CN105028213A - Tissue-culturing rapid propagation method for dendrobium officinale - Google Patents
Tissue-culturing rapid propagation method for dendrobium officinale Download PDFInfo
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Abstract
The invention discloses a tissue-culturing rapid propagation method for dendrobium officinale. The tissue-culturing rapid propagation method includes the steps of primary culturing, wherein seed culturing is adopted, stem tip and caulicle culturing is carried out, and protocorm and lateral bud induction is carried out; secondary culturing, wherein repeated transferring culturing is carried out on buds or protocorm obtained through the primary culturing on culturing media comprising 1/2MS, 0.5 mg/L of 6_BA, 0.2 mg/L of NAA, 5% of mashed potatoes, 5% of mashed bananas and 2% of sugar; rooting and hardening-off culturing, wherein culturing media comprise 1/2MS, 0.2 mg/L of NAA, 0.2 mg/L of 6_BIBA, 5% of mashed potatoes, 10% of mashed bananas and 7.5% of sugar, and the pH value is 5.8; inoculation is carried out, and seedling hardening is carried out through media prepared in the mode that pure pine bark is adopted and soaked in 2,000-fold carbendazim liquid for 24 hours, and then piling fermentation is carried out for two months to three months. The seed germination rate is high, the bud-seedling growing speed is high, seedlings are thick, blade surfaces are unfolded, root systems are developed, and the survival rate is high; the aim of rapid and nontoxic propagation of dendrobium officinale seedlings is achieved.
Description
Technical field
The present invention relates to a kind of candidum tissue culturing quick-breeding method.
Background technology
Dendrobium candidum (formal name used at school: Dendrobiumofficinale), also known as ribbed hedyotis herb, belongs to aerial orchid section herbaceous plant epiphytic orchid class, and the stem of noble dendrobium can be divided into tens of kinds, yellow grass, Jin Chai, horsewhip etc., and dendrobium candidum is the superfine product of the stem of noble dendrobium, and it is gained the name because epidermis is iron green.Dendrobium officinale only grows between the back precipice seam on overhanging cliff or on the large tree of virgin forest, and is close to harsh to the requirement of the microclimates such as temperature, humidity, illumination, and an annual plant branch once, is about one-inch, at least takes 2-3 and just can gather.Be traditional rare traditional Chinese medicine, in supplementary Amplifications of the Compendium of Materia Medica, say that " clearing stomach removes abnormal heat, promotes the production of body fluid the stem of noble dendrobium, strains.With for tea, Appetizing spleen-tonifying.Clinically, the stem of noble dendrobium is used for the diseases such as treatment the moon hinders body fluid deficiency, dry polydipsia, food is retched less, abnormal heat, and order after being ill is secretly failed to understand.
The seed of dendrobium candidum is superfine little, and embryonic development is incomplete, and without endosperm tissue, germination rate is extremely low in its natural state.In addition predatoryly for a long time to excavate, cause wild dendrobium candidum resource closely exhausted, be in Critical Condition.Now still there is provenance confusion, planting technology is uneven, and standardized level is low, and imitating wild planting area is little, and product quality is difficult to ensure.
Summary of the invention
The object of the invention is open a kind of candidum tissue culturing quick-breeding method, to solve the problem of seedlings of Dendrobium officinale, for the artificial planting developing dendrobium candidum provides basic condition.
