CN101455180A - Open type plant tissue culture seedlings-raising method - Google Patents
Open type plant tissue culture seedlings-raising method Download PDFInfo
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- CN101455180A CN101455180A CNA2009100604848A CN200910060484A CN101455180A CN 101455180 A CN101455180 A CN 101455180A CN A2009100604848 A CNA2009100604848 A CN A2009100604848A CN 200910060484 A CN200910060484 A CN 200910060484A CN 101455180 A CN101455180 A CN 101455180A
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Abstract
The present invention relates to an open type plant tissue seedling cultivation method pertaining to the agroforestry field of plant tissue seedling cultivation technology. The method can be performed in a nonsterile natural environmental condition in a room temperature and the method comprises steps of adding plant extracts bacteriostatic agent in a culture medium, in each 1L culture medium, adding bacteriostatic agent of 4-15g, manufacturing a bacteriostasis culture medium, and performing an inoculated culture by utilizing the bacteriostasis culture medium. The bacteriostatic agent comprises two kinds one of which comprises garlic extracts, coptis root extracts and honeysuckle leaf extracts with a mass percent of 20-100%:0-40%:0-50%; the other one of which comprises liquorice root extracts, osmanthus extracts and lycoris radiata extracts with a mass percent of 10-100%:0-80%:0-50%. By utilizing the method of the present invention, the plant tissue seedling cultivation process can be divorced from a stringent axenic operation environment and can be performed in an open nonsterile environment, therefore, links in the plant tissue seedling cultivation are simplified radically, the working efficiency is improved and the breeding cost is greatly reduced.
Description
Technical field
The invention belongs to the plant tissue culture seedlings-raising technology in agricultural field, be specifically related to a kind of open type plant tissue and cultivate factorial seedling-culturing method.Promptly in medium, add under the condition that adopts the formulated bacteriostatic agent of plant extracts, make the plant tissue culture seedlings-raising process break away from strict aseptic operating environment, especially do not need autoclaving, can in the open environment that carries disease germs, carry out plant tissue culture seedlings-raising, fundamentally simplified the plant tissue culture seedlings-raising link, improve the operating efficiency of growing seedlings, greatly reduced the plant tissue culture seedlings-raising cost.
Background technology
Plant Tissue Breeding is the beginning of this century, is the plant propagation technology that base growth is got up with plant physiology.Plant Tissue Breeding claims plant cloning again, finger by sterile working any organ, tissue or the cell inoculation of plant containing on the medium that nutriment and plant growth regulating substance etc. form in artificial preparation, under the manual control environment, carry out cultured in vitro, make it grow, be differentiated to form the process of whole plant.Plant Tissue Breeding provides new means for the quick breeding of plant, and a little explant of inoculation can be induced to form callus and regeneration bud and root, subculture repeatedly after, can set up clone, produce test-tube plantlet in a large number.Set up plant test-tube plantlet breeding of method, very important meaning is arranged, one, breeding is not subject to seasonal restrictions fast, can obtain a large amount of individualities in a short time; Its two, it is numerous to select fine individual plant to expand, and can guarantee the homozygosity of kind, can promote fast high yield, high anti-, new quality variety; Its three, by the detoxification cultured in vitro, can cultivate the test-tube plantlet of not being with any pathogen and virus, reduce the generation of disease.Therefore, utilize the Plant Tissue Breeding fast breeding technique, improving reproduction coefficient, effectively preventing kind of sexual involution and produce in the high-quality seedling and played decisive role.Development through a nearly century, plant tissue culture technique is with its special advantages, now obtained using widely in fields such as the preservation of factorial seedling growth, seedling detoxification rejuvenation, genetic breeding, germ plasm resource and exchanges as an important means of modern biotechnology.At present, in the diversified economy plant, set up corresponding breeding fast breeding system.
Develop so far, the theoretical research of Plant Tissue Breeding is own through quite profound, but also there are many problems in its sport technique segment.Present widely used plant tissue culture seedlings-raising is to carry out under airtight, gnotobasis, comprises that inoculation explant, evoked callus, callus are differentiated to form the several steps such as transplanting of test-tube plantlet, test-tube plantlet.There are four defectives in this tissue culture mode: the medium pollution is difficult to solve, the test-tube plantlet cost is too high, the large-scale production technology is immature and the test tube production technology is loaded down with trivial details.
