CN105941158A - Method for culturing strawberry virus-free seedlings through tissue culture technology - Google Patents
Method for culturing strawberry virus-free seedlings through tissue culture technology Download PDFInfo
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- 235000016623 Fragaria vesca Nutrition 0.000 title claims abstract description 28
- 235000011363 Fragaria x ananassa Nutrition 0.000 title claims abstract description 28
- 238000005516 engineering process Methods 0.000 title claims abstract description 28
- 238000012258 culturing Methods 0.000 title abstract description 3
- 240000009088 Fragaria x ananassa Species 0.000 title 1
- 239000001963 growth medium Substances 0.000 claims abstract description 57
- 241000220223 Fragaria Species 0.000 claims abstract description 29
- 230000001954 sterilising effect Effects 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 230000001939 inductive effect Effects 0.000 claims abstract description 10
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims abstract description 9
- 238000005286 illumination Methods 0.000 claims description 41
- 239000007943 implant Substances 0.000 claims description 30
- 239000002609 medium Substances 0.000 claims description 24
- 241000196324 Embryophyta Species 0.000 claims description 21
- 239000013589 supplement Substances 0.000 claims description 11
- 229960002523 mercuric chloride Drugs 0.000 claims description 8
- 239000000575 pesticide Substances 0.000 claims description 8
- 229920006395 saturated elastomer Polymers 0.000 claims description 6
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 claims 1
- 229910052753 mercury Inorganic materials 0.000 claims 1
- 238000004659 sterilization and disinfection Methods 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract 1
- 241000700605 Viruses Species 0.000 description 19
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- 206010020649 Hyperkeratosis Diseases 0.000 description 13
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- 238000009395 breeding Methods 0.000 description 10
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- 102000018997 Growth Hormone Human genes 0.000 description 6
- 108010051696 Growth Hormone Proteins 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 239000000122 growth hormone Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000001784 detoxification Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000004899 motility Effects 0.000 description 3
- 239000002574 poison Substances 0.000 description 3
- 231100000614 poison Toxicity 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193738 Bacillus anthracis Species 0.000 description 2
- 241000218632 Strawberry vein banding virus Species 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
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- 206010013911 Dysgeusia Diseases 0.000 description 1
- 241000221785 Erysiphales Species 0.000 description 1
- 244000307700 Fragaria vesca Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 244000170916 Paeonia officinalis Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 241000710036 Strawberry mild yellow edge virus Species 0.000 description 1
- 241000413494 Strawberry mottle virus Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
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- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
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Classifications
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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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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for culturing strawberry virus-free seedlings through a tissue culture technology. The method includes the steps of firstly, selecting and sterilizing an explant, wherein unopened buds of a strawberry plant are collected, after the buds are sterilized and processed with alcohol and corrosive sublimate, anther is taken out, and the explant is obtained; secondly, inducing cluster buds, wherein the explant obtained in the first step is inoculated in a culture medium M1 to be cultured in the dark for 8-10 days and then cultured in light for 80-90 days till the cluster buds are produced, and the bud height of the cluster buds is not smaller than 2 cm; thirdly, transferring the cluster buds for culture, wherein the cluster buds obtained in the second step are transferred into a culture medium M2 to be cultured for 15-25 days till the cluster buds grow to strawberry seedlings which are 3-5 cm high, and then the strawberry seedlings are transferred into a culture medium M3 to be subjected to rooting culture to obtain the strawberry virus-free seedlings. The applied culture media are simple in formula, the tissue culture process is simple, the virus-free rate reaches 100%, the reproduction coefficient is high, the transplanting survival rate is high, seedlings are neat, later unified transplanting and planting management is convenient, and industrial production can be achieved.
Description
Technical field
The present invention relates to biological tissue culture technical field, be specifically related to one and utilize tissue culture technology to cultivate Fructus Fragariae Ananssae detoxification
The method of Seedling.