Candidum tissue culturing quick-breeding method, comprise Initial culture, squamous subculture, Rooting and hardening-off culture, concrete steps are as follows:
One, Initial culture:
(1), seed culture
A, fruit collection, gather the ripe capsule do not split 8 ~ September;
B, sterilizing, carry out surface sterilizing in aseptic working platform, and capsule surface cleaned by the alcohol with 70%, then soaks 10 minutes with the mercuric chloride of 0.1%, aseptic water washing 6 times;
C, sowing, suck capsule surface moisture with aseptic filter paper, cut pericarp, evenly sowed by Powdered seed and shake up in media surface or be directly sprinkling upon in sterile water by seed, then get this mixed liquor with aseptic straw and be placed on aseptic culture medium;
D, condition of culture, intensity of illumination is 1600 ~ 2000LX, light application time 10h/d, cultivation temperature 25 ± 2 DEG C;
(2), stem apex, young stem culture:
A, stem apex or young stem collection, select growing way, proterties good, the stem apex of anosis worm or the young stem of band axillalry bud, clean with tap water, washing agent;
B, sterilizing, with alcohol-pickled 30 seconds of 70% in aseptic working platform, then soak 15 minutes with 0.1% mercuric chloride solution, afterwards with aseptic water washing at least 6 times;
C, inoculation, suck surface moisture by the stem apex aseptic filter paper of sterilizing, and cut into 3 ~ 5mm length and the stem section saved with, is inoculated on inducing culture;
(3), protocorm, the induction of the lateral bud
A, two kinds of medium
A:1/2MS+BA0.2 ~ 0.5mg/L+NAA2 ~ 0.5mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%;
B:1/2MS+6_BA1mg/L+NAA0.2mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%, pH value is 5.8;
B, induction step: I, stem apex, stem section are inoculated into induction culture medium A being carried out lateral bud, when inducing lateral bud to grow up to ball-type, cut flakey leaf quilt and surrounding browning is organized; II or the branch cutting that will newly send out, repeatedly cultivate, be inoculated into induction B medium carrying out protocorm; III, be divided into by protocorm fritter to continue to be transferred on B medium after 60 days, later every 50 ~ 60 days subcultures once, make protocorm constantly breed;
Two, dendrobium candidum squamous subculture
(1) medium, the protocorm obtained in Initial culture or bud being transferred to 1/2MS+6_BA0.5mg/L+NAA0.2mg/L+ mashed potato 5%+ banana puree 5%+ sugar 2% to be transferred repeatedly cultivation; The number of times of repeatedly transferring is determined by the seedling amount needed;
(2), inoculation time according to the growing state of seedling, according to large, medium and small classification switching, the quantity that blake bottle is inoculated is determined because of container size;
Switching in (3) 40 ~ 60 days once;
(4) condition of culture: temperature 25 ~ 27 DEG C, illumination 8 ~ 10h/d intensity of illumination 1600 ~ 2000LX.
Three, Rooting and hardening-off culture:
(1), medium, 1/2MS++NAA0.2mg/L+6_BIBA0.2mg/L++ mashed potato 5%+ banana puree 10%+ sugar 7.5%, pH value is 5.8;
(2), inoculate, height 2 ~ 3cm seedling in squamous subculture is separated into individual plant, is inoculated on aforementioned root media;
(3), condition of culture, light application time 10 ~ 12h/d, intensity of illumination: the first two months 1600 ~ 2000LX, later 4000LX, cultivation temperature 25 ± 2 DEG C; When seedling of taking root has 5 ~ 6 exhibition leaves, highly reach 6cm, can supply to transplant training by bottle outlet when root system is normal;
(4) seedling hardening of, taking root training:
A, hardening matrix are selected, and use pure pine bark, specification size is 0.5 × 0.5cm, and through sterilize and fully moistening, the fermentation of spray water obtains, namely employing 2000 times of carbendazim liquids soak 24 hours, then pile and seal fermentation 2 ~ 3 months, accomplish that namely hold agglomerating loosing one's grip falls apart.
B, transplant planting, adopt the cave dish field planting of sterilization, transplanting depth controls at the base portion not burying seedling.
Manage after c, transplant planting, after transplant planting, irrigate normal root water, with shading net shading 80% and according to aerial temperature and humidity, foliar spray, prevents blade face from dewatering, and after 7 days, is not burnt as degree, progressively strengthen illumination with blade face; Daytime keeps 27 DEG C in canopy, be not less than 15 DEG C night, shading 70% after 15 days, and moisturizing prevents drying; Spray once with tpn 600 times of liquid of 75% weekly, diseases prevention is kept a full stand of seedings.
D, the extermination of disease and insect pest, do the temperature humidity regulation and control of booth and ventilated work well, relative moisture controls 60% ~ 65%, and temperature controls at 20 DEG C ~ 30 DEG C; To black rot, anthracnose, the 600 times of liquid of the tpn with 75% or 25% auspicious mycin, 600 times of liquid, continuous spraying 2 ~ 3 times, 7 ~ 10 days, interval; Quicklime control snail is spread around seedbed.
In described embryo culture method, on medium, seeding method is: ripe planting seed is on the medium of 1/2MS+ mashed potato 5%; Immature seed, adds exogenous hormone and the basic element of cell division in the medium, growth hormone sows, or broadcast on the medium of 1/2MS+NAAO0.2mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%.