Both be fit to the plant tissue growth growth owing to be rich in the medium of nutrition, be more suitable for growing of assorted bacterium.Therefore; no matter be in the experimental study of Plant Tissue Breeding or in batch production is produced; pollution is ubiquitous problem, and it has seriously influenced the large-scale production of plant nursery stock, structure, production cost and the valuable materials of plant test tube gene pool and the aspect such as has saved from damage.Therefore, control has been polluted into the primary technology in the tissue culture.And the factor that influence is polluted is varied, as kind, sterilization method, medium and the vessel of the kind of explant, the time of drawing materials, preprocess method, disinfectant go out mattress, operating personnel and working environment requirement etc. all with pollute closely related, therefore, make pollution problem be difficult to control, become one of major reason of restriction tissue culture industrialization.
In addition, traditional Plant Tissue Breeding mode all is to carry out under the aseptic sealing condition of strictness, sterile working is a strict demand of conventional plant tissue culture, as will be to medium, cultivate the vessel high-temperature sterilization, any vessel that are used to operate and handle plant tissue all will be sterilized, and its operating process all must be carried out in gnotobasis such as superclean bench.If can set up unsterilised tissue culture technique, pollution rate can also be reduced in the tolerance interval, tissue culture technique will obtain huge leap.Therefore, it is extremely important to seek a kind of open tissue culture technique that need not sterilizing program.The open tissue culture of plant promptly under the effect of bacteriostatic agent, makes Plant Tissue Breeding break away from strict aseptic operating environment, do not need autoclaving and superclean bench, carry out Plant Tissue Breeding in the collarium border in open having, fundamentally simplify the tissue culture link, reduce cost.
As everyone knows, the pollution in traditional tissue culture procedures mainly shows as the pollution of medium, and nutritious medium provides the suitable place that grows for bacterium, fungi.Therefore, can guarantee the plant tissue normal growth as long as medium both is transformed into, and have the medium of bacteria resistance function again, in a single day mushroom loses and grows the place, just can not produce harm to tissue culture procedures.The key of transforming medium is to find a kind of broad-spectrum antibacterial agent that can add medium to.At present, the screening of broad-spectrum antibacterial agent is puzzlement Plant Tissue Breeding expert's a difficult problem always, has the scholar that bacteriostatic agent and tissue culture relation were carried out research.A large amount of chemical sterilization disinfectant benzoic acids, sorb acids, phenols, chlorine-containing compound class, quaternary ammonium salts, ureas, guanidine class or natural disinfectant bacteriostatic agent are added in employings such as Dan Wen repaiies in medium, set up the method for plant tissue culture (application number: 200410024035.5 publication numbers: CN1628507A) under a kind of non-sterile condition.They have done a large amount of single or combinations to common antibiotics, antibiotic, preservative and have added test in the medium to, experimental results show that, certain preventive and therapeutic effect that common antibiotics, antibiotic, preservative have the pollution in the Plant Tissue Breeding, but wanting to find a kind of suitable treatment combination at different plant varieties is the comparison difficulty, often when medium was antibiotic, the growth of plant tissue also was suppressed; And plant tissue but can't obtain satisfied antibacterial effect can normal growth the time.This also is the obstacle that domestic and international experts and scholars generally run into.One of its reason is also not have a kind of antibiotic all effective to all bacteriums, and the drug effect phase is short; The 2nd, some antibiotic, preservative are under valid density, and its sterilization, antibiotic ion pair plant tissue directly produce injury.
For this reason, we extract from various plants has the bacteriostatic activity material, set up plant extracts resources bank with good fungistatic effect, developed the bacteriostatic agent that is suitable for open tissue culture, the plant tissue culture seedlings-raising process can be carried out in the open environment that carries disease germs, fundamentally simplify the link of growing seedlings, improved operating efficiency, greatly reduced the plant tissue culture seedlings-raising cost.
Summary of the invention
The objective of the invention is to set up the open type plant tissue culture seedlings-raising method of a kind of low cost, high benefit, under the condition of adding the formulated bacteriostatic agent of plant extracts, make the plant tissue culture seedlings-raising process break away from strict aseptic operating environment, especially do not need autoclaving, can in the open environment that carries disease germs, carry out plant tissue culture seedlings-raising, simplify the plant tissue culture seedlings-raising link, improved operating efficiency, reduced the plant tissue culture seedlings-raising cost.
