CN106171985A - A kind of liquid shallow quick-breeding method of Rhizoma Zingiberis Recens - Google Patents
A kind of liquid shallow quick-breeding method of Rhizoma Zingiberis Recens Download PDFInfo
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- CN106171985A CN106171985A CN201610546635.0A CN201610546635A CN106171985A CN 106171985 A CN106171985 A CN 106171985A CN 201610546635 A CN201610546635 A CN 201610546635A CN 106171985 A CN106171985 A CN 106171985A
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- zingiberis recens
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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
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention discloses the liquid shallow quick-breeding method of a kind of Rhizoma Zingiberis Recens, comprise the following steps: 1) material heat treatment；2) sterilization of outer implant and inoculation；3) initial culture；4) successive transfer culture；5) root culture；6) seedling exercising；7) transplant.The present invention cultivates using Rhizoma Zingiberis Recens bud as outer implant, uses stem apex heat treatment detoxification technology, it is thus achieved that detoxification takes off bacterium Rhizoma Zingiberis Recens seedling, only uses ethanol, HgCl with traditional2Comparing etc. disinfectant external implant disinfection way, virus elimination rate is high, and survival rate is high.Using fluid medium to carry out shallow-layer cultivation, preferably condition of culture, be greatly saved cost, the tissue culture quick breeding system for Rhizoma Zingiberis Recens breeding provides Technical Reference.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to the liquid shallow quick-breeding method of a kind of Rhizoma Zingiberis Recens.
Background technology
Rhizoma Zingiberis Recens is Zingiberaceae perennial unifacial leaf herbaceous plant, production mostly is raw cultivation in 1 year, extensively cultivates in China.Raw
Rhizoma Zingiberis Recens is nutritious due to it, and medicinal effects is good, it has also become important seasoning vegetable and medicine, chemical industry and raw materials for food industry；Add
Its economic worth upper is high, storage tolerance, has the most become the main special product of China and has earned foreign exchange one of vegetable.
Do not bloom due to Rhizoma Zingiberis Recens or seldom bloom, breeding being difficult with sexual propagation, and minority kind can only be leaned on long
Phase asexual propagation, thus cause the Rhizoma Zingiberis Recens multiple virus of plant cylinder accumulation, yield declines, quality deterioration, and resistance declines.In recent years
Coming, along with the development of biotechnology, plant tissue culture technique has the most gradually been applied in the production of Rhizoma Zingiberis Recens, achieves good
Economic benefit.After detoxicated ginger, plant growing way is strong, and yield significantly improves, and disease resistance strengthens.Rhizoma Zingiberis Recens tissue cultured seedling is used to substitute Rhizoma Zingiberis Recens
Block, can be greatly saved kind of a Rhizoma Zingiberis Recens consumption, improves the yield and quality of Rhizoma Zingiberis Recens, it is thus achieved that preferably economic benefit.Meanwhile, tissue training is used
Support the loss that Ginger Germplasm can be overcome to be caused because of natural disaster and pest and disease damage, and plant quality can be changed
Good, form and gene structure by changing Rhizoma Zingiberis Recens chromosome, reach to improve the purpose of Quality of Ginger.
Summary of the invention
For problems of the prior art, the present invention provides the liquid shallow quick-breeding method of a kind of Rhizoma Zingiberis Recens.
The present invention is realized by the following technical programs:
The liquid shallow quick-breeding method of described a kind of Rhizoma Zingiberis Recens, it is characterised in that comprise the following steps:
1) material heat treatment: selected piece of big, meat thickness, bright yellow color, eye healthy and strong Rhizoma Zingiberis Recens block full, unputrefied, under tap water
Clean earth, steep 30 min sterilizations by 500 times of immersions of 50 % carbendazim, then cover with the fine sand after high temperature sterilize, be placed in perseverance
Accelerating germination in temperature incubator, condition of culture is temperature 36-38 DEG C, and air humidity is 70-80 %, and whole process need not illumination, Rhizoma Zingiberis Recens
After bud grows, taking the Rhizoma Zingiberis Recens of accelerating germination in incubator, 50 DEG C of superhigh temperature process 5 min for inoculating；
2) sterilization of outer implant is with inoculation: cutting the Rhizoma Zingiberis Recens bud of after heat treatment 1-2 cm, tap water rinses 30 min, is subsequently placed in
The ethanol of 70% soaks 30 s, discards ethanol, aseptic water washing 1 time, then with 10% NaClO sterilization 10-15 min, discard
NaClO solution, aseptic water washing 5 times, after draining the water, takes out and utilizes binocular anatomical lens to strip 0.2-0.4 on superclean bench
The shoot apical meristem of mm；