Background technology
Fructus Fragariae Ananssae is Rosaceae Fragaria perennial root perennial evergreen herbaceous plant, belongs to polyploid, is a kind of by crawling
Stem carries out vegetative crop, and the method for tradition seed cultivation is to use the vegetative manner of stolon breeding,
Inefficient, it is unfavorable for the popularization of improved seeds, and pole easily infected virus.China has identified that clear and definite Fructus Fragariae Ananssae is sick
Poison has four kinds, i.e. Strawberry mottle virus (SMOV), strawberry light yellow edge virus (SMYEV), Fructus Fragariae Ananssae wrinkle
Contracting virus (SCRV) and strawberry veinbanding virus (SVBV).It is the bottle in strawberry production that seedling generally infects virus
Neck problem, the Fructus Fragariae Ananssae of infection virus is more weak than the Fructus Fragariae Ananssae growing way of uninfecting virus, resistance is poor, yield is decreased obviously, really
Product quality deteriorates with commodity property.For virosis, there is presently no medicament can administer, and therefore, cultivates anosis
Poison female parent seedling, cultivates virus-free nursery stock, is the fundamental measure of preventing and treating Strawberry Virus.At present, virus-free female parent seedling
Mainly by strawberry detoxification tissue culture technology, i.e. sloughed by technology such as anther tissue cultivation, Micro-stem tip tissue cultures
Virus causing disease obtain nontoxic Fructus Fragariae Ananssae regeneration plant, make strawberry seedling recover kind original seed good strains of seeds, with reach high-quality,
The purpose of high yield.But the virus-free female parent seedling of Fructus Fragariae Ananssae is cultivated and is difficult to, the outer implant of tissue culture is selected defective, training
Condition during Yanging controls not enough etc., will result in that detoxification efficiency is poor, pollution, vitrification, breed coefficient low,
The problems such as transplanting survival rate is the highest, cause the biggest loss to producing.
Summary of the invention
In view of this, the application provides a kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling, used medium
Formula is simple, group training simple flow, and virus elimination rate reaches 100%, breeds coefficient height, and transplanting survival rate is high, seedling
Neatly, it is simple to transplanting that the later stage is unified and Cultivate administration, reduce cost of labor, industrialization production can be carried out.
For solving above technical problem, the technical scheme that the present invention provides is that one utilizes tissue culture technology cultivation Fructus Fragariae Ananssae to take off
The method of poison Seedling, said method comprising the steps of:
(1) outer implant is chosen and sterilizing: the unopened alabastrum of strawberrying plant, uses at ethanol and mercuric chloride sterilizing
After reason, strip off alabastrum, takes out flower pesticide, obtains outer implant；
(2) inducing clumping bud: outer for step (1) gained implant is inoculated in culture medium M1, first light culture 8
10 days, then illumination cultivation 80 90 days, until producing Multiple Buds, and the bud height of Multiple Buds is not less than 2cm；
Consisting of of described culture medium M1: MS minimal medium, supplements 0.5 3.0mg/L TDZ, 0.05 0.30mg/L
IAA；
(3) Multiple Buds switching is cultivated: be forwarded in culture medium M2 by step (2) gained Multiple Buds, cultivates 15
25 days, until Multiple Buds grows up to the Strawberry seedlings that 3 5cm are high, more described Strawberry seedlings is forwarded to culture medium
In M3, carry out root culture, obtain Fructus Fragariae Ananssae detoxic seedling；Described culture medium M2 is MS minimal medium, institute
Stating culture medium M3 is 1/2MS minimal medium.
Preferably, in step (1), the sterilising conditions of described alabastrum is: first with 75% ethanol sterilizing 18 25s,
Again with 0.1% mercuric chloride solution sterilizing 12 15min.
Being more highly preferred to, in step (1), the sterilising conditions of described flower pesticide is: first with 75% ethanol sterilizing 20s,
Again with 0.1% mercuric chloride solution sterilizing 13min.
Preferably, in step (2), described outer implant is inoculated in culture medium M1, first light culture 9 days, then light
According to cultivating 80 days.
Preferably, in step (2), consisting of of described culture medium M1: MS minimal medium, supplement 1.0
—2.0mg/L TDZ、0.1—0.2mg/L IAA。
Preferably, in step (2), described condition of culture is intensity of illumination 2000 3000LX, light irradiation time 16h/
My god, cultivation temperature 24 26 DEG C.
Preferably, in step (3), described Multiple Buds is forwarded in culture medium M2, cultivates 20 days.
