CN105613301B - In skill Chunlan thoroughly tissue culture method - Google Patents

In skill Chunlan thoroughly tissue culture method Download PDF

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Publication number
CN105613301B
CN105613301B CN201610135692.XA CN201610135692A CN105613301B CN 105613301 B CN105613301 B CN 105613301B CN 201610135692 A CN201610135692 A CN 201610135692A CN 105613301 B CN105613301 B CN 105613301B
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culture
skill
chunlan
saturating
stock
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CN105613301A (en
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孙叶芳
郑琪
邢海
赵虎
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SHAOXING ACADEMY OF AGRICULTURAL SCIENCES
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SHAOXING ACADEMY OF AGRICULTURAL SCIENCES
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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
    • 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 belongs to plant tissue culture field, be related to it is a kind of in skill Chunlan thoroughly tissue culture method.Comprise the following steps：A, selection, the whole piece obtained in laboratory cultures all in flaxen thoroughly skill Chunlan root-like stock as group training material, B, Multiplying culture, with the saturating skill Chunlan root-like stock of culture medium centering containing MS, TDZ, 6 BA and coconut palm breast Multiplying culture is carried out in culturing room, C, differentiation culture, with the saturating skill Chunlan root-like stock of culture medium centering containing MS, 6 BA, IBA and coconut palm breast differentiation culture is carried out in culturing room, D, culture of rootage, culture of rootage is carried out with the saturating skill Chunlan root-like stock of the culture medium centering containing MS, 6 BA, IBA, NAA, AC and PVP in culturing room.Tissue culture of the present invention saturating skill Chunlan in obtaining, skill is to stabilization, and the cultivation and batch production of saturating skill Chunlan provide reliable method in.

Description

In skill Chunlan thoroughly tissue culture method
Technical field
The invention belongs to plant tissue culture technical field, be related to it is a kind of in skill Chunlan thoroughly tissue culture method.
Background technology
In thoroughly skill be orchid leaf skill one kind, in orchid leaf skill kind, it has more ornamental value than general orchid.In Thoroughly, refer to blade and white the wide line bar in insertion leaf occur, green white or greenish-yellow alternate thin white silk used in ancient China skill, the i.e. leaf in orchid occurs in floral leaf face Occurs length and inconsistent white or yellow color " entity " lines of width on face.
One plant of skill it is blue from flower seed to highest skill to boundary, at least about need the time by 5 years to 15 years or longer. And seed is incomplete under regular situation, it is extremely difficult to sprout, if continuing to use traditional division propagation method, breeding coefficient is again extremely low, causes Many treasure kind can not amount reproduction to meet the market demand.Chunlan root-like stock is the favourable stage of China blue shoot proliferation, It is also the key that quick breeding improves growth coefficient.Due to those the beautiful thin white silk used in ancient China skill characters occurred on orchid blade face, do not have Higher genetic stability, it is generally the case that tissue-cultured seedling is under the influence of by culture environment, and skill is to unstability.Grind Study carefully the blue tissue cultures of hybrid orchid and line skill it has been reported that but the tissue culture of the saturating skill Chunlan of centering and Ye Yilan skill to culture not yet Appear in the newspapers.
Chinese patent literature [application number：201010127788.4] a kind of orchid germchit propagating method is disclosed, its seedling Obtaining mainly includes seed sprouting, protocorm shoot proliferation and process of rooting culture, it is characterised in that the propagation method is in seedling The hardening of rooted seedling and the Mycorrhizal process with mycorrhizal fungi are also carried out after acquisition.Above-mentioned use Chunlan seed asepsis sprouting and bacterium The Mycorrhizal Cultivating techniques of mycorrhiza fungi symbiosis, can both breed improved seeds, and excellent blue strain is selected from seedling from seed, and can be fast Speed improves Chunlan and breeds speed, overcomes that pure tissue-cultured seedling is slow-growing, and the cycle is long, the shortcoming do not bloomed even, to the big face of Chunlan Product industrialization is expanded numerous with important value.But the program does not disclose related art scheme of the skill to culture.
The content of the invention
The purpose of the present invention is in view of the shortcomings of the prior art there is provided a kind of tissue-culturing rapid propagation and skill to skill spring thoroughly in stable Blue tissue culture method.