Also need in described seedling hardening training step of taking root seed and seedling disinfection, cave dish sterilization, nursery bed disinfection, seed and seedling disinfection adopts 2000 times of carbendazim liquids to soak 10 minutes, and must clean up before the dish sowing of cave, rear employing 0.1% potassium permanganate liquid carries out disinfection; Seedbed all must adopt 2000 times of carbendazim liquid medicine jets to drench after often educating a collection of seedling, repeatedly disinfects for 2-3 time.
Adopt Initial culture, squamous subculture (Multiplying culture), Rooting and hardening-off culture three section type incubation step, namely Seeds of Dendrobium Candidum is cultivated and is formed protocorm and form budlet further by Initial culture in seed culture medium, then dendrobium candidum budlet is transferred to be trained in subculture medium and there are 2 ~ 3 true leaves and with the seedling of 1 ~ 2 young root, seedling is transferred in Rooting and hardening-off culture base to be again trained and there are 2 ~ 3 roots, 4 blades, plant height >=2.5cm, stem is thick >=the commodity plantlet in vitro of 0.2cm, then commodity plantlet in vitro is transplanted and be cultured to and can gather to the matrix on seedbed in booth.
By implementing the present invention, seed germination rate is high, bud seedling produce fast, pollute low, commodity plantlet in vitro is sturdy, blade face expansion, well developed root system, survival rate are high; Thus realize the object that seedlings of Dendrobium officinale rapid nontoxic breeds, solve the rare difficult problem of seedling.For Extend culture provides strong scientific basis and guidance, dendrobium candidum resource in imminent danger is made to obtain rational exploitation and utilization.
Embodiment
Following as embodiments of the invention, technical scheme is described further.
One, dendrobium candidum Initial culture: this is the basis of whole incubation, its objective is and explant is carried out to surface sterilizing, sets up sterile system, and the effect of this one-phase is different according to the composition of the sterilizing methods of explant, inoculation operational means and medium.The foundation of dendrobium candidum axenic breeding system, realizes by two kinds of methods (capsule, stem apex or stem section).
1. seed (embryo) is cultivated, the fruit of dendrobium candidum is capsule, the inner seed (10 ~ 30 ten thousand even more) tiny containing extremely many dust, seed development does not entirely only have a simple embryo, lacks endosperm tissue, under natural conditions seed do not germinate or germination rate extremely low.A, fruit collection, 8 ~ September gathers mature seed, the capsule also do not split.At this moment the seed in capsule is separated from each other easy sowing, sterilizing, only can carry out surface sterilizing to capsule.B, sterilizing, remove perianth, carpopodium that capsule place is deposited, in aseptic working platform, carry out surface sterilizing.Capsule surface cleaned by alcohol with 70%, then soaks 10 minutes with the mercuric chloride of 0.1%, aseptic water washing 6 times.C, sowing, suck capsule surface moisture with aseptic filter paper, cut pericarp, Powdered seed is evenly sowed and shakes up in media surface or seed is directly sprinkling upon in a certain amount of sterile water, then get this mixed liquor (the method sowing comparatively evenly, emerge more neat) on aseptic culture medium with aseptic straw.D, medium, ripe planting seed, on the medium of 1/2MS+ mashed potato 5%, does not need to add exogenous hormone.Immature seed, medium needs to add exogenous hormone, can broadcast on the medium of 1/2MS+NAAO0.2mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%.E, condition of culture, intensity of illumination is 1600 ~ 2000LX, light application time 10h/d, cultivation temperature 25 ± 2 DEG C.Cultivate 15 ~ 20 days seeds and turn increase green, spherical in shape by yellow, base portion has the protocorm of filiform hair; Within 50 ~ 60 days, grow a large amount of protocorms and differentiate seedling.When temperature is between 25 ~ 27 DEG C, seed germination growth is very fast; When temperature is lower than 15 DEG C, even if it is also lower to meet nutritional condition germination rate; When Wen Di is at 23 ~ 25 DEG C, protocorm well-grown, propagation are comparatively fast.
