 10 days, about 29.4% bud begins to sprout; To inoculation about 25 days, the germination rate of bud reached 67.1% (seeing table).Along with the prolongation of time, blastogenesis is long very fast and vigorous, and survival rate is also than higher.When blastogenesis was long, basal part of stem also expanded gradually and forms callus, and in the callus production process, what at first see is that the explant incision callus occurs and sprouts, and then expands, and otch becomes transparence by brown, and epidermis begins distending.
The tissue culture information slip of fruit of a cubeb litsea tree bud
Two, Lei Ming, Li Wei, the optimization of fruit of a cubeb litsea tree sterile system, Hubei Institute For Nationalities's journal (natural science edition), the 26th the 1st phase of volume of 200 year March.
Material: wild plant; Cultivate vessel and adopt plastic bottle I (330ml), plastic bottle II (580ml), triangular flask (250ml), Cans diameter 6.0cm); Joint filling material is for sealing film, five grades of ventilating covers, plastic bottle, seals film, five grades of ventilating covers and produced by Beijing safe horticultural facility company of shaking; The explant disinfectant is mercury chloride (Beijing dicyclo chemical reagent factory), alcohol.
The condition of supporting is trained: the inoculation back changes the light cultivation over to after secretly cultivating for 1～2 week. and cultivation temperature is 25 ± 2 ℃, light 1500～2000Lux, light application time 12h/d carries out the differentiation of inducing of callus and bud.
Method: materials disinfection: select the good solid individual plant of robust growth, no damage by disease and insect, semi-lignified branch, stem, the leaf of cut-off footpath 0.3～0.5cm, with running water rinse well, washing powder, Efficacious Disinfeitant immersion 10～15min; Flowing water flushing 1h; Get its stem with bud under aseptic condition,, handle 6～10min with 0.1% mercuric chloride again with 75% alcohol disinfecting 20s; Aseptic water washing 5～6 times, the segment confession inoculation use of then material being cut into 1～2cm.Each culture materials all is inoculated in MS+6-BA210mg/L+210mg/L 214-D medium (adding the agar of 3.0% sugar and 0.7%).With the stem is explant; With reference to top condition; (plastic bottle I (330ml), plastic bottle II (580ml), triangular flask (250ml), Cans (diameter 6.0cm) and joint filling material (seal film, five grades of ventilating covers, plastic bottle, seal film, five grades of ventilating covers) and joint filling material access times are to the influence of fruit of a cubeb litsea tree test-tube plantlet pollution for research different culture vessel.
The tissue culture of fruit of a cubeb litsea tree different explants:
1. the grey tissue culture of giving bud in mountain.Bud produces brown stain behind inoculation 3～10d, and some is penetrated in the medium, makes medium present brown in various degree.Behind inoculation 7d, bud begins to sprout; Behind the 12d, about 27.2% bud begins to sprout; During to inoculation 22d, the germination rate of bud reaches 57.3%.Simultaneously, base portion also expands gradually and forms callus, and in the callus production process, what at first see is that the explant incision callus occurs and sprouts, and the otch brown becomes transparence, and epidermis begins distending;
2. the callus induction of fruit of a cubeb litsea tree stem.Fruit of a cubeb litsea tree stem section just has stem section lateral bud to begin to sprout in inoculation behind the 10d, and while explant incision is expanded and white callus occurred; After 3～4 weeks of inoculation, white callus changes into green kick gradually and differentiates green Xiao Cong bud gradually, and inductivity is up to 43.0%.Some callus agglomerate can produce some faint yellow secretion; These faint yellow secretion infiltrate in the medium gradually; Form the zone of a dark color on surface that callus contacts with medium, and to around diffusion, and the prolongation of asking when cultivating; The secretion that the callus agglomerate produces is transformed into brown, and it is thirsty and dead that last this callus is easy to become; The callus induction of fruit of a cubeb litsea tree leaf.10d after inoculation, blade then curl, thicken, but induced velocity is slow, and after 2～3 weeks, some explant forms the callus of bulk gradually by white point, and inductivity is up to 11.0%.Leaf brown stain speed is fast than stem and bud.The survival rate of blade is lower, and growth speed is sneezed slowly.About inoculation 20d, blade edge begins to occur the graininess projection, and celestial body shape point continues to cultivate the back particle and enlarges gradually.