Preferably, in step (3), described Multiple Buds is in culture medium M2 or described Strawberry seedlings is in culture medium
Condition of culture in M3 is: intensity of illumination 2000 3000LX, light irradiation time 16h/ days, cultivation temperature 24
26℃。
Preferably, in step (1), the kind of described strawberry be any one be selected from liquor-saturated chivalrous, snow younger sister, a chapter Ji,
The most beautiful.
In technical scheme, described TDZ is a plant growth regulators, has the strongest basic element of cell division
Activity, can promote regeneration and the breeding of plant sprout, and that breaks bud stops eye, promotes seed germination, promotes wound healing group
Knit growth, delay plant senescence etc., the effect of other phytohormone and biological active substances can be regulated and plant
The growth and development process of thing, can be used as plant tissue culture.
Described IAA is heteroauxing, is a kind of auxin, it is possible to the growth of regulation plant, can not only promote
Enter growth, and there is the effect of Developing restraint and Apparatuses formation.On a cellular level, cambial cell can be stimulated
Division, the cell elongation of stimulation branch, suppression root cell growth, promotion xylem, phloem cell break up, promote
Cutting root of hair, the morphogenesis of regulation callus, therefore can be used as plant tissue culture.
Described MS minimal medium has higher inorganic salt concentration, it is possible to ensure the mineral nitrogen battalion needed for tissue growth
Supporting, moreover it is possible to accelerate the growth of callus, be more stable ionic equilibrium solution, its nitrate content is high, its
The quantity of nutrient and ratio are suitable, can meet nutrition and the physiological need of plant cell, thus the scope of application is relatively wider,
Most plants tissue-culturing quick-propagation uses it as the minimal medium of culture medium.
Described 1/2MS minimal medium is that in MS minimal medium, a great number of elements halves, remaining constant formation
Culture medium.
Technical scheme provides a kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling, including outer implant
Choose and sterilizing, inducing clumping bud, the step of Multiple Buds switching cultivation, use flower pesticide to organize as outer implant
Cultivate, flower pesticide is inoculated in culture medium M1 when cultivating, first carries out light culture, then carry out illumination cultivation, light
According to cultivating to about 10 days, start that callus occurs, about illumination cultivation to 40 day, callus length to 1cm
Left and right, about illumination cultivation to 60 day, callus length is to diameter 2 3cm size, and has budlet point to sprout,
Illumination cultivation was to about 80 days, and budlet point grows blade, bud height about 2cm, was forwarded to the bud grown cultivate
In base M2, until bud grows up to the Strawberry seedlings that 3 5cm are high, then it is forwarded to culture medium M3 and carries out root culture,
Obtain strawberry detoxification tissue culture Seedling completely.
Snow younger sister's kind described herein, really shape is good, color is beautiful, sugariness is high, manageability, with plantation tradition Fructus Fragariae Ananssae
Comparing, thin leaf amount is little, without flower thinning, vegetables and fruits, can save artificial 20%；Disease resistance is strong, malformed fruit rate is low, can
Volume increase 20%.
Described chapter Ji's kind, fruit is neatly in conico-acuminate, and fruit is healthy and strong, light bright in colour, good smell；
Sarcocarp pale red, delicate succulence, dense sweet taste, enjoy endless aftertastes, the superfine product in Japan is described as Fructus Fragariae Ananssae；Bear fruit
By force, ripe morning, the most anti-anthrax.
Described liquor-saturated chivalrous kind, plant strain growth gesture is strong, and stolon generating capacity is few, and fruit is peony, and really shape is good；
Yielding ability is good, and fruit is conico-acuminate, and really shape is big, without ditch, without malformed fruit, fruit ruby, good luster, sarcocarp
White, sweet crisp and refreshing mouth；High botrytis resistant, powdery mildew and anthrax.
The most beautiful described kind, shallow dormancy, precocity, plant type are compacter；Early yield is high, and commodity is good, quality
Close with " red cheek " kind；Sense anthrax, mildew-resistance.
Above-mentioned strawberry cultivars all can use the method described in technical scheme to carry out the cultivation of Fructus Fragariae Ananssae detoxic seedling.
Compared to existing tissue culture technology, technical scheme used medium formula is simple, group training simple flow,
Virus elimination rate reaches 100%, breeds coefficient height, and transplanting survival rate is high, and seedling is neat, it is simple to the transplanting that the later stage is unified
And Cultivate administration, reduce cost of labor, industrialization production can be carried out.
