CN105638463A - Tissue-culture rapid propagation method for succulents - Google Patents
Tissue-culture rapid propagation method for succulents Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention discloses a tissue-culture rapid propagation method for succulents. The method comprises the following steps: (1) selecting and sterilizing an explant: cutting tender leaves or stems of the succulents, and washing the cut leaves or stems cleanly; carrying out sterilizing treatment by adopting spirit and mercury bichloride, so as to obtain the explant; (2) carrying out primary culture: inoculating the explant obtained in the step (1) to a primary culture medium for primary culture until buds are differentiated from the explant; (3) carrying out propagation culture: cutting off the buds differentiated from the explant in the step (2), and inoculating the buds to a propagation culture medium for propagation culture until the length of a new bud germinated from each growth point is 3cm to 5cm and the multiplication coefficient is greater than 2; (4) carrying out rooting culture: separating new buds germinated in the step (3) into single plants, and inoculating the single plants to a rooting culture medium for rooting culture until each single plant grows 2 to 3 new roots and the length of each new root is greater than 3cm. According to the method, the culture time is short, the propagation frequency is high, young seedlings are regular, and the labor cost is reduced, so that the method can be used for carrying out industrialized production.
Description
Technical field
The present invention relates to biological tissue culture technical field, be specifically related to the tissue culture and rapid propagation method of a kind of succulent.
Background technology
Succulent refers to certain part of vegetable nutritorium, as stem or leaf or root (a few species has two parts concurrently) have the parenchyma of prosperity in order to preserve moisture, and a class plant of the plump succulence that seems in shape. They major parts are grown in arid or area arid for some time in a year, have long time root absorption every year less than moisture, and only the moisture by internal storage sustains life.
Succulent is little potted plant, because it is easily cultivated, and global shape, color and leaf various, ornamental value is high, by consumers, the on-line shop of succulent of sale at present, solid shop/brick and mortar store gather way quickly, and the market demand is huge, but major part succulent manufacturer relies on blade cottage propagation at present, it is inefficient, and breeding coefficient is low, and the breeding cycle is long, site requirements is big, and cost of labor is higher. Therefore, improve the production efficiency of succulent, shorten the cultivation time, reduce cost of labor and become current problem demanding prompt solution.
Summary of the invention
In view of this, the application provides the tissue culture and rapid propagation method of a kind of succulent, its incubation time is short, proliferation frequency is high, and seedling is neat, it is simple to transplanting that the later stage is unified and Cultivate administration, reduce cost of labor, can guarantee that the seedling plants that grows concordance in shape, it is easy to operation, industrialization production can be carried out.
For solving above technical problem, technical scheme provided by the invention is the tissue culture and rapid propagation method of a kind of succulent, comprises the following steps:
(1) outer implant is chosen and sterilizing: cuts the tender blade of the children of succulent or stem section, cleans up; After adopting ethanol and mercuric chloride sterilization treatment, obtain outer implant;
(2) initial culture: outer for step (1) gained implant is inoculated on initial culture base and carries out initial culture, until described outer implant differentiates bud, consisting of of described initial culture base: MS minimal medium, supplements 0.01 0.08mg/LNAA, 2.0 5.0mg/L6-BA, 0.1 0.8g/L caseinhydrolysate, 10 50g/L sucrose, 2 10g/L agar;
(3) enrichment culture: cut by the bud that step (2) China and foreign countries implant differentiates, it is inoculated on proliferated culture medium and carries out enrichment culture, until growing point sprouts the sprouting length that to 3 5cm, growth coefficient is more than 2, consisting of of described proliferated culture medium: MS minimal medium, supplement 0.5 2.0mg/LKT, 0.1 0.4mg/L2,4-D, 1.0 5.0mg/LGA3,10 50g/L sucrose, 2 10g/L agar;
(4) root culture: be separated into individual plant by sprouting, in step (3), the sprouting, it is inoculated on root media and carries out root culture, until individual plant grows 23 Gen Xingen, new root length degree is more than 3cm, consisting of of described root media: 1/4MS minimal medium, supplements 0.01 0.08mg/LNAA, 10 50g/L sucrose, 2 10g/L agar, 0.1 0.5mg/L activated carbon.