Three, Chinese patent CN 100563431C, application number 200710170434.6, the method for inducing litsea cubeta multiple shoot.It is characterized in that comprising the steps:
1) explant sterilization: choose fruit of a cubeb litsea tree butt portion children shoot, remove blade, clean the back so to go into mass concentration be 3% H
2O
2The middle immersion 15～20 minutes, taking-up changes over to immediately and contains the 0.1%HgCl that mass concentration is 1～5% Tween-80
2Soaked in the solution 10～20 minutes; Constantly use banister brush grooming branch therebetween; Take out then with aseptic water washing 5～6 times and with the blotting paper of sterilizing water is blotted, cut then and contain stem segment and make explant, be seeded in rapidly on the MS medium; Put into the dark place and cultivate, explant was transferred on the new MS medium in second day;
2) bud is induced: will be on the MS medium no longer the explant of brownization change in the inducing culture, inducing culture is MS+6-BA0.5～1.0mg/L+IBA 0.1～0.5mg/L+GA 1.0～2.0mg/L+ agar 7.0g/L+ sucrose 30g/L, pH6.0; Cultivation temperature is 25 ± 2 ℃, periodicity of illumination 12～14h.d-l, and luminous intensity is 1200～2000lux; Behind the inducing culture 21 days, obtain induced bud;
3) bud propagation: cut single induced bud, remove 2/3 blade, cut the stem section that contains joint and be inoculated on the proliferated culture medium, proliferated culture medium is MS+6-BA 1.0～2.5mg/L+IBA 0.2～0.5mg/L+ agar 7.0g/L+ sucrose 30g/L, pH6.0; Cultivated 25～30 days, and obtained the propagation bud;
4, will breed bud repeating step 2, step 3 is carried out successive transfer culture, obtains healthy and strong litsea cubeta multiple shoot.
Summary of the invention
The object of the invention: in order to solve the problem that exists in the above-mentioned prior art, the efficient Fast Asexual Propagation Technique of exploitation fruit of a cubeb litsea tree choiceness aims to provide the asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree.
Technical scheme of the present invention is: utilize the totipotent characteristics of plant cell tool, spray is under isolated condition, and the people provides particular environment condition and medium to cultivate the mechanism that forms whole plant.Giving birth to spray then with the individual plant of fruit yield >=40kg is explant, and under aseptic condition, the people is environmental condition and the medium culture subculture bud that provides the suitable fruit of a cubeb litsea tree to grow; With group training subculture bud is the fringe bar, and yellow soil is a matrix, and growth hormone is that rooting promoter is handled the fringe bar, and taking root through callus forms the method for whole plant.
The present invention is achieved in that
The asexual rapid seedling cultivation method of a kind of fruit of a cubeb litsea tree; It is characterized in that: take root outward with fruit of a cubeb litsea tree group training subculture bud test tube; With fruit of a cubeb litsea tree choiceness group training subculture bud be to insert fringe, and the excellent strain group training of fruit of a cubeb litsea tree subculture bud test tube is grown seedlings outward or the method for tissue cultivating seedling juvenile form branch cuttage and seedling culture, cultivation fruit of a cubeb litsea tree subculture bud cuttage seeding or fruit of a cubeb litsea tree tissue cultivating seedling juvenile form branch cuttage seeding; Comprise that the subculture bud cultivates/build garden, matrix, the processing of fringe bar and cuttage, grows seedlings and go out the garden operational sequence, its operating procedure is following:
(1) garden is cultivated/built to the subculture bud: with fruit yield 40kg >=individual plant to give birth to spray then be explant, aseptic bud is obtained in the sterilization inoculation, aseptic bud is transferred on shoot proliferation medium and the subculture strong seedling culture base bred and strong seedling culture; Or set up tissue cultivating seedling juvenile form branch cuttage cutting orchard with routine;
(2) matrix: the matrix dark 30cm in 8 * 12mm plastic containers that after pulverizer is pulverized, packs into, preceding 1 day of cuttage is with 0.3～0.5% disinfecting solution of potassium permanganate, 12～24h, the running water cleaning medicine and the matrix of drenching before the cuttage;
(3) fringe bar processing/cuttage: with the subculture simple bud is the fringe bar, the clip robust growth, the subculture bud of height >=2cm, the 1mm place shears under the end segment, keeps 3～4 blades, place the solution of taking root to handle 15～30min after, vertically insert in the matrix, degree of depth bud grows 1/4; Or the collection robust growth, be semi-lignified or lignified tissue cultivating seedling juvenile form branch, through the processing of finite concentration rooting promoter, in vertically inserting in the matrix May of September to next year then;
(4) grow seedlings: build the high shading shed of 200cm, degree of shading 75% is set up the little shed of 50～60cm high sealing under the cool canopy, cultivates fruit of a cubeb litsea tree subculture bud cuttage seeding or group training juvenile form branch cuttage seeding, and natural scattering illumination is penetrated and grown seedlings;
(5) go out the garden: can go out garden afforestation when the high 20cm of growth of cuttage seedlings is above.
Above-described subculture medium and strong seedling culture base adopt MSC (MS+Ca (NO
3)
2380mg/L+H
3BO
38.2mg/L)+BA 1.6mg/L+IAA 0.1m g/L+VC 10mg/L is the shoot proliferation medium by, MSC (MS+Ca (NO
3)
2380mg/L+H
3BO
38.2mg/L)+BA 0.6mg/L+IAA 0.3mg/L+VC 15mg/L is a subculture strong seedling culture base.