3) initial culture: cultivate in shoot apical meristem is inoculated into initial culture base, described initial culture base is: MS
The agar of naphthalene acetic acid (NAA)+7 g/L of 6-benzyl aminoadenine (the 6-BA)+0.1-0.3 mg/L of culture medium+2-3 mg/L+
The edible sugar of 30 g/L, pH value range is 5.5-5.8；The culture medium prepared is divided in the tissue culture bottle of 240 mL, every bottle
25 mL, 121 DEG C of sterilizing 15 min；
4) successive transfer culture: select after initial culture height of seedling at 3 more than cm, and there is the outer implant of 3-6 axillalry bud, be inoculated in and continue
Culture base is cultivated, after outer implant produces Multiple Buds, from each clump of bud, selects 1 strain, with the shears of sterilizing, clip bastem
The vegetable material of portion 0.5 more than cm, carries out cucumber mosaic virus and tobacco mosaic virus (TMV) detection by enzyme linked immunosorbent assay；So
Cutting the two or more buds of band with the dissecting knife after sterilization from nontoxic Rhizoma Zingiberis Recens Multiple Buds afterwards, related base portion cuts away all of
And 0.5 stem section of cm above section, it is forwarded in initial culture base make axillalry bud extend, after height of seedling 4 more than cm, is forwarded to liquid again
Continuing propagation in body subculture medium, described subculture medium is: the edible sugar+2-5 of MS culture medium+10-30 g/L
The antioxidant of the naphthalene acetic acid+0.1-1.7 g/L of the 6-benzyl aminoadenine+0.1-0.5 mg/L of mg/L, pH value range is
5.5-5.8；The culture medium prepared is divided in the tissue culture bottle of 240 mL, and every bottle of 10 mL, in incubation, culture medium must not have
Cross culture materials, otherwise affect propagation, 121 DEG C of sterilizing 15 min；
5) root culture: select height of seedling 3 more than cm, there is the outer implant of 3-6 axillalry bud, be inoculated in liquid root media
Cultivating, described root media is: the 6-benzyl amino gland of the edible sugar+0.1-0.4 mg/L of MS culture medium+5-20 mg/L is fast
The murphy juice of paclobutrazol+100 g/L of the naphthalene acetic acid+0.4-2.0 mg/L of purine+0.2-0.6 mg/L, pH value range is 5.5-
5.8；The culture medium prepared is divided in the tissue culture bottle of 240 mL, every bottle of 10 mL；
6) seedling exercising: being taken out from culturing room by tissue cultured seedling, be positioned in seeding room, temperature is maintained at 22-29 DEG C, and humidity is maintained at
70-75 %, it is to avoid direct sunlight；After placing 3-4 d, open bottle cap, allow seedling gradually adapt to external environment condition, morning and evening aerosol apparatus
Seedling and indoor are sprayed, through the seedling exercising process of about 1 week, can transplant；
7) transplant: be Vermiculitum weight ratio: the substrate of perlite=1:1 is put in the nutrient bag of sterilization, each process 100
Bag.With tweezers, each tissue cultured seedling is taken out from culture bottle during transplanting, be divided into individual plant, rinse the cultivation of residual on root well with clear water
Liquid, accomplish not hinder root, soaks 20 min with 500 times of carbendazim solutions, the most carefully plants in nutrient bag, waters foot and determines root water,
Shading with sunshade net after transplanting, often water and spray, humidity is maintained at 75-85 %, between temperature 22-25 DEG C；Execute weekly 1
The carbamide of secondary 0.1 % and 0.2 % KH2PO4, carry out the prevention and control of plant diseases, pest control simultaneously.
The liquid shallow quick-breeding method of described a kind of Rhizoma Zingiberis Recens, it is characterised in that the condition of culture of step 3) initial culture
For: cultivation temperature 25-30 DEG C, light application time is to irradiate 12-14 h every day, and intensity of illumination is 1200-1500 Lx, incubation time
For 15-30 d.
The liquid shallow quick-breeding method of described a kind of Rhizoma Zingiberis Recens, it is characterised in that the condition of culture of step 4) successive transfer culture
For: cultivation temperature 25-30 DEG C, light application time is to irradiate 12-14 h every day, and intensity of illumination is 1200-1500 Lx.
The liquid shallow quick-breeding method of described a kind of Rhizoma Zingiberis Recens, it is characterised in that the condition of culture of step 5) root culture
For: cultivation temperature 25-30 DEG C, light application time is to irradiate 12-14 h every day, and intensity of illumination is 1200-1500 Lx.
The liquid shallow quick-breeding method of described a kind of Rhizoma Zingiberis Recens, it is characterised in that the antioxidant in step 4) is 0.5 g/
The sodium thiosulfate of L, the thiourea of 0.1 g/L, the dithiothreitol, DTT of 0.5 g/L, the cysteine of 0.5 g/L, 0.1 g/L anti-
One or more in bad hematic acid.
The liquid shallow quick-breeding method of described a kind of Rhizoma Zingiberis Recens, it is characterised in that the root media in step 5) also includes
Organic additive, described organic additive be the murphy juice of 100 g/L, the corn juice of 100 g/L, the Semen Tritici aestivi flour of 100 g/L,
The caseinhydrolysate of 0.2 g/L, 0.2 g/L yeast extract in any one.