2, stem apex, young stem culture, utilizes the stem section of stem apex or the tender band axillalry bud of children to cultivate for propagating materials.Seedling better can keep maternal plant proterties, growth is neat, growing way is prosperous, be easy to standardized production, to the seed selection of new varieties with to expand numerous be unusual effective method; But technical requirement is high, the cycle is long, cost is high, difficulty is large, select suitable medium and method most important.A, explant (stem apex or young stem) gather, and select growing way, proterties good, and the stem apex of anosis worm or the young stem of band axillalry bud, remove leaf, leaf sheath etc. not, clean with tap water, washing agent, described washing agent is the articles for washing of tableware or fruit.B, sterilizing, with alcohol-pickled 30 seconds of 70% in aseptic working platform, then soak 15 minutes with 0.1% mercuric chloride solution, use aseptic water washing afterwards 6 times.C, inoculation, suck surface moisture by the explant aseptic filter paper of sterilizing, and the stem section cutting into 3_5mm long band and have to save is inoculated on inducing culture.
3, protocorm, the induction of the lateral bud, A:1/2MS+BA0.2_0.5mg/L+NAA2_0.5mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%.B:1/2MS+6_BA1mg/L+NAA0.2mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%, pH value is 5.8; Stem apex, stem section are inoculated into induction culture medium A being carried out lateral bud, when inducing lateral bud to grow up to ball-type, cut flakey leaf quilt and surrounding browning is organized; Also the branch cutting can will newly sent out, repeatedly cultivates, is inoculated into induction B medium carrying out protocorm.Be divided into by protocorm fritter to continue to be transferred on B medium after 60 days, later every 50 ~ 60 days subcultures once, make protocorm constantly breed.Protocorm is cultivated about 60 days on inducing culture B without cutting, and indefinite bud can produce gradually, finally develops into band leaf young shoot.
Low concentration sugar (2%) is relatively applicable to differential growth.Condition of culture: intensity of illumination is 1600 ~ 2000LX, light application time 10h/d, cultivation temperature 25 ± 2 DEG C.
Two, dendrobium candidum squamous subculture (Multiplying culture): this one-phase cultivates the bud point or seedling that object is breeding mass efficient, makes it quantity and expands, obtain a large amount of sterile propagation systems rapidly, reach tissue-culturing rapid propagation object.1, the medium protocorm obtained in Initial culture or bud being transferred to 1/2MS+6_BA0.5mg/L+NAA0.2mg/L+ mashed potato 5%+ banana puree 5%+ sugar 2% is transferred repeatedly cultivation, the quantity of female bottle switching is according to production scale, the production schedule and determining.According to the growing state of seedling when 2, inoculating, according to large, medium and small classification switching, the quantity of blake bottle inoculation is determined because of container size, and density is evenly moderate, and size wants consistent.3, switching in 40 ~ 60 days once, and early ageing, the phenomenon such as aging easily appear in overlong time.4, condition of culture: temperature 25 ~ 27 DEG C, illumination 8 ~ 10h/d intensity of illumination 1600 ~ 2000LX.
Three, Rooting and hardening-off culture: bottle seedling rises in value to some, will enter strong sprout to the shunting of part seedling and take root the stage, comprise medium, inoculation, condition of culture, seedling hardening of taking root training link.1, medium, 1/2MS++NAA0.2mg/L+6_IBA0.2mg/L++ mashed potato 5%+ banana puree 10%+ sugar 7-5%, pH value is 5 ~ 8.2, inoculate, 2-3cm seedling highly large in squamous subculture is separated into individual plant, and be inoculated on root media, inoculum density is determined because of culture vessel.3, condition of culture, light application time 10 ~ 12h/d, intensity of illumination: the first two months 1600 ~ 2000LX, later 4000LX, cultivation temperature 25 ± 2 DEG C.When seedling of taking root has 5 ~ 6 exhibition leaves, highly reach 6cm, can supply to transplant training by bottle outlet when root system is normal.3, seedling hardening of taking root training, a, cave dish are selected, and the cave dish of different size is widely different to the growth effect of seedling, should according to seedling size, growth rate etc. because usually selecting the cave dish be applicable in production, to take into account prouctiveness and seedling quality, select 50 hole dishes.B, sterilization, comprise seed and seedling disinfection, cave dish sterilization, matrix sterilization, nursery bed disinfection etc.Seed and seedling disinfection adopts 2000 times of carbendazim liquids to soak 10 minutes, must clean up, the disinfection of rear employing 0.1% potassium permanganate liquid before the dish sowing of cave, and Reusability forbidden by the cave dish of non-sterile.Disinfection is all wanted in seedbed after often educating a collection of seedling.Matrix adopts 2000 times of carbendazim liquids to soak 24 hours, and then heap seals fermentation 2 ~ 3 months.C, substrate preparation, the pure pine bark of hardening matrix, specification size is 0.5 × 0.5cm, and through sterilization, fermentation, and water spray fully moisteningly can not have bulk, accomplishes that namely hold agglomerating loosing one's grip falls apart.D, transplant planting, ensure that each cavities