Preferably, described outer implant is chosen and the concrete operations of sterilizing are: cuts the tender blade of the children of succulent or stem section, first soaks 10 30min with detergent solution, then scrub axillalry bud position with hairbrush, after scrubbing clean under flowing water flushed night; On aseptic operating platform, ethanol postincubation 5 15s with 75%, with aseptic water washing 34 times, then process 8 12min, aseptic water washing 57 times with the mercuric chloride of 0.1%, dry. Wherein, the time that mercuric chloride processes is determined according to the old tender degree of outer implant.
Preferably, consisting of of described initial culture base: MS minimal medium, supplement 0.03 0.06mg/LNAA, 3.0 5.0mg/L6-BA, 0.3 0.6g/L caseinhydrolysate, 20 40g/L sucrose, 5 8g/L agar.
It is more highly preferred to, consisting of of described initial culture base: MS minimal medium, supplements 0.05mg/LNAA, 4.0mg/L6-BA, 0.5g/L caseinhydrolysate, 30g/L sucrose, 6g/L agar.
Preferably, consisting of of described proliferated culture medium: MS minimal medium, supplement 0.5 1.5mg/LKT, 0.2 0.3mg/L2,4-D, 2.0 4.0mg/LGA3,20 40g/L sucrose, 5 8g/L agar.
It is more highly preferred to, consisting of of described proliferated culture medium: MS minimal medium, supplements 1.0mg/LKT, 0.25mg/L2,4-D, 3.0mg/LGA3,30g/L sucrose, 6g/L agar.
Preferably, consisting of of described root media: 1/4MS minimal medium, supplement 0.03 0.06mg/LNAA, 20 40g/L sucrose, 5 8g/L agar, 0.2 0.3mg/L activated carbon.
It is more highly preferred to, consisting of of described root media: 1/4MS minimal medium, supplements 0.05mg/LNAA, 30g/L sucrose, 6g/L agar, 0.2mg/L activated carbon.
Preferably, in described initial culture, enrichment culture, root culture step, cultivation temperature is 23 27 DEG C, and intensity of illumination is 2400 2500Lx, and light application time is 10 12h/d.
Preferably, described initial culture base, proliferated culture medium, root media pH value be 6.0 6.5.
Herein described MS minimal medium has higher inorganic salt concentration, ensure that the mineral nutrition needed for tissue growth, the growth of callus can also be accelerated, it is more stable ionic equilibrium solution, its nitrate content is high, and the quantity of its nutrient and ratio are suitable, can meet nutrition and the physiological need of plant cell, thus the scope of application is relatively wider, most plants tissue-culturing quick-propagation uses it as the minimal medium of culture medium.
Described 1/4MS minimal medium is on MS minimal medium basis, extract a great number of elements of 1/4, reduce the content of its nitrogen, potassium element, and guarantee that there is in culture medium higher inorganic salt concentration, ensure that the mineral nutrition needed for tissue growth, the growth of callus can also be accelerated, it is more stable ionic equilibrium solution, its nitrate content is high, quantity and the ratio of its nutrient are suitable, meeting nutrition and the physiological need of plant cell, thus the scope of application is relatively wider, most plants tissue-culturing quick-propagation uses it as the minimal medium of culture medium.
Described NAA is naphthalene acetic acid, it it is a kind of auxin, use when plant uses cuttage breeding, it is also possible in plant tissue culture, cell division and expansion can be promoted, induced synthesis adventitious root increases setting, prevent shedding, change female, male flower ratio etc., can through the tender epidermis of blade, branch, seed enters in plant, with nutritional flow transporting to Herb.