The present invention cultivates using Rhizoma Zingiberis Recens bud as outer implant, uses stem apex-heat treatment detoxification technology, it is thus achieved that detoxification takes off bacterium
Rhizoma Zingiberis Recens seedling, only uses ethanol, HgCl with traditional2Comparing etc. disinfectant external implant disinfection way, virus elimination rate is high, survival rate
High.And use preferred cultivating system to carry out tissue culture, and it being greatly saved cost, the Fast-propagation for Rhizoma Zingiberis Recens breeding provides technology
Reference.
In the present invention, the tissue culture of Rhizoma Zingiberis Recens uses liquid culture system, either takes root examination at Rhizoma Zingiberis Recens subculture or Rhizoma Zingiberis Recens
In testing, the liquid culture medium propagation with identical hormonal readiness is the most all better than solid medium with taking root, with
Time liquid culture medium save a large amount of manpower and financial resources to a great extent.Additionally, in the present invention, group is replaced with edible sugar
Knit and cultivate conventional sucrose, use the reduction of liquid culture system, beneficially production cost simultaneously.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail, and provides detailed description of the invention.
Embodiment 1:
1) material heat treatment: selected piece of big, meat thickness, bright yellow color, eye healthy and strong Rhizoma Zingiberis Recens block full, unputrefied, under tap water
Clean earth, steep 30 min sterilizations by 500 times of immersions of 50% carbendazim, then cover with the fine sand after high temperature sterilize, be placed in constant temperature
Accelerating germination in incubator, condition of culture is temperature 36-38 DEG C, and air humidity is 70-80%, and whole process need not illumination, and Rhizoma Zingiberis Recens bud is long
After going out, taking the Rhizoma Zingiberis Recens of accelerating germination in incubator, 50 DEG C of superhigh temperature process 5 min for inoculating；
2) sterilization of outer implant is with inoculation: cutting the Rhizoma Zingiberis Recens bud of after heat treatment 1-2 cm, tap water rinses 30 min, is subsequently placed in
The ethanol of 70% soaks 30 s, discards ethanol, aseptic water washing 1 time, then with 10 % NaClO sterilization 10-15 min, discard
NaClO solution, aseptic water washing 5 times, after draining the water, takes out and utilizes binocular anatomical lens to strip 0.2-0.4 on superclean bench
The shoot apical meristem of mm；
3) initial culture: shoot apical meristem is inoculated into 121 DEG C of sterilizing 15 min, the initial culture base (MS of every bottle of 20 mL
The edible sugar of agar+30 g/L of naphthalene acetic acid+7 g/L of 6-benzyl aminoadenine+0.25 mg/L of culture medium+2 mg/L,
PH 5.6), 25-30 DEG C, illumination 12-14 h/d, intensity of illumination is 1200-1500 Lx, cultivates 28 d, and bud ratio is 98.2
%, and bud quality is good, aetiolation is few, uses heat treatment sterilization, and the pollution rate of bud is low, survival rate 90 %.
4) successive transfer culture: select after initial culture height of seedling at 3 more than cm, and there is the outer implant of 3-6 axillalry bud, inoculate
Liquid subculture medium (edible sugar+3 mg/L of MS culture medium+15 g/L in 121 DEG C of sterilizing 15 min, every bottle of 10 mL
6-benzyl aminoadenine+0.3 mg/L naphthalene acetic acid+0.5 g/L sodium thiosulfate+0.1 g/L thiourea+0.5 g/L bis-sulfur Soviet Union
Sugar alcohol+0.5 g/L cysteine+0.1 g/L ascorbic acid, pH 5.6) in, 25-30 DEG C, illumination 12-14 h/d, illumination is strong
Degree for 1200-1500 Lx, cultivates 30 d, after outer implant produces Multiple Buds, with the shears of sterilizing, clip bastem portion 0.5 cm with
On vegetable material, with sterilization after dissecting knife from nontoxic Rhizoma Zingiberis Recens Multiple Buds cut band two or more buds, related base portion
Cut away all of and the stem section of 0.5 cm above section, be forwarded in initial culture base make axillalry bud extend, height of seedling 4 more than cm
After be forwarded to again in liquid subculture medium continue propagation, proliferation times is 6.58, melting brown rate 6.5 % of material, browning
Substantially reduce.
5) root culture: select height of seedling 3 more than cm, there is the outer implant of 3-6 axillalry bud, be inoculated in 121 DEG C of sterilizings 15
Liquid root media (the 6-benzyl amino gland of edible sugar+0.2 mg/L of MS culture medium+10 mg/L of min, every bottle of 10 mL
The murphy juice of paclobutrazol+100 g/L of naphthalene acetic acid+1.6 mg/L of purine+0.4 mg/L, pH 5.6) in, 25-30 DEG C, light
According to 12-14 h/d, intensity of illumination is 1200-1500 Lx, cultivates 30 d.Going out radical is 8.3, and root length is 39.0 mm, Se Bai and
Slightly, thick 0.32 cm of stem, blade is big, and plump, color is dark green.
