CN107980631A - A kind of tissue culture and rapid propagation method of four seasons orchid - Google Patents
A kind of tissue culture and rapid propagation method of four seasons 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
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Abstract
The present invention provides a kind of tissue culture and rapid propagation method of four seasons orchid, belongs to field of plant tissue culture technique.Disinfection, Fiber differentiation, squamous subculture and strong seedling culture of this method including explant and etc..Wherein, by the way of alcohol disinfecting and plant disinfectant coordinate disinfection, the active ingredient of plant disinfectant is glycyrrhizic acid, isatis root extract, bupleurum extract for the disinfection of explant；Moringa leaf juice and white mulberry vein-juice be 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 four seasons orchid are remarkably improved using culture medium prescription provided by the invention, shortens cultivation cycle, improves the quality of four seasons 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 four seasons orchid.
【Background technology】
Four seasons orchid is one kind of Chinese cymbidium, gains the name because the four seasons can bloom, belongs to Terrestrial orchid, mainly in annual 7-
August part is bloomed, main product on city of Leshan sichuan Meishan City, Guangdong, Fujian, Taiwan and other places, be born on steep cliff more and sparse woods in, it is local
People claims Chillon.Four seasons orchid plant is robust, and root is thick and long.It is slightly more per basin seedling number suitably with the blue potted plant plant of more than five, it is placed in woods
Between, flower garden or the hall, flourishing with flowers and leaves, daring it is very big, it is also possible to larger high waist lot pot it is potted plant plant number seedling, it is verdant steep when long,
There is very much expression.The flowers are in blossom midsummer, and cool breeze blows Lan Xiang, makes one to feel more quiet and beautiful.Sword-leaved cymbidium cultivation history is long, various in style, in China
South cultivation is very universal, is balcony, parlor, flower stand and garth step furnishings good merchantable brand, it appears pure and fresh Gao Zhi.
For a long time, for four seasons orchid based on division propagation, division propagation speed is slower.Want to obtain in a short time more
Plant, can utilize tissue cultures mode breed, at present, many researchs have been carried out to four seasons orchid tissue cultures both at home and abroad,
Four seasons orchid tissue cultures program is generally：(1) selected seed or stem apex do explant；(2) induced synthesis protocorm；(3) protocorm
Stem is largely bred；(4) plantlet is differentiated；(5) strong plantlets and rootage；(6) greenhouse is transplanted.But the kind that aseptically sowing seeds method is bred
Seedling variation is more, and seedling early growth is inconsistent, and takes stem apex to have certain injury to maternal plant as explant.Bennet bud is as explant
Body is put into practice on some cymbidium varieties, and the bennet bud of four seasons orchid is not utilized also in terms of tissue culture.By
In each cymbidium variety is having its own in habit the characteristics of, when carrying out tissue culture to four seasons orchid stalk bud explant, using existing
Some explant disinfection way, using common culture medium carry out tissue culture, cannot still obtain satisfied tissue culture effect, cause to breed
Cycle is grown, and appreciation rate is low, the poor quality of gained tissue-cultured seedling.