Described 6-BA is 6-benzyl aminoadenine, it it is a kind of auxin, its Main Function is the formation promoting bud, can also occur by callus induction, promote cell division, promote undifferentiated histo-differentiation, promote the accumulation of biological substance in vivo, promote that lateral bud occurs, it is prevented that aging, be the basic element of cell division in plant tissue and cell culture.
Described KT is CPPU, is a kind of phenylurea class plant growth regulator with cytokine activity, and its biological activity relatively 6-benzyl aminopurine is high 10 100 times. It can affect the growth of plant sprout, accelerate cell mitogen, promotion cell increase and differentiation, it is prevented that fruit comes off with flower, also can as the basic element of cell division of plant tissue culture.
Described 2,4-D is the one of auxin analog formed for callus induction, can as plant growth regulating hormone, for tissue culture.
Described GA3 is gibberellins, is a plant growth regulators, is primarily to facilitate the growth promoter of crop, maturation ahead of time, improves yield and breaks the dormancy of the organs such as seed, tuber, bulb, stratification, tiller, bolting, improve fruit fruiting rate, it is also possible in plant tissue culture.
Compared with prior art, its detailed description is as follows: technical scheme provides the tissue culture and rapid propagation method of a kind of succulent for the application, including outer implant choose and sterilizing, initial culture, enrichment culture, root culture step. By to initial culture base, proliferated culture medium, root media screening, obtain nutrient media components and the proportioning of the best, adopt the culture medium that aforementioned growth hormone combination becomes, the seedling incubation time obtained is short, and proliferation frequency is high, and seedling is neat, it is easy to later stage unified transplanting and Cultivate administration, can guarantee that the seedling plants that grows concordance in shape, it is easy to operation, industrialization production can be carried out.
Detailed description of the invention
In order to make those skilled in the art be more fully understood that technical scheme, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, comprises the following steps:
(1) outer implant is chosen and sterilizing: cuts the tender blade of the children of succulent or stem section, first soaks 10 30min with detergent solution, then scrub axillalry bud position with hairbrush, after scrubbing clean under flowing water flushed night; On aseptic operating platform, ethanol postincubation 5 15s with 75%, with aseptic water washing 34 times, then process 8 12min, aseptic water washing 57 times with the mercuric chloride of 0.1%, dry. Wherein, the time that mercuric chloride processes is determined according to the old tender degree of outer implant;
(2) initial culture: outer for step (1) gained implant is inoculated on initial culture base and carries out initial culture, until described outer implant differentiates bud, consisting of of described initial culture base: MS minimal medium, supplements 0.05mg/LNAA, 4.0mg/L6-BA, 0.5g/L caseinhydrolysate, 30g/L sucrose, 6g/L agar;
(3) enrichment culture: cut by the bud that step (2) China and foreign countries implant differentiates, it is inoculated on proliferated culture medium and carries out enrichment culture, until growing point sprouts the sprouting length that to 3 5cm, growth coefficient is more than 2, consisting of of described proliferated culture medium: MS minimal medium, supplement 1.0mg/LKT, 0.25mg/L2,4-D, 3.0mg/LGA3,30g/L sucrose, 6g/L agar;When described enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 28 days, the length of sprouting reaches 5cm, and sprouting is sturdy, now can carry out root culture;
(4) root culture: be separated into individual plant by sprouting, in step (3), the sprouting, it is inoculated on root media and carries out root culture, until individual plant grows 23 Gen Xingen, new root length degree is more than 3cm, consisting of of described root media: 1/4MS minimal medium, supplementing 0.05mg/LNAA, 30g/L sucrose, 6g/L agar, 0.2mg/L activated carbon, described process of rooting culture starts to take root when proceeding to 15 days.
In wherein said initial culture, enrichment culture, root culture step, cultivation temperature is 23 27 DEG C, and intensity of illumination is 2400 2500Lx, and light application time is 10 12h/d, described initial culture base, proliferated culture medium, root media pH value be 6.0 6.5.