6) seedling exercising: being taken out from culturing room by tissue cultured seedling, be positioned in seeding room, temperature is maintained at 22-29 DEG C, humidity keeps
At 70-75%, it is to avoid direct sunlight；After placing 3-4d, open bottle cap, allow seedling gradually adapt to external environment condition, morning and evening aerosol apparatus
Seedling and indoor are sprayed, through the seedling exercising process of about 1 week, can transplant.
7) transplant: be Vermiculitum weight ratio: the substrate of perlite=1:1 is put in the nutrient bag of sterilization, each process
100 bags.With tweezers, each tissue cultured seedling is taken out from culture bottle during transplanting, be divided into individual plant, rinse residual on root well with clear water
Culture fluid, accomplish not hinder root, soaks 20 min with 500 times of carbendazim solutions, the most carefully plants in nutrient bag, waters foot and determines root
Water, shades with sunshade net after transplanting, often waters and spray, and humidity is maintained at 75-85 %, between temperature 22-25 DEG C；Weekly
Execute carbamide and the 0.2 % KH of 1 0.1 %2PO4, carry out the prevention and control of plant diseases, pest control, tissue cultured seedling survival rate 98 % simultaneously.
Embodiment 2: Stem tip induction bud ratio
In MS culture medium, add the agar of 7 g/L, the edible sugar of 30 g/L, change hormone combination, at 27 ± 2 DEG C, illumination
Every day time irradiates 12-14 h, and intensity of illumination is 1200-1500 Lx, after cultivating 25 d, observes hormon proportioning to sprouting
The impact of rate.
The impact on bud ratio of the table 1 hormon proportioning
From table 1 it will be seen that after hormone 6-BA concentration is more than 2 mg/L, induction bud ratio reduces, and along with hormone
The increasing of NAA concentration, induction bud ratio raises, and most suitable hormone combination is 6-BA concentration 2 mg/L, NAA concentration 0.25 mg/
L。
Embodiment 3: heat treatment detoxification efficiency and initial culture survival rate
It is respectively adopted tradition HgCl2Sterilization and the method for heat treatment sterilization, inducing culture detects its pollution rate after 10 days respectively, and
After initial culture, detect its survival rate, contrast the detoxification efficiency of the two and the impact on initial culture survival rate, the results are shown in Table
2.From table 2 it can be seen that the material processed through heat treatment sterilization method of the present invention, pollution rate is only sterilized through mercuric chloride less than tradition
Method, and in initial culture, its survival rate, apparently higher than the material processed through traditional disinfection methods, further illustrates, this invention
Heat treatment sterilization method is less to the damage of stem apex, it is easy to survive.
The different sterilization method of table 2 is on initial culture pollution rate and the impact of survival rate
| Sterilization method | Process bottle number | Pollution rate (%) | Survival rate (%) |
| Traditional disinfection methods | 100 | 8 | 71 |
| Heat treatment sterilization method | 100 | 6 | 90 |
Embodiment 4: the impact that successive transfer culture is bred by hormone combination
The impact that successive transfer culture is bred by table 3 hormone combination
Being inoculated in the subculture medium of different hormone combinations by bud and cultivate, proliferation times is shown in Table 3.As seen from the table, swash
The concentration of element has significant impact to the proliferation times of successive transfer culture, along with the increase of 6-BA concentration, on its proliferation times is notable
Rise, but when increasing to 3 mg/L, its increment multiple reaches maximum, then increases 6-BA concentration, on the contrary can Inhibit proliferaton；
NAA increasing concentrations, can promote propagation, reach maximum when 0.3 mg/L, then begin to decline, optimal proportioning is that 6-BA is dense
Spend 3 mg/L, NAA concentration 0.3 mg/L, i.e. No. 8 culture medium.
Embodiment 5: add antioxidant to the impact of brown stain in Subculture
Brownization refers to the just differentiation of implant induction outside or again in atomization, autologous tissue discharges brown thing from surface to culture medium
Matter gradually becomes brown down to culture medium, the outer implant also phenomenon of death therewith browning further.Brownization has a strong impact on outer planting
The differentiation of body, even results in the failure of cultivation, and effective Browning control has the effect of key to liquid culture.In culture fluid
Adding antioxidant, aminoacid etc. can effective Browning control.This experiment adds thiourea, dithiothreitol, DTT, cysteine,
Sodium thiosulfate, ascorbic acid and combinations thereof have investigated the inhibitory action to brownization, observe outer planting bulk-growth feelings after cultivating 30 d
Condition, the results are shown in Table 4.Compared with the control, add antioxidant in the medium and can substantially reduce stem culm base callus browning
Rate, in 4 kinds of antioxidants, 0.1 g/L ascorbic acid is little compared with remaining 3 kinds of antioxidant usage amount, and effect is good.And in training
Support and base adds ascorbic acid, sodium thiosulfate, thiourea, the antioxidant combination composition of dithiothreitol, DTT, add half Guang simultaneously
Propylhomoserin, can reduce stem culm base callus browning rate, can be greatly improved again Multiple Buds increment multiple, be more beneficial for tissue cultured seedling
Successive transfer culture.
