CN107711506A - A kind of tissue culture and rapid propagation method of floribunda orchid - Google Patents
A kind of tissue culture and rapid propagation method of floribunda orchid Download PDFInfo
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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
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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
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
- A01N65/42—Aloeaceae [Aloe family] or Liliaceae [Lily family], e.g. aloe, veratrum, onion, garlic or chives
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Abstract
The present invention provides a kind of tissue culture and rapid propagation method of floribunda orchid, belongs to field of plant tissue culture technique.This method includes the steps such as sterilization, Fiber differentiation, squamous subculture and the strong seedling culture of explant.Wherein, by the way of alcohol disinfecting and plant disinfectant coordinate sterilization, the active ingredient of plant disinfectant is Acetylshikonin, asparagus juice, Extract from Nerium indicum Mill. Leaf for the sterilization of explant；Moringa leaf juice and matrimony vine leaf juice are with the addition of in culture medium used in each cultivation stage as nutritional agents.Injury of the method for the present invention to explant is small, and induction differentiation rate, explant growth coefficient and the rooting rate of floribunda orchid are remarkably improved using culture medium prescription provided by the invention, shortens cultivation cycle, improves the quality of floribunda orchid tissue-cultured seedling.
Description
【Technical field】
The present invention relates to field of plant tissue culture technique, and in particular to a kind of tissue culture and rapid propagation method of floribunda orchid.
【Background technology】
Floribunda orchid (Cymbidium floribundum) is that orchid family Cymbidium grows nonparasitically upon another plant class plant, category《Endangered species of wild fauna and flora kind
International trade pact》Appendix II species, it is China second class protection plant.Floribunda orchid is distributed mainly on Yunnan, Guangxi, Guangdong, good fortune
Each province on the south Jian Deng China the Changjiang river；It is born in height above sea level 100-3300m woods, on border tree, or along trench palisades.Its leaf banding pattern
Keratin, there is gloss, the cold-proof power of drought resisting is strong；Lace fragrance, the florescence is longer, and pattern is rich and flowery colourful, has higher ornamental value,
It is one of comparatively ideal pot flowers.
Floribunda orchid bud easily breaks up, and florescence length, cultivation is easy, and growth is fast, strong adaptability, is good ornamental flower.It is long
Since phase, floribunda orchid is based on division propagation, but reproduction speed is slow.Want to obtain more plant, Ke Yili in a short time
Bred with the mode of tissue cultures, the domestic report to floribunda orchid progress tissue culture propagation is seldom.At present, also there are some scholar's research
Explant done using floribunda orchid stem apex carry out tissue cultures and obtain the method for floribunda orchid seedling, but the easy microbiological contamination of floribunda orchid stem apex,
It is sensitive to chemosterilant and its stem apex children is tender, easily come to harm, cause proliferation rate and differentiation rate in incubation low；Separately
Outside, because the research of correlation is less, the culture medium for being more suitable for the growth of stem apex differentiated tissue is not found, causes tissue culture procedures
In, cultivation cycle length, it is unsatisfactory to cultivate effect.
【The content of the invention】
The goal of the invention of the present invention is：For above-mentioned problem, there is provided a kind of tissue culture and rapid propagation method of floribunda orchid,
Injury of the method for the present invention to explant is small, and is remarkably improved luring for floribunda orchid using culture medium prescription provided by the invention
Differentiation rate, explant growth coefficient and rooting rate are led, shortens cultivation cycle, improves the quality of floribunda orchid tissue-cultured seedling.
