CN104160959B - A kind of method of rattan water spinach tissue cultures - Google Patents
A kind of method of rattan water spinach tissue cultures Download PDFInfo
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- CN104160959B CN104160959B CN201410386348.9A CN201410386348A CN104160959B CN 104160959 B CN104160959 B CN 104160959B CN 201410386348 A CN201410386348 A CN 201410386348A CN 104160959 B CN104160959 B CN 104160959B
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- rattan
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- water spinach
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- axillalry bud
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- 238000000034 method Methods 0.000 title claims abstract description 36
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Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention provides a kind of method of rattan water spinach tissue cultures, comprising: A) obtain stem section does not at least sprout axillalry bud rattan water spinach rattan with 1; B) Initial culture, Initial culture base is the agar powder of MS medium+2.0-3.0mg/L6-benzyl aminoadenine+0.1-0.3mg/L methyl α-naphthyl acetate+30g/L carbon source+6g/L, and pH value is 5.5-5.8; C) subculture medium is MS medium+0.5-1.5mg/L6-benzyl aminoadenine+0.3-0.7mg/L gibberellin+30g/L carbon source+6g/L agar powder, and pH value is 5.5-5.8; D) culture of rootage, root media is 1/2MS medium+0.3-0.6mg/L methyl α-naphthyl acetate+15g/L carbon source+6g/L agar powder, and pH value is 5.5-5.8; E) plantlet in vitro of having taken root is carried out hardening.
Description
Technical field
The present invention relates to field of plant tissue culture, particularly a kind of method of rattan water spinach tissue cultures.
Background technology
Water spinach (Lpomoea aquatica) belongs to the annual or herbaceos perennial of Convolvulaceae (Convolvulaceae) sweet potato genus (Lpomoea), have another name called bengal dayflower herb, water spinach, logical heart dish etc., originate in China's tropical rainy area, advantages such as there is fast growth, output is high, picking time is long, high-temp resisting high-humidity resisting, resistance, damage by disease and insect are less, generally cultivate in China Yangtze river basin and areas to the south, for one of important leafy vegetable summer and autumn, existing very high nutritive value, has again clearing heat and detoxicating, the medical value such as promoting urination and removing dampness, reduction blood sugar.Water spinach divides seed water spinach and rattan water spinach two kinds, and the more sub-water spinach of rattan water spinach quality is better, particularly Changsha rattan water spinach, and stem is overgrow, internode brachymedial empty, stem stalk is sturdy, good heat resistance, quality are more tender, and vegetative period is longer, and output is higher, more firmly gets liking of people.
In recent years, although rattan water spinach is as main leaf vegetables spring and summer, there is certain planting scale, also achieve good economic benefit, but do not bloom in the Yangtze river basin due to rattan water spinach, difficulty is set seeds, for vegetative plant, on producing at present, the method for the climing cottage propagation of stem is mainly taked to breed female seedling.The method has many drawbacks, seriously constrains the development of production.Be mainly reflected in following several respects: first, rattan water spinach cannot obtain seed, the tradition mode of reserving seed for planting can only select old stem stalk, re-started cottage breeding tissue before the beginning of autumn to enrich healthy and strong tendril and do kind climing, then before first frost, took climing being stored in cellar of stem reserve seed for planting, the process of reserving seed for planting need keep the low temperature of 10-15 DEG C and the air humidity of 75%, spring in next year temperature reaches 15 DEG C of ability and takes out kind of a climing nursery, employing the method wastes time and energy, and requires strict, plant climing survival rate lower than 80% to growing environment.The second, cottage propagation makes rattan water spinach reproduction speed slack-off, and usual 1 water spinach bar can only breed 1 strain plant, and transplanting seedling time is longer, and field management is comparatively complicated.3rd, the whole body that vegetative crop is all vulnerable to one or more pathogens is infected, pathogen infect the death that not necessarily all can cause plant, a lot of virus even may not show any symptom, but the existence of virus can reduce crop yield and quality.Rattan water spinach equally easily causes the accumulation of virus in cottage propagation process, causes kind of matter to be degenerated, has a strong impact on the proterties such as rattan water spinach output, quality and resistance.By inquiry, the disease of current rattan water spinach mainly contains the viruses such as zonate spot, brown spot and mosaic virus, and people adopt medicine to control in the course of cultivation usually, and ignore renewal female seedling, both contaminated environment, have buried again food security hidden danger.
