CN109349103A - A kind of New Zealand spinach quick breeding method for tissue culture - Google Patents
A kind of New Zealand spinach quick breeding method for tissue culture Download PDFInfo
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- CN109349103A CN109349103A CN201811083287.3A CN201811083287A CN109349103A CN 109349103 A CN109349103 A CN 109349103A CN 201811083287 A CN201811083287 A CN 201811083287A CN 109349103 A CN109349103 A CN 109349103A
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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 a kind of New Zealand spinach quick breeding method for tissue culture.The present invention obtains aseptic seedling from seed sprouting and only needs 90d to cultivation seedling is obtained, greatly accelerate the reproduction speed of New Zealand spinach, pass through shoot proliferation, every month can be proliferated 10.33 times or so, therefore a large amount of tissue-cultured seedling can be obtained in a short time, tissue-cultured seedling is after transplanting, and high survival rate is up to 86%, so as to enable this traditional Chinese herbal medicine of New Zealand spinach and novel vegetables all preferably to develop, further to be laid a good foundation from now on using the species.Method of the invention is easy to operate, easy to implement, and culture medium used and reagent easily configure, at low cost, easily a wide range of to promote.
Description
Technical field:
The invention belongs to field of plant reproduction, and in particular to a kind of New Zealand spinach quick breeding method for tissue culture.
Background technique:
New Zealand spinach (Tetragonia tetragonioides) is Aizoaceae (Aizoaceae) New Zealand spinach category (Tetragonia)
Meat draft is mainly distributed on the ground such as Jiangsu, Zhejiang, Fujian, Guangdong, Yunnan at present.Certain ground is also presented in its application method
Domain property, in Yunnan mainly as a kind of traditional herbal medicines, clearing heat and detoxicating, wind-dispelling detumescence, in In Guangdong Province then mostly as a kind of wild
Vegetables, integration of drinking and medicinal herbs, treatment enteritis, septicemia etc..Modern research shows that New Zealand spinach has good anticancer, anti-inflammatory, immune tune
Section, reducing blood lipid and other effects.
It is cultivated on solid medium using plant explant, obtains callus or intact plant, as plant tissue
Culture, also referred to as in vitro culture or explant culture.As different types of plant condition of culture required in vitro culture and
There is very big difference in method etc., according to specific plant, adjust type of culture medium, culture illumination and temperature, different trainings
Stage culture medium growth regulator proportion is supported, the formation of evoking adventive bud, establishes effective bud brood body on this basis respectively
System and plant regeneration system.Carry out the protection of plant with plant tissue culture technique at present and breeds on many plant species
It succeeds.
But up to the present, the research in terms of the tissue cultures of New Zealand spinach, country's report is seldom, and this research and forefathers
Research have many differences.
Summary of the invention:
It is an object of the invention to overcome the deficiencies of existing technologies, provide a kind of can obtain the cultivation of a large amount of New Zealand spinach in a short time
The New Zealand spinach quick breeding method for tissue culture of seedling.
New Zealand spinach quick breeding method for tissue culture of the invention, comprising the following steps:
The acquisition of S1, aseptic seedling: New Zealand spinach (Tetragonia tetragonioides) seed is carried out disinfection processing, will
New Zealand spinach seed after disinfection treatment is inoculated in germination medium, and condition of culture is 22~28 DEG C, and intensity of illumination is 50~60 μ
mol m-2s-1, 12~13h/d of illumination, until growing aseptic seedling, the germination medium are as follows: every liter fast containing 6- benzyl amino
1.0~3.0mg of purine, remaining composition are MS culture medium, pH 5.9；
The induction of S2, adventitious bud: using the stem section of aseptic seedling as explant, explant is carried out disinfection processing, will be sterilized
Treated, and explant is inoculated in induced medium cultivates, and condition of culture is 22~28 DEG C, and intensity of illumination is 50~60 μm of ol
m-2s-1, 12~13h/d of illumination, until inducing adventitious bud, the induced medium are as follows: and every liter contains N- phenyl-N ' -1,
2,3- thiadiazoles 0.5~1.0mg of -5- urea or 6-benzyl aminopurine 1.0mg, α-naphthylacetic acid 0.1mg or indole -3-butyric acid 0.1mg,
Remaining composition is 1/2MS culture medium, pH 5.9；