Embodiment 2
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, is distinctive in that with embodiment 1:
In step (2), consisting of of initial culture base: MS minimal medium, supplement 0.01mg/LNAA, 2.0mg/L6-BA, 0.1g/L caseinhydrolysate, 10g/L sucrose, 2g/L agar;
In step (3), consisting of of proliferated culture medium: MS minimal medium, supplement 0.5mg/LKT, 0.1mg/L2,4-D, 1.0mg/LGA3,10g/L sucrose, 2g/L agar;
In step (4), consisting of of root media: 1/4MS minimal medium, supplement 0.08mg/LNAA, 50g/L sucrose, 10g/L agar, 0.5mg/L activated carbon.
In the present embodiment, when enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 30 days, the length of sprouting reaches 5cm, and sprouting is sturdy; Process of rooting culture starts to take root when proceeding to 15 days.
Embodiment 3
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, is distinctive in that with embodiment 1:
In step (2), consisting of of initial culture base: MS minimal medium, supplement 0.08mg/LNAA, 5.0mg/L6-BA, 0.8g/L caseinhydrolysate, 50g/L sucrose, 10g/L agar;
In step (3), consisting of of proliferated culture medium: MS minimal medium, supplement 2.0mg/LKT, 0.4mg/L2,4-D, 5.0mg/LGA3,50g/L sucrose, 10g/L agar;
In step (4), consisting of of root media: 1/4MS minimal medium, supplement 0.01mg/LNAA, 10g/L sucrose, 2g/L agar, 0.1mg/L activated carbon.
In the present embodiment, when enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 28 days, the length of sprouting reaches 5cm, and sprouting is sturdy; Process of rooting culture starts to take root when proceeding to 17 days.
Embodiment 4
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, is distinctive in that with embodiment 1:
In step (2), consisting of of initial culture base: MS minimal medium, supplement 0.01mg/LNAA, 2.0mg/L6-BA, 0.1g/L caseinhydrolysate, 10g/L sucrose, 2g/L agar;
In step (3), consisting of of proliferated culture medium: MS minimal medium, supplement 2.0mg/LKT, 0.4mg/L2,4-D, 5.0mg/LGA3,50g/L sucrose, 10g/L agar;
In step (4), consisting of of root media: 1/4MS minimal medium, supplement 0.01mg/LNAA, 10g/L sucrose, 2g/L agar, 0.1mg/L activated carbon.
In the present embodiment, when enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 28 days, the length of sprouting reaches 5cm, and sprouting is sturdy;Process of rooting culture starts to take root when proceeding to 17 days.
Embodiment 5
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, is distinctive in that with embodiment 1:
In step (2), consisting of of initial culture base: MS minimal medium, supplement 0.08mg/LNAA, 5.0mg/L6-BA, 0.8g/L caseinhydrolysate, 50g/L sucrose, 10g/L agar;
In step (3), consisting of of proliferated culture medium: MS minimal medium, supplement 0.5mg/LKT, 0.1mg/L2,4-D, 1.0mg/LGA3,10g/L sucrose, 2g/L agar;
In step (4), consisting of of root media: 1/4MS minimal medium, supplement 0.01mg/LNAA, 10g/L sucrose, 2g/L agar, 0.1mg/L activated carbon.
In the present embodiment, when enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 30 days, the length of sprouting reaches 5cm, and sprouting is sturdy; Process of rooting culture starts to take root when proceeding to 17 days.
Embodiment 6
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, is distinctive in that with embodiment 1:
In step (2), consisting of of initial culture base: MS minimal medium, supplement 0.03mg/LNAA, 3.0mg/L6-BA, 0.3g/L caseinhydrolysate, 20g/L sucrose, 5g/L agar;
In step (3), consisting of of proliferated culture medium: MS minimal medium, supplement 0.5mg/LKT, 0.2mg/L2,4-D, 2.0mg/LGA3,20g/L sucrose, 5g/L agar;
In step (4), consisting of of root media: 1/4MS minimal medium, supplement 0.06mg/LNAA, 40g/L sucrose, 8g/L agar, 0.3mg/L activated carbon.