Summary of the invention
The present invention utilizes plant tissue culture technique, a kind of method of rattan water spinach tissue cultures is provided, while producing high quality seedling in a large number at short notice, cause of disease harm can also be reduced, to the development promoting Hunan Province this traditional characteristics vegetables industry, solving the problems such as vegetables dull seasons is under-supply has very important meaning.
Particularly, the invention provides a kind of method of rattan water spinach tissue cultures, described method comprises the steps:
A) pretreatment: obtain rattan water spinach rattan, rinse well under flowing water, be cut into the stem section of 3-4cm, and ensure the axillalry bud at least do not sprouted with 1 in described stem section, and to the sterilization of described stem section surface, such as can with the alcohol surface sterilization of 70%, with aseptic water washing repeatedly, again with antiseptic solution sterilization, and then with aseptic water washing repeatedly;
B) Initial culture: be inoculated in Initial culture base and cultivate, described Initial culture base is the agar powder of the carbon source+6g/L of the methyl α-naphthyl acetate+30g/L of the 6-benzyl aminoadenine+0.1-0.3mg/L of MS medium+2.0-3.0mg/L, and pH value range is 5.5-5.8;
C) squamous subculture: more than height of seedling 4cm after selection Initial culture, and there is the explant of 3-5 axillalry bud, be cut into the stem section with 1-2 axillalry bud, be inoculated in subculture medium and cultivate, when the Multiple Buds length that explant produces has 3-5 axillalry bud, be separated described Multiple Buds, being forwarded in Initial culture base makes axillalry bud extend, be forwarded to again after more than height of seedling 4cm in subculture medium and continue propagation, described subculture medium is the agar powder of the carbon source+6g/L of the gibberellin+30g/L of the 6-benzyl aminoadenine+0.3-0.7mg/L of MS medium+0.5-1.5mg/L, pH value range is 5.5-5.8,
D) culture of rootage: select more than height of seedling 4cm, there is the explant of 3-5 axillalry bud, be inoculated in root media and cultivate, described root media is the agar powder of the carbon source+6g/L of the methyl α-naphthyl acetate+15g/L of 1/2MS medium+0.3-0.6mg/L, and pH value range is 5.5-5.8;
E) select according to described step D) more than height of seedling 4cm, there is the plantlet in vitro of 3-5 axillalry bud, to have taken root and the plantlet in vitro of well developed root system carries out hardening.
Confirm through experiment, compared with side runner rattan, select main rattan rattan as experiment material during group training, its explant more easily tolerates the erosion of thimerosal, and growth potential is stronger, and therefore, in one preferred embodiment, rattan water spinach rattan is selected from main rattan rattan.
Terminal bud described herein refers to the bud grown at plant trunk or side shoot top.Axillalry bud normally points to the raw bud at axil place.Unless otherwise indicated, otherwise, axillalry bud can comprise terminal bud to axillalry bud in the present invention.Multiple Buds refers to the colony's crowd shoots come by plant organ and healing tissue development, the shape indefinite bud that clusters formed by Calli Differentiation after referring to rattan water spinach stem segment base portion callus in the present invention.Explant refers to from living plant body and cuts the part tissue or organ that get off to carry out tissue cultures, is specially the rattan water spinach stem section containing axillalry bud.
In a concrete embodiment, described steps A) in thimerosal be add polysorbas20 5% liquor natrii hypochloritis or add polysorbas20 0.1% mercuric chloride solution, and instilling 50 μ L i.e. 1 polysorbas20 in every 100mL thimerosal, disinfecting time is 5-8 minute.
In a concrete embodiment, in described Initial culture base, also comprise the ribonucleic acid yeast of 0.3-1.0g/L.The beneficial effect adding ribonucleic acid yeast is mainly ribonucleic acid yeast can make rattan water spinach explant stem stalk xanthate ratio significantly reduce, and axillary bud growth speed is relatively very fast, and plant strain growth gesture is more vigorous.
In one preferred embodiment, the ribonucleic acid yeast of 0.5g/L is also comprised in described Initial culture base.
In a concrete embodiment, in described subculture medium, also comprise the one in the citric acid of the ditert-butylhydro quinone of 0.02g/L, the silver nitrate of 0.05g/L, Vc and 0.1g/L of 0.05g/L.Effect, antioxidant ditert-butylhydro quinone significantly can reduce the melting brown rate of stem culm base callus, and effect is better than the g/L citric acid of the silver nitrate of 0.05g/L, the Vc of 0.05g/L and 0.1, the therefore ditert-butylhydro quinone of preferred 0.02g/L.