Adventitious bud: being transferred to the proliferation that adventitious bud is carried out in proliferated culture medium by S3, shoot proliferation, and condition of culture is 22~28
DEG C, intensity of illumination is 50~60 μm of ol m-2s-1, 12~13h/d of illumination, the adventitious bud that shoot proliferation obtains can be used for training of taking root
Support or continue shoot proliferation, the proliferated culture medium are as follows: every liter contains 0.5~2.0mg of 6-benzyl aminopurine and α-naphthalene second
0.1~0.3mg of acid, remaining composition are 1/2MS culture medium, pH 5.9；
S4, culture of rootage: the adventitious bud that shoot proliferation is obtained is transferred in root media, and condition of culture is 22~28
DEG C, intensity of illumination is 50~60 μm of ol m-2s-1, illumination 12-13h/d makes adventitious bud rooting, grows up to tissue-cultured seedling, and described takes root
Culture medium are as follows: every liter contains 1.0~2.0mg of indole -3-butyric acid or α-naphthylacetic acid 0.5mg, remaining composition is 1/2MS culture medium,
PH is 5.9；
S5, tissue culture transplantation of seedlings: tissue-cultured seedling is transplanted in cultivation matrix, be placed in it is wet, shade in the environment of cultivate, pour
Water, fertilising obtain cultivation seedling to meet tissue-cultured seedling growth required moisture and nutritional need after routine culture.
Described is transplanted to tissue-cultured seedling in cultivation matrix, be placed in it is wet, shade in the environment of cultivate, water, fertilising with
Moisture and nutritional need needed for meeting tissue-cultured seedling growth obtain cultivation seedling specifically: transplant tissue-cultured seedling after routine culture
Into cultivation matrix, it is placed in that relative humidity is 50~70%, light transmittance is cultivated in the environment of being 40~50%, 20~25 DEG C, the phase
Between it is per week application 1 ‰ compound fertilizer it is primary, obtain cultivation seedling；The mass ratio of N:P:K is 1:1:1 in the compound fertilizer.
It by the processing that carries out disinfection of New Zealand spinach (Tetragonia tetragonioides) seed is by New Zealand spinach it is preferred that described
(Tetragonia tetragonioides) seed is cleaned with tap water, the alcohol water blend table for being first 75% with volume fraction
Disinfection 10s, then aseptic water washing 2 times are cleaned in face, then impregnate 8min in the mercuric chloride aqueous solution that mass fraction is 0.1%, so
It uses aseptic water washing 3 times afterwards.
It is preferred that described using the stem section of aseptic seedling as explant, explant is carried out disinfection processing, after disinfection treatment
Explant to be inoculated in induced medium culture be using the stem section of aseptic seedling as explant, first with volume fraction for 75% wine
Disinfection 10s is cleaned on smart aqueous solution surface, and sterile water elution 2 times is later to impregnate in 0.1% mercuric chloride aqueous solution in mass fraction
8min, and with sterile water washing 3 times, stem section is finally cut into 1cm size, is inoculated in induced medium and cultivates.
The cultivation matrix is preferably that peat soil, vermiculite and river sand are the mixed mixed base in 1:2:1~2 by volume
Matter；Or preferably peat soil and vermiculite are the mixed-matrix that 1:2 is mixed by volume；Or preferably peat soil and river sand is pressed
The mixed-matrix of volume ratio 1:2 mixing.
MS culture medium in the present invention is international culture medium, ingredient and preparation method can referring to Tan Wencheng, wear
Plan is just edited, " ornamental plant tissue culture technique ", Beijing: China Forestry Publishing House, and 1991.1/2MS culture medium is to train MS
The a great number of elements and microelement supported in base halve, other components unchangeds and the culture medium that is formed.
Since New Zealand spinach is meat draft, and directly, the disinfection of acquisition stem section explant is more difficult.Therefore of the invention by New Zealand spinach
The aseptic seedling stem sections induced after seed disinfection are used for evoking adventive bud as explant, and effect is very ideal.By sterilization
New Zealand spinach seed be inoculated in germination medium, general 30d is obtained with complete aseptic seedling plant.By the stem section with site
It is inoculated in induced medium, general 20d can induce adventitious bud, switch to shoot proliferation, and general 20d can subculture one
It is secondary.Adventitious bud is transferred in root media, 20d can take root for forming tissue-cultured seedling, and tissue culture transplantation of seedlings is into matrix general one
It can grow within a month to 5~6cm high, obtain cultivation seedling.