Above-described matrix adopts yellow soil.
Above-described natural scattering illumination is penetrated and is grown seedlings, and is 800～6000Lx natural scattering light, irradiation 8～10h/d.
The above-described solution of taking root adopts ABT
6The mixed solution of 400～600ppm and IBA 100～300ppm.
The method that above-described training subculture bud test tube is grown seedlings outward is that every bottle of 60～80ml seals with cotton balls with the triangular flask packing of medium with 500ml, again with brown paper wrapping cotton balls and bottleneck, at 120 ℃ and 1.1kg.cm
2Under the condition, 15min is subsequent use with the high-pressure steam sterilizing pan sterilization; With every clump 5～8 strain, the mode that every bottle graft kind is 6～8 clumps is inoculated on the new subculture medium with the tender shoots of robust growth, and 25 ± 5 ℃ temperature, natural scattering illumination is penetrated and grown seedlings; Cultivate 25～30d, height of seedling 2.5～5cm, 4～6 in blade; With the outer cottage method of conventional bottle, will handle the vertical cuttage of subculture bud in matrix through rooting promoter, be sealed to little shed with plastic film then; Temperature in the canopy is controlled at 15～35 ℃, air humidity 90%, 15d after the cuttage; Every separated 3d opens plastic film 1 time, and time 15～30min opens 1 every day backward; Time 60～120min, slotting back 25～40d rooting percent reaches at 80% o'clock and can open film, looks strong and weak the shade net that suitably hides 75% shade density of sunlight; Cuttage finishes with the carbendazim spray nursery stock of 0.1% concentration, whenever at a distance from carbendazim, tpn and the topsin of 7～10d spray 1 time 0.1%, is used alternatingly later on; Timely trickle, the withered blade of removing and plant; Behind cuttage 15～20d, every at a distance from 10～15d spray foliage fertilizer 1 time, concentration 0.05～0.1%, the nursery stock tender leaf grows 2～3 to begin to topdress, every at a distance from 15～25d sealing fertilizer 1 time, liquid manure, concentration 0.05～3% is looked the growth of nursery stock, from thin to thick.When the high 20cm of growth of cuttage seedlings is above, can go out garden afforestation.
The method that above-described tissue cultivating seedling juvenile form branch cuttage seeding is grown seedlings is to set up earth seedbed or the high overhead husky bed of liftoff 30～50cm by the specification that is about 1000cm, wide 120cm, high 30cm, draining furrow width 50cm; Select the tissue cultivating seedling of high 15～20cm, by the seeding row spacing field planting of 10～15cm * 20～25cm, 20～30d is liftoff, and 15cm place truncation is urged to sprout; Nursery stock recovers growth, puts forth new leaves to begin to topdress 2～3 the time, whenever topdress 1 time at a distance from 15～30d, composite fertilizer, liquid manure concentration 0.2～0.5%, the growth and the climatic variation situation of looking nursery stock, the concentration of applying fertilizer from thin to thick, number of times is cultivated tissue cultivating seedling from more to less; The fringe bar is handled and cuttage is to gather robust growth, and 10～15cm is long, is semi-lignified or lignification tissue cultivating seedling spray; All the other are wiped out to keep 3～4 blades in top, and the branch base portion is cut sth. askew into level and smooth inclined-plane with sharp cutter, places the solution of taking root to handle 30～50min; The vertical matrix of inserting is packed into after cracker is pulverized in the plastic containers of 8 * 12cm; Degree of depth bud is long by 1/3, and preceding 1 day 0.3～0.5% disinfecting solution of potassium permanganate 12～24h of cuttage cleans the medicine matrix of drenching with running water before the cuttage; It is that the cuttage of fringe bar is drenched and is sealed to the high little shed of 60cm with plastic film behind the water that cuttage seeding is cultivated; Natural scattering illumination is penetrated and is grown seedlings, and temperature of shed is controlled at 15～35 ℃, air humidity 80～90%, and 15d after the cuttage, every separated 3d opens plastic film 1 time, and time 30～60min opens time 60～120min every day 1 time backward; Slotting back 25～40d rooting rate reaches at 80% o'clock and can open film, looks strong and weak the shade net that suitably hides 75% shade density of sunlight.Insert the back a kind of germ killing drugs in carbendazim, tpn and the topsin of 7～15d spray 1 time 0.1% whenever, and be used alternatingly; Timely trickle, the withered blade of removing and plant; Behind cuttage 6～10d, whenever spray foliage fertilizer 1 time at a distance from 10～15d, concentration 0.05～0.1%, the nursery stock tender leaf grows 2～3 to begin to topdress; Whenever at a distance from 15～30d sealing fertilizer 1 time, liquid manure, concentration 0.05～3%; Look the growth of nursery stock, apply fertilizer from thin to thick, number of times has many to few; When the high 20cm of growth of cuttage seedlings is above, can go out garden afforestation.