Table 4 antioxidant, aminoacid are on the impact of brown stain in incubation
| Numbering | Additive | Material melting brown rate | Proliferation times |
| 0 | Nothing | 32.5 % | 4.16 |
| 1 | 0.5g/L sodium thiosulfate | 28.9 % | 4.57 |
| 2 | 0.1 g/L thiourea | 29.2 % | 4.38 |
| 3 | 0.5 g/L dithiothreitol, DTT | 19.8 % | 4.97 |
| 4 | 0.1 g/L ascorbic acid | 14.2 % | 5.11 |
| 5 | 0.5 g/L cysteine | 16.5 % | 5.06 |
| 6 | 0.5g/L sodium thiosulfate+0.1 g/L thiourea | 23.6 % | 4.65 |
| 7 | 0.5 g/L cysteine+0.5 g/L dithiothreitol, DTT | 12.5 % | 5.34 |
| 8 | 0.5 g/L sodium thiosulfate+0.1 g/L thiourea+0.5 g/L cysteine+0.5 g/L dithiothreitol, DTT | 10.8 % | 5.49 |
| 9 | 0.5 g/L sodium thiosulfate+0.5 g/L cysteine+0.5 g/L dithiothreitol, DTT+0.1 g/L ascorbic acid | 7.5 % | 5.73 |
| 10 | 0.5 g/L sodium thiosulfate+0.1 g/L thiourea+0.5 g/L dithiothreitol, DTT+0.5 g/L cysteine+0.1 g/L ascorbic acid | 6.5 % | 6.50 |
Embodiment 6: the impact that successive transfer culture is bred by cultivating system type
Based on No. 8 culture medium in embodiment 3, adding 7 g/L agar, be configured to solid medium, cultivation temperature is 27
± 2 DEG C, light application time irradiates 12-14 h every day, and intensity of illumination is 1200-1500 Lx, and liquid, solid culture system are respectively provided with
Three groups parallel, cultivates 28 days and observes increment multiple, the results are shown in Table 5.
The table 5 liquid/solid cultivating system successive transfer culture rate of increase
From table 5 it will be seen that compared with solid medium, liquid culture system to the propagation of successive transfer culture almost without shadow
Ring, and average increment multiple is slightly higher compared with solid culture.Because fluid medium is more beneficial for the absorption of Rhizoma Zingiberis Recens tissue cultured seedling, therefore growth is fast
Degree is very fast, and proliferation times relatively solid culture is high.
Embodiment 7: the impact that successive transfer culture is bred by sugar concentration, the results are shown in Table 6.
Carbon source is the indispensable material of plant tissue culture, and it provides energy, Er Qieneng can not only to outer implant
Maintain certain osmotic pressure, and the sugared concentration required for each stage of tissue culture is different.
The table 6 sugar concentration impact on successive transfer culture proliferation times
| Sugar concentration (g/L) | Sprout number (individual) |
| 0 | 0.6 |
| 10 | 3.9 |
| 15 | 4.9 |
| 20 | 4.8 |
| 25 | 4.8 |
| 30 | 4.6 |
As can be seen from Table 6, sugar concentration is when 15 g/L, and the number that sprouts is most, and sugar concentration continues to raise, and Bud polarization number has dropped
Low.
Embodiment 8: the impact that Rhizoma Zingiberis Recens is taken root by hormone combination
The impact that Rhizoma Zingiberis Recens is taken root by table 7 hormone combination
As shown in Table 7, add exogenous hormone in the medium and be easy to make Rhizoma Zingiberis Recens take root, simply take root in different culture medium
Root length, quantity are different, and quantity of taking root when 6-BA concentration 0.2 mg/L, NAA concentration 0.4 mg/L is most, and root length is the most relatively
Long, Gen Sebai and thick, although height of seedling not maximum, but after transplanting, survival rate is higher.
Embodiment 9: the impact that Rhizoma Zingiberis Recens is taken root by liquid culture system
6-BA(concentration 0.2 mg/L it is added in MS culture medium), NAA concentration (0.4 mg/L), it is configured to liquid culture respectively
Base and solid medium (adding the agar of 7 g/L), observe its situation of taking root, each do three groups parallel, the results are shown in Table 8.Rhizoma Zingiberis Recens
The radical that goes out in Seedling liquid medium within affects inconspicuous higher than solid medium, root length and plant height.And solid medium is in refining
Miao Shixu washes the agar being bonded on root, and this can cause the damage of root, causes survival rate to decline.And fluid medium reduces
The consumption of agar, reduces culture medium cost.
The impact that Rhizoma Zingiberis Recens is taken root by table 8 liquid culture system
Embodiment 10: the impact that Rhizoma Zingiberis Recens is taken root by white sugar concentration
The Rhizoma Zingiberis Recens Seedling of successive transfer culture is inoculated in the root media of different sugar concentration cultivation, the results are shown in Table 9.Sugar concentration is 10
During g/L, it is most that every strain goes out radical, and root is the longest.Along with the increase of sugar concentration, go out radical and root length all declines.