To reach above-mentioned purpose, present invention employs following technical proposal：The tissue culture method of saturating skill Chunlan in a kind of, including Following steps：
A, selection, the whole piece obtained in laboratory cultures is all for skill Chunlan root-like stock is used as tissue culture material thoroughly in flaxen Material,
B, Multiplying culture, with the saturating skill Chunlan root-like stock of culture medium centering containing MS, TDZ, 6-BA and coconut palm breast in culturing room Carry out Multiplying culture,
C, differentiation culture, with the saturating skill Chunlan root-like stock of culture medium centering containing MS, 6-BA, IBA and coconut palm breast in culturing room Differentiation culture is carried out,
D, culture of rootage, are existed with the saturating skill Chunlan root-like stock of the culture medium centering containing MS, 6-BA, IBA, NAA, AC and PVP Culturing room carries out culture of rootage.
In above-mentioned thoroughly in the tissue culture method of skill Chunlan, in stepb, described culture medium is 3/4MS+TDZ 0.5mg·L-1+6-BA 1.0mg·L-1+50mL·L-1Coconut palm breast, in step C, described culture medium is 3/4MS+6- BA1.0mg·L-1+IBA 1.0mg·L-1+50mL·L-1Coconut palm breast, in step D, described culture medium are 1/2MS+6- BA1.0mg·L-1+IBA 1.0mg·L-1+NAA 1.0mg·L-1+2g·L-1AC+5g·L-1PVP。
In above-mentioned thoroughly in the tissue culture method of skill Chunlan, described culture room temperature is 25 DEG C ± 2 DEG C, and relative humidity is 60%-80%, intensity of illumination is 1000-3000Lx, and light application time is 10-14h/d.
In above-mentioned thoroughly in the tissue culture method of skill Chunlan, described culture room temperature is 25 DEG C, and relative humidity is 70%, Intensity of illumination is 2000Lx, and light application time is 12h/d.
Compared with prior art, the advantage of the invention is that：Skill Chunlan thoroughly during this method tissue culture is obtained, skill is to steady Fixed, the cultivation and batch production of saturating skill Chunlan provide reliable method in.
Embodiment
Reagent used, unless otherwise specified, can be commercially available from routine biochemistry reagent shop in following embodiments.With Quantitative data in lower embodiment, is respectively provided with three repetition experiments, results averaged.
Embodiment 1
Saturating skill Chunlan root-like stock is material in being produced using after plum valve bud mutation, and it is all skill thoroughly in flaxen to choose whole piece Chunlan root-like stock is placed in the blake bottle for being placed with culture medium as group training material, and in carrying out Multiplying culture in culturing room, is cultivated Base is 3/4MS+TDZ 0.5mgL-1+6-BA 1.0mg·L-1+50mL·L-1Coconut palm breast, culture room temperature is 25 DEG C, relatively wet Spend for 70%, intensity of illumination is 2000Lx, and light application time is 12h/d, after Multiplying culture terminates, and saturating skill Chunlan root-like stock is put by Enter in the blake bottle equipped with culture medium, and in carrying out differentiation culture in culturing room, culture medium is 3/4MS+6-BA1.0mgL-1+ IBA 1.0mg·L-1+50mL·L-1Coconut palm breast, culture room temperature is 25 DEG C, and relative humidity is 70%, and intensity of illumination is 2000Lx, Light application time is 12h/d, and after differentiation culture terminates, saturating skill Chunlan root-like stock is put into the blake bottle equipped with culture medium and carried out by Culture of rootage, culture medium is 1/2MS+6-BA1.0mgL-1+IBA 1.0mg·L-1+NAA 1.0mg·L-1+2g·L-1AC+ 5g·L-1PVP, culture room temperature is 25 DEG C, and relative humidity is 70%, and intensity of illumination is 2000Lx, and light application time is 12h/d.It is raw Root culture terminates, that is, the tissue culture of saturating skill Chunlan in completing.