In a concrete embodiment, also comprise in the L-cysteine hydrochloride of the L-hydroxy-proline of 0.1-0.5g/L, 0.3-0.7g/L Serine and 0.1-0.5g/L in described subculture medium one or more.The described amino acid whose Multiple Buds of raising greatly reproduction coefficient used in combination, is more conducive to the squamous subculture of plantlet in vitro.
In a concrete embodiment, in described subculture medium, also comprise the rattan water spinach bleeding sap of 15%.The rattan water spinach bleeding sap of 15% to improve Multiple Buds breeding effect and described antioxidant and amino acid whosely with the used in combination of optium concentration, there is the effect be equal to.
In a concrete embodiment, in described subculture medium, MS medium is the MS medium of improvement, wherein, the MS medium of described improvement is specially the ammonium nitrate of 2475mg/L, the potassium nitrate of 1900mg/L, the calcium chloride dihydrate of 440mg/L, the epsom salt of 370mg/L, the potassium dihydrogen phosphate of 225mg/L, the potassium chloride of 70mg/L, the potassium iodide of 0.83mg/L, the boric acid of 6.2mg/L, the four water manganese sulphates of 22.3mg/L, the white vitriol of 8.6mg/L, the Sodium Molybdate Dihydrate of 0.25mg/L, the cupric sulfate pentahydrate of 0.025mg/L, the CoCL2 6H2O of 0.025mg/L, the inositol of 100mg/L, the nicotinic acid of 0.5mg/L, the puridoxine hydrochloride of 0.5mg/L, the thiamine hydrochloride of 0.1mg/L, the glycine of 2.0mg/L, the calcium pantothenate of 0.3mg/L, the ferrous sulfate heptahydrate of 139.0mg/L and the disodium ethylene diamine tetraacetate of 186.5mg/L.
In a concrete embodiment, at described step B) in the condition of cultivation be: the described temperature do not sprouted before axillalry bud is stretched to 1cm is 30.0 ± 0.5 DEG C; Described axillalry bud be stretched to 1cm after temperature be 25.0 ± 0.5 DEG C, intensity of illumination is 2000lx, and light application time is 16 hours/day;
And/or at described step C) in the condition of cultivation be: temperature during illumination is 28.0 ± 0.5 DEG C, and temperature time dark is 18.0 ± 0.5 DEG C, and intensity of illumination is 3000lx, and light application time is 16 hours/day;
And/or at described step D) in the condition of cultivation be: temperature during illumination is 28.0 ± 0.5 DEG C, and temperature time dark is 18.0 ± 0.5 DEG C, and intensity of illumination is 3000lx, and light application time is 16 hours/day;
And/or in described step e) in, during described hardening, temperature is 25.0 ± 0.5 DEG C, and intensity of illumination is 2000lx, and light application time is 16 hours/day, and humid control is more than 85%.
In a concrete embodiment, described step B) in incubation time at 7-10 days; And/or described step C) in subculture 1 incubation time 12-16 days; And/or described step D) in incubation time 8-12 days; And/or described step e) in the hardening time be 10-14 days.
In one preferred embodiment, described step B) in incubation time at 7 days; And/or described step C) in subculture 1 incubation time 12 days; And/or described step D) in incubation time 8 days; And/or described step e) in the hardening time be 10 days.
In a concrete embodiment, described carbon source be selected from glucose, fructose, maltose, sucrose and white sugar one or more, preferred white sugar.
In a concrete embodiment, also comprise the step I of rattan water spinach virus-free culture), described step I) at described step B) after and in step C) before, after getting Initial culture, axillalry bud is stretched to the terminal bud of the explant of more than 3cm, or directly acquisition is grown on terminal bud on the outdoor main rattan of rattan water spinach, stripping length is after 0.8-1.2mm stem apex, described terminal bud stem apex is seeded in Primary culture base and carries out virus-free culture, describedly be grown on terminal bud on the outdoor main rattan of rattan water spinach and need sterilization, sterilization method is with described steps A) pretreatment in described sterilization method; Described Primary culture base is the active carbon of the agar powder+1.5g/L of the carbon source+6g/L of the gibberellin+30g/L of the methyl α-naphthyl acetate+0.05-0.20mg/L of MS medium+0.1-0.3mg/L; And/or described step I) in the virus-free culture time be 14-18 days, be preferably 14 days.For the maternal plant being subject to pathogen infection, by the plantlet in vitro that stem apex detoxify obtains, relative to the female seedling without detoxification, its disease resistance, resistance, adaptability strengthen all greatly, and vegetative period is shorter, and output is higher, the amount of application of agricultural chemicals, chemical fertilizer reduces, both reduced production cost, and alleviated pollution on the environment again, the safety nuisance free vegetables of producing have good economic benefit and ecological benefits.In addition, after rattan water spinach detoxification 1 time, directly nutrition can be carried out in laboratory or outdoor numerous soon, without the need to again carrying out detoxification; For the maternal plant not by virus infection, without the need to considering detoxification step, directly carrying out Fast-propagation, within shorter time and less space, a large amount of plant can be bred by 1 individuality.