Due to the tissue-cultured seedling very little of New Zealand spinach, it is usually no more than 3cm, dehydration death is easy to after being transplanted to outdoor.Therefore
By tissue culture transplantation of seedlings to mixed-matrix, (mixed-matrix is that peat soil, perlite and river sand are 1:2:1~2 by volume to the present invention
Mixed mixed-matrix；It or is by volume the mixed-matrix of 1:2 mixing for peat soil and perlite；Or for perlite and
River sand by volume 1:2 mixing mixed-matrix) in, be placed in it is wet, cover in the environment of cultivate, water, apply fertilizer to meet group
Moisture and nutritional need needed for training seedling growth, guarantee that tissue-cultured seedling has higher survival rate.
Culture medium in the present invention, other than germination medium is MS culture medium, remaining is 1/2MS culture medium, this measure
The vitrification phenomenon in New Zealand spinach tissue culture procedures can be largely reduced.
Compared with prior art, the invention has the following advantages that
The present invention obtains aseptic seedling from seed sprouting and only needs 90d to cultivation seedling is obtained, and greatly accelerates the breeding of New Zealand spinach
Speed can be proliferated 10.33 times or so every month, therefore can obtain a large amount of tissue culture in a short time by shoot proliferation
Seedling, tissue-cultured seedling is after transplanting, and high survival rate is up to 86%, so as to make this traditional Chinese herbal medicine of New Zealand spinach and novel vegetables
All can preferably it develop, further to be laid a good foundation from now on using the species.
The method of the present invention is easy to operate, easy to implement, and culture medium used and reagent easily configure, at low cost, easily a wide range of
It promotes.
Detailed description of the invention:
Fig. 1 is the tissue cultures of New Zealand spinach；A: the culture of New Zealand spinach aseptic seedling；B: New Zealand spinach stem section culture 20 days or so, formation
Bud clump；C: stem section is proliferated on proliferated culture medium；D: inducing on 3/4MS culture medium, the bud clump of partial vitrification；E: New Zealand spinach stem
Section cultivates 20 days or so the bud clumps formed on the proliferated culture medium of pH5.9；F: sprout cultivates 20 days left sides on root media
The right side, root system are formed；G: the root system of sprout in root media；H: New Zealand spinach tissue culture transplantation of seedlings is into mixed-matrix.
Specific embodiment:
It the following examples are further illustrations of the invention, is not limitation of the present invention.
Embodiment 1:
The acquisition of S1, aseptic seedling: the New Zealand spinach (Tetragonia acquired from South China Botanical Garden Chinese Academy of Sciences
Tetragonioides) plant seed cleans New Zealand spinach seed with tap water, the alcohol water blend for being first 75% with volume fraction
Disinfection 10s, then aseptic water washing 2 times are cleaned in surface, then impregnate 8min in the mercuric chloride aqueous solution that mass fraction is 0.1%,
Then it uses aseptic water washing 3 times, is finally inoculated in germination medium, condition of culture is 22~26 DEG C, and intensity of illumination is
55μmol m-2s-1, obtain aseptic seedling after illumination 12h/d, 30d (see Figure 1A)；Germination medium are as follows: contain in every liter of culture medium
6-benzyl aminopurine (BAP) 1.0mg, remaining composition are MS culture medium, pH 5.9.
The induction of S2, adventitious bud: using the stem section of aseptic seedling as explant, first with volume fraction for 75% alcohol water blend table
Disinfection 10s is cleaned in face, and sterile water elution 2 times is later to impregnate 8min in 0.1% mercuric chloride aqueous solution in mass fraction, and with nothing
Bacterium water washing 3 times, stem section is finally cut into 1cm size, is inoculated in induced medium and cultivates, condition of culture is 22~26 DEG C,
Intensity of illumination is 55 μm of ol m-2s-1, induce adventitious bud after cultivating 20d under conditions of illumination 12h/d (see Figure 1B)；Induction training
Support base are as follows: contain N- phenyl-N ' -1,2,3- thiadiazoles -5- urea (TDZ) 1.0mg and α-naphthylacetic acid (NAA) in every liter of culture medium
0.1mg, remaining composition are 1/2MS culture medium, pH 5.9.