Embodiment 2
Saturating skill Chunlan root-like stock is material in being produced using after plum valve bud mutation, and it is all skill thoroughly in flaxen to choose whole piece Chunlan root-like stock is placed in the blake bottle for being placed with culture medium as group training material, and in carrying out Multiplying culture in culturing room, is cultivated Base is 3/4MS+TDZ 0.5mgL-1+6-BA 1.0mg·L-1+50mL·L-1Coconut palm breast, culture room temperature is 23 DEG C, relatively wet Spend for 60%, intensity of illumination is 1000Lx, and light application time is 10h/d, after Multiplying culture terminates, and saturating skill Chunlan root-like stock is put by Enter in the blake bottle equipped with culture medium, and in carrying out differentiation culture in culturing room, culture medium is 3/4MS+6-BA1.0mgL-1+ IBA 1.0mg·L-1+50mL·L-1Coconut palm breast, culture room temperature is 23 DEG C, and relative humidity is 60%, and intensity of illumination is 1000Lx, Light application time is 10h/d, and after differentiation culture terminates, saturating skill Chunlan root-like stock is put into the blake bottle equipped with culture medium and carried out by Culture of rootage, culture medium is 1/2MS+6-BA1.0mgL-1+IBA 1.0mg·L-1+NAA 1.0mg·L-1+2g·L-1AC+ 5g·L-1PVP, culture room temperature is 23 DEG C, and relative humidity is 60%, and intensity of illumination is 1000Lx, and light application time is 10h/d.It is raw Root culture terminates, that is, the tissue culture of saturating skill Chunlan in completing.
Embodiment 3
Saturating skill Chunlan root-like stock is material in being produced using after plum valve bud mutation, and it is all skill thoroughly in flaxen to choose whole piece Chunlan root-like stock is placed in the blake bottle for being placed with culture medium as group training material, and in carrying out Multiplying culture in culturing room, is cultivated Base is 3/4MS+TDZ 0.5mgL-1+6-BA 1.0mg·L-1+50mL·L-1Coconut palm breast, culture room temperature is 27 DEG C, relatively wet Spend for 80%, intensity of illumination is 3000Lx, and light application time is 14h/d, after Multiplying culture terminates, and saturating skill Chunlan root-like stock is put by Enter in the blake bottle equipped with culture medium, and in carrying out differentiation culture in culturing room, culture medium is 3/4MS+6-BA1.0mgL-1+ IBA 1.0mg·L-1+50mL·L-1Coconut palm breast, culture room temperature is 27 DEG C, and relative humidity is 80%, and intensity of illumination is 3000Lx, Light application time is 10h/d, and after differentiation culture terminates, saturating skill Chunlan root-like stock is put into the blake bottle equipped with culture medium and carried out by Culture of rootage, culture medium is 1/2MS+6-BA1.0mgL-1+IBA 1.0mg·L-1+NAA 1.0mg·L-1+2g·L-1AC+ 5g·L-1PVP, culture room temperature is 27 DEG C, and relative humidity is 80%, and intensity of illumination is 3000Lx, and light application time is 14h/d.It is raw Root culture terminates, that is, the tissue culture of saturating skill Chunlan in completing.
Embodiment 4-8
The present embodiment is substantially the same manner as Example 1, and difference is, the culture medium in Multiplying culture is different, specifically It see the table below：
As seen from the above table, the more thick shape of density of rhizome on 3/4MS culture mediums, growing way is best, hence it is evident that be better than other 5 Culture medium is planted, therefore it is more suitable to choose 3/4MS culture mediums.It should be noted that B5 medium propagation is very fast, but experimental observation, With the growth of incubation time, plant growing way dies down, and browning is obvious.
Embodiment 9-13
The present embodiment is substantially the same manner as Example 1, and difference is, the hormone concentration and combination in Multiplying culture are not Together, specifically it see the table below：
As can be seen from the above table, individually addition BA and TDZ is obviously promoted effect to the propagation of root-like stock, but TDZ Proliferation times are more slightly higher than BA.Likewise, in same concentrations BA, addition TDZ can more promote root-like stock than addition NAA Growth and propagation.When NAA or TDZ concentration is identical, the BA of low concentration can more promote propagation and the growth of root-like stock, and root shape Stem is all light yellow or bottle green, and high concentration BA makes root-like stock growth retardation on the contrary, and browning occurs.TDZ 0.5mg· L-1+6-BA 1.0mgL-1 and 6-BA1.0mgL-1+NAA 0.3mgL-1 combine most bright to the cultivation effect of root-like stock It is aobvious, and upgrowth situation is preferably, therefore preferably select saturating skill Chunlan root-like stock in one of team in the two culture mediums to be cultivated.