To achieve these goals, the technical solution adopted by the present invention is as follows：
A kind of tissue culture and rapid propagation method of floribunda orchid, include sterilization, Fiber differentiation, squamous subculture and the strong sprout training of explant
Support, concretely comprise the following steps：
(1) sterilization of explant：The stem apex of floribunda orchid is gathered as explant, the long 2cm-4cm of explant, uses flowing water first
Rinse, then with surface portion blade is peelled off after the alcohol-pickled 1min that volumetric concentration is 75%, then soaked in plant disinfectant
Continue to peel off partial blade after bubble 8min-15min, then continue to soak 5-10min in the plant disinfectant, use sterilized water
Rinse 2-4 times, finally move into superclean bench, remaining blade is peelled off under anatomical lens, exposes growing point, is cut with scalpel
Take the shoot apical meristem of 2-5mm sizes；The plant disinfectant is mixed by the raw material of following parts by weight：Acetylshikonin
0.05-0.08 parts, asparagus juice 5-8 parts, Extract from Nerium indicum Mill. Leaf 0.1-0.2 parts, Tween-60 2-4 parts, water 95-100 parts；
(2) Fiber differentiation：The shoot apical meristem is seeded in inducing culture, is 25 ± 1 DEG C in temperature, illumination
For 2000LX, cultivated 48-52 days under conditions of daily illumination 10-14h, obtain floribunda orchid garden bulb；The group of the inducing culture
Turn into：Spend No. 1 3g/L+ activated carbon 1g/L+ gibberellin 1mg/L-2mg/L+ agar 5.0g/L-8.0g/L+ sucrose 2.0g/L- of treasured
4.0g/L+ Moringa leaf juice 5.0g/L-8.0g/L+ matrimony vine leaf juices 3.0g/L-5.0g/L；
(3) squamous subculture：The floribunda orchid garden bulb that growth obtains in step (2) is transferred in subculture medium, in temperature
Spend for 25 ± 1 DEG C, cultivate under conditions of illumination 2000LX, daily illumination 10-14h, constantly changed with the growth of garden bulb
Culture medium, squamous subculture obtain the floribunda orchid shoot of long root long leaf after 75-85 days；
(4) strong seedling culture：Floribunda orchid shoot is transferred in strong seedling culture base, it is 3-5 bars to cultivate to radical, blade 3-4
Piece, height of seedling 4-8cm, that is, obtain the floribunda orchid seedling that can be transplanted.
In the present invention, it is preferable that the composition of the subculture medium is：Spend precious No. 1 3g/L+ activated carbon 1g/L+ methyl α-naphthyl acetate
2mg/L-3mg/L+ agar 5.0g/L-8.0g/L+ Moringa leaf juice 5.0g/L-8.0g/L+ matrimony vine leaf juice 3.0g/L-5.0g/L+ sugarcanes
Sugared 2.0g/L-4.0g/L.
In the present invention, it is preferable that the composition of the strong seedling culture base is：1/2MS+ activated carbon 1g/L+ indolebutyric acids
0.4mg/L-0.6mg/L+ agar 5.0g/L-8.0g/L+ Moringa leaf juice 5.0g/L-8.0g/L+ matrimony vine leaf juices 3.0g/L-5.0g/L
+ sucrose 2.0g/L-4.0g/L.
In the present invention, it is preferable that the extracting method of the oleander extract is：Smash into fresh sweetscented oleander leaf to pieces slurry,
The alcohol reflux for being 70%-75% with volumetric concentration extracts 2-6 times, and the liquid ratio of ethanol and the sweetscented oleander leaf is 5-8：1；Close
And filtrate, concentration, dry sweetscented oleander leaf alcohol extract；Using acetone: n-butanol volume ratio is 3-4：1 mixed liquor is extractant,
Extractant is added into the sweetscented oleander leaf alcohol extract, is extracted 2-5 times, merges extract solution, the extractant is reclaimed and obtains pressing from both sides bamboo
Peach leaf antipathogenic composition extract.
In the present invention, it is preferable that the preparation method of the Moringa leaf juice and matrimony vine leaf juice is：It is then abundant first to clean blade
Smash to pieces, then with filtered through gauze, the filtrate be corresponding juice.The asparagus juice is after aloe is removed into epidermis, to be stirred with machine
Break into what juice obtained.
In summary, by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
(1) present invention is mainly entered using the stem apex of floribunda orchid as explant when being carried out disinfection to it using plant disinfectant
Row immersion, the plant disinfectant is to add emulsifier tween -60 and water to mix by Acetylshikonin, asparagus juice, Extract from Nerium indicum Mill. Leaf
Made of conjunction；Wherein asparagus juice and sweetscented oleander leaf are respectively provided with the effect of good sterilizing and anti-virus, but disappear during both compound uses
Toxic effect fruit and fungicidal spectrum can not meet the needs of explant sterilization, find to add a small amount of second after the applicant's repetition test
Acyl alkannin, which coordinates, can play synergistic effect, can greatly increase the effect of sterilization；It can be substantially reduced using the plant disinfectant
The damage ratio of explant and the pollution rate for reducing inoculation.