Adventitious bud: being transferred to the proliferation that adventitious bud is carried out in proliferated culture medium by S3, shoot proliferation, and condition of culture is 22~26
DEG C, intensity of illumination is 55 μm of ol m-2s-1, illumination 12h/d, 20 days are a subculture cycle, and each subculture cycle is proliferated 9.76 times
(see Fig. 1 C-E)；Proliferated culture medium are as follows: contain 6-benzyl aminopurine (BAP) 0.5mg, α-naphthylacetic acid (NAA) in every liter of culture medium
0.1mg, remaining composition are 1/2MS culture medium, pH 5.9.
S4, culture of rootage: the adventitious bud that shoot proliferation is obtained is transferred in root media, and condition of culture is 22~26
DEG C, intensity of illumination is 55 μm of ol m-2s-1, illumination 12h/d makes it take root, tissue-cultured seedling grown up to after 20d (see Fig. 1 F-G)；It takes root training
Support base are as follows: contain indole -3-butyric acid (IBA) 1.0mg in every liter of culture medium, remaining composition is 1/2MS culture medium, pH 5.9.
S5, tissue culture transplantation of seedlings: it is what 1:2:1 was mixed that tissue-cultured seedling, which is transplanted to peat soil, perlite and river sand by volume,
(see Fig. 1 H) in mixed-matrix, it is placed in the environment of wet, shade (relative humidity is 50~70%, and light transmittance is 40~50%)
Culture, outdoor temperature are watered between 20~25 DEG C, the fertilising (compound fertilizer (N:P:K 1:1:1) one of application 1 ‰ per week
It is secondary), to meet tissue-cultured seedling growth required moisture and nutritional need, through routine culture, tissue-cultured seedling grows to 6cm high after one month,
Cultivation seedling is obtained, high survival rate is up to 85%.
Embodiment 2:
The acquisition of S1, aseptic seedling: the New Zealand spinach (Tetragonia acquired from South China Botanical Garden Chinese Academy of Sciences
Tetragonioides) plant seed cleans New Zealand spinach seed with tap water, the alcohol water blend for being first 75% with volume fraction
Disinfection 10s, then aseptic water washing 2 times are cleaned in surface, then impregnate 8min in the mercuric chloride aqueous solution that mass fraction is 0.1%,
Then it uses aseptic water washing 3 times, is finally inoculated in germination medium, condition of culture is 23~28 DEG C, and intensity of illumination is
50μmol m-2s-1, aseptic seedling is obtained after illumination 12h/d, 30d；Germination medium are as follows: contain 6- benzyl amino in every liter of culture medium
Purine (BAP) 2.0mg, remaining composition are MS culture medium, pH 5.9.
The induction of S2, adventitious bud: using the stem section of aseptic seedling as explant, first with volume fraction for 75% alcohol water blend table
Disinfection 10s is cleaned in face, and sterile water elution 2 times is later to impregnate 8min in 0.1% mercuric chloride aqueous solution in mass fraction, and with nothing
Bacterium water washing 3 times, stem section is finally cut into 1cm size, is inoculated in induced medium and cultivates, condition of culture is 23~28 DEG C,
Intensity of illumination is 50 μm of ol m-2s-1, adventitious bud is induced after cultivating 20d under conditions of illumination 12h/d；Induced medium are as follows: every
It rises and contains N- phenyl-N ' -1,2,3- thiadiazoles -5- urea (TDZ) 0.5mg and α-naphthylacetic acid (NAA) 0.1mg in culture medium, remaining
Composition is 1/2MS culture medium, pH 5.9.
Adventitious bud: being transferred to the proliferation that adventitious bud is carried out in proliferated culture medium by S3, shoot proliferation, and condition of culture is 23~28
DEG C, intensity of illumination is 50 μm of ol m-2s-1, illumination 12h/d, 22 days are a subculture cycle, each subculture cycle proliferation 10.33
Times；Proliferated culture medium are as follows: contain 6-benzyl aminopurine (BAP) 1.0mg, α-naphthylacetic acid (NAA) 0.2mg in every liter of culture medium,
Remaining composition is 1/2MS culture medium, pH 5.9.