Advantage of the present invention and good effect:
1, the group training subculture bud cultivated with the excellent strain of fruit of a cubeb litsea tree fruit high yield of method of the present invention be slotting fringe, is matrix with the yellow soil, ABT
6With the mixed solution of IBA finite concentration proportioning be rooting promoter, 15～35 ℃ of temperature, air humidity is more than 90%, the 60cm higher primary school shed control light of preserving moisture, environment form whole plant, planting percent 85～95% down.Tissue cultivating seedling is taken root and is accomplished with transplanting a step, has reduced the program of taking root in the bottle, has shortened seedling raise period; Reduced cost simultaneously; Can produce the higher group training subculture blastogenesis offspring wood of genetic stability in a short time in batches and be applied to market, tree growth is good, and forest form is neat.
2, for adopting the fringe maternal plant, it sprouts healthy and strong spray is the fringe bar to the present invention's method, is matrix with the yellow soil, ABT with the excellent strain clonal tissue culture of the fruit of a cubeb litsea tree fruit high yield seedling that is colonizated in the land for growing field crops
6With IBA finite concentration proportioning mixed solution be root-growing agent, 15～35 ℃ of temperature, air humidity 80～90%, the preserve moisture environment of control light of 60cm higher primary school shed forms down whole plant, planting percent 70%～85%.Under the large natural environment condition, grow seedlings, consumed power has not obviously reduced the clone seedling cost, can produce the higher group training subculture blastogenesis offspring of genetic stability simultaneously in a short time in batches and be applied to growth, and tree growth is good, and forest form is neat.
3, the asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree can reach rapid batch and produce high yield, high-quality clone nursery stock purpose; Wherein group is cultivated seedling-growing method and is taken up an area of fewly, and growing seedlings not limited by season and region, artificially develops promoting the wild good resource of the fruit of a cubeb litsea tree, improve the litsea cubeba oil unit are output and quality, export goods and earn foreign currency all significant.
Embodiment
Below in conjunction with embodiment, the method that the fruit of a cubeb litsea tree of the present invention group training subculture bud bottle is grown seedlings outward with tissue cultivating seedling juvenile form branch cuttage and seedling culture further describes.
Embodiment 1
The time of taking root is April to fruit of a cubeb litsea tree group training subculture bud test tube outward.Use the individual plant bud organ of fruit of a cubeb litsea tree fruit yield>=40kg to be explant, aseptic bud is obtained in sterilization, aseptic bud is transferred to bred on the subculture medium and strong seedling culture. and the shoot proliferation medium is by MSC (MS+Ca (NO
3)
2380mg/L+H
3BO
38.2mg/L)+BA 1.6mg/L+IAA 0.1mg/L+VC 10mg/L, strong subculture seedling medium is by MSC (MS+Ca (NO
3)
2380mg/L+H
3BO
38.2mg/L)+BA 0.6mg/L+IAA0.3mg/L+VC 15mg/L.Medium is with the triangular flask packing of 500ml, and every bottle of 65ml seals with cotton balls, again with brown paper wrapping cotton balls and bottleneck, at 120 ℃ and 1.1kg.cm
2Under the condition, 15min is for use with the high-pressure steam sterilizing pan sterilization.In between sterile working with the tender shoots of robust growth with every clump 5 strain, the mode that every bottle graft kind is 6 clumps is inoculated on the new subculture medium, 25 ± 5 ℃ temperature in culturing room; The illumination of 800～6000Lx natural scattering is penetrated the condition of 8～10h/d and is cultivated down; Cultivate 28d, bud growth coefficient 3.5, height of seedling is more than 2.5; 4 in blade can carry out the outer cuttage of test tube.