In a concrete embodiment, described rattan water spinach is selected from Changsha rattan water spinach.
Compared with traditional seedling raising manners, beneficial effect of the present invention:
(1) method provided by the invention is not subject to seasonal restrictions, and each month all can emerge.If start January to carry out rattan water spinach tissue cultures, March can emerge, and just has rattan water spinach to go on the market by the end of March.Surviving the winter with traditional buried cellar for storing things of rattan water spinach preserves compared with female seedling, and method provided by the invention can make Time To Market 1 month ahead of time nearly.And for the embodiment optimized, the time required for whole incubation can do sth. in advance time of two weeks than the embodiment do not optimized, this is more conducive to rattan water spinach and goes on the market ahead of time.
(2) a large amount of plant can be obtained in the method short time provided by the invention.Be inoculated into the numerous seedling in land for growing field crops from explant and only need 55 days, plantlet in vitro bottle outlet only needs 10 days to immigration land for growing field crops, and induction survival rate is up to 97.59%.Compared with traditional cottage propagation, method band provided by the invention 1 axillalry bud rattan stem section can breed 140 strain rattan water spinach tissue culture plant by tissue culture technique, and the plant quantity obtained in the short time is large.
(3) method provided by the invention saves place.Whole incubation is carried out in the illumination cultivation room of temperature control control light, 1 blake bottle can cultivate 6 strain plantlet in vitro, 1 layer of culturing rack (specification is long 1250mm × wide 500mm × high 360mm) can cultivate 672 strain plantlet in vitro, and compared with cottage propagation, occupied ground area is little.
(4) method provided by the invention can save labour cost.Without the need to whole, epiphragma, pouring rich water in the whole incubation of rattan water spinach, without the need to being equipped with the personnel of concrete management, compared with traditional cottage propagation, a large amount of labour and cost be saved.
Accompanying drawing explanation
The rattan water spinach plantlet in vitro of Fig. 1 hardening.
Embodiment
Below in conjunction with specific embodiment, the present invention is elaborated, but scope of the present invention is not limited to this.
MS medium used is Murashige and Skoog, and formula is in table 1, and the formula of 1/2MS medium is in table 2.Wherein, FeSO
47H
2o can use FeSO
47H
2o and Na
2eDTA2H
2o prepares, form FeEDTA chelated forms, its reason only could be organized when about pH5.2 by ferrous iron and be utilized, and the iron provided with chelated forms until when medium pH in incubation rises to 7.6-8.0 still can utilize by plant tissue.
Table 1 MS medium component (unit: mg/L)
| Macroelement | Content | Trace element | Content | Organic substance | Content | Molysite | Content |
| NH 4NO 3 | 1650 | KI | 0.83 | Inositol | 100 | FeSO 4·7H 2O | 27.8 |
| KNO 3 | 1900 | H 3BO 3 | 6.2 | Nicotinic acid | 0.5 | Na 2·EDTA·2H 2O | 37.3 |
| CaCl 2·2H 2O | 440 | MnSO 4·4H 2O | 22.3 | Puridoxine hydrochloride | 0.5 | ||
| MgSO 4·7H 2O | 370 | ZnSO 4·7H 2O | 8.6 | Thiamine hydrochloride | 0.1 | ||
| KH 2PO 4 | 170 | Na 2MoO 4·2H 2O | 0.25 | Glycine | 2.0 | ||
| CuSO 4·5H 2O | 0.025 | ||||||
| CoCl 2·6H 2O | 0.025 |
Table 2 1/2MS medium component (unit: mg/L)
| Macroelement | Content | Trace element | Content | Organic substance | Content | Molysite | Content |
| NH 4NO 3 | 825 | KI | 0.83 | Inositol | 100 | FeSO 4·7H 2O | 27.8 |
| KNO 3 | 950 | H 3BO 3 | 6.2 | Nicotinic acid | 0.5 | Na 2·EDTA·2H 2O | 37.3 |
| CaCl 2·2H 2O | 220 | MnSO 4·4H 2O | 22.3 | Puridoxine hydrochloride | 0.5 | ||
| MgSO 4·7H 2O | 185 | ZnSO 4·7H 2O | 8.6 | Thiamine hydrochloride | 0.1 | ||
| KH 2PO 4 | 85 | Na 2MoO 4·2H 2O | 0.25 | Glycine | 2.0 | ||
| CuSO 4·5H 2O | 0.025 | ||||||
| CoCl 2·6H 2O | 0.025 |
Embodiment 1
(1) draw materials: with Changsha rattan water spinach for experiment material, morning, 7:00-8:00 got the main rattan rattan of rattan water spinach in large Tanaka, rattan water spinach blade and petiole is removed with the scissors crossed through 70% alcohol wipe, about 5min is rinsed under flowing water, rattan water spinach rattan is cut into the stem section that 3-4cm is long, and ensures the axillalry bud at least do not sprouted with 1 in each stem section.