S4, culture of rootage: the adventitious bud that shoot proliferation is obtained is transferred in root media, and condition of culture is 23~28
DEG C, intensity of illumination is 50 μm of ol m-2s-1, illumination 12h/d makes it take root, tissue-cultured seedling grown up to after 22d；Root media are as follows: every
It rises and contains indole -3-butyric acid (IBA) 2.0mg in culture medium, remaining composition is 1/2MS culture medium, pH 5.9.
S5, tissue culture transplantation of seedlings: tissue-cultured seedling is transplanted to perlite and river sand by volume it is the mixed-matrix of 1:2 mixing
In, be placed in it is wet, shade (relative humidity be 50~70%, light transmittance be 40~50%) in the environment of cultivate, outdoor temperature be situated between
In 20~25 DEG C, watered, fertilising (compound fertilizer (N:P:K 1:1:1) of application 1 ‰ per week is primary), to meet tissue-cultured seedling
Moisture and nutritional need needed for growth, through routine culture, tissue-cultured seedling grows to 5~6cm high after one month, obtains cultivation seedling, at
Motility rate is up to 86%.
Embodiment 3:
The acquisition of S1, aseptic seedling: the New Zealand spinach (Tetragonia acquired from South China Botanical Garden Chinese Academy of Sciences
Tetragonioides) plant seed cleans New Zealand spinach seed with tap water, the alcohol water blend for being first 75% with volume fraction
Disinfection 10s, then aseptic water washing 2 times are cleaned in surface, then impregnate 8min in the mercuric chloride aqueous solution that mass fraction is 0.1%,
Then it uses aseptic water washing 3 times, is finally inoculated in germination medium, condition of culture is 23~28 DEG C, and intensity of illumination is
60μmol m-2s-1, aseptic seedling is obtained after illumination 12h/d, 30d；Germination medium are as follows: contain 6- benzyl amino in every liter of culture medium
Purine (BAP) 3.0mg, remaining composition are MS culture medium, pH 5.9.
The induction of S2, adventitious bud: using the stem section of aseptic seedling as explant, first with volume fraction for 75% alcohol water blend table
Disinfection 10s is cleaned in face, and sterile water elution 2 times is later to impregnate 8min in 0.1% mercuric chloride aqueous solution in mass fraction, and with nothing
Bacterium water washing 3 times, stem section is finally cut into 1cm size, is inoculated in induced medium and cultivates, condition of culture is 23~28 DEG C,
Intensity of illumination is 60 μm of ol m-2s-1, adventitious bud is induced after cultivating 20d under conditions of illumination 12h/d；Induced medium are as follows: every
It rises and contains N- phenyl-N ' -1,2,3- thiadiazoles -5- urea (TDZ) 0.5mg and indole -3-butyric acid (IBA) 0.1mg in culture medium,
Remaining composition is 1/2MS culture medium, pH 5.9.
Adventitious bud: being transferred to the proliferation that adventitious bud is carried out in proliferated culture medium by S3, shoot proliferation, and condition of culture is 23~28
DEG C, intensity of illumination is 60 μm of ol m-2s-1, illumination 12h/d, 20 days are a subculture cycle, each subculture cycle proliferation 10.12
Times；Proliferated culture medium are as follows: contain 6-benzyl aminopurine (BAP) 2.0mg, α-naphthylacetic acid (NAA) 0.1mg in every liter of culture medium,
Remaining composition is 1/2MS culture medium, pH 5.9.
S4, culture of rootage: the adventitious bud that shoot proliferation is obtained is transferred in root media, and condition of culture is 23~28
DEG C, intensity of illumination is 60 μm of ol m-2s-1, illumination 12h/d makes it take root, tissue-cultured seedling grown up to after 20d；Root media are as follows: every
It rises and contains indole -3-butyric acid (IBA) 1.5mg in culture medium, remaining composition is 1/2MS culture medium, pH 5.9.
S5, tissue culture transplantation of seedlings: tissue-cultured seedling is transplanted to peat soil and perlite by volume it is the mixed-matrix of 1:2 mixing
In, be placed in it is wet, shade (relative humidity be 50~70%, light transmittance be 40~50%) in the environment of cultivate, outdoor temperature be situated between
In 20~25 DEG C, watered, fertilising (compound fertilizer (N:P:K 1:1:1) of application 1 ‰ per week is primary), to meet tissue-cultured seedling
Moisture and nutritional need needed for growth, through routine culture, tissue-cultured seedling grows to 5~6cm high after one month, obtains cultivation seedling, at
Motility rate is up to 80%.