With fruit of a cubeb litsea tree group training subculture bud is to insert fringe, and yellow soil is a matrix.The subculture bud is cultivated 30d in the strong seedling culture base, when transferring medium new simple bud stalwartness >=2.5cm is sheared, and puts into the bottle of sterile water and preserves moisture.Yellow soil is after pulverizer is pulverized, and dark 30cm is placed on and puts up the 200cm height in 8 * 12cm plastic containers of packing into, is equipped with in the canopy of 75% shading screen, with 0.5% concentration potassium permanganate sterilization 24h, inserts preceding with the running water matrix of drenching.In building canopy, prune simple bud, cut angle in the place with 1mm under the sharp cutter end segment, tangent plane is straight, smooth, cleaning, and the branch skin can not perk, removes base portion leaf and petiole, keeps 3 leaves, base portion insertion root-inducing promoter ABT
6Handle 20min in the 400ppm+IBA 2000ppm mixed solution, take out budling and vertically insert in the matrix, the degree of depth is that bud is long by 1/4, puts up the shed plastic film sealing of 50cm higher primary school.Insert in the preceding 15d in back, every separated 3d opens plastic film 1 time, and the time is 15min, opens every day 1 time backward; Time is 80min, and in time water spray keeps about 25 ℃ of temperature in the plastic greenhouse; More than 90% the air humidity, in time remove withered plant, cultivate 25d; When having 80% bud seedling rooting to form whole plant, can open plastic film, look strong and weak the shade net that suitably hides 75% shade density of sunlight.Cuttage finishes with the carbendazim spray nursery stock of 0.1% concentration, and every later on carbendazim, tpn and topsin at a distance from 7d spray 1 time 0.1% are used alternatingly.Timely trickle, the withered blade of removing and plant; Behind the cuttage 15d, every separated 10d spray foliage fertilizer 1 time, concentration 0.05%, the nursery stock tender leaf grows 3 to begin to topdress, every separated 20d sealing fertilizer 1 time, liquid manure, concentration 0.1～3% is looked the growth of nursery stock, from thin to thick.The bud seedling is grown high 25cm when above, can go out garden afforestation.Subculture bud cuttage survival rate reaches 95%.
Embodiment 2
The time of taking root is October to fruit of a cubeb litsea tree group training subculture bud test tube outward.Use the individual plant bud organ of fruit of a cubeb litsea tree fruit yield >=40kg to be explant, aseptic bud is obtained in sterilization, aseptic bud is transferred to bred on the subculture medium and strong seedling culture.The shoot proliferation medium is by MSC (MS+Ca (NO
3)
2380mg/L+H
3BO
38.2mg/L)+BA 1.6mg/L+IAA 0.1mg/L+VC 10mg/L, subculture strong seedling culture base is by MSC (MS+Ca (NO
3)
2380mg/L+H
3BO
38.2mg/L)+BA 0.6mg/L+IAA 0.3mg/L+VC 15mg/L.Medium is with the triangular flask packing of 500ml, and every bottle of 65ml seals with cotton balls, again with brown paper wrapping cotton balls and bottleneck, at 120 ℃ and 1.1kg.cm
2Under the condition, 15min is for use with the high-pressure steam sterilizing pan sterilization.In between sterile working with the tender shoots of robust growth with every clump 8 strain; The mode that every bottle graft kind is 7 clumps is inoculated on the new subculture medium, the temperature 25 ± 5 ℃ of culturing room, and the natural scattering illumination of 800～5000Lx left and right sides is penetrated the condition of 8～10h/d and is cultivated down; Cultivate 30d; Height of seedling is more than 2.5, and 4 in blade can carry out the outer cuttage of test tube.
With fruit of a cubeb litsea tree group training subculture bud is to insert fringe, and yellow soil is a matrix.The subculture bud is cultivated 30d in the strong seedling culture base, when transferring medium new simple bud stalwartness >=2.5cm is sheared, and puts into the bottle of sterile water and preserves moisture.Yellow soil is after pulverizer is pulverized, and dark 30cm is placed on and puts up the 200cm height in 8 * 12cm plastic containers of packing into, is equipped with in the canopy of 75% shading screen, with 0.4% concentration potassium permanganate sterilization 22h, inserts preceding with the running water matrix of drenching.In building canopy, prune simple bud, cut angle in the place with 1mm under the sharp cutter end segment, tangent plane is straight, smooth, cleaning, and the branch skin can not perk, removes base portion leaf and petiole, keeps 4 leaves, base portion insertion root-inducing promoter ABT
6Handle 30min in the 500ppm+IBA 200ppm mixed solution, take out budling and vertically insert in the matrix, the degree of depth is that bud is long by 1/4, puts up the shed plastic film sealing of 50cm higher primary school.Insert in the preceding 15d in back, every separated 3d opens plastic film 1 time, and the time is 20min, opens every day 1 time backward; Time is 120min, and in time water spray keeps 20～30 ℃ of temperature in the plastic greenhouse; More than 90% the air humidity, in time remove withered plant, cultivate 40d; When having 80% bud seedling rooting to form whole plant, can open plastic film, look strong and weak the shade net that suitably hides 75% shade density of sunlight.Cuttage finishes with the carbendazim spray nursery stock of 0.1% concentration, and every later on carbendazim, tpn and topsin at a distance from 10d spray 1 time 0.1% are used alternatingly.Timely trickle, the withered blade of removing and plant; Behind the cuttage 18d, every separated 13d spray foliage fertilizer 1 time, concentration 0.05%, the nursery stock tender leaf grows 3 to begin to topdress, every separated 25d sealing fertilizer 1 time, liquid manure, concentration 0.1～3% is looked the growth of nursery stock, from thin to thick.The bud seedling is grown high 25cm when above, can go out garden afforestation.Subculture bud cuttage survival rate reaches 85%.