has seedling, and all will in the center in hole, and transplanting depth will be suitable for, and can not bury base portion.Manage after e, transplant planting, after transplant planting, irrigate normal root water, with shading net shading 80% and according to aerial temperature and humidity, suitably carry out foliar spray, prevent blade face from dewatering, after 7 days, progressively suitably strengthen illumination, avoid high light direct projection.General daytime keeps 27 DEG C in canopy, and be not less than 15 DEG C night, shading 70% after 15 days, timely and appropriate discovery moisturizing, prevents drying.Spray once with tpn 600 times of liquid of 75% weekly, diseases prevention is kept a full stand of seedings.Water and fertilizer management is the key factor of cave dish hardening domestication success, and frequently water and can accelerate matrix leaching loss of nutrient, reduce gas permeability, be unfavorable for root growth, humidity is excessive simultaneously, and induction disease is grown, and occurs situations such as falling leaves, bind.Therefore Plug seedling sprinkling method is very important, should water foot in the morning, water permeable, not all not water every day, accomplish that timely and appropriate discovery waters permeable, to the hole of water deficient, can carry out spot film moisturizing in the afternoon.After hardening tames 20 days, particulate application slow-release fertilizer 2kg/ mu, 01% flower the more carries out foliar spray, to ensure that seedling is healthy and strong, neat.F, the extermination of disease and insect pest, do the temperature humidity regulation and control of booth well, and ventilated work, occur with preventing disease and pest, if there is black rot, anthracnose etc., tpn 600 times of liquid of conventional 75% or 25% auspicious mycin, 600 times of liquid, continuous spraying 2 ~ 3 times, 7 ~ 10 days, interval.To the control of snail, around seedbed, spread the control of quicklime method.
Influence factor in production process and solution: 1, medium, in large-scale production, preparation and the use of medium are very crucial.Suitably should adjust the concentration of growth regulator in medium according to the growing way situation of seedling, otherwise can cause and have a strong impact on Growth and Reproduction because growth regulator is too high or too low.2, condition of culture, the growth and breeding of light to seedling has a significant effect, and shows the aspect such as light intensity, light quality, photoperiod, in conjunction with the situation of oneself, will do the adjustment adapted to.The ventilative situation of blake bottle also has considerable influence to seedling growth.3, subculture cycle, Subculture Time is not unalterable, will consider according to the growing state cultivating object, condition of culture, the medium used and seedling.Numerous stage is expanded, for accelerating reproduction speed, when the firm differentiation phase of seedling just cuts subculture in early stage; Later stage under the prerequisite keeping certain breeding radix, when quantitatively producing, in order to more seedlings can be used for taking root, can the subculture of interval long period, reach and can maintain the object that certain breeding amount can improve again plantlet in vitro.4, subculture number, subculture number is unsuitable too many, is embodied in: degradation under undergrowth, regeneration capacity and the rate of increase.5, pollute, it is recurrent for polluting in tissue cultures, badly will increase production cost, cause causing destructive loss time serious if control.Therefore should strengthen management in whole production process, strengthen aseptic consciousness, in strict accordance with sterile working program and the strict operation of requirement.
In height above sea level 2050 meter Jing Wai village, the object trained by group, scale carry out design and the layout in workshop, be successively arranged to a continuous print production line, avoid link to arrange, cause work load in the future and cause confusion by the natural working procedure of group training.
According to the technological process that expected volume and scale of investment and Plant Tissue Breeding are produced, comprise between draining room, medium preparation workshop (weighing room, preparation room, sterilizing chamber), seed receiving vehicle, training workshop, sunlight booth etc.
Adopt Initial culture, squamous subculture (Multiplying culture), Rooting and hardening-off culture three section type incubation step, namely Seeds of Dendrobium Candidum is cultivated and is formed protocorm and form budlet further by Initial culture in seed culture medium, then dendrobium candidum budlet is transferred to be trained in subculture medium and there are 2 ~ 3 true leaves and with the seedling of 1 ~ 2 young root, seedling is transferred in Rooting and hardening-off culture base to be again trained and there are 2 ~ 3 roots, 4 blades, plant height >=2.5cm, stem is thick >=the commodity plantlet in vitro of 0.2cm, then commodity plantlet in vitro is transplanted and be cultured to and can gather to the matrix on seedbed in booth.By implementing the present invention, seed germination rate is high, bud seedling produce fast, pollute low, commodity plantlet in vitro is sturdy, blade face expansion, well developed root system, survival rate are high; Thus realize the object that seedlings of Dendrobium officinale rapid nontoxic breeds, solve the rare difficult problem of seedling.