Embodiment 14-21
The present embodiment is substantially the same manner as Example 1, and difference is, the hormone concentration when breaking up culture is different, tool Body see the table below：
As seen from the above table, with the growth of incubation time, root-like stock budding number and number of seedling are all in rising trend, wherein The root-like stock of tri- combinations of 19-21 and the seedling differentiated have browning.Addition 6-BA and NAA can make root-like stock germination simultaneously Seedling, and as 6-BA and IBA concentration rises, budding number and number of seedling decline on the contrary, the 6-BA and IBA of this explanation high concentration are not Beneficial to the differentiation of root-like stock, it is suppressed that growth.6-BA 1.0mgL-1+IBA1.0mgL-1 combination inductivity reaches 97.5%, number of seedling is 336 seedlings.
Embodiment 22-23
The present embodiment is substantially the same manner as Example 1, and difference is, intensity of illumination is different, shown in table specific as follows：
As seen from the above table, compared to 1000lux and 3000lux, under 2000lux illumination, the emergence rate of root-like stock and go out Skill rate is all best, too low and too high intensity of illumination, the trend of saturating skill Chunlan in being unfavorable for.When illumination is too low, or Perhaps because it needs substantial amounts of chlorophyll to ensure growth, the yellow skill on leaf is caused to be degenerated.And when illumination is too high, seedling growth by Resistance.
Embodiment 24-25
The present embodiment is substantially the same manner as Example 1, and difference is, root-like stock selection is different, shown in table specific as follows：
As can be seen from the above table, the skill of the saturating skill Chunlan of different root-like stock material centerings is very big to influence is moved towards, and works as selection During the faint yellow root-like stock of whole piece, go out skill rate and be up to 89.8%, and the skill rate that goes out that whole piece is the root-like stock of green is then zero, this explanation Root-like stock does not have skill, and the seedling differentiated also is difficult to skill occur.In the seedling that the yellowish green root-like stock having concurrently is differentiated, with skill Essentially root-like stock material band is slightly green.
Specific embodiment described herein is only to spirit explanation for example of the invention.Technology neck belonging to of the invention The technical staff in domain can be made various modifications or supplement to described specific embodiment or be replaced using similar mode Generation, but without departing from the spiritual of the present invention or surmount scope defined in appended claims.

Claims (4)

1. the tissue culture method of saturating skill Chunlan in a kind of, it is characterised in that comprise the following steps：
A, selection, the whole piece obtained in laboratory cultures all in flaxen thoroughly skill Chunlan root-like stock as group training material,
B, Multiplying culture, are carried out with the saturating skill Chunlan root-like stock of culture medium centering containing MS, TDZ, 6-BA and coconut palm breast in culturing room Multiplying culture,
C, differentiation culture, are carried out with the saturating skill Chunlan root-like stock of culture medium centering containing MS, 6-BA, IBA and coconut palm breast in culturing room Differentiation culture,
D, culture of rootage, with the saturating skill Chunlan root-like stock of the culture medium centering containing MS, 6-BA, IBA, NAA, AC and PVP in culture Room carries out culture of rootage.
2. the tissue culture method of saturating skill Chunlan according to claim 1, it is characterised in that in stepb, described culture Base is 3/4MS+TDZ 0.5mgL-1+6-BA 1.0mg·L-1+50mL·L-1Coconut palm breast, in step C, described culture medium is 3/4MS+6-BA1.0mg·L-1+IBA 1.0mg·L-1+50mL·L-1Coconut palm breast, in step D, described culture medium are 1/2MS +6-BA1.0mg·L-1+IBA 1.0mg·L-1+NAA 1.0mg·L-1+2g·L-1AC+5g·L-1PVP。
3. the tissue culture method of saturating skill Chunlan according to claim 1 or 2, it is characterised in that described culture room temperature For 25 DEG C ± 2 DEG C, relative humidity is 60%-80%, and intensity of illumination is 1000-3000Lx, and light application time is 10-14h/d.
4. the tissue culture method of saturating skill Chunlan according to claim 3, it is characterised in that described culture room temperature is 25 DEG C, relative humidity is 70%, and intensity of illumination is 2000Lx, and light application time is 12h/d.
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