Open type plant tissue culture seedlings-raising method provided by the invention, it is characterized in that, this method is carried out having under the room temperature natural environmental condition of bacterium: add the plant extracts bacteriostatic agent in medium, calculate with the 1L medium, the addition of plant extracts bacteriostatic agent is 4～15g, prepare antibacterial medium, utilize this antibacterial medium to carry out inoculated and cultured again.
Above-mentioned plant extracts bacteriostatic agent can adopt following two classes, and one comprises garlic P.E, coptis extract and folium lonicerae extract, and its mass percent is: 20～100%:0～40%:0～50%; It two comprises licorice root extract, Flos Osmanthi Fragrantis extract and short-tube lycoris extract, and its mass ratio is: 10～100%:0～80%:0～50%.
Use antibacterial medium provided by the present invention, can be used for inoculation and the cultivations such as seed, root, stem, leaf and callus of plant, and the transplanting of test-tube plantlet, can reduce contamination by micro in the plant tissue culture course effectively, guarantee the normal development growth of plant tissue, organ, cell and test-tube plantlet.
Inoculation of the present invention is meant to be had under the natural environmental condition of bacterium, and above-mentioned plant tissue or test-tube plantlet are seeded in the antibacterial medium, and except need not to carry out the aseptic process program, it is identical with the inoculation formality of conventional organization cultivation specifically to inoculate operating performance.
Owing to used antibacterial medium, thereby the inoculation in the open type plant tissue cultural method of the present invention and incubation need not to carry out strict aseptic process again and (need not autoclaving, aseptic inoculation and aseptic culture), changed the traditional concept that to carry out aseptic process in the conventional plant tissue culture overall process, can omit high-pressure sterilizing pot, superclean bench, equipment and strict aseptic environments facilities such as uviol lamp, and can be replaced by the glassware of rapid wear cheap, plastic cup easy and simple to handle, omit loaded down with trivial details sterilization formality, greatly reduced production cost.
The present invention cultivates with traditional sterile tissue and compares, and has the simplification link, saves characteristics such as cost, and is specific as follows:
1. the selection of culture vessel: in traditional tissue culture, culture vessel need select the vial of high temperature high voltage resistant and polypropylene to seal film.And in open tissue culture, in medium, add bacteriostatic agent, and allow antibacterial medium replace autoclaving, and the disposal plastic cup of available cheapness seal with preservative film as culture vessel, this greatly reduces cost.
2. the sterilization of inoculation utensil: in traditional tissue culture, the scissors of inoculation usefulness, tweezers, blade etc. all adopt autoclaving or alcolhol burner calcination sterilization, and in open tissue culture, after the inoculation utensil is cleaned with 75% alcohol, can effectively prevent because the pollution that the inoculation utensil causes.
3. inoculation method: in transfer room, the alcohol with 75% will inoculate table top and hand is wiped clean, and opens and seal one jiao of film, with the inoculation utensil plant is inoculated on the medium, and rim of a cup is obturaged gets final product again.
4. sport technique segment and cost analysis: open tissue culture has been saved autoclaving and superclean bench inoculation, has simplified the tissue culture link.Because the rich material resources of preparation bacteriostatic agent, production technology is simple, and thimerosal also can reuse, and therefore open tissue culture is compared with traditional tissue culture, saves cost greatly.
Embodiment
Below by will describing the present invention more in detail by following examples, and following examples only are illustrative, and the present invention is not subjected to the restriction of these embodiment.
Open type plant tissue culture seedlings-raising can carry out according to following steps:
(1) antibacterial culture medium preparation
The employed kinds of culture medium of Plant Tissue Breeding is a lot, and as MS, N6, White etc., Plant Tissue Breeding is that example describes with the MS medium in this patent.Preparation 1L MS medium, add 4～15g bacteriostatic agent standard according to every liter of medium then, bacteriostatic agent is added in the minimal medium, regulates pH to 6.0, be mixed with antibacterial medium (adding 1% agar in the solid culture medium) with 0.1mol/L NaOH or 0.1mol/L HCl.Adopt transparent plastic cup packing, branch is installed on the container cover of medium tampon or seal, can inoculate after the medium cooling with preservative film.