The key technology of exploratory development large area standardized planting, sums up cultivation management technology, makes dendrobium candidum land for growing field crops popularizing planting evidence-based.Dendrobium candidum resource in imminent danger is made to obtain rational exploitation and utilization.
Inside and outside adopting, screening plastic membrane booth, frame flower beds high density non-soil substrate culture pattern are cultivated in flakes, improve survival rate more than 98%, dendrobium candidum Soilless Culture Methods does not limit by place, no matter be balcony, windowsill, desktop, or ground, field, on tree, room etc. all can use the plantation such as flowerpot, cave dish, a people just can manage very like a cork.The most important thing is that its growth cycle is short, plantation can be gathered about half a year.
Claims (3)
1. candidum tissue culturing quick-breeding method, comprises Initial culture, squamous subculture, Rooting and hardening-off culture, it is characterized in that concrete steps are as follows:
One, Initial culture
(1), seed culture:
a,fruit gathers, and gathers the ripe capsule do not split 8 ~ September;
b,sterilizing, carries out surface sterilizing in aseptic working platform, and capsule surface cleaned by the alcohol with 70%, then soaks 10 minutes with the mercuric chloride of 0.1%, aseptic water washing 6 times;
c,sowing, sucks capsule surface moisture with aseptic filter paper, cuts pericarp, evenly sowed by Powdered seed and shake up in media surface or be directly sprinkling upon in sterile water by seed, then gets this mixed liquor with aseptic straw and be placed on aseptic culture medium;
d,condition of culture, intensity of illumination is 1600 ~ 2000LX, light application time 10h/d, cultivation temperature 25 ± 2 DEG C;
(2), stem apex, young stem culture:
a,stem apex or young stem collection, select growing way, proterties good, and the stem apex of anosis worm or the young stem of band axillalry bud, clean with tap water, washing agent;
b,sterilizing, with alcohol-pickled 30 seconds of 70% in aseptic working platform, then soaks 15 minutes with 0.1% mercuric chloride solution, afterwards with aseptic water washing at least 6 times;
c,inoculation, sucks surface moisture by the stem apex aseptic filter paper of sterilizing, and cuts into 3 ~ 5mm length and the stem section saved with, is inoculated on inducing culture described later;
(3), protocorm, the induction of the lateral bud:
a,two kinds of medium: A:1/2MS+BA0.2 ~ 0.5mg/L+NAA2 ~ 0.5mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%; B:1/2MS+6_BA1mg/L+NAA0.2mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%, pH value is 5.8;
b,induction step: I, stem apex, stem section are inoculated into induction culture medium A being carried out lateral bud, when inducing lateral bud to grow up to ball-type, cuts flakey leaf quilt and surrounding browning is organized; II or the branch cutting that will newly send out, repeatedly cultivate, be inoculated into induction B medium carrying out protocorm; III, be divided into by protocorm fritter to continue to be transferred on B medium after 60 days, later every 50 ~ 60 days subcultures once, make protocorm constantly breed;
Two, dendrobium candidum squamous subculture
(1) medium, the protocorm obtained in Initial culture or bud being transferred to 1/2MS+6_BA0.5mg/L+NAA0.2mg/L+ mashed potato 5%+ banana puree 5%+ sugar 2% to be transferred repeatedly cultivation; The number of times of repeatedly transferring is determined by the seedling amount needed;
(2), inoculation time according to the growing state of seedling, according to large, medium and small classification switching, the quantity that blake bottle is inoculated is determined because of container size;
Switching in (3) 40 ~ 60 days once;
(4) condition of culture: temperature 25 ~ 27 DEG C, illumination 8 ~ 10h/d intensity of illumination 1600 ~ 2000LX;
Three, Rooting and hardening-off culture
(1), medium, 1/2MS++NAA0.2mg/L+6_BIBA0.2mg/L++ mashed potato 5%+ banana puree 10%+ sugar 7.5%, pH value is 5.8;
(2), inoculate, height 2 ~ 3cm seedling in squamous subculture is separated into individual plant, is inoculated on step (1) described root media;