The impact that Rhizoma Zingiberis Recens is taken root by table 9 white sugar concentration
| White sugar concentration (g/L) | Go out root rate (%) | Go out radical (bar/strain) | Root length (mm) |
| 0 | 100 | 1.67 | 0.26 |
| 5 | 100 | 6.85 | 26.3 |
| 10 | 100 | 7.00 | 29.5 |
| 15 | 100 | 6.62 | 25.5 |
| 20 | 100 | 6.48 | 25.2 |
| 25 | 100 | 6.33 | 24.8 |
Embodiment 11: selecting detoxicated ginger seedling subculture and the impact taken root of carbon source
All being replaced with sucrose by white sugar in initial culture base, subculture medium and root media, other operations and condition are constant,
The results are shown in Table 10.Selection edible sugar or sucrose are as carbon source, several at the proliferation times of successive transfer culture and the aspect of taking root of Seedling
Not impact, and white sugar more takes advantage in price than sucrose, and white sugar can be used completely to reduce group training cost.
Table 10 edible sugar and sucrose are to detoxicated ginger seedling subculture and the comparison taken root
Embodiment 12: the paclobutrazol of variable concentrations is on Rhizoma Zingiberis Recens strong sprout and the impact taken root
Certain density paclobutrazol can suppress the longitudinal growth of Rhizoma Zingiberis Recens Seedling, promotes its cross growth, stem overstriking, and blade broadens and adds
Thickness, plays the effect in strong sprout, can promote that root system development grows simultaneously, make Seedling be prone to transplant survival.At MS culture medium+6-BA
In 0.2 mg/L+NAA 0.4 mg/L culture fluid, it is separately added into 0.4,0.8,1.2,1.6,2.0 mg/L paclobutrazols, not add
Adding paclobutrazol is control experiment, observe its on Rhizoma Zingiberis Recens strong sprout and the impact taken root, the results are shown in Table 11.
Table 11 paclobutrazol is on Rhizoma Zingiberis Recens strong sprout and the impact taken root
From table 11 it will be seen that 0.4-2.0 mg/L Concentraton gradient, paclobutrazol has the effect in strong sprout, 1.6
Below mg/L, paclobutrazol has the effect promoting root system development, considers, and uses the paclobutrazol of 1.2-1.6 mg/L concentration to have
It is beneficial to strong sprout and hestening rooting simultaneously.
Embodiment 13: the different organic additive impacts on Rhizoma Zingiberis Recens tissue cultured seedling strong plantlets and rootage,
Being added in root media by different types of organic additive, 30 d observe, and the results are shown in Table 12.Add 100 g/
The tissue cultured seedling of L murphy juice grows vigorous, bottle green, and robustness is high, and the effect of strong plantlets and rootage is best.Organic additive juice contains
Having the Organic substances such as aminoacid, hormone, enzyme, it is nutritious, is complicated nutritional blend, can be that plant growing provides some raw
Reason active substance, supplements the micro constitutent of some the unknowns.And add natural organic matter culture medium easily prepare, with low cost,
Organ Differentiation, cell proliferation there is obvious facilitation, in can be applicable to produce.
The impact on Rhizoma Zingiberis Recens tissue cultured seedling strong plantlets and rootage of table 12 organic additive
Embodiment 14: different substrates is on Rhizoma Zingiberis Recens tissue cultured seedling plant height, root length and the impact of survival rate
Table 13 different substrates is on Rhizoma Zingiberis Recens tissue cultured seedling plant height, root length and the impact of survival rate
| Matrix type | Average plant height (cm) | Average root length (cm) | Survival rate (%) |
| Vermiculitum: perlite=1:1 | 8.9 | 6.7 | 98 |
| Perlite | 8.0 | 6.1 | 88 |
| Vermiculitum | 7.8 | 5.7 | 83 |
Transplant the different substrates impact on Rhizoma Zingiberis Recens tissue cultured seedling after 30 d, the results are shown in Table 13.Survival rate is the highest, transplants the most successful.Raw
Survival rate after Rhizoma Zingiberis Recens test tube transplantation of seedlings is at perlite: be up to 98 % in the substrate of Vermiculitum=1:1, and plant height, root length are higher than treasure
Zhu Yan and the growth conditions of Vermiculitum condition.Just because of different substrates, there is different physicochemical properties, just result in Rhizoma Zingiberis Recens test tube
The difference that Seedling grows after transplanting.Vermiculitum acts primarily as water retention, and its porosity is big, and water holding capacity is strong, and perlite acts primarily as
Ventilation effect.Therefore, in conjunction with both advantages, Vermiculitum: the transplanting that the substrate of perlite=1:1 is most suitable for Rhizoma Zingiberis Recens test tube Seedling is raw
Long.