In the present embodiment, when enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 29 days, the length of sprouting reaches 5cm, and sprouting is sturdy; Process of rooting culture starts to take root when proceeding to 15 days.
Embodiment 7
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, is distinctive in that with embodiment 1:
In step (2), consisting of of initial culture base: MS minimal medium, supplement 0.06mg/LNAA, 5.0mg/L6-BA, 0.6g/L caseinhydrolysate, 40g/L sucrose, 8g/L agar;
In step (3), consisting of of proliferated culture medium: MS minimal medium, supplement 1.5mg/LKT, 0.3mg/L2,4-D, 4.0mg/LGA3,40g/L sucrose, 8g/L agar;
In step (4), consisting of of root media: 1/4MS minimal medium, supplement 0.03mg/LNAA, 20g/L sucrose, 5g/L agar, 0.2mg/L activated carbon.
In the present embodiment, when enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 28 days, the length of sprouting reaches 5cm, and sprouting is sturdy; Process of rooting culture starts to take root when proceeding to 16 days.
Embodiment 8
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, is distinctive in that with embodiment 1:
In step (2), consisting of of initial culture base: MS minimal medium, supplement 0.03mg/LNAA, 3.0mg/L6-BA, 0.3g/L caseinhydrolysate, 20g/L sucrose, 5g/L agar;
In step (3), consisting of of proliferated culture medium: MS minimal medium, supplement 1.5mg/LKT, 0.3mg/L2,4-D, 4.0mg/LGA3,40g/L sucrose, 8g/L agar;
In step (4), consisting of of root media: 1/4MS minimal medium, supplement 0.03mg/LNAA, 20g/L sucrose, 5g/L agar, 0.2mg/L activated carbon.
In the present embodiment, when enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 28 days, the length of sprouting reaches 5cm, and sprouting is sturdy; Process of rooting culture starts to take root when proceeding to 16 days.
Embodiment 9
The tissue culture and rapid propagation method of a kind of succulent described in the present embodiment, is distinctive in that with embodiment 1:
In step (2), consisting of of initial culture base: MS minimal medium, supplement 0.06mg/LNAA, 5.0mg/L6-BA, 0.6g/L caseinhydrolysate, 40g/L sucrose, 8g/L agar;
In step (3), consisting of of proliferated culture medium: MS minimal medium, supplement 0.5mg/LKT, 0.2mg/L2,4-D, 2.0mg/LGA3,20g/L sucrose, 5g/L agar;
In step (4), consisting of of root media: 1/4MS minimal medium, supplement 0.03mg/LNAA, 20g/L sucrose, 5g/L agar, 0.2mg/L activated carbon.
In the present embodiment, when enrichment culture process proceeds to 15 days, sprouting sprouts in growing point place, and when 29 days, the length of sprouting reaches 5cm, and sprouting is sturdy; Process of rooting culture starts to take root when proceeding to 16 days.
Embodiment 10
The impact that in initial culture base, succulent is broken up by growth hormone composition and concentration
The outer implant taking growing state consistent is some, is inoculated on the initial culture base that pH value is 6.0 6.5 after sterilizing is dried, and cultivation temperature is 23 27 DEG C, and intensity of illumination is 2400 2500Lx, and light application time is 10 12h/d. Wherein initial culture base adopts MS minimal medium, supplementation with growth hormones, caseinhydrolysate, sucrose, agar. At MS minimal medium, and when the caseinhydrolysate supplemented, sucrose, agar are all identical, being grouped according to the composition of growth hormone and concentration, observe and record the cultivation situation of culture medium China and foreign countries implant, concrete packet and cultivation results are in Table 1.