Embodiment 4:
The acquisition of S1, aseptic seedling: the New Zealand spinach (Tetragonia acquired from South China Botanical Garden Chinese Academy of Sciences
Tetragonioides) plant seed cleans New Zealand spinach seed with tap water, the alcohol water blend for being first 75% with volume fraction
Disinfection 10s, then aseptic water washing 2 times are cleaned in surface, then impregnate 8min in the mercuric chloride aqueous solution that mass fraction is 0.1%,
Then it uses aseptic water washing 3 times, is finally inoculated in germination medium, condition of culture is 23~28 DEG C, and intensity of illumination is
50μmol m-2s-1, aseptic seedling is obtained after illumination 13h/d, 30d；Germination medium are as follows: contain 6- benzyl amino in every liter of culture medium
Purine (BAP) 2.0mg, remaining composition are MS culture medium, pH 5.9.
The induction of S2, adventitious bud: using the stem section of aseptic seedling as explant, first with volume fraction for 75% alcohol water blend table
Disinfection 10s is cleaned in face, and sterile water elution 2 times is later to impregnate 8min in 0.1% mercuric chloride aqueous solution in mass fraction, and with nothing
Bacterium water washing 3 times, stem section is finally cut into 1cm size, is inoculated in induced medium and cultivates, condition of culture is 23~28 DEG C,
Light intensity is 50 μm of ol m-2s-1, adventitious bud is induced after cultivating 20d under conditions of illumination 13h/d；Induced medium are as follows: every liter of training
It supports and contains 6-benzyl aminopurine (BAP) 1.0mg and indole -3-butyric acid (IBA) 0.1mg in base, remaining composition is 1/2MS culture
Base, pH 5.9.
Adventitious bud: being transferred to the proliferation that adventitious bud is carried out in proliferated culture medium by S3, shoot proliferation, and condition of culture is 23~28
DEG C, light intensity is 50 μm of ol m-2s-1, illumination 13h/d, 20 days are a subculture cycle, and each subculture cycle is proliferated 9.86 times；Increase
Grow culture medium are as follows: contain 6-benzyl aminopurine (BAP) 1.0mg, α-naphthylacetic acid (NAA) 0.3mg, remaining composition in every liter of culture medium
For 1/2MS culture medium, pH 5.9.
S4, culture of rootage: the adventitious bud that shoot proliferation is obtained is transferred in root media, and condition of culture is 23~28
DEG C, intensity of illumination is 50 μm of ol m-2s-1, illumination 13h/d makes it take root, tissue-cultured seedling grown up to after 20d；Root media are as follows: every
It rises and contains α-naphthylacetic acid (NAA) 0.5mg in culture medium, remaining composition is 1/2MS culture medium, pH 5.9.
S5, tissue culture transplantation of seedlings: it is what 1:2:2 was mixed that tissue-cultured seedling, which is transplanted to peat soil, river sand and perlite by volume,
In mixed-matrix, be placed in it is wet, shade (relative humidity be 50~70%, light transmittance be 40~50%) in the environment of cultivate, room
Outer temperature is watered between 20~25 DEG C, fertilising (compound fertilizer (N:P:K 1:1:1) of application 1 ‰ per week is primary), with full
Moisture and nutritional need needed for sufficient tissue-cultured seedling growth, through routine culture, tissue-cultured seedling grows to 5cm high after one month, is cultivated
Seedling, high survival rate is up to 82%.