【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 four seasons orchid,
The method of the present invention carries out tissue culture propagation using the bennet bud of four seasons orchid as explant, and using the plant small to explant injury
Thimerosal carries out disinfection, and using culture medium provided by the invention carry out tissue culture, be remarkably improved four seasons orchid induction differentiation rate,
Explant growth coefficient and rooting rate, shorten cultivation cycle, improve the quality of four seasons 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 four seasons orchid, includes disinfection, Fiber differentiation, squamous subculture and the strong sprout training of explant
Support, concretely comprise the following steps：
(1) disinfection of explant：To bloom, four seasons of robust growth, no disease and pests harm it is blue as maternal, cut female parent
Bennet bud on bennet rinses bennet bud, then soak 1- with the alcohol that volumetric concentration is 75% with flowing water first as explant
2min, then peels off the bract on bennet bud surface, continues to peel off one after then soaking 8min-15min in plant disinfectant
Layer bract, then continues to soak 5-10min in the plant disinfectant, with sterile water 3-5 times, the explant after must sterilizing；
The plant disinfectant is mixed by the raw material of following parts by weight：0.05-0.08 parts of glycyrrhizic acid, isatis root extract 0.8-
1.2 parts, 0.5-0.8 parts of bupleurum extract, 6-8 parts of OP-10,150-200 parts of water；
(2) Fiber differentiation：By the both ends notch of the explant after the disinfection, explant is seeded in inducing culture
In, light culture 10 days under conditions of being 23-25 DEG C in temperature, then move to temperature be 23-25 DEG C, intensity of illumination be 1500lx's
Interior, daily illumination 10-14h are cultivated, and incubation time is 40-45 days；
(3) squamous subculture：The budlet that Fiber differentiation is sprouted, from after base portion stripping, is inoculated on subculture medium, trains
Support 23-25 DEG C of temperature, light application time 12-14h/d, intensity of illumination 1500lx；
(4) strong seedling culture：After shoot proliferation culture 4-6 generations, select high more than 2cm, have the small of obvious projection on explant
Seedling is transferred in strong seedling culture base and cultivates, 23-25 DEG C of cultivation temperature, light application time 12-14h/d, intensity of illumination 2500lx, training
It is 3-5 bars to support to radical, blade 3-4 pieces, height of seedling 4-8cm, you can hardening, obtains the four seasons orchid seedling that can be transplanted after hardening.
More preferably, the inducing culture is：MS+0.3-0.5mg/L6-BA+0.2-0.4mg/LNAA+0.2-0.4mg/L
Kinetin+40-55g/L Moringa leaf juice+30-40g/L white mulberry vein-juice+40-50mg/L cysteine+20-25g/L sucrose+6-8g/L
Agar, pH 5.4-5.5.
More preferably, the composition of the subculture medium is：Miller+0.2-0.3mg/L6-BA+0.1-0.2mg/LNAA+
0.1-0.2mg/L kinetin+0.05-0.08mg/L zeatin+40-55g/L Moringa leaf juice+30-40g/L white mulberry vein-juices+20-25g/
L sucrose+6-8g/L agar, pH 5.5-5.6.
More preferably, the composition of the strong seedling culture base is：Miller+0.2-0.3mg/L6-BA+0.1-0.2mg/LNAA+
2-4mg/L indolebutyric acid+40-55g/L Moringa leaf juice+30-40g/L white mulberry vein-juice+20-25g/L sucrose+6-8g/L agar, pH are
5.6-5.7。
More preferably, the bupleurum extract is to decoct radix bupleuri 2 times with the 8-10 times of water measured, and each 1.5-2h, merging carries
Liquid is taken, filtering and concentrating is obtained to dry.
More preferably, the isatis root extract is to decoct Radix Isatidis 2 times, each 1.5-2h with the 8-10 times of water measured, and is closed
And extracting solution, filtering and concentrating are obtained to dry.
More preferably, the preparation method of the Moringa leaf juice is：First clean and blade and then fully smash to pieces, then with filtered through gauze,
The filtrate is white mulberry vein-juice.
More preferably, the white mulberry vein-juice is fully smashed to pieces after new fresh mulberry leaf is cleaned, then is with filtered through gauze, the filtrate
White mulberry vein-juice.
In conclusion by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
(1) present invention mainly uses plant disinfectant using the bennet bud of four seasons orchid as explant when carrying out disinfection to it
Soaked, which is to add emulsifier op-10 and water to mix by glycyrrhizic acid, isatis root extract, bupleurum extract
It is manufactured；Wherein isatis root extract and radix bupleuri are respectively provided with the effect of good sterilizing and anti-virus, but disappear during both compound uses
Toxic effect fruit and fungicidal spectrum cannot meet the needs of explant disinfection, find to add on a small quantity sweet after the applicant's repetition test
Oxalic acid, which coordinates, can play synergistic effect, can greatly increase the effect of disinfection；Explant can be substantially reduced using the plant disinfectant
The damage ratio of body and the pollution rate for reducing inoculation.