(2) inoculation
Seeded process is carried out in open environment.Except strict aseptic oese need not sterilized, do not needed to medium overseas, other inoculation step is the same with traditional vaccine program, that is: with disinfectant explant is carried out disinfection earlier, obtain aseptic explant, then on the inoculation table top, explant after the sterilization is seeded in the medium that is added with bacteriostatic agent, seals with preservative film.
(3) initial culture
Place culturing room to cultivate (chamber of cultivating need not sterilization) inoculation material,, behind the one-period, carry out successive transfer culture according to aforesaid operations to obtain callus.
(4) differentiation of calli
Callus is inoculated in the antibacterial differential medium cultivates, taking root sprouts, and forms test-tube plantlet.
(5) transplanting of test-tube plantlet
Because the whole incubation of test-tube plantlet is open cultivation, so the transplanting of test-tube plantlet does not need hardening.After the test-tube plantlet taking-up, with clear water its root is rinsed well, soak the test-tube plantlet root with bacteriostatic agent again, transplant with the clean back of flushing with clean water after 30 minutes.
The open tissue culture of embodiment one konjaku
The open tissue culture of konjaku is carried out according to following steps:
(1) preparation of bacteriostatic agent:
1. the preparation of garlic P.E
Fresh white skin garlic 1kg after the peeling, add entry 300ml after cleaning, beat 20 minutes with beater and obtain mashed garlic, pour in the hermetically sealed can, drip the hydrochloric acid of 0.02mol/L and constantly be stirred to mashed garlic pH=6, leave standstill at 40 ℃ of lower seals of temperature, allow endogenous allinase in the mashed garlic separate alliin 4 hours to produce allicin.After enzymolysis is finished, the garlic slurry is transferred in the agitator tank, add the 4L95% edible ethanol, after fully stirring, plate-frame filtering obtains the allicin extract, transfers in the vacuum decompressioning and concentrating tank, and 60 ℃ of following decompression distillation are to doing, obtain yellow garlic P.E, detect allicin content through high performance liquid chromatography and equal 6.98%.
2. the preparation of coptis extract
Take by weighing dry coptis rhizome 1kg, cracker is pulverized the back and is crossed 80 eye mesh screens, Golden Thread places extractor to add 3L 95% industrial alcohol, 95 ℃ of lixiviates 5 hours, obtain rhizoma extracting liquid by plate-frame filtering, transfer in the vacuum decompressioning and concentrating tank, 55 ℃ of following decompression distillation are to doing, obtain coptis extract, detect berberine content through high performance liquid chromatography and equal 20%.
3. the preparation of folium lonicerae extract
Take by weighing dry folium lonicerae 1kg, cracker is pulverized the back and is crossed 60 eye mesh screens, the folium lonicerae powder places extractor to add 2L 80% industrial alcohol, regulate pH value 5.0,95 ℃ of lixiviates 3 hours obtain the folium lonicerae extract by plate-frame filtering, transfer in the vacuum decompressioning and concentrating tank, decompression distillation obtains the folium lonicerae extract to doing.Detect chlorogenic acid content through high performance liquid chromatography and equal 10%.
Three kinds of extracts are mixed respectively according to subordinate list 1 described 5 kinds of ratios, be prepared into 5 kinds of bacteriostatic agents.
(2) antibacterial culture medium preparation
Prepare 5 parts of 1L MS medium, in wherein adding 1% agar, fully after the dissolving, add hormone: 6-BA 2mg/L+NAA 0.1mg/L+IBA 0.2mg/L, add 5 kinds of bacteriostatic agents described in (1) then respectively, addition is 10g/L, is mixed with 5 kinds of antibacterial medium of solid.Adopt transparent plastic cup packing, the container that branch is installed medium seals with preservative film, can inoculate after the medium cooling.
(3) inoculation konjak corm
The konjak corm of inoculation after the routine sterilization cultivated.Contrast 1 is the solid culture medium that does not add bacteriostatic agent described in (2); Contrast 2 is the solid culture medium that does not add bacteriostatic agent described in (2), and medium needs sterilization.20 bottles of every winding kinds, 5 explants of every bottle graft kind, totally 100 explants.Antibacterial medium sees attached list 1 to the influence of konjaku callus induction and bud formation.