In table 1 initial culture base, growth hormone composition and concentration affect result to what succulent broke up
| Packet | NAA(mg/L) | 6-BA(mg/L) | Inoculation number (individual) | Differentiation number (individual) | Differentiation rate (%) |
| 1 | 0.01 | -- | 30 | 21 | 70.00 |
| 2 | 0.05 | -- | 30 | 22 | 73.33 |
| 3 | 0.08 | -- | 30 | 23 | 76.67 |
| 4 | -- | 2.0 | 30 | 22 | 73.33 |
| 5 | -- | 4.0 | 30 | 20 | 66.67 |
| 6 | -- | 5.0 | 30 | 24 | 80.00 |
| 7 | 0.01 | 2.0 | 30 | 27 | 90.00 |
| 8 | 0.05 | 4.0 | 30 | 29 | 96.67 |
| 9 | 0.08 | 5.0 | 30 | 28 | 93.33 |
As can be seen from the above table, NAA and 6-BA use simultaneously than the two arbitrary alone time differentiation rate much higher, and can be seen that from 79 groups, both when using simultaneously, and NAA is 0.05mg/L, 6-BA when being 4.0mg/L, differentiation rate is the highest, and the optimal growth hormone for initial culture base forms condition.
Embodiment 11
The impact that in proliferated culture medium, succulent is bred by growth hormone composition and concentration
The Bud Differentiation obtained after initial culture taking growing state consistent is some, is inoculated on the proliferated culture medium that pH value is 6.0 6.5, and cultivation temperature is 23 27 DEG C, and intensity of illumination is 2400 2500Lx, and light application time is 10 12h/d. Wherein proliferated culture medium adopts MS minimal medium, supplementation with growth hormones, sucrose, agar. At MS minimal medium, and when the sucrose supplemented, agar are all identical, being grouped according to the composition of growth hormone and concentration, observe and record the cultivation situation of Bud Differentiation in culture medium, concrete packet and cultivation results are in Table 2.
In table 2 proliferated culture medium, growth hormone composition and concentration affect result to what succulent bred
As can be seen from the above table, KT, 2,4-D, GA3 use simultaneously than three arbitrary alone time the rate of increase high, sprouting is longer, growing way is better, from 10 12 groups it can be seen that when three uses simultaneously, KT is 1.0mg/L, 2,4-D when be 0.25mg/L, GA3 being 3.0mg/L, its rate of increase is the highest, and the optimal growth hormone for proliferated culture medium forms condition.
Embodiment 12
The impact that in root media, succulent is taken root by growth hormone composition and concentration
The plumelet obtained after enrichment culture taking growing state consistent is some, is inoculated on the root media that pH value is 6.0 6.5, and cultivation temperature is 23 27 DEG C, and intensity of illumination is 2400 2500Lx, and light application time is 10 12h/d. Wherein root media adopts MS minimal medium, supplementation with growth hormones, sucrose, agar, activated carbon. At MS minimal medium, and when the sucrose supplemented, agar, activated carbon are all identical, being grouped according to the composition of growth hormone and concentration, observe and record the cultivation situation of plumelet in culture medium, concrete packet and cultivation results are in Table 3.
What in table 3 root media, succulent was taken root by growth hormone composition and concentration affects result
As can be seen from the above data, NAA is when 0.05mg/L, and its rooting rate reaches 100%, and growing way is better, and number of on average taking root is all higher with average root length, for the optimal growth hormone condition of root media.