(2) Moringa leaf juice and matrimony vine leaf juice are with the addition of in the culture medium that uses of the present invention as nutriment, in leaf of Moringa
Containing several mineral materials, vitamin, 20 kinds of amino acid, 46 kinds of antioxygens element and 36 kinds of naturally anti-scorching bodies and mineral matter, every 100 grams
The vitamin C contained in Moringa is 7 times of citrus, and iron is 3 times of spinach, and vitamin A is 4 times of carrot, and calcareous is milk
4 times, potassium is 3 times of banana, and protein is 2 times of Yoghourt；Folium lycii is rich in glycine betaine, rutin and several amino acids and micro
Element etc., the joint addition of both materials, works with growth regulator, basal medium one, can significantly improve and spend more
Blue induction differentiation rate, explant growth coefficient and rooting rate, shorten cultivation cycle, and improve the quality of floribunda orchid tissue-cultured seedling.
(3) Extract from Nerium indicum Mill. Leaf is in preparation process in the present invention, and the extracting method of use is first alcohol extracting, then with third
The intermixture of ketone and n-butanol is extracted, and can pointedly obtain the antibacterial activity composition in sweetscented oleander leaf, and what is obtained carries
Take thing antibacterial activity preferable.
【Embodiment】
In order to more clearly express the present invention, below by way of specific embodiment, the invention will be further described.
In an embodiment of the present invention, the preparation method of Moringa leaf juice, matrimony vine leaf juice and asparagus juice is as follows：
Moringa leaf juice：First clean and blade and then fully smash to pieces, then with filtered through gauze, the filtrate be Moringa leaf juice.
Matrimony vine leaf juice：First clean and blade and then fully smash to pieces, then with filtered through gauze, the filtrate be Moringa leaf juice.
Asparagus juice：After aloe first is removed into epidermis, beaten into juice and produced with machine.
In some embodiments of the invention, oleander extract obtains by the following method：Fresh sweetscented oleander leaf is smash
Slurry is broken into, is extracted 2 times with the alcohol reflux that volumetric concentration is 70%, the liquid ratio of ethanol and the sweetscented oleander leaf is 8：1；Merge
Filtrate, concentration, dry sweetscented oleander leaf alcohol extract；Using acetone: n-butanol volume ratio is 3：1 mixed liquor is extractant, to institute
State and extractant is added in sweetscented oleander leaf alcohol extract, extract 2 times, merge extract solution, reclaim the extractant and obtain sweetscented oleander leaf suppression
Bacterium component extract.
In other embodiments of the present invention, oleander extract obtains by the following method：By fresh sweetscented oleander leaf
Smash into slurry to pieces, extracted 6 times with the alcohol reflux that volumetric concentration is 75%, the liquid ratio of ethanol and the sweetscented oleander leaf is 5：1；Close
And filtrate, concentration, dry sweetscented oleander leaf alcohol extract；Using acetone: n-butanol volume ratio is 4：1 mixed liquor is extractant, to
Extractant is added in the sweetscented oleander leaf alcohol extract, is extracted 5 times, merges extract solution, reclaims the extractant and obtain sweetscented oleander leaf
Antipathogenic composition extract.
The antibacterial activity for the oleander extract that the above method obtains does not have significant difference, therefore in the examples below,
Do not repeated by specific embodiment.
In an embodiment of the present invention, culture medium prepares is transferred to 5.4- according to the method for prior art by pH value afterwards
5.6.