Detailed description of the invention
In order to make those skilled in the art be more fully understood that technical scheme, below in conjunction with specific embodiment
The present invention is described in further detail.
Embodiment 1
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling described in the present embodiment, comprises the following steps:
(1) outer implant is chosen and sterilizing: gather the unopened alabastrum of the strawberry that kind is snow younger sister, alabastrum
Size is at 0.4 0.6mm, first with 75% ethanol sterilizing 20s, then with 0.1% mercuric chloride solution sterilizing 13min, takes out
Flower pesticide, obtains outer implant；
(2) inducing clumping bud: outer for step (1) gained implant is inoculated in culture medium M1, first light culture 9
My god, then illumination cultivation 80 days, until producing Multiple Buds, and the bud height of Multiple Buds is not less than 2cm, and condition of culture is
Cultivate illumination 2000 3000LX, cultivation temperature 24 26 DEG C, light irradiation time 16h/ days；Described culture medium M1
Consist of: MS minimal medium, supplement 1.0mg/L TDZ, 0.2mg/L IAA；
(3) Multiple Buds switching is cultivated: be forwarded in culture medium M2 by step (2) gained Multiple Buds, cultivates 20
My god, until Multiple Buds grows up to the Strawberry seedlings that 3 5cm are high, more described Strawberry seedlings is forwarded to culture medium M3
In, carry out root culture, obtain Fructus Fragariae Ananssae detoxic seedling；Described culture medium M2 is MS minimal medium, cultivates bar
Part is for cultivating illumination 2000 3000LX, cultivation temperature 24 26 DEG C, light irradiation time 16h/ days, described culture medium
M3 is 1/2MS minimal medium, and condition of culture is cultivation illumination 2000 3000LX, cultivation temperature 24 26 DEG C,
Light irradiation time 16h/ days.
Described in the present embodiment method cultivate Fructus Fragariae Ananssae detoxic seedling, its callus generate time be light culture after 9 days again
Illumination cultivation 10 days, illumination cultivation occurs bud point for about 60 days, cultivates about 80 days bud point length to about 2cm,
Wound healing rate is 71.1%, and differentiation rate is 52.3%, and virus elimination rate is 100%, and breeding coefficient is 7, and plant becomes
Motility rate is 92%.
Embodiment 2
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling described in the present embodiment, with side described in embodiment 1
The difference of method is:
Step (2) inducing clumping bud is: outer for step (1) gained implant be inoculated in culture medium M1, the darkest
Cultivate 9 days, then illumination cultivation 80 days, until producing Multiple Buds, and the bud height of Multiple Buds is not less than 2cm, cultivates
Condition for cultivate illumination 2000 3000LX, cultivation temperature 24 26 DEG C, light irradiation time 16h/ days；Described training
Foster base M1 consists of: MS minimal medium, supplements 1.5mg/L TDZ, 0.1mg/L IAA.
Method described in the present embodiment cultivates Fructus Fragariae Ananssae detoxic seedling, and outer implant is inoculated in when cultivating in culture medium M1,
The time that callus generates is that after 9 days illumination cultivation 10 days again for light culture, and illumination cultivation bud occurs in about 60 days
Point, cultivates about 80 days bud point length to about 2cm, and wound healing rate is 71.1%, and differentiation rate is 52.3%,
Virus elimination rate is 100%, and breeding coefficient is 7, and survival rate of plant is 92%.
Embodiment 3
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling described in the present embodiment, with side described in embodiment 1
The difference of method is:
Step (2) inducing clumping bud is: outer for step (1) gained implant be inoculated in culture medium M1, the darkest
Cultivate 9 days, then illumination cultivation 80 days, until producing Multiple Buds, and the bud height of Multiple Buds is not less than 2cm, cultivates
Condition for cultivate illumination 2000 3000LX, cultivation temperature 24 26 DEG C, light irradiation time 16h/ days；Described training
Foster base M1 consists of: MS minimal medium, supplements 1.0mg/L TDZ, 0.2mg/L IAA.
Method described in the present embodiment cultivates Fructus Fragariae Ananssae detoxic seedling, and outer implant is inoculated in when cultivating in culture medium M1,
The time that callus generates is that after 9 days illumination cultivation 10 days again for light culture, and illumination cultivation bud occurs in about 60 days
Point, cultivates about 80 days bud point length to about 2cm, and wound healing rate is 79.2%, and differentiation rate is 55.6%,
Virus elimination rate is 100%, and breeding coefficient is 8, and survival rate of plant is 95.6%.