Embodiment 3
Fruit of a cubeb litsea tree tissue cultivating seedling juvenile form branch cutting time is November.Select physical features smooth, have a good transport service, water supply and drainage location in order makes ground, garden; Confirm manual work or tractor-ploughing soil behind the ground, garden, dark 30cm plays furrow.Set up earth seedbed or the high overhead husky bed of liftoff 50cm by the specification that is about 1000cm, wide 120cm, high 30cm, draining furrow width 50cm; Select the tissue cultivating seedling of high 20cm, press the seeding row spacing field planting of 15cm * 20cm, the normal root water of drenching, 30d is liftoff, and 15cm place truncation is urged to sprout; Nursery stock recovers growth, puts forth new leaves to begin to topdress 3 the time, whenever topdress 1 time at a distance from 20d, composite fertilizer, liquid manure concentration 0.2～0.5%, the growth and the climatic variation situation of looking nursery stock, fertilising concentration from thin to thick, number of times is answered application in time after gathering the fringe bar from more to less.
9 points are gathered robust growth in the morning, and 13cm is long, is semi-lignified or lignification tissue cultivating seedling spray, and the lower end is inserted in the basin and preserved moisture in the clear water.Yellow soil in the plastic containers of the 8 * 12cm that after cracker is pulverized, packs into, is placed in the land for growing field crops, and preceding 1 day 0.5% disinfecting solution of potassium permanganate 12h of cuttage cleans the medicine matrix of drenching with running water before the cuttage.The branch of will preserving moisture takes out to be pruned, and all the other are wiped out to keep 3 blades in top, and the branch base portion is cut sth. askew into level and smooth inclined-plane with sharp cutter, places to contain 600ppmABT
6Handle 40min in the+200ppm IBA mixed solution, vertically insert in the yellow soil matrix, degree of depth bud long 1/3.The cuttage of fringe bar is drenched and is sealed to the high little shed of 60cm with plastic film behind the water; Temperature of shed is controlled at about 30 ℃, air humidity about 85%, and 15d after the cuttage, every separated 3d opens plastic film 1 time, and time 40min opens time 60min every day 1 time backward; Slotting back 36d rooting rate reaches at 80% o'clock and can open film, looks strong and weak the shade net that suitably hides 75% shade density of sunlight.Insert the every germ killing drugs (carbendazim, tpn and topsin are used alternatingly) in back at a distance from 10d spray 1 time 0.1%.Timely trickle, the withered blade of removing and plant; Behind the cuttage 8d, whenever spray foliage fertilizer 1 time at a distance from 15d, concentration 0.05%, the nursery stock tender leaf grows 3 to begin to topdress, whenever separated 20d sealing fertilizer 1 time, liquid manure, concentration 0.05～3% is looked the growth of nursery stock, applies fertilizer from thin to thick, and number of times has arrive to lack more.The bud seedling is grown high 25cm when above, can go out garden afforestation.Tissue cultivating seedling juvenile form branch cuttage survival rate reaches 75%.
Embodiment 4
Fruit of a cubeb litsea tree tissue cultivating seedling juvenile form branch cutting time is March.Select physical features smooth, have a good transport service, water supply and drainage location in order makes ground, garden; Confirm manual work or tractor-ploughing soil behind the ground, garden, dark 25cm plays furrow.Set up earth seedbed or the high overhead husky bed of liftoff 35cm by the specification that is about 1000cm, wide 120cm, high 30cm, draining furrow width 50cm; Select the tissue cultivating seedling of high 15cm, press the seeding row spacing field planting of 15cm * 20cm, the normal root water of drenching, 25d is liftoff, and 15cm place truncation is urged to sprout; Nursery stock recovers growth, puts forth new leaves to begin to topdress 2 the time, whenever topdress 1 time at a distance from 15d, composite fertilizer, liquid manure concentration 0.2～0.5%, the growth and the climatic variation situation of looking nursery stock, fertilising concentration from thin to thick, number of times is answered application in time after gathering the fringe bar from more to less.