Embodiment 1
A kind of tissue culture and rapid propagation method of floribunda orchid, include sterilization, Fiber differentiation, squamous subculture and the strong sprout training of explant
Support, concretely comprise the following steps：
(1) sterilization of explant：The stem apex of floribunda orchid is gathered as explant, the long 2cm-4cm of explant, uses flowing water first
Rinse, then with surface portion blade is peelled off after the alcohol-pickled 1min that volumetric concentration is 75%, then soaked in plant disinfectant
Continue to peel off partial blade after bubble 8min, then continue to soak 5min in the plant disinfectant, with aseptic water washing 2 times,
Finally move into superclean bench, remaining blade is peelled off under anatomical lens, exposes growing point, it is big to cut 2-5mm with scalpel
Small shoot apical meristem；Plant disinfectant is mixed by the raw material of following parts by weight：0.05 part of Acetylshikonin, asparagus juice
5 parts, 0.1 part of Extract from Nerium indicum Mill. Leaf, 2 parts of Tween-60,95 parts of water；
(2) Fiber differentiation：The shoot apical meristem is seeded in inducing culture to (composition of inducing culture is：
Spend precious No. 1 3g/L+ activated carbon 1g/L+ gibberellin 1mg/L+ agar 5.0g/L+ sucrose 2.0g/L+ Moringa leaf juice 5.0g/L+ matrimony vine
Leaf juice 5.0g/L)；It is 25 ± 1 DEG C in temperature, is cultivated 52 days under conditions of illumination 2000LX, daily illumination 10h, obtain floribunda orchid
Garden bulb；
(3) squamous subculture：The floribunda orchid garden bulb that growth obtains in step (2) is transferred in subculture medium (subculture
The composition of culture medium is：Spend No. 1 3g/L+ activated carbon 1g/L+ methyl α-naphthyl acetate 2mg/L+ agar 5.0g/L+ Moringa leaf juices 5.0g/L+ of treasured
Matrimony vine leaf juice 5.0g/L+ sucrose 2.0g/L)；It is 25 ± 1 DEG C in temperature, is trained under conditions of illumination 2000LX, daily illumination 10h
Support, as culture medium is constantly changed in the growth of garden bulb, squamous subculture obtains the floribunda orchid shoot of long root long leaf after 85 days；
(4) strong seedling culture：Floribunda orchid shoot is transferred in strong seedling culture base to (composition of strong seedling culture base is：1/2MS+
Activated carbon 1g/L+ indolebutyric acid 0.4mg/L+ agar 5.0g/L+ Moringa leaf juice 5.0g/L+ matrimony vine leaf juice 5.0g/L+ sucrose 2.0g/
L；) to obtain within 68 days radical be 3-5 bars for culture, blade 3-4 pieces, height of seedling 4-8cm floribunda orchid seedling.
Embodiment 2
A kind of tissue culture and rapid propagation method of floribunda orchid, include sterilization, Fiber differentiation, squamous subculture and the strong sprout training of explant
Support, concretely comprise the following steps：
(1) sterilization of explant：The stem apex of floribunda orchid is gathered as explant, the long 2cm-4cm of explant, uses flowing water first
Rinse, then with surface portion blade is peelled off after the alcohol-pickled 1min that volumetric concentration is 75%, then soaked in plant disinfectant
Continue to peel off partial blade after bubble 12min, then continue to soak 8min in the plant disinfectant, with aseptic water washing 3 times,
Finally move into superclean bench, remaining blade is peelled off under anatomical lens, exposes growing point, it is big to cut 2-5mm with scalpel
Small shoot apical meristem；Plant disinfectant is mixed by the raw material of following parts by weight：0.06 part of Acetylshikonin, asparagus juice
6 parts, 0.15 part of Extract from Nerium indicum Mill. Leaf, 3 parts of Tween-60,98 parts of water；
(2) Fiber differentiation：The shoot apical meristem is seeded in inducing culture to (composition of inducing culture is：
Spend precious No. 1 3g/L+ activated carbon 1g/L+ gibberellin 1.5mg/L+ agar 6g/L+ sucrose 3g/L+ Moringa leaf juice 6g/L+ matrimony vine leaf juice
4g/L)；It is 25 ± 1 DEG C in temperature, is cultivated 48 days under conditions of illumination 2000LX, daily illumination 12h, obtain floribunda orchid garden ball
Stem；
(3) squamous subculture：The floribunda orchid garden bulb that growth obtains in step (2) is transferred in subculture medium (subculture
The composition of culture medium is：Spend precious No. 1 3g/L+ activated carbon 1g/L+ methyl α-naphthyl acetate 2.5mg/L+ agar 6g/L+ Moringa leaf juice 7g/L+ Chinese holly
Qi leaf juice 4.0g/L+ sucrose 3.0g/L)；It is 25 ± 1 DEG C in temperature, is trained under conditions of illumination 2000LX, daily illumination 12h
Support, as culture medium is constantly changed in the growth of garden bulb, squamous subculture obtains the floribunda orchid shoot of long root long leaf after 75 days；
(4) strong seedling culture：Floribunda orchid shoot is transferred in strong seedling culture base to (composition of strong seedling culture base is：1/2MS+
Activated carbon 1g/L+ indolebutyric acid 0.5mg/L+ agar 6.0g/L+ Moringa leaf juice 7.0g/L+ matrimony vine leaf juice 4.0g/L+ sucrose 3.0g/
L；) to obtain within 66 days radical be 3-5 bars for culture, blade 3-4 pieces, height of seedling 4-8cm floribunda orchid seedling.