Below being only the preferred embodiment of the present invention, it is noted that above-mentioned preferred implementation is not construed as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range. For those skilled in the art, without departing from the spirit and scope of the present invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. the tissue culture and rapid propagation method of a succulent, it is characterised in that: comprise the following steps:
(1) outer implant is chosen and sterilizing: cuts the tender blade of the children of succulent or stem section, cleans up; After adopting ethanol and mercuric chloride sterilization treatment, obtain outer implant;
(2) initial culture: outer for step (1) gained implant is inoculated on initial culture base and carries out initial culture, until described outer implant differentiates bud, consisting of of described initial culture base: MS minimal medium, supplements 0.01 0.08mg/LNAA, 2.0 5.0mg/L6-BA, 0.1 0.8g/L caseinhydrolysate, 10 50g/L sucrose, 2 10g/L agar;
(3) enrichment culture: cut by the bud that step (2) China and foreign countries implant differentiates, it is inoculated on proliferated culture medium and carries out enrichment culture, until growing point sprouts the sprouting length that to 3 5cm, growth coefficient is more than 2, consisting of of described proliferated culture medium: MS minimal medium, supplement 0.5 2.0mg/LKT, 0.1 0.4mg/L2,4-D, 1.0 5.0mg/LGA3,10 50g/L sucrose, 2 10g/L agar;
(4) root culture: be separated into individual plant by sprouting, in step (3), the sprouting, it is inoculated on root media and carries out root culture, until individual plant grows 23 Gen Xingen, new root length degree is more than 3cm, consisting of of described root media: 1/4MS minimal medium, supplements 0.01 0.08mg/LNAA, 10 50g/L sucrose, 2 10g/L agar, 0.1 0.5mg/L activated carbon.
2. the tissue culture and rapid propagation method of a kind of succulent according to claim 1, it is characterized in that: described outer implant is chosen and the concrete operations of sterilizing are: cut the tender blade of the children of succulent or stem section, first soak 10 30min with detergent solution, scrub axillalry bud position with hairbrush again, after scrubbing clean under flowing water flushed night; On aseptic operating platform, ethanol postincubation 5 15s with 75%, with aseptic water washing 34 times, then process 8 12min, aseptic water washing 57 times with the mercuric chloride of 0.1%, dry.
3. the tissue culture and rapid propagation method of a kind of succulent according to claim 1, it is characterized in that: consisting of of described initial culture base: MS minimal medium, supplement 0.03 0.06mg/LNAA, 3.0 5.0mg/L6-BA, 0.3 0.6g/L caseinhydrolysate, 20 40g/L sucrose, 5 8g/L agar.
4. the tissue culture and rapid propagation method of a kind of succulent according to claim 3, it is characterized in that: consisting of of described initial culture base: MS minimal medium, supplement 0.05mg/LNAA, 4.0mg/L6-BA, 0.5g/L caseinhydrolysate, 30g/L sucrose, 6g/L agar.
5. the tissue culture and rapid propagation method of a kind of succulent according to claim 1, it is characterized in that: consisting of of described proliferated culture medium: MS minimal medium, supplement 0.5 1.5mg/LKT, 0.2 0.3mg/L2,4-D, 2.0 4.0mg/LGA3,20 40g/L sucrose, 5 8g/L agar.
6. the tissue culture and rapid propagation method of a kind of succulent according to claim 5, it is characterized in that: consisting of of described proliferated culture medium: MS minimal medium, supplement 1.0mg/LKT, 0.25mg/L2,4-D, 3.0mg/LGA3,30g/L sucrose, 6g/L agar.
7. the tissue culture and rapid propagation method of a kind of succulent according to claim 1, it is characterized in that: consisting of of described root media: 1/4MS minimal medium, supplement 0.03 0.06mg/LNAA, 20 40g/L sucrose, 5 8g/L agar, 0.2 0.3mg/L activated carbon.
8. the tissue culture and rapid propagation method of a kind of succulent according to claim 7, it is characterised in that: consisting of of described root media: 1/4MS minimal medium, supplement 0.05mg/LNAA, 30g/L sucrose, 6g/L agar, 0.2mg/L activated carbon.
9. the tissue culture and rapid propagation method of a kind of succulent according to claim 1, it is characterised in that: in described initial culture, enrichment culture, root culture step, cultivation temperature is 23 27 DEG C, and intensity of illumination is 2400 2500Lx, and light application time is 10 12h/d.
10. the tissue culture and rapid propagation method of a kind of succulent according to claim 1, it is characterised in that: described initial culture base, proliferated culture medium, root media pH value be 6.0 6.5.
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