Glycyrrhiza glabra, glycyrrhiza inflate bat and Ural Radix Glycyrrhizae seed germination under the embodiment two open conditions
Glycyrrhiza glabra, glycyrrhiza inflate bat and Ural Radix Glycyrrhizae seed germination carry out according to following steps under the open condition:
(1) preparation of bacteriostatic agent:
Prepare garlic P.E, coptis extract and folium lonicerae extract according to the preparation method described in the embodiment one, and three kinds of extracts are mixed respectively according to subordinate list 2 described 5 kinds of ratios, be prepared into 5 kinds of bacteriostatic agents.
(2) antibacterial culture medium preparation
Prepare 5 parts of 1L MS medium,, be the seed germination medium in wherein adding 1% agar.Add 5 kinds of bacteriostatic agents described in (1) respectively, addition is 4g/L, is mixed with 5 kinds of antibacterial medium of solid.Adopt transparent plastic cup packing, the container that branch is installed medium seals with preservative film, can inoculate after the medium cooling.
(3) Radix Glycyrrhizae seed inoculation
After soaking glycyrrhiza glabra, glycyrrhiza inflate bat and Ural Radix Glycyrrhizae seed 1h respectively with the concentrated sulfuric acid, clean with flushing with clean water, after the routine sterilization, be inoculated in respectively in the seed germination medium, each handles 20 bottles of inoculations, 10 seeds of every bottle graft kind, totally 200 seeds.Contrast 1 is the seed germination medium described in (2), and medium does not add bacteriostatic agent; Contrast 2 be the seed germination medium described in (2), and medium do not add bacteriostatic agent, but medium needs to sterilize.Antibacterial medium sees attached list 2 to the influence of three kinds of Radix Glycyrrhizae seed germinations.
The open tissue cultivating and seedling of embodiment three Radix Glycyrrhizaes
The open tissue cultivating and seedling of Radix Glycyrrhizae carries out according to following steps:
(1) preparation of bacteriostatic agent:
1. the preparation of licorice root extract
Accurately take by weighing dry Radix Glycyrrhizae rhizome 1kg, cracker is pulverized the back and is crossed 80 eye mesh screens, the Radix Glycyrrhizae powder places extractor to add 3L 50% industrial alcohol, ultrasonic Extraction 1 hour, obtain the licoflavone extract by plate-frame filtering, transfer in the vacuum decompressioning and concentrating tank, 55 ℃ of following decompression distillation are to doing, obtain licorice root extract, wherein the purity of licoflavone equals 70%.
2. the preparation of Flos Osmanthi Fragrantis extract
Accurately take by weighing dry sweet osmanthus 1kg, add 500ml 90% ethanol, desorb 15 minutes.The alcohol reflux that adds 4L 80% again extracted 15 minutes, extracted 2 times, filtered, and filtrate is transferred in the vacuum decompressioning and concentrating tank, and 55 ℃ of following decompression distillation obtain Flos Osmanthi Fragrantis extract to doing, and wherein the purity of sweet osmanthus flavones equals 40%.
3. the preparation of short-tube lycoris extract
Accurately take by weighing dry short-tube lycoris 1kg, cracker is pulverized the back and is crossed 80 eye mesh screens, the short-tube lycoris powder places extractor to add 10L methyl alcohol, 75 ℃ of refluxing extraction 3 hours, obtain the short-tube lycoris extract by plate-frame filtering, transfer in the vacuum decompressioning and concentrating tank, 55 ℃ of following decompression distillation are to doing, obtain the short-tube lycoris extract, the purity that detects maryllidaceous alkaloid through high performance liquid chromatography equals 2%.
Three kinds of extracts are mixed respectively according to subordinate list 3 described 5 kinds of ratios, be prepared into 5 kinds of bacteriostatic agents.
(2) antibacterial culture medium preparation
Prepare 5 parts of 1L MS medium, in wherein adding 1% agar, fully after the dissolving, add hormone: behind the 6-BA 2mg/L+KT 0.5mg/L+IBA 0.5mg/L, add 5 kinds of bacteriostatic agents described in (1) then respectively, addition is 4g/L, is mixed with 5 kinds of antibacterial medium.Adopt transparent plastic cup packing, branch is installed tampon on the container cover of medium, can inoculate after the medium cooling.