Embodiment 3
A kind of tissue culture and rapid propagation method of floribunda orchid, include sterilization, Fiber differentiation, squamous subculture and the strong sprout training of explant
Support, concretely comprise the following steps：
(1) sterilization of explant：The stem apex of floribunda orchid is gathered as explant, the long 2cm-4cm of explant, uses flowing water first
Rinse, then with surface portion blade is peelled off after the alcohol-pickled 1min that volumetric concentration is 75%, then soaked in plant disinfectant
Continue to peel off partial blade after bubble 15min, then continue to soak 10min in the plant disinfectant, with aseptic water washing 4
It is secondary, finally move into superclean bench, remaining blade is peelled off under anatomical lens, exposes growing point, 2-5mm is cut with scalpel
The shoot apical meristem of size；Plant disinfectant is mixed by the raw material of following parts by weight：0.08 part of Acetylshikonin, aloe
8 parts of juice, 0.2 part of Extract from Nerium indicum Mill. Leaf, 4 parts of Tween-60,100 parts of water；
(2) Fiber differentiation：The shoot apical meristem is seeded in inducing culture to (composition of inducing culture is：
Spend precious No. 1 3g/L+ activated carbon 1g/L+ gibberellin 2mg/L+ agar 8.0g/L+ sucrose 4.0g/L+ Moringa leaf juice 8.0g/L+ matrimony vine
Leaf juice 3.0g/L)；It is 25 ± 1 DEG C in temperature, cultivates 50 days under conditions of illumination 2000LX, daily illumination 10-14h, much
Hua Lan gardens bulb；
(3) squamous subculture：The floribunda orchid garden bulb that growth obtains in step (2) is transferred in subculture medium (subculture
The composition of culture medium is：Spend No. 1 3g/L+ activated carbon 1g/L+ methyl α-naphthyl acetate 3mg/L+ agar 8.0g/L+ Moringa leaf juices 8.0g/L+ of treasured
Matrimony vine leaf juice 3.0g/L+ sucrose 4.0g/L)；It is 25 ± 1 DEG C in temperature, is trained under conditions of illumination 2000LX, daily illumination 14h
Support, as culture medium is constantly changed in the growth of garden bulb, squamous subculture obtains the floribunda orchid shoot of long root long leaf after 78 days；
(4) strong seedling culture：Floribunda orchid shoot is transferred in strong seedling culture base to (composition of strong seedling culture base is：1/2MS+
Activated carbon 1g/L+ indolebutyric acid 0.6mg/L+ agar 8.0g/L+ Moringa leaf juice 8.0g/L+ matrimony vine leaf juice 3.0g/L+ sucrose 4.0g/
L；) to obtain within 68 days radical be 3-5 bars for culture, blade 3-4 pieces, height of seedling 4-8cm floribunda orchid seedling.
Comparative example 1
Mass concentration is used to be carried out disinfection for 0.1% sodium hypochlorite instead of plant disinfectant, other conditions and embodiment 2
It is identical, investigate influence of the disinfection way to induction differentiation rate, explant growth coefficient and rooting rate.
Comparative example 2
Moringa juice and matrimony vine leaf juice are replaced using banana puree, other conditions are same as Example 2, i.e. the group of inducing culture
Turn into：Spend No. 1 3g/L+ activated carbon 1g/L+ gibberellin 1.5mg/L+ agar 6g/L+ sucrose 3g/L+ banana purees 10g/L of treasured；Subculture
The composition of culture medium is：Spend precious No. 1 3g/L+ activated carbon 1g/L+ methyl α-naphthyl acetate 2.5mg/L+ agar 6g/L+ banana puree 10g/L+ sucrose
3.0g/L；The composition of strong seedling culture base is：1/2MS+ activated carbon 1g/L+ indolebutyric acid 0.5mg/L+ agar 6.0g/L+ banana purees
10g/L+ sucrose 3.0g/L；Using the identical method culture floribunda orchid seedling of embodiment 2, statistics induction differentiation rate in incubation,
Explant growth coefficient and rooting rate.