9 points are gathered robust growth in the morning, and 12cm is long, is semi-lignified or lignification tissue cultivating seedling spray, and the lower end is inserted in the basin and preserved moisture in the clear water.Yellow soil in the plastic containers of the 8 * 12cm that after cracker is pulverized, packs into, is placed in the land for growing field crops, and preceding 1 day 0.3% disinfecting solution of potassium permanganate 22h of cuttage cleans the medicine matrix of drenching with running water before the cuttage.The branch of will preserving moisture takes out to be pruned, and all the other are wiped out to keep 3 blades in top, and the branch base portion is cut sth. askew into level and smooth inclined-plane with sharp cutter, places to contain 500ppm ABT
6Handle 45min in the+300ppm IBA mixed solution, vertically insert in the yellow soil matrix, degree of depth bud long 1/3.The cuttage of fringe bar is drenched and is sealed to the high little shed of 60cm with plastic film behind the water; Temperature of shed is controlled at about 20 ℃, air humidity about 85%, and 15d after the cuttage, every separated 3d opens plastic film 1 time, and time 40min opens time 120min every day 1 time backward; Slotting back 25d rooting rate reaches at 80% o'clock and can open film, looks strong and weak the shade net that suitably hides 75% shade density of sunlight.Insert the every germ killing drugs (carbendazim, tpn and topsin are used alternatingly) in back at a distance from 8d spray 1 time 0.1%.Timely trickle, the withered blade of removing and plant; Behind the cuttage 10d, whenever spray foliage fertilizer 1 time at a distance from 15d, concentration 0.05%, the nursery stock tender leaf grows 2 to begin to topdress, whenever separated 25d sealing fertilizer 1 time, liquid manure, concentration 0.05～3.0% is looked the growth of nursery stock, applies fertilizer from thin to thick, and number of times has arrive to lack more.The bud seedling is grown high 25cm when above, can go out garden afforestation.Tissue cultivating seedling juvenile form branch cuttage survival rate reaches 85%.
Claims (7)
1. asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree; It is characterized in that: take root outward with fruit of a cubeb litsea tree group training subculture bud test tube; With fruit of a cubeb litsea tree choiceness group training subculture bud is to insert fringe, and the excellent strain group training of fruit of a cubeb litsea tree subculture bud test tube is grown seedlings outward or tissue cultivating seedling juvenile form branch cuttage and seedling culture, cultivates fruit of a cubeb litsea tree subculture bud cuttage seeding or fruit of a cubeb litsea tree tissue cultivating seedling juvenile form branch cuttage seeding; Comprise that the subculture bud cultivates/build garden, matrix, the processing of fringe bar and cuttage, grows seedlings and go out the garden operational sequence, its operating procedure is following:
(1) garden is cultivated/built to the subculture bud: giving birth to spray then with the individual plant of fruit yield >=40kg is explant, and aseptic bud is obtained in the sterilization inoculation, aseptic bud is transferred on shoot proliferation medium and the subculture strong seedling culture base bred and strong seedling culture; Or set up tissue cultivating seedling juvenile form branch cuttage cutting orchard with routine;
(2) matrix: the matrix dark 30cm in 8 * 12mm plastic containers that after pulverizer is pulverized, packs into, preceding 1 day of cuttage is with 0.3～0.5% disinfecting solution of potassium permanganate, 12～24h, the running water cleaning medicine and the matrix of drenching before the cuttage;
(3) fringe bar processing/cuttage: with the subculture simple bud is the fringe bar, the clip robust growth, the subculture bud of height >=2cm, the 1mm place shears under the end segment, keeps 3～4 blades, place the solution of taking root to handle 15～30min after, vertically insert in the matrix, degree of depth bud grows 1/4; Or the collection robust growth, be semi-lignified or lignified tissue cultivating seedling juvenile form branch, through the processing of finite concentration rooting promoter, in vertically inserting in the matrix May of September to next year then;
(4) grow seedlings: build the high shading shed of 200cm, degree of shading 75% is set up the little shed of 50～60cm high sealing under the cool canopy, cultivates fruit of a cubeb litsea tree subculture bud cuttage seeding or group training juvenile form branch cuttage seeding, and natural scattering illumination is penetrated and grown seedlings;
(5) go out the garden: the high 20cm of growth of cuttage seedlings goes out garden afforestation when above.
2. the asexual rapid seedling cultivation method of a kind of fruit of a cubeb litsea tree according to claim 1 is characterized in that: described subculture medium and strong seedling culture base, adopt MSC (MS+Ca (NO
3)
2380mg/L+H
3BO
38.2mg/L)+BA 1.6mg/L+IAA 0.1mg/L+VC 10mg/L is the shoot proliferation medium, MSC (MS+Ca (NO
3)
2380mg/L+H
3BO
38.2mg/L)+BA 0.6mg/L+IAA 0.3mg/L+VC 15mg/L is a subculture strong seedling culture base.
3. the asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree according to claim 1 is characterized in that: described matrix, adopt yellow soil.
4. the asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree according to claim 1 is characterized in that: described natural scattering illumination is penetrated and is grown seedlings, and is 800～6000Lx natural scattering light, irradiation 8～10h/d.
5. the asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree according to claim 1 is characterized in that: the described solution of taking root, adopt ABT
6The mixed solution of 400～600ppm and IBA 100～300ppm.