(2) Moringa leaf juice and white mulberry vein-juice be with the addition of in the culture medium that uses of the present invention as nutriment, contained in leaf of Moringa
There are several mineral materials, vitamin, 20 kinds of amino acid, 46 kinds of antioxygens element and 36 kinds of naturally anti-a scorching bodies and mineral matter, every 100 grams peppery
The vitamin C contained in wood is 7 times of citrus, and iron is 3 times of spinach, and vitamin A is 4 times of carrot, and calcareous is the 4 of milk
Times, potassium is 3 times of banana, and protein is 2 times of Yoghourt；In mulberry leaf containing abundant protein, carbohydrate and it is inorganic into
Point, containing more folic acid, the chromocor compound of high level and mulberry glycosides, abundant potassium, calcium, Iron and Vitamin A, B1, B2,
The elements such as B3, C, folic acid and copper zinc, the joint addition of both materials, work with growth regulator, basal medium one,
Induction differentiation rate, explant growth coefficient and the rooting rate of four seasons orchid can be significantly improved, shortens cultivation cycle, and improve the four seasons
The quality of blue tissue-cultured seedling.
(3) for the present invention using bennet bud as explant, the growth of gained tissue-cultured seedling is consistent, will not produce variating seedling, and energy
The maternal plant caused by taking stem apex explant is reduced to lose；Due to the sterilization method using science and special culture medium is developed,
In shoot proliferation incubation, the propagation of protocorm can reach 8-10 times, and tissue-cultured seedling root system is healthy and strong, survival rate 95%.
【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, Moringa leaf juice, white mulberry vein-juice, bupleurum extract, the preparation method of isatis root extract
It is as follows：
Bupleurum extract is to decoct radix bupleuri 2 times, each 1.5-2h with the 8-10 times of water measured, and merges extracting solution, and filtering is dense
It is reduced to dry obtain.
Isatis root extract is to decoct Radix Isatidis 2 times, each 1.5-2h with the 8-10 times of water measured, and merges extracting solution, mistake
Filter is concentrated to dryness to obtain.
Moringa leaf juice：First clean and blade and then fully smash to pieces, then with filtered through gauze, the filtrate be white mulberry vein-juice.
White mulberry vein-juice：Fully smashed to pieces after new fresh mulberry leaf is cleaned, then with filtered through gauze, the filtrate be white mulberry vein-juice.
Embodiment 1
A kind of tissue culture and rapid propagation method of four seasons orchid, includes disinfection, Fiber differentiation, squamous subculture and the strong sprout training of explant
Support, concretely comprise the following steps：
(1) disinfection of explant：To bloom, four seasons of robust growth, no disease and pests harm it is blue as maternal, cut female parent
Bennet bud on bennet rinses bennet bud, then soaked with the alcohol that volumetric concentration is 75% with flowing water first as explant
1min, then peels off the bract on bennet bud surface, continues the bract that peels a layer from after then soaking 8min in plant disinfectant,
Then continue to soak 5min in the plant disinfectant, with sterile water 3 times, the explant after must sterilizing；The plant disinfection
Liquid is mixed by the raw material of following parts by weight：0.05 part of glycyrrhizic acid, 0.8 part of isatis root extract, 0.5 part of bupleurum extract,
OP-106 parts, 150 parts of water；
(2) Fiber differentiation：By the both ends notch of the explant after the disinfection, explant is seeded in inducing culture
In, light culture 10 days under conditions of being 23-25 DEG C in temperature, then move to temperature be 23-25 DEG C, intensity of illumination be 1500lx's
Interior, daily illumination 10h are cultivated, and incubation time is 45 days；Inducing culture is：MS+0.3mg/L6-BA+0.2mg/
LNAA+0.2mg/L kinetin+40g/L Moringa leaf juice+30/L white mulberry vein-juice+40mg/L cysteine+20g/L sucrose+6g/L fine jades
Fat, pH 5.4.