Comparative example 3
Different from the culture medium used in embodiment 2, specific culture medium is：The composition of inducing culture is：1/2MS+ lives
Property charcoal 1g/L+6-BA1.0mg/L+NAA0.1mg/L+ agar 6g/L+ sucrose 20g/L；The composition of subculture medium is：1/2MS+
Activated carbon 1g/L+6-BA1.0mg/L+NAA0.5mg/L+ agar 6g/L+ sucrose 20g/L；The composition of strong seedling culture base is：1/2MS
+ activated carbon 1g/L+6-BA1.0mg/L+NAA0.5mg/L+ agar 6g/L+ sucrose 20g/L.Trained using the identical method of embodiment 2
Floribunda orchid seedling is supported, statistics induces differentiation rate, explant growth coefficient and rooting rate in incubation.
Tissue culture test method and result：
Every group takes 200 explant samples, and the method that embodiment 1-3 and comparative example 1-3 is respectively adopted carries out tissue culture, examination
During testing, count the microbiological contamination situation of each group, proliferative conditions, differentiation are taken root situation, investigate shadow of each factor to floribunda orchid tissue culture
Ring.
1st, the microbiological contamination situation after explant inoculation：
After Fiber differentiation 10 days, the microbiological contamination quantity of each group, its result such as table 1 below are counted：
Table 1
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Microbiological contamination number | 4 | 3 | 5 | 6 | 5 | 4 |
From the results shown in Table 1, adopted using the plant disinfectant of the present invention, the microbiological contamination situation ratio of floribunda orchid explant
It is slightly good with the hypochlorous acid sterilization of routine.
2nd, the growing state during tissue culture：
After the explant Fiber differentiation 68 days of floribunda orchid, the differentiation and proliferation situation of each group explant is counted, will induction training
Foster protocorm squamous subculture is after 85 days, statistical average inductivity and average rooting rate；Continue strong seedling culture to count after 65 days
Average root long, the result of each group are as shown in table 2.
Table 2
From the results shown in Table 2, embodiment 1-3 average stem length, growth coefficient, average inductivity, average life
Root rate, average root long are better than comparative example 1, illustrate the change due to disinfection way, although the control to microbiological contamination number and existing skill
Art is suitable, but by the way of the present invention, does not injure explant, and during tissue culture, the growing state in each stage is all relative
Preferably.Relative to comparative example 2, the average inductivity of embodiment 1-3 average stem length, growth coefficient, average rooting rate, average root
It is long obvious preferable, illustrate to compare addition banana puree, be engaged using Moringa leaf juice and matrimony vine leaf juice as nutritional agents, make culture
The composition of base is more suitable for the growth of floribunda orchid explant.Relative to comparative example 3, embodiment 3 is because the culture medium used is different, respectively
The growing state in individual stage is also obvious preferable.
Described above is the detailed description for the present invention preferably possible embodiments, but embodiment is not limited to this hair
Bright patent claim, the equal change completed or modification change under the technical spirit suggested by all present invention, all should belong to
Cover the scope of the claims in the present invention.