6. the asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree according to claim 1; It is characterized in that: the described group of method that training subculture bud test tube is grown seedlings outward; Be that every bottle of 60～80ml seals with cotton balls with the triangular flask packing of medium with 500ml; Again with brown paper wrapping cotton balls and bottleneck, at 120 ℃ and 1.1kg/cm
2Under the condition, 15min is subsequent use with the high-pressure steam sterilizing pan sterilization; With every clump 5～8 strain, the mode that every bottle graft kind is 6～8 clumps is inoculated on the new subculture medium with the tender shoots of robust growth, and 25 ± 5 ℃ temperature, natural scattering illumination is penetrated and grown seedlings; Cultivate 25～30d, height of seedling 2.5～5cm, 4～6 in blade; With the outer cottage method of conventional bottle, will handle the vertical cuttage of subculture bud in matrix through rooting promoter, be sealed to little shed with plastic film then; Temperature in the canopy is controlled at 15～35 ℃, air humidity 90%, 15d after the cuttage; Every separated 3d opens plastic film 1 time, and time 15～30min opens 1 every day backward; Time 60～120min, slotting back 25～40d rooting percent reaches at 80% o'clock and opens film, looks strong and weak the shade net that suitably hides 75% shade density of sunlight; Cuttage finishes with the carbendazim spray nursery stock of 0.1% concentration, whenever at a distance from carbendazim, tpn and the topsin of 7～10d spray 1 time 0.1%, is used alternatingly later on; Timely trickle, the withered blade of removing and plant; Behind cuttage 15～20d, every at a distance from 10～15d spray foliage fertilizer 1 time, concentration 0.05～0.1%, the nursery stock tender leaf grows 2～3 to begin to topdress, every at a distance from 15～25d sealing fertilizer 1 time, liquid manure, concentration 0.05～3% is looked the growth of nursery stock, and from thin to thick, number of times is from more to less; When the high 20cm of growth of cuttage seedlings is above, can go out garden afforestation.
7. the asexual rapid seedling cultivation method of the fruit of a cubeb litsea tree according to claim 1; It is characterized in that: the method that described tissue cultivating seedling juvenile form branch cuttage seeding is grown seedlings is to set up earth seedbed or the high overhead husky bed of liftoff 30～50cm by the specification that is about 1000cm, wide 120cm, high 30cm, draining furrow width 50cm; Select the tissue cultivating seedling of high 15～20cm, by the seeding row spacing field planting of 10～15cm * 20～25cm, 20～30d is liftoff, and 15cm place truncation is urged to sprout; Nursery stock recovers growth, puts forth new leaves to begin to topdress 2～3 the time, whenever topdress 1 time at a distance from 15～30d, composite fertilizer, liquid manure concentration 0.2～0.5%, the growth and the climatic variation situation of looking nursery stock, the concentration of applying fertilizer from thin to thick, number of times is cultivated tissue cultivating seedling from more to less; The fringe bar is handled and cuttage is to gather robust growth, and 10～15cm is long, is semi-lignified or lignification tissue cultivating seedling spray; All the other are wiped out to keep 3～4 blades in top, and the branch base portion is cut sth. askew into level and smooth inclined-plane with sharp cutter, places the solution of taking root to handle 30～50min; The vertical matrix of inserting is packed into after cracker is pulverized in the plastic containers of 8 * 12cm; Degree of depth bud is long by 1/3, and preceding 1 day 0.3～0.5% disinfecting solution of potassium permanganate 12～24h of cuttage cleans the medicine matrix of drenching with running water before the cuttage; It is that the cuttage of fringe bar is drenched and is sealed to the high little shed of 60cm with plastic film behind the water that cuttage seeding is cultivated; Natural scattering illumination is penetrated and is grown seedlings, and temperature of shed is controlled at 15～35 ℃, air humidity 80～90%, and 15d after the cuttage, every separated 3d opens plastic film 1 time, and time 30～60min opens time 60～120min every day 1 time backward; Slotting back 25～40d rooting rate reaches at 80% o'clock and opens film, looks strong and weak the shade net that suitably hides 75% shade density of sunlight, and slotting back is a kind of germ killing drugs in carbendazim, tpn and the topsin of 7～15d spray 1 time 0.1% whenever, and are used alternatingly; Timely trickle, the withered blade of removing and plant; Behind cuttage 6～10d, whenever spray foliage fertilizer 1 time at a distance from 10～15d, concentration 0.05～0.1%, the nursery stock tender leaf grows 2～3 to begin to topdress; Whenever at a distance from 15～30d sealing fertilizer 1 time, liquid manure, concentration 0.05～3%; Look the growth of nursery stock, apply fertilizer from thin to thick, number of times from more to less; When the high 20cm of growth of cuttage seedlings is above, can go out garden afforestation.
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| CN107223565A (en) * | 2017-06-13 | 2017-10-03 | 中国林业科学研究院亚热带林业研究所 | Cubeb litsen tree tissue-cultured seedling root media and cultural method |
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