(3) squamous subculture：The budlet that Fiber differentiation is sprouted, from after base portion stripping, is inoculated on subculture medium, trains
Support 23-25 DEG C of temperature, light application time 12h/d, intensity of illumination 1500lx；The composition of subculture medium is：Miller+0.2m
G/L6-BA+0.1mg/LNAA+0.1mg/L kinetin+0.05mg/L zeatin+40g/L Moringa leaf juice+30g/L white mulberry vein-juices+
20g/L sucrose+6g/L agar, pH 5.5.
(4) strong seedling culture：After 4 generation of shoot proliferation culture, select high more than 2cm, have the seedling of obvious projection on explant
It is transferred in strong seedling culture base and cultivates, 23-25 DEG C of cultivation temperature, light application time 12h/d, intensity of illumination 2500lx, culture is extremely
Radical is 3-5 bars, blade 3-4 pieces, height of seedling 4-8cm, you can hardening, obtains the four seasons orchid seedling that can be transplanted after hardening；Strong sprout
The composition of culture medium is：Miller+0.2mg/L6-BA+0.1mg/LNAA+4mg/L indolebutyric acid+40g/L Moringa leaf juices+30g/
L white mulberry vein-juice+20g/L sucrose+6g/L agar, pH 5.6.
Embodiment 2
A kind of tissue culture and rapid propagation method of four seasons orchid, includes disinfection, Fiber differentiation, squamous subculture and the strong sprout training of explant
Support, concretely comprise the following steps：
(1) disinfection of explant：To bloom, four seasons of robust growth, no disease and pests harm it is blue as maternal, cut female parent
Bennet bud on bennet rinses bennet bud, then soaked with the alcohol that volumetric concentration is 75% with flowing water first as explant
1min, then peels off the bract on bennet bud surface, continues the bract that peels a layer from after then soaking 8min in plant disinfectant,
Then continue to soak 5min in the plant disinfectant, with sterile water 4 times, the explant after must sterilizing；The plant disinfection
Liquid is mixed by the raw material of following parts by weight：0.06 part of glycyrrhizic acid, 1.0 parts of isatis root extract, 0.6 part of bupleurum extract,
OP-107 parts, 180 parts of water；
(2) Fiber differentiation：By the both ends notch of the explant after the disinfection, explant is seeded in inducing culture
In, light culture 10 days under conditions of being 23-25 DEG C in temperature, then move to temperature be 23-25 DEG C, intensity of illumination be 1500lx's
Interior, daily illumination 12h are cultivated, and incubation time is 40 days；Inducing culture is：MS+0.4mg/L6-BA+0.3mg/
LNAA+0.3mg/L kinetin+48g/L Moringa leaf juice+35g/L white mulberry vein-juice+45mg/L cysteine+22/L sucrose+7g/L fine jades
Fat, pH 5.5.
(3) squamous subculture：The budlet that Fiber differentiation is sprouted, from after base portion stripping, is inoculated on subculture medium, trains
Support 23-25 DEG C of temperature, light application time 13h/d, intensity of illumination 1500lx；The composition of subculture medium is：Miller+
0.25mg/L6-BA+0.15mg/LNAA+0.15mg/L kinetin+0.06mg/L zeatin+48/L Moringa leaf juice+35g/L mulberry leaf
Juice+22g/L sucrose+7g/L agar, pH 5.6.
(4) strong seedling culture：After 5 generation of shoot proliferation culture, select high more than 2cm, have the seedling of obvious projection on explant
It is transferred in strong seedling culture base and cultivates, 23-25 DEG C of cultivation temperature, light application time 13h/d, intensity of illumination 2500lx, culture is extremely
Radical is 3-5 bars, blade 3-4 pieces, height of seedling 4-8cm, you can hardening, obtains