Embodiment 4
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling described in the present embodiment, with side described in embodiment 1
The difference of method is:
Step (2) inducing clumping bud is: outer for step (1) gained implant be inoculated in culture medium M1, the darkest
Cultivate 9 days, then illumination cultivation 80 days, until producing Multiple Buds, and the bud height of Multiple Buds is not less than 2cm, cultivates
Condition for cultivate illumination 2000 3000LX, cultivation temperature 24 26 DEG C, light irradiation time 16h/ days；Described training
Foster base M1 consists of: MS minimal medium, supplements 1.5mg/L TDZ, 0.1mg/L IAA.
Method described in the present embodiment cultivates Fructus Fragariae Ananssae detoxic seedling, and outer implant is inoculated in when cultivating in culture medium M1,
The time that callus generates is that after 9 days illumination cultivation 10 days again for light culture, and illumination cultivation bud occurs in about 60 days
Point, cultivates about 80 days bud point length to about 2cm, and wound healing rate is 79.2%, and differentiation rate is 55.6%,
Virus elimination rate is 100%, and breeding coefficient is 8, and survival rate of plant is 95.6%.
Embodiment 5
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling described in the present embodiment, with side described in embodiment 1
The difference of method is:
Step (2) inducing clumping bud is: outer for step (1) gained implant be inoculated in culture medium M1, the darkest
Cultivate 9 days, then illumination cultivation 80 days, until producing Multiple Buds, and the bud height of Multiple Buds is not less than 2cm, cultivates
Condition for cultivate illumination 2000 3000LX, cultivation temperature 24 26 DEG C, light irradiation time 16h/ days；Described training
Foster base M1 consists of: MS minimal medium, supplements 1.5mg/L TDZ, 0.2mg/L IAA.
Method described in the present embodiment cultivates Fructus Fragariae Ananssae detoxic seedling, and outer implant is inoculated in when cultivating in culture medium M1,
The time that callus generates is that after 9 days illumination cultivation 10 days again for light culture, and illumination cultivation bud occurs in about 60 days
Point, cultivates about 80 days bud point length to about 2cm, and wound healing rate is 82.2%, and differentiation rate is 59.8%,
Virus elimination rate is 100%, and breeding coefficient is 7, and survival rate of plant is 96.7%.
Embodiment 6
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling described in the present embodiment, with side described in embodiment 1
The difference of method is:
Step (2) inducing clumping bud is: outer for step (1) gained implant be inoculated in culture medium M1, the darkest
Cultivate 9 days, then illumination cultivation 80 days, until producing Multiple Buds, and the bud height of Multiple Buds is not less than 2cm, cultivates
Condition for cultivate illumination 2000 3000LX, cultivation temperature 24 26 DEG C, light irradiation time 16h/ days；Described training
Foster base M1 consists of: MS minimal medium, supplements 0.5mg/L TDZ, 0.10mg/L IAA.
Method described in the present embodiment cultivates Fructus Fragariae Ananssae detoxic seedling, and outer implant is inoculated in when cultivating in culture medium M1,
The time that callus generates is that after 9 days illumination cultivation 10 days again for light culture, and illumination cultivation bud occurs in about 60 days
Point, cultivates about 80 days bud point length to about 2cm, and wound healing rate is 83.6%, and differentiation rate is 60.5%,
Virus elimination rate is 100%, and breeding coefficient is 7, and survival rate of plant is 96.8%.
Embodiment 7
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling described in the present embodiment, with side described in embodiment 1
The difference of method is:
Step (1) outer implant is chosen and sterilizing is: gather the unopened alabastrum that kind is liquor-saturated chivalrous strawberry,
The size of alabastrum is at 0.4 0.6mm, with 75% ethanol sterilizing 18s, then with 0.1% mercuric chloride solution sterilizing 12min,
Take out flower pesticide, obtain outer implant.