(3) inoculation glycyrrhiza inflate bat hypocotyl
Glycyrrhiza inflate bat, the glycyrrhiza glabra hypocotyl of inoculation after the routine sterilization cultivated.Contrast 1 is the solid culture medium that does not add bacteriostatic agent described in (2); Contrast 2 is the solid culture medium that does not add bacteriostatic agent described in (2), and medium needs sterilization.20 bottles of every winding kinds, 6 stem sections of every bottle graft kind, totally 120 stem sections.Antibacterial medium sees attached list 3 to the influence of glycyrrhiza inflate bat, glycyrrhiza glabra callus induction rate and differentiation rate.
Caper seed germination and induce hypocotyl differentiation under the embodiment four open conditions
Caper seed germination and induce hypocotyl differentiation to carry out under the open condition according to following steps:
(1) preparation of bacteriostatic agent:
Prepare garlic P.E, coptis extract and folium lonicerae extract according to the preparation method described in the embodiment one, and three kinds of extracts are mixed respectively according to subordinate list 4 described 5 kinds of ratios, be prepared into 5 kinds of bacteriostatic agents.
(2) antibacterial culture medium preparation
Preparation MS medium in wherein adding 1% agar, after the dissolving, adds hormone fully.Adding hormone in the seed germination medium is 6-BA 1.0mg/L, induces that to add hormone in the hypocotyl differential medium be 6-BA 0.1mg/L+NAA 0.01mg/L.Add 5 kinds of bacteriostatic agents described in (1) respectively, addition is 15g/L, is mixed with antibacterial medium.Adopt transparent plastic cup packing, the container that branch is installed medium seals with preservative film, can inoculate after the medium cooling.
(3) caper seed inoculation
Behind concentrated sulfuric acid immersion caper seed 1h, clean with flushing with clean water, after the routine sterilization, be inoculated in the seed germination medium, each handles 10 bottles of inoculations, 15 seeds of every bottle graft kind, totally 150 seeds.Behind seed germination, cut hypocotyl, place differential medium, 20 bottles of every winding kinds, 6 stem sections of every bottle graft kind, totally 120 stem sections.Contrast 1 is the medium that does not add bacteriostatic agent described in (2); Contrast 2 is the medium that does not add bacteriostatic agent described in (2), but medium needs sterilization.Antibacterial medium sees attached list 4 to the influence of caper seed germination and hypocotyl differentiation.
The formation and the transplanting of konjaku test-tube plantlet under the embodiment five open conditions
The formation of konjaku test-tube plantlet and transplanting are carried out according to following steps under the open condition:
(1) preparation of bacteriostatic agent:
Prepare licorice root extract, Flos Osmanthi Fragrantis extract and short-tube lycoris extract according to the preparation method described in the embodiment three, and three kinds of extracts are mixed respectively according to subordinate list 5 described 5 kinds of ratios, be prepared into 5 kinds of bacteriostatic agents.
(2) antibacterial culture medium preparation
Preparation 1L MS medium, in wherein adding 1% agar, after the dissolving, add hormone fully: behind the 6-BA1.5mg/L+NAA 0.3mg/L, add 5 kinds of bacteriostatic agents described in (1) respectively, addition is 15g/L, is mixed with antibacterial medium.Adopt transparent plastic cup packing, the container that branch is installed medium seals with preservative film, can inoculate after the medium cooling.
(3) inoculation konjaku indefinite bud
The konjaku indefinite bud of inoculation after the routine sterilization cultivated, and makes it to be differentiated to form test-tube plantlet.Contrast 1 is the medium that does not add bacteriostatic agent described in (2); Contrast 2 is the medium that does not add bacteriostatic agent described in (2), and medium needs sterilization, 20 bottles of every winding kinds, 5 indefinite buds of every bottle graft kind, totally 100 indefinite buds.
(4) preparation of liquid bacteriostatic agent
Get 5 kinds of bacteriostatic agents described in (1) respectively, every kind takes by weighing 5g and is dissolved in the 1L water, makes 5 kinds of liquid bacteriostatic agents after stirring, and is respectively applied for the immersion of test-tube plantlet root.