(3), condition of culture, light application time 10 ~ 12h/d, intensity of illumination: the first two months 1600 ~ 2000LX, later 4000LX, cultivation temperature 25 ± 2 DEG C; When seedling of taking root has 5 ~ 6 exhibition leaves, highly reach 6cm, can supply to transplant training by bottle outlet when root system is normal;
(4) seedling hardening of, taking root training
a,hardening matrix is selected, and use pure pine bark, specification size is 0.5 × 0.5cm, and through sterilize and fully moistening, the fermentation of spray water obtains, namely employing 2000 times of carbendazim liquids soak 24 hours, then pile and seal fermentation 2 ~ 3 months, accomplish that namely hold agglomerating loosing one's grip falls apart;
b,transplant planting, adopt the cave dish field planting of sterilization, transplanting depth controls at the base portion not burying seedling;
c,manage after transplant planting, after transplant planting, irrigate normal root water, with shading net shading 80% and according to aerial temperature and humidity, foliar spray, prevents blade face from dewatering, and after 7 days, is not burnt as degree, progressively strengthen illumination with blade face; Daytime keeps 27 DEG C in canopy, be not less than 15 DEG C night, shading 70% after 15 days, and moisturizing prevents drying; Spray once with tpn 600 times of liquid of 75% weekly, diseases prevention is kept a full stand of seedings;
d,the extermination of disease and insect pest, do the temperature humidity regulation and control of booth and ventilated work well, relative moisture controls 60% ~ 65%, and temperature controls at 20 DEG C ~ 30 DEG C; To black rot, anthracnose, the 600 times of liquid of the tpn with 75% or 25% auspicious mycin, 600 times of liquid, continuous spraying 2 ~ 3 times, 7 ~ 10 days, interval; Quicklime control snail is spread around seedbed.
2. candidum tissue culturing quick-breeding method according to claim 1, is characterized in that the seeding method on medium in embryo culture method is: ripe planting seed is on the medium of 1/2MS+ mashed potato 5%; Immature seed, adds exogenous hormone and the basic element of cell division in the medium, growth hormone sows, or broadcast on the medium of 1/2MS+NAAO0.2mg/L+ sugar 2%+ mashed potato 5%+ banana puree 5%.
3. candidum tissue culturing quick-breeding method according to claim 1, it is characterized in that taking root in seedling hardening training step also needs seed and seedling disinfection, cave dish sterilization, nursery bed disinfection, seed and seedling disinfection adopts 2000 times of carbendazim liquids to soak 10 minutes, must clean up before the dish sowing of cave, rear employing 0.1% potassium permanganate liquid carries out disinfection; Seedbed all must adopt 2000 times of carbendazim liquid medicine jets to drench after often educating a collection of seedling, repeatedly disinfects for 2-3 time.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105613296A (en) * | 2016-01-25 | 2016-06-01 | 浙江欧银农业发展有限公司 | Tissue culture inoculating method for dendrobium officinale |
| CN107432240A (en) * | 2017-08-01 | 2017-12-05 | 霍山县大化坪秀峰茶业精制厂 | A kind of method for culturing seedlings of Dendrobidium huoshanness |
| CN113598045A (en) * | 2021-07-16 | 2021-11-05 | 上海植物园 | Culture medium for dendrobium yunnanense and rapid propagation method |
| CN115176688A (en) * | 2022-07-12 | 2022-10-14 | 神农架国家公园科学研究院 | Wild-imitating planting method for dendrobium kojima |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105613296A (en) * | 2016-01-25 | 2016-06-01 | 浙江欧银农业发展有限公司 | Tissue culture inoculating method for dendrobium officinale |
| CN107432240A (en) * | 2017-08-01 | 2017-12-05 | 霍山县大化坪秀峰茶业精制厂 | A kind of method for culturing seedlings of Dendrobidium huoshanness |
| CN113598045A (en) * | 2021-07-16 | 2021-11-05 | 上海植物园 | Culture medium for dendrobium yunnanense and rapid propagation method |
| CN115176688A (en) * | 2022-07-12 | 2022-10-14 | 神农架国家公园科学研究院 | Wild-imitating planting method for dendrobium kojima |
| CN115176688B (en) * | 2022-07-12 | 2023-08-11 | 神农架国家公园科学研究院 | Wild-imitating planting method for dendrobium candidum |
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