Method described in the present embodiment cultivates Fructus Fragariae Ananssae detoxic seedling, and the time that callus generates is light culture light again after 9 days
According to cultivating 10 days, illumination cultivation occurs bud point for about 60 days, cultivates about 80 days bud point length to about 2cm,
Wound healing rate is 88.9%, and differentiation rate is 61.1%, and virus elimination rate is 100%, and breeding coefficient is 8, and plant becomes
Motility rate is 100%.
Embodiment 8
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling described in the present embodiment, with side described in embodiment 1
The difference of method is:
Step (1) outer implant is chosen and sterilizing is: gather the unopened alabastrum that kind is the most beautiful strawberry,
The size of alabastrum is at 0.4 0.6mm, first with 75% ethanol sterilizing 25s, then with 0.1% mercuric chloride solution sterilizing 15min,
Take out flower pesticide, obtain outer implant.
Method described in the present embodiment cultivates Fructus Fragariae Ananssae detoxic seedling, and the time that callus generates is light culture light again after 9 days
According to cultivating 10 days, illumination cultivation occurs bud point for about 60 days, cultivates about 80 days bud point length to about 2cm,
Wound healing rate is 84.5%, and differentiation rate is 60.2%, and virus elimination rate is 100%, and breeding coefficient is 7, and plant becomes
Motility rate is 97.2%.
Embodiment 9
Growth hormone composition and the concentration impact on cultivating in culture medium M1
The outer implant taking the growing state of different strawberry cultivars (snow younger sister, chivalrous, a chapter Ji the most beautiful, liquor-saturated) consistent is some,
It is inoculated into respectively in culture medium M1, first light culture 9 days, then illumination cultivation 80 days, until producing Multiple Buds, and
The bud height of Multiple Buds is not less than 2cm, and the condition of cultivation is for cultivating illumination 2000 3000LX, cultivation temperature 24
26 DEG C, light irradiation time 16h/ days；Described culture medium M1 uses MS minimal medium, supplements certain life
Long hormone is formed.Under conditions of described MS minimal medium is identical, composition and concentration according to growth hormone enter
Row culture medium is numbered, and respectively A, B, C, D, E, F, G, concrete numbering situation is shown in Table 1, observes and remember
Recording each strawberry cultivars cultivation situation in each culture medium China and foreign countries implant, cultivation results is shown in Table 25.
The hormonal components of each culture medium and concentration in table 1 embodiment 9
| Culture medium is numbered | TDZ(mg/L) | IAA(mg/L) |
| A | -- | 0.15 |
| B | 1.5 | -- |
| C | 0.5 | 0.05 |
| D | 1.0 | 0.1 |
| E | 1.5 | 0.15 |
| F | 2.0 | 0.2 |
| G | 3.0 | 0.3 |
Table 2 avenges younger sister's kind growth hormone composition and concentration in each culture medium affects result to cultivate
| A | B | C | D | E | F | G | |
| Inoculation number () | 18 | 18 | 18 | 18 | 18 | 18 | 18 |
| Wound healing number (individual) | 3 | 4 | 8 | 11 | 14 | 12 | 9 |
| Wound healing rate (%) | 16.7 | 22.2 | 44.4 | 61.1 | 77.8 | 66.7 | 50.0 |
| Multiple Buds number (individual) | 0 | 0 | 1 | 5 | 11 | 4 | 3 |
| Differentiation rate (%) | 0.0 | 0.0 | 5.6 | 27.8 | 61.1 | 22.2 | 16.7 |
| Death toll () | 15 | 14 | 10 | 7 | 4 | 6 | 9 |
| Mortality rate (%) | 83.3 | 77.8 | 55.6 | 38.9 | 22.2 | 33.3 | 50.0 |
The most beautiful kind of table 3 growth hormone composition and concentration in each culture medium affect result to cultivate
The liquor-saturated chivalrous kind of table 4 growth hormone composition and concentration in each culture medium affect result to cultivate
| A | B | C | D | E | F | G | |
| Inoculation number () | 18 | 18 | 18 | 18 | 18 | 18 | 18 |
| Wound healing number (individual) | 1 | 2 | 4 | 10 | 16 | 11 | 6 |
| Wound healing rate (%) | 5.6 | 11.1 | 22.2 | 55.6 | 88.9 | 61.1 | 33.3 |
| Multiple Buds number (individual) | 0 | 0 | 1 | 5 | 11 | 4 | 0 |
| Differentiation rate (%) | 0.0 | 0.0 | 5.6 | 27.8 | 61.1 | 22.2 | 0.0 |
| Death toll () | 17 | 16 | 14 | 8 | 2 | 7 | 12 |
| Mortality rate (%) | 94.4 | 88.9 | 77.8 | 44.4 | 11.1 | 38.9 | 66.7 |
A table 5 chapter Ji's kind growth hormone composition and concentration in each culture medium affect result to cultivate
As can be seen from the above data, TDZ and IAA uses more preferable than alone one of which simultaneously, its wound healing rate,
Growing thickly and sprout differentiation rate and survival rate of plant is intended to higher, wherein TDZ is at 1.0 2.0mg/L, and IAA is 0.1
During 0.2mg/L, particularly TDZ its indices when 1.5mg/L, IAA are at 0.15mg/L is all preferable, for this
The preferred version of application.
Below it is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred implementation is not construed as
Limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For this skill
For the those of ordinary skill in art field, without departing from the spirit and scope of the present invention, it is also possible to make some changing
Entering and retouch, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (9)
1. one kind utilizes the method that tissue culture technology cultivates Fructus Fragariae Ananssae detoxic seedling, it is characterised in that: described method includes following
Step:
(1) outer implant is chosen and sterilizing: the unopened alabastrum of strawberrying plant, by ethanol and mercuric chloride sterilization treatment
After, take out flower pesticide, obtain outer implant；
(2) inducing clumping bud: outer for step (1) gained implant is inoculated in culture medium M1, first light culture 8
10 days, then illumination cultivation 80 90 days, until producing Multiple Buds, and the bud height of Multiple Buds is not less than 2cm；
Consisting of of described culture medium M1: MS minimal medium, supplements 0.5 3.0mg/L TDZ, 0.05 0.30mg/L
IAA；
(3) Multiple Buds switching is cultivated: be forwarded in culture medium M2 by step (2) gained Multiple Buds, cultivates 15
25 days, until Multiple Buds grows up to the Strawberry seedlings that 3 5cm are high, more described Strawberry seedlings is forwarded to culture medium
In M3, carry out root culture, obtain Fructus Fragariae Ananssae detoxic seedling；Described culture medium M2 is MS minimal medium, institute
Stating culture medium M3 is 1/2MS minimal medium.
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling the most according to claim 1, its feature exists
In: in step (1), the sterilising conditions of described alabastrum is: first with 75% ethanol sterilizing 18 25s, then with 0.1%
Mercuric chloride solution sterilizing 12 15min.
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling the most according to claim 2, its feature exists
In: in step (1), the sterilising conditions of described alabastrum is: first with 75% ethanol sterilizing 20s, then with 0.1% liter
Mercury solution sterilizing 13min.
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling the most according to claim 1, its feature exists
In: in step (2), described outer implant is inoculated in culture medium M1, first light culture 9 days, then illumination cultivation
80 days.
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling the most according to claim 1, its feature exists
In: in step (2), consisting of of described culture medium M1: MS minimal medium, supplement 1.0 2.0mg/L
TDZ、0.1—0.2mg/L IAA。
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling the most according to claim 1, its feature exists
In: in step (2), described condition of culture is intensity of illumination 2000 3000LX, light irradiation time 16h/ days, training
Support temperature 24 26 DEG C.
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling the most according to claim 1, its feature exists
In: in step (3), described Multiple Buds is forwarded in culture medium M2, cultivates 20 days.
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling the most according to claim 1, its feature exists
In: in step (3), the training that described Multiple Buds is in culture medium M2 or described Strawberry seedlings is in culture medium M3
Foster condition is: intensity of illumination 2000 3000LX, light irradiation time 16h/ days, cultivation temperature 24 26 DEG C.
A kind of method utilizing tissue culture technology to cultivate Fructus Fragariae Ananssae detoxic seedling the most according to claim 1, its feature exists
In: in step (1), the kind of described strawberry is that any one is selected from liquor-saturated chivalrous, snow younger sister, a chapter Ji, the most beautiful.
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Non-Patent Citations (2)
| Title |
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| 任建宏: ""草莓"拉松7号"花药培养研究"", 《安徽农业科学》 * |
| 王敬东: "植物生长调节剂TDZ在草莓花药培养中的应用", 《安徽农业科学》 * |
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