(5) transplanting of test-tube plantlet
After treating that indefinite bud forms sturdy test-tube plantlet, test-tube plantlet is taken out, its root is rinsed well, again its root is placed 5 kinds of liquid bacteriostatic agents of (4) preparation respectively, soak after 30 minutes with clear water, clean with flushing with clean water, transplant.Antibacterial medium sees attached list 5 to the formation of konjaku test-tube plantlet and the influence of transplanting.
The Chinese yew explant induction forms callus under the embodiment six open conditions
Chinese yew explant induction formation callus carries out according to following steps under the open condition:
(1) preparation of bacteriostatic agent:
Prepare licorice root extract, Flos Osmanthi Fragrantis extract and short-tube lycoris extract according to the preparation method described in the embodiment three, and three kinds of extracts are mixed respectively according to subordinate list 6 described 5 kinds of ratios, be prepared into 5 kinds of bacteriostatic agents.
(2) antibacterial culture medium preparation
Prepare 5 parts of 1L MS medium, in wherein adding 1% agar, fully after the dissolving, add hormone: dichlorphenoxyacetic acid 0.2mg/L and α-Nai Yisuan 0.5mg/L, add 5 kinds of bacteriostatic agents described in (1) then respectively, addition is 8g/L, is mixed with 5 kinds of antibacterial medium.Adopt transparent plastic cup packing, branch is installed tampon on the container cover of medium, can inoculate after the medium cooling.
(3) inoculation Chinese yew explant
Chinese Chinese yew, taxus chinensis in northeast and the Taxus x media tender stem of inoculation after the routine sterilization cultivated.Contrast 1 is the solid culture medium that does not add bacteriostatic agent described in (2); Contrast 2 is the solid culture medium that does not add bacteriostatic agent described in (2), and medium needs sterilization.20 bottles of every winding kinds, 6 stem sections of every bottle graft kind, totally 120 stem sections.Antibacterial medium sees attached list 6 to the influence of Chinese Chinese yew, taxus chinensis in northeast and Taxus x media callus induction rate.
The formation of konjaku callus induction and bud under the subordinate list 1 open condition
| Garlic, the coptis, folium lonicerae extract quality ratio | Pollution bottle number (bottle) | Callus induction rate (%) | The quantity of sprouting (individual) |
| 100％：0：0 | 1 | 92 | 373 |
| 20％：30％：50％ | 4 | 70 | 271 |
| 40％：30％：30％ | 2 | 85 | 336 |
| 60％：20％：20％ | 1 | 94 | 375 |
| 40％：40％：20％ | 2 | 89 | 358 |
| Contrast 1 | 20 | 0 | 0 |
| Contrast 2 | 1 | 93 | 361 |
Glycyrrhiza glabra, glycyrrhiza inflate bat and Ural Radix Glycyrrhizae seed germination under the subordinate list 2 open conditions
| Garlic, the coptis, folium lonicerae extract quality ratio | Glycyrrhiza glabra seed germination rate % | Glycyrrhiza inflate bat seed germination rate % | Ural Radix Glycyrrhizae seed germination rate % |
| 100％：0：0 | 88 | 97 | 91 |
| 20％：30％：50％ | 62 | 71 | 65 |
| 40％：30％：30％ | 77 | 79 | 83 |
| 60％：20％：20％ | 83 | 92 | 88 |
| 40％：40％：20％ | 79 | 84 | 81 |
| Contrast 1 | 51 | 75 | 59 |
| Contrast 2 | 89 | 95 | 93 |
Glycyrrhiza inflate bat and glycyrrhiza glabra callus induction and differentiation under the subordinate list 3 open conditions
| Licorice, sweet osmanthus, short-tube lycoris extract quality ratio | Glycyrrhiza inflate bat callus induction rate % | Glycyrrhiza inflate bat differentiation rate % | Glycyrrhiza glabra callus induction rate % | Glycyrrhiza glabra differentiation rate % |
| 100％：0：0 | 97 | 51 | 92 | 43 |
| 10％：80％：10％ | 91 | 44 | 88 | 34 |
| 30％：20％：50％ | 83 | 38 | 78 | 37 |
| 60％：20％：20％ | 97 | 55 | 91 | 46 |
| 40％：30％：30％ | 93 | 47 | 89 | 39 |
| Contrast 1 | 0 | 0 | 0 | 0 |
| Contrast 2 | 95 | 49 | 89 | 43 |
Caper seed germination and induce hypocotyl differentiation under the subordinate list 4 open conditions