Claims (7)
1. the liquid shallow quick-breeding method of a Rhizoma Zingiberis Recens, it is characterised in that comprise the following steps:
1) material heat treatment: selected piece of big, meat thickness, bright yellow color, eye healthy and strong Rhizoma Zingiberis Recens block full, unputrefied, under tap water
Clean earth, steep 30 min sterilizations by 500 times of immersions of 50% carbendazim, then cover with the fine sand after high temperature sterilize, be placed in constant temperature
Accelerating germination in incubator, condition of culture is temperature 36-38 DEG C, and air humidity is 70-80%, and whole process need not illumination, and Rhizoma Zingiberis Recens bud is long
After going out, taking the Rhizoma Zingiberis Recens of accelerating germination in incubator, 50 DEG C of superhigh temperature process 5 min for inoculating；
2) sterilization of outer implant is with inoculation: cutting the Rhizoma Zingiberis Recens bud of after heat treatment 1-2 cm, tap water rinses 30 min, is subsequently placed in
The ethanol of 70% soaks 30 s, discards ethanol, aseptic water washing 1 time, then with 10 % NaClO sterilization 10-15 min, discard
NaClO solution, aseptic water washing 5 times, after draining the water, takes out and utilizes binocular anatomical lens to strip 0.2-0.4 on superclean bench
The shoot apical meristem of mm；
3) initial culture: cultivate in shoot apical meristem is inoculated into initial culture base, described initial culture base is: MS
Agar+30 g/L's of naphthalene acetic acid+7 g/L of the 6-benzyl aminoadenine+0.1-0.3 mg/L of culture medium+2-3 mg/L is edible
White sugar, pH value range is 5.5-5.8；
4) successive transfer culture: select after initial culture height of seedling at 3 more than cm, and there is the outer implant of 3-6 axillalry bud, be inoculated in and continue
Culture base is cultivated, after outer implant produces Multiple Buds, from each clump of bud, selects 1 strain, with the shears of sterilizing, clip bastem
The vegetable material of portion 0.5 more than cm, carries out cucumber mosaic virus and tobacco mosaic virus (TMV) detection by enzyme linked immunosorbent assay；So
Cutting the two or more buds of band with the dissecting knife after sterilization from nontoxic Rhizoma Zingiberis Recens Multiple Buds afterwards, related base portion cuts away all of
And 0.5 stem section of cm above section, it is forwarded in initial culture base make axillalry bud extend, after height of seedling 4 more than cm, is forwarded to liquid again
Continuing propagation in body subculture medium, described subculture medium is: the edible sugar+2-5 mg/ of MS culture medium+10-30g/L
The antioxidant of the naphthalene acetic acid+0.1-1.7 g/L of the 6-benzyl aminoadenine+0.1-0.5 mg/L of L, pH value range is 5.5-
5.8；
5) root culture: select height of seedling 3 more than cm, there is the outer implant of 3-6 axillalry bud, be inoculated in liquid root media
Cultivating, described root media is: the 6-benzyl amino gland of the edible sugar+0.1-0.4 mg/L of MS culture medium+5-20 mg/L is fast
The murphy juice of paclobutrazol+100 g/L of the naphthalene acetic acid+0.4-2.0 mg/L of purine+0.2-0.6 mg/L, pH value range is 5.5-
5.8；
6) seedling exercising: being taken out from culturing room by tissue cultured seedling, be positioned in seeding room, temperature is maintained at 22-29 DEG C, and humidity is maintained at
70-75 %, it is to avoid direct sunlight；After placing 3-4 d, open bottle cap, allow seedling gradually adapt to external environment condition, morning and evening aerosol apparatus
Seedling and indoor being carried out humidity environment of spraying. through the seedling exercising process of about 1 week, can transplant；
7) transplant: be Vermiculitum weight ratio: the substrate of perlite=1:1 is put in the nutrient bag of sterilization, each process 100
Bag.
2. with tweezers, each tissue cultured seedling is taken out from culture bottle when transplanting, be divided into individual plant, rinse residual on root well with clear water
Culture fluid, accomplish not hinder root, soaks 20 min with 500 times of carbendazim solutions, the most carefully plants in nutrient bag, waters foot and determines root
Water, shades with sunshade net after transplanting, often waters and spray, and humidity is maintained at 75-85 %, between temperature 22-25 DEG C；Weekly
Execute carbamide and 0.2 %KH of 1 0.1 %2PO4, carry out the prevention and control of plant diseases, pest control simultaneously.
The liquid shallow quick-breeding method of a kind of Rhizoma Zingiberis Recens the most as claimed in claim 1, it is characterised in that step 3) initial culture
Condition of culture is: cultivation temperature 25-30 DEG C, and light application time is to irradiate 12-14 h every day, and intensity of illumination is 1200-1500 Lx,
Incubation time is 15-30 d.
The liquid shallow quick-breeding method of a kind of Rhizoma Zingiberis Recens the most as claimed in claim 1, it is characterised in that step 4) successive transfer culture
Condition of culture is: cultivation temperature 25-30 DEG C, and light application time is to irradiate 12-14 h every day, and intensity of illumination is 1200-1500 Lx.