(2) explant sterilization: stem section is loaded in nylon net bag, put into triangular flask, on superclean bench, first use 70% alcohol disinfecting 30 seconds, discard alcohol, aseptic water washing 1 time, then with add polysorbas20 5% liquor natrii hypochloritis or add polysorbas20 0.1% mercuric chloride solution (instilling 50 μ L polysorbas20s in every 100mL thimerosal) sterilizing 6 minutes, abandon liquor natrii hypochloritis or mercuric chloride solution, aseptic water washing 3-5 time.Press from both sides out the explant after sterilization with aseptic nipper, put into the sterilized culture dish being lined with filter paper, blot stem section surface moisture, for subsequent use.
(3) explant inoculation and Initial culture: cut stem section end a little and make axillalry bud be in 2/3 position from stem segment base portion with the scalpel after sterilizing, with the tweezers after sterilization, stem section is seeded to (formula is the agar powder of the sucrose+6g/L of the methyl α-naphthyl acetate+30g/L of the 6-benzyl aminoadenine+0.2mg/L of MS medium (see table 1)+2.0mg/L, and pH value range is 5.5-5.8) in Initial culture base to cultivate.Condition of culture: when the axillalry bud do not sprouted is stretched to 1cm, temperature is 30.0 ± 0.5 DEG C, and temperature is 25.0 ± 0.5 DEG C afterwards, and intensity of illumination is 2000lx, and light application time is 16 hours/day, and incubation time is 15 days.
(4) explant squamous subculture: with the scalpel after sterilization by more than the height of seedling 4cm in Initial culture base, and there is the explant of 3-5 axillalry bud, be cut into the stem section with 1-2 axillalry bud, and with the tweezers after sterilization, stem section is transferred to subculture medium (the i.e. agar powder of the sucrose+6g/L of the gibberellin+30g/L of the 6-benzyl aminoadenine+0.5mg/L of MS medium (see table 1)+1.0mg/L, pH value range is 5.5-5.8) in carry out Multiplying culture, when the Multiple Buds length that explant produces has 3-5 axillalry bud, be separated described Multiple Buds, being forwarded in Initial culture base makes axillalry bud extend, be forwarded to again after more than height of seedling 4cm in subculture medium and continue propagation, can Cyclic culture 10-15 time like this, condition of culture: during illumination, temperature is 28.0 ± 0.5 DEG C, time dark, temperature is 18.0 ± 0.5 DEG C, intensity of illumination is 3000lx, light application time is 16 hours/day, incubation time is 15 days.
(5) explant culture of rootage: explant Multiple Buds is separated with the scalpel after sterilization and tweezers, select more than height of seedling 4cm, with the explant of 3-5 axillalry bud, be forwarded in root media (formula is the agar powder of the sucrose+6g/L of the methyl α-naphthyl acetate+15g/L of 1/2MS medium+1.0mg/L, and pH value range is 5.5-5.8) and carry out culture of rootage.Condition of culture: during illumination, temperature is 28.0 ± 0.5 DEG C, time dark, temperature is 18.0 ± 0.5 DEG C, and intensity of illumination is 3000lx, and light application time is 16 hours/day, and incubation time is 10 days.
(6) training tissue culture seedling and transplanting: will to take root and the plantlet in vitro of well developed root system takes out after 2 days in the uncork of illumination cultivation room, careful clean plantlet in vitro root medium under flowing water, move in the nutritive cube being equipped with and being made up of the peat composed of rotten mosses, vermiculite and perlite (volume ratio is 3:1:2) mixed-matrix again, water sufficient clear water, 25 DEG C of lower refining seedlings 8 days, intensity of illumination is 2000lx, and light application time is 16 hours/day, and humidity should control more than 85%.Treat that plantlet growth potential becomes prosperous and moves into large Tanaka.