| Garlic, the coptis, folium lonicerae extract quality ratio | Caper seed germination rate % | Hypocotyl differentiation bud ratio % | The growing state of test-tube plantlet |
| 100％：0：0 | 33 | 9 | Seedling is sturdy dark green |
| 20％：30％：50％ | 19 | 3 | Seedling is very thin dark green |
| 40％：30％：30％ | 28 | 5 | Seedling is very thin dark green |
| 60％：20％：20％ | 37 | 11 | Seedling is sturdy dark green |
| 40％：40％：20％ | 33 | 5 | Seedling is slim and frahile |
| Contrast 1 | 15 | 0 | No seedling |
| Contrast 2 | 36 | 11 | Seedling is sturdy dark green |
The formation and the transplanting of konjaku test-tube plantlet under the subordinate list 5 open conditions
| Licorice, sweet osmanthus, short-tube lycoris extract quality ratio | Planting percent (%) | The survival rate of test-tube plantlet (%) |
| 100％：0：0 | 46 | 41 |
| 10％：80％：10％ | 33 | 25 |
| 30％：20％：50％ | 42 | 36 |
| 60％：20％：20％ | 43 | 36 |
| 40％：30％：30％ | 39 | 33 |
| Contrast 1 | 0 | 0 |
| Contrast 2 | 47 | 38 |
Chinese Chinese yew, taxus chinensis in northeast and Taxus x media explant form the influence of callus under the subordinate list 6 open conditions
| Licorice, sweet osmanthus, short-tube lycoris extract quality ratio | China Chinese yew callus induction rate % | Taxus chinensis in northeast callus induction rate % | Taxus x media callus induction rate % |
| 100％：0：0 | 97 | 91 | 92 |
| 10％：80％：10％ | 88 | 86 | 81 |
| 30％：20％：50％ | 85 | 88 | 83 |
| 60％：20％：20％ | 97 | 92 | 91 |
| 40％：30％：30％ | 93 | 95 | 89 |
| Contrast 1 | 0 | 0 | 0 |
| Contrast 2 | 95 | 91 | 89 |
The present invention not only is confined to above-mentioned embodiment; persons skilled in the art are according to content disclosed by the invention; can adopt other multiple embodiment to implement the present invention; therefore; every employing technical scheme of the present invention and thinking; do some simple designs that change or change, all fall into the scope of protection of the invention.
Claims (5)
1, a kind of open type plant tissue culture seedlings-raising method, it is characterized in that, this method is carried out having under the room temperature natural environmental condition of bacterium: add the plant extracts bacteriostatic agent in medium, calculate with the 1L medium, the addition of plant extracts bacteriostatic agent is 4～15g, prepare antibacterial medium, utilize this antibacterial medium to carry out inoculated and cultured again.
2, open type plant tissue culture seedlings-raising method as claimed in claim 1, it is characterized in that, described plant extracts bacteriostatic agent comprises garlic P.E, coptis extract and folium lonicerae extract, and its mass percent is: 20～100%:0～40%:0～50%.
3, open type plant tissue culture seedlings-raising method as claimed in claim 2, it is characterized in that, the percentage by weight of the allicin in the garlic P.E is more than or equal to 6.0%, the percentage by weight of berberine is more than or equal to 20.0% in the coptis extract, and the percentage by weight of chlorogenic acid is more than or equal to 10.0% in the folium lonicerae extract.
4, open type plant tissue culture seedlings-raising method as claimed in claim 1, it is characterized in that, described plant extracts bacteriostatic agent comprises licorice root extract, Flos Osmanthi Fragrantis extract and short-tube lycoris extract, and its mass percent is: 10～100%:0～80%:0～50%.
5, open type plant tissue culture seedlings-raising method as claimed in claim 4, it is characterized in that, licoflavone purity in the licorice root extract is more than or equal to 70%, sweet osmanthus flavones purity in the Flos Osmanthi Fragrantis extract is more than or equal to 40%, and the maryllidaceous alkaloid purity in the short-tube lycoris extract is more than or equal to 2%.
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