The liquid shallow quick-breeding method of a kind of Rhizoma Zingiberis Recens the most as claimed in claim 1, it is characterised in that step 5) root culture
Condition of culture is: cultivation temperature 25-30 DEG C, and light application time is to irradiate 12-14 h every day, and intensity of illumination is 1200-1500 Lx.
The liquid shallow quick-breeding method of a kind of Rhizoma Zingiberis Recens the most as claimed in claim 1, it is characterised in that the antioxidation in step 4)
Agent be the sodium thiosulfate of 0.5 g/L, the thiourea of 0.1 g/L, the dithiothreitol, DTT of 0.5 g/L, the cysteine of 0.5 g/L,
One or more in the ascorbic acid of 0.1 g/L.
The liquid shallow quick-breeding method of a kind of Rhizoma Zingiberis Recens the most as claimed in claim 1, it is characterised in that the training of taking root in step 5)
Foster base also includes that organic additive, described organic additive are the murphy juice of 100 g/L, the corn juice of 100 g/L, 100 g/L
Semen Tritici aestivi flour, the caseinhydrolysate of 0.2 g/L, 0.2 g/L yeast extract in any one.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107006375A (en) * | 2017-06-02 | 2017-08-04 | 合肥华盖生物科技有限公司 | A kind of orchid fast propagating culture medium |
| CN108812315A (en) * | 2018-06-27 | 2018-11-16 | 芜湖东源新农村开发股份有限公司 | The method for tissue culture of ginger |
| CN110521594A (en) * | 2019-07-22 | 2019-12-03 | 佛山市粤山生物科技有限公司 | A kind of cultural method improving beautiful millettia root rooting rate |
| CN111771725A (en) * | 2020-07-31 | 2020-10-16 | 潍坊兴旺生物种业有限公司 | Optimized ginger virus-free seedling regeneration propagation method and development of industrialization process thereof |
| CN111837959A (en) * | 2020-08-05 | 2020-10-30 | 四川农业大学 | Micro ginger block induction method based on ginger test-tube plantlet and application |
| CN115769786A (en) * | 2022-11-30 | 2023-03-10 | 安徽农业大学 | Method for obtaining regeneration seedlings of drynaria fortunei by alternative tissue culture |
| CN117158323A (en) * | 2023-11-02 | 2023-12-05 | 潍坊理工学院 | Tissue culture breeding and rapid propagation seedling method based on ginger tubers |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102144565A (en) * | 2011-03-03 | 2011-08-10 | 中国药科大学 | Method for cultivating ginger-free viruses and cultivating virus-free seedlings by means of propagating and rooting synchronously |
-
2016
- 2016-07-13 CN CN201610546635.0A patent/CN106171985B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102144565A (en) * | 2011-03-03 | 2011-08-10 | 中国药科大学 | Method for cultivating ginger-free viruses and cultivating virus-free seedlings by means of propagating and rooting synchronously |
Non-Patent Citations (5)
| Title |
|---|
| 吴家丽: "白水生姜组培脱毒及离体快繁体系建立研究", 《长江蔬菜》 * |
| 王兆升: "鲜切生姜褐变机理及保险关键技术研究", 《中国博士学位论文全文数据库 工程科技辑Ⅰ辑》 * |
| 罗天宽: "生姜脱毒快繁体系研究与脱毒姜推广体系探讨", 《中国优秀博硕士学位论文全文数据库（硕士）农业科技辑》 * |
| 陈传红等: "多效唑在生姜组织培养中的应用研究", 《长江蔬菜》 * |
| 韦金洋: "生姜的组织培养与农杆菌介导遗传转化体系建立的研究", 《中国优秀博硕士学位论文全文数据库（硕士）农业科技辑》 * |
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| CN108812315A (en) * | 2018-06-27 | 2018-11-16 | 芜湖东源新农村开发股份有限公司 | The method for tissue culture of ginger |
| CN110521594A (en) * | 2019-07-22 | 2019-12-03 | 佛山市粤山生物科技有限公司 | A kind of cultural method improving beautiful millettia root rooting rate |
| CN111771725A (en) * | 2020-07-31 | 2020-10-16 | 潍坊兴旺生物种业有限公司 | Optimized ginger virus-free seedling regeneration propagation method and development of industrialization process thereof |
| CN111837959A (en) * | 2020-08-05 | 2020-10-30 | 四川农业大学 | Micro ginger block induction method based on ginger test-tube plantlet and application |
| CN115769786A (en) * | 2022-11-30 | 2023-03-10 | 安徽农业大学 | Method for obtaining regeneration seedlings of drynaria fortunei by alternative tissue culture |
| CN115769786B (en) * | 2022-11-30 | 2023-12-19 | 安徽农业大学 | Method for obtaining copper tomb-white Jiang Zaisheng seedlings in alternating tissue culture mode |
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| CN117158323B (en) * | 2023-11-02 | 2024-02-13 | 潍坊理工学院 | Tissue culture breeding and rapid propagation seedling method based on ginger tubers |
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