Embodiment 2-5
Embodiment 2-5 the rattan water spinach explant after sterilization is inoculated in respectively add 0.1 respectively, 0.3,0.5, in the Initial culture base of the ribonucleic acid yeast of 0.7g/L concentration, other operations and condition are with embodiment 1.Cultivate and observe explant growth situation afterwards in 7 days, the results are shown in Table 3.The beneficial effect adding ribonucleic acid yeast is as shown in Table 3 mainly ribonucleic acid yeast can make rattan water spinach explant stem stalk xanthate ratio significantly reduce, and axillary bud growth speed is relatively very fast, and plant strain growth gesture is more vigorous.When ribonucleic acid yeast concentration is more than or equal to 0.5g/L, explant growth effect is without significant difference, considers production cost, selects in medium, add 0.5g/L ribonucleic acid yeast.
The result (incubation time is observable result after 7 days) of explant growth situation observed by table 3 add the ribonucleic acid yeast of variable concentrations in Initial culture base after
* yellow refers in tissue culture procedures, and the impact due to medium component, outside environmental elements makes the whole strain of explant or partial organ's chlorosis, final withered dead phenomenon.
Embodiment 6-16
The result (incubation time is observable result after 10 days) of explant growth situation observed by table 4 add antioxidant, ispol and rattan water spinach bleeding sap in subculture medium after
1--brownization: in plant tissue culture course, explant autologous tissue discharges brown material so that medium overstrike gradually from surface to medium, the phenomenon of explant also further browning thereupon, refers to the phenomenon of stem culm base callus browning in the present invention.
2--15% rattan water spinach bleeding sap: overhead cut off at 15-20cm place by rattan water spinach stem, section is poured into plastic bottle, and bleeding sap and nature flow out from wound, and the liquid distilled water diluting collected, to 15% of original solution, can use after 0.22 μm of filter membrane.
3--Multiple Buds reproduction coefficient refers to the number by the shape indefinite bud that clusters formed by Calli Differentiation after 1 stem segment base portion callus.Occur that decimal point is because the mean value calculated herein.
Explant after Initial culture is cut into the stem section with 1-2 axillalry bud, be seeded in the subculture medium that with the addition of antioxidant (silver nitrate of 0.05g/L, the citric acid of Vc, 0.1g/L of 0.05g/L and the ditert-butylhydro quinone of 0.02g/L) respectively, other operations and condition are with embodiment 1.
Cultivate and observe explant growth situation afterwards in 10 days, the results are shown in Table 4.As shown in Table 4, compared with embodiment 1, add antioxidant in the medium and obviously can reduce stem culm base callus browning rate, but Multiple Buds reproduction coefficient is not improved, in 4 kinds of antioxidants, the antioxidant usage amount that the ditert-butylhydro quinone of 0.02g/L is conventional compared with all the other 3 kinds is little, and effect is better.And while adding antioxidant ditert-butylhydro quinone in the medium, also add the amino acid blending constituent of L-hydroxy-proline, Serine and L-cysteine hydrochloride, stem culm base callus browning rate can be reduced, can greatly improve Multiple Buds reproduction coefficient again, more be conducive to the squamous subculture of plantlet in vitro.In addition, its effect of rattan water spinach bleeding sap of adding 15% in the medium can be considered with the L-cysteine hydrochloride of the ditert-butylhydro quinone adding 0.02g/L, the L-hydroxy-proline of 0.5g/L, the Serine of 0.05g/L and 0.1g/L and is equal to.
Embodiment 17
Table 5 modified MS medium composition (unit: mg/L)
| Macroelement | Content | Trace element | Content | Organic substance | Content | Molysite | Content |
| NH 4NO 3 | 2475 | KI | 0.83 | Inositol | 100 | FeSO 4·7H 2O | 139 |
| KNO 3 | 1900 | H 3BO 3 | 6.2 | Nicotinic acid | 0.5 | Na 2·EDTA·2H 2O | 186.5 |
| CaCl 2·2H 2O | 440 | MnSO 4·4H 2O | 22.3 | Puridoxine hydrochloride | 0.5 | ||
| MgSO 4·7H 2O | 370 | ZnSO 4·7H 2O | 8.6 | Thiamine hydrochloride | 0.1 | ||
| KH 2PO 4 | 255 | Na 2MoO 42H 2O | 0.25 | Glycine | 2.0 | ||
| KCl | 70 | CuSO 4·5H 2O | 0.025 | Calcium pantothenate | 0.3 | ||
| CoCl 2·6H 2O | 0.025 |
Explant after squamous subculture is cut into the stem section with 1-2 axillalry bud, is seeded in the MS medium (see table 5) improved, other operations and condition are with embodiment 1.Cultivate and observe explant growth situation afterwards in 10 days, the results are shown in Table 6.As shown in Table 6, adopt the MS medium beneficial effect after improvement to be mainly and explant blade can be made to increase, leaf look darker, and stem stalk is more healthy and stronger, and axillalry bud is loose, and growth potential is better, and growth rate is faster.
The result (incubation time is observable result after 10 days) of explant growing state in table 6 MS and modified MS medium
Embodiment 18
All replaced with white sugar by sucrose in Initial culture base, subculture medium and root media, other operations and condition are with embodiment 1.Result shows, compared with embodiment 1, adopts white sugar do not slow down growth rate, the Multiple Buds reproduction coefficient of rattan water spinach explant as carbon source and reduce robust plant degree.And white sugar more takes advantage in price than sucrose, white sugar can be used reduce group training cost completely.
Embodiment 19
The step I of rattan water spinach virus-free culture), described step I) in described step 3) after and in step 4) before, after getting Initial culture, axillalry bud is stretched to the explant terminal bud of more than 3cm or directly obtains and be grown on terminal bud on the outdoor main rattan of rattan water spinach, the terminal bud stem apex that length is 1.0mm is stripped under disecting microscope, be seeded in Primary culture base and carry out virus-free culture, described Primary culture base is the active carbon of the agar powder+1.5g/L of the carbon source+6g/L of the gibberellin+30g/L of the methyl α-naphthyl acetate+0.05-0.20mg/L of MS medium+0.1-0.3mg/L; The virus-free culture time is 14-18 days.Other operations and condition are with embodiment 1.
The rattan water spinach rattan of the rattan water spinach tissue cultural seedlings of free after hardening and cellar being reserved seed for planting is transplanted in booth simultaneously, and field management is consistent, grows and carries out Agronomic characteristic and Pests to it after 1 month, the results are shown in Table 7.As shown in Table 7, compared with the non-detoxic seedling of normal cutting propagation, main rattan average length amplification is 15.28%, the thick amplification of the average stem of main rattan is 22.56%, the main rattan of every root sprouts number of branch and on average increases 3, and average leaf area amplification is 14.26%, and detoxic seedling advantage main manifestations is detoxic seedling fast growth, growth potential is prosperous, disease-free generation.
Table 7 detoxic seedling and normal cutting propagation seedling Agronomic characteristic and disease survey (growing observable result after 1 month)
Claims (12)
1. a method for rattan water spinach tissue cultures, is characterized in that, comprises the steps:
A) pretreatment: obtain rattan water spinach rattan, rinse well under flowing water, be cut into the stem section of 3-4cm, the axillalry bud at least do not sprouted with 1 in described stem section, and to described stem section surface sterilization;
B) Initial culture: be inoculated in Initial culture base and cultivate, described Initial culture base is the agar powder of the carbon source+6g/L of the methyl α-naphthyl acetate+30g/L of the 6-benzyl aminoadenine+0.1-0.3mg/L of MS medium+2.0-3.0mg/L, and pH value range is 5.5-5.8;
C) squamous subculture: after selecting Initial culture, height of seedling is at more than 4cm, and there is the explant of 3-5 axillalry bud, be cut into the stem section with 1-2 axillalry bud, be inoculated in subculture medium and cultivate, when the Multiple Buds length that explant produces has 3-5 axillalry bud, be separated described Multiple Buds, be forwarded in Initial culture base and axillalry bud is extended, be forwarded to again after more than height of seedling 4cm in subculture medium and continue propagation; Described subculture medium is the agar powder of the carbon source+6g/L of the gibberellin+30g/L of the 6-benzyl aminoadenine+0.3-0.7mg/L of MS medium+0.5-1.5mg/L, and pH value range is 5.5-5.8;
D) culture of rootage: select more than height of seedling 4cm, there is the explant of 3-5 axillalry bud, be inoculated in root media and cultivate, described root media is the agar powder of the carbon source+6g/L of the methyl α-naphthyl acetate+15g/L of 1/2MS medium+0.3-0.6mg/L, and pH value range is 5.5-5.8;
E) select to have taken root and the plantlet in vitro of well developed root system carries out hardening.
2. method according to claim 1, it is characterized in that, described steps A) in thimerosal be add polysorbas20 5% liquor natrii hypochloritis or add polysorbas20 0.1% mercuric chloride solution, and instill 50 μ L polysorbas20s in every 100mL thimerosal, disinfecting time is 5-8 minute.
3. method according to claim 1, is characterized in that, also comprises the ribonucleic acid yeast of 0.3-1.0g/L in described Initial culture base.
4. method according to claim 3, is characterized in that, described Initial culture base comprises the ribonucleic acid yeast of 0.5g/L.
5. method according to claim 1, is characterized in that, also comprises the one in the citric acid of the ditert-butylhydro quinone of 0.02g/L, the silver nitrate of 0.05g/L, Vc and 0.1g/L of 0.05g/L in described subculture medium.
6. method according to claim 5, is characterized in that, described subculture medium comprises the ditert-butylhydro quinone of 0.02g/L.
7. the method according to claim 1-6 any one, is characterized in that, also comprises one or more in the L-cysteine hydrochloride of the L-hydroxy-proline of 0.1-0.5g/L, the Serine of 0.3-0.7g/L and 0.1-0.5g/L in described subculture medium.
8. the method according to claim 1-6 any one, is characterized in that, also comprises the rattan water spinach bleeding sap of 15% in described subculture medium.
9. method according to claim 1, it is characterized in that, in described subculture medium, MS medium is the MS medium of improvement, wherein, the MS medium of described improvement is the ammonium nitrate of 2475mg/L, the potassium nitrate of 1900mg/L, the calcium chloride dihydrate of 440mg/L, the epsom salt of 370mg/L, the potassium dihydrogen phosphate of 225mg/L, the potassium chloride of 70mg/L, the potassium iodide of 0.83mg/L, the boric acid of 6.2mg/L, the four water manganese sulphates of 22.3mg/L, the white vitriol of 8.6mg/L, the Sodium Molybdate Dihydrate of 0.25mg/L, the cupric sulfate pentahydrate of 0.025mg/L, the CoCL2 6H2O of 0.025mg/L, the inositol of 100mg/L, the nicotinic acid of 0.5mg/L, the puridoxine hydrochloride of 0.5mg/L, the thiamine hydrochloride of 0.1mg/L, the glycine of 2.0mg/L, the calcium pantothenate of 0.3mg/L, the ferrous sulfate heptahydrate of 139.0mg/L and the disodium ethylene diamine tetraacetate of 186.5mg/L.
10. method according to claim 1, is characterized in that, at described step B) in the condition of cultivation be: the described axillalry bud do not sprouted be stretched to 1cm before temperature be 30.0 ± 0.5 DEG C; Described axillalry bud be stretched to 1cm after temperature be 25.0 ± 0.5 DEG C, intensity of illumination is 2000lx, and light application time is 16 hours/day;
And/or at described step C) in the condition of cultivation be: during illumination, temperature is 28.0 ± 0.5 DEG C, and time dark, temperature is 18.0 ± 0.5 DEG C, and intensity of illumination is 3000lx, and light application time is 16 hours/day;
And/or at described step D) in the condition of cultivation be: during illumination, temperature is 28.0 ± 0.5 DEG C, and time dark, temperature is 18.0 ± 0.5 DEG C, and intensity of illumination is 3000lx, and light application time is 16 hours/day;
And/or in described step e) in, during described hardening, temperature is 25.0 ± 0.5 DEG C, and intensity of illumination is 2000lx, and light application time is 16 hours/day, and humid control is more than 85%.
11. methods according to claim 1, it is characterized in that, also comprise the step I of rattan water spinach virus-free culture), described step I) at described step B) after and in step C) before, after getting Initial culture, axillalry bud is stretched to the terminal bud of the explant of more than 3cm, or directly acquisition is grown on terminal bud on the outdoor main rattan of rattan water spinach, stripping length is after 0.8-1.2mm terminal bud stem apex, described terminal bud stem apex is seeded in Primary culture base and carries out virus-free culture, on the described main rattan of rattan water spinach being grown on outdoor, terminal bud needs sterilization, sterilization method is with described steps A) pretreatment in sterilization method, described Primary culture base is the active carbon of the agar powder+1.5g/L of the carbon source+6g/L of the gibberellin+30g/L of the methyl α-naphthyl acetate+0.05-0.20mg/L of MS medium+0.1-0.3mg/L.
12. methods according to claim 1, is characterized in that, described rattan water spinach is selected from Changsha rattan water spinach.
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