Claims (6)
1. a kind of tissue culture and rapid propagation method of floribunda orchid, include sterilization, Fiber differentiation, squamous subculture and the strong seedling culture of explant,
It is characterized in that concretely comprise the following steps：
(1) sterilization of explant：The stem apex of floribunda orchid is gathered as explant, the long 2cm-4cm of explant, is rushed first with flowing water
Wash, then with surface portion blade is peelled off after the alcohol-pickled 1min that volumetric concentration is 75%, then soaked in plant disinfectant
Continue to peel off partial blade after 8min-15min, then continue to soak 5-10min in the plant disinfectant, rushed with sterilized water
Wash 2-4 times, finally move into superclean bench, remaining blade is peelled off under anatomical lens, exposes growing point, is cut with scalpel
The shoot apical meristem of 2-5mm sizes；The plant disinfectant is mixed by the raw material of following parts by weight：Acetylshikonin
0.05-0.08 parts, asparagus juice 5-8 parts, Extract from Nerium indicum Mill. Leaf 0.1-0.2 parts, Tween-60 2-4 parts, water 95-100 parts；
(2) Fiber differentiation：The shoot apical meristem is seeded in inducing culture, is 25 ± 1 DEG C in temperature, illumination is
Cultivated 48-52 days under conditions of 2000LX, daily illumination 10-14h, obtain floribunda orchid garden bulb；The composition of the inducing culture
For：Spend No. 1 3g/L+ activated carbon 1g/L+ gibberellin 1mg/L-2mg/L+ agar 5.0g/L-8.0g/L+ sucrose 2.0g/L- of treasured
4.0g/L+ Moringa leaf juice 5.0g/L-8.0g/L+ matrimony vine leaf juices 3.0g/L-5.0g/L；
(3) squamous subculture：The floribunda orchid garden bulb that growth obtains in step (2) is transferred in subculture medium, is in temperature
25 ± 1 DEG C, cultivate under conditions of illumination 2000LX, daily illumination 10-14h, culture is constantly changed with the growth of garden bulb
Base, squamous subculture obtain the floribunda orchid shoot of long root long leaf after 75-85 days；
(4) strong seedling culture：Floribunda orchid shoot is transferred in strong seedling culture base, it is 3-5 bars to cultivate to radical, blade 3-4 pieces, seedling
High 4-8cm, that is, obtain the floribunda orchid seedling that can be transplanted.
2. the tissue culture and rapid propagation method of floribunda orchid according to claim 1, it is characterised in that：The composition of the subculture medium
For：Spend No. 1 3g/L+ activated carbon 1g/L+ methyl α-naphthyl acetate 2mg/L-3mg/L+ agar 5.0g/L-8.0g/L+ Moringa leaf juices 5.0g/L- of treasured
8.0g/L+ matrimony vine leaf juice 3.0g/L-5.0g/L+ sucrose 2.0g/L-4.0g/L.
3. the tissue culture and rapid propagation method of floribunda orchid according to claim 1, it is characterised in that：The composition of the strong seedling culture base
For：1/2MS+ activated carbon 1g/L+ indolebutyric acid 0.4mg/L-0.6mg/L+ agar 5.0g/L-8.0g/L+ Moringa leaf juices 5.0g/L-
8.0g/L+ matrimony vine leaf juice 3.0g/L-5.0g/L+ sucrose 2.0g/L-4.0g/L.
4. the tissue culture and rapid propagation method of floribunda orchid according to claim 1, it is characterised in that：The oleander extract carries
The method is taken to be：Smash into fresh sweetscented oleander leaf to pieces slurry, the alcohol reflux for being 70%-75% with volumetric concentration extracts 2-6 times, ethanol
Liquid ratio with the sweetscented oleander leaf is 5-8：1；Merging filtrate, concentration, dry sweetscented oleander leaf alcohol extract；With acetone: positive fourth
Alcohol volume ratio is 3-4：1 mixed liquor is extractant, and extractant is added into the sweetscented oleander leaf alcohol extract, is extracted 2-5 times, is closed
And extract solution, reclaim the extractant and obtain sweetscented oleander leaf antipathogenic composition extract.
5. the tissue culture and rapid propagation method of floribunda orchid according to claim 1, it is characterised in that：The Moringa leaf juice and folium lycii
The preparation method of juice is：First clean and blade and then fully smash to pieces, then with filtered through gauze, the filtrate be corresponding juice.
6. the tissue culture and rapid propagation method of floribunda orchid according to claim 1, it is characterised in that：The asparagus juice is to remove aloe
After removing epidermis, beat what is obtained into juice with machine.
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| CN116034874A (en) * | 2023-01-09 | 2023-05-02 | 西安市农业技术推广中心 | Method for proliferation and differentiation of cymbidium rhizome into seedlings |
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| CN104285804A (en) * | 2014-10-24 | 2015-01-21 | 柳州市天姿园艺有限公司 | Special culture medium for cymbidium floribundum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104285804A (en) * | 2014-10-24 | 2015-01-21 | 柳州市天姿园艺有限公司 | Special culture medium for cymbidium floribundum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116034874A (en) * | 2023-01-09 | 2023-05-02 | 西安市农业技术推广中心 | Method for proliferation and differentiation of cymbidium rhizome into seedlings |
| CN116034874B (en) * | 2023-01-09 | 2023-12-19 | 西安市农业技术推广中心 | Method for proliferation and differentiation of cymbidium rhizome into seedlings |
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