Claims (4)
1. a kind of New Zealand spinach quick breeding method for tissue culture, which comprises the following steps:
The acquisition of S1, aseptic seedling: New Zealand spinach (Tetragonia tetragonioides) seed is carried out disinfection processing, will be sterilized
Treated, and New Zealand spinach seed is inoculated in germination medium, and condition of culture is 22~28 DEG C, and intensity of illumination is 50~60 μm of ol m- 2s-1, 12~13h/d of illumination, until grow aseptic seedling, the germination medium are as follows: every liter containing 6-benzyl aminopurine 1.0~
3.0mg, remaining composition are MS culture medium, pH 5.9；
The induction of S2, adventitious bud: using the stem section of aseptic seedling as explant, explant is carried out disinfection processing, will be disinfected
Explant afterwards, which is inoculated in induced medium, to be cultivated, and condition of culture is 22~28 DEG C, and intensity of illumination is 50~60 μm of ol m-2s-1, 12~13h/d of illumination, until induce adventitious bud, the induced medium are as follows: and every liter contains N- phenyl-N ' -1, and 2,3-
Thiadiazoles 0.5~1.0mg of -5- urea or 6-benzyl aminopurine 1.0mg, α-naphthylacetic acid 0.1mg or indole -3-butyric acid 0.1mg, remaining
Composition is 1/2MS culture medium, pH 5.9；
Adventitious bud: being transferred to the proliferation that adventitious bud is carried out in proliferated culture medium by S3, shoot proliferation, and condition of culture is 22~28 DEG C,
Intensity of illumination is 50~60 μm of ol m-2s-1, 12~13h/d of illumination, the adventitious bud that shoot proliferation obtains can be used for culture of rootage or
Person continues shoot proliferation, the proliferated culture medium are as follows: every liter contains 0.5~2.0mg of 6-benzyl aminopurine and α-naphthylacetic acid 0.1
~0.3mg, remaining composition are 1/2MS culture medium, pH 5.9；
S4, culture of rootage: the adventitious bud that shoot proliferation is obtained is transferred in root media, and condition of culture is 22~28 DEG C, light
It is 50~60 μm of ol m according to intensity-2s-1, illumination 12-13h/d makes adventitious bud rooting, grows up to tissue-cultured seedling, the culture of rootage
Base are as follows: every liter contains 1.0~2.0mg of indole -3-butyric acid or α-naphthylacetic acid 0.5mg, remaining composition is 1/2MS culture medium, and pH is
5.9；
S5, tissue culture transplantation of seedlings: tissue-cultured seedling is transplanted in cultivation matrix, be placed in it is wet, shade in the environment of cultivate, water, apply
Fertilizer obtains cultivation seedling to meet tissue-cultured seedling growth required moisture and nutritional need after routine culture.
2. New Zealand spinach quick breeding method for tissue culture according to claim 1, which is characterized in that described by New Zealand spinach
The processing that carries out disinfection of (Tetragonia tetragonioides) seed is by New Zealand spinach (Tetragonia
Tetragonioides) seed is cleaned with tap water, first cleans disinfection with the alcohol water blend surface that volume fraction is 75%
10s, then aseptic water washing 2 times, then 8min is impregnated in the mercuric chloride aqueous solution that mass fraction is 0.1%, then use sterile water
It rinses 3 times.
3. New Zealand spinach quick breeding method for tissue culture according to claim 1, which is characterized in that described by aseptic seedling
Explant is carried out disinfection processing, the explant after disinfection treatment is inoculated in induced medium and is trained as explant by stem section
Supporting is using the stem section of aseptic seedling as explant, is first that disinfection 10s is cleaned on 75% alcohol water blend surface with volume fraction, sterile
Water elution 2 times, be to impregnate 8min in 0.1% mercuric chloride aqueous solution, and with sterile water washing 3 times, finally will in mass fraction later
Stem section is cut into 1cm size, is inoculated in induced medium and cultivates.
4. described in any item New Zealand spinach quick breeding method for tissue culture according to claim 1~3, which is characterized in that described
Cultivation matrix is that peat soil, perlite and river sand are the mixed mixed-matrix in 1:2:1~2 by volume；Or for peat soil and
Perlite is the mixed-matrix of 1:2 mixing by volume；Or the mixed base for perlite and river sand 1:2 mixing by volume
Matter.
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| CN103583357A (en) * | 2013-10-08 | 2014-02-19 | 浙江省农业科学院 | Method for sterile seeding of lithops and establishing regeneration system |
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| CN103583357A (en) * | 2013-10-08 | 2014-02-19 | 浙江省农业科学院 | Method for sterile seeding of lithops and establishing regeneration system |
| CN106748117A (en) * | 2016-12-16 | 2017-05-31 | 重庆市江津区森德家庭农场 | A kind of Aizoaceae succulent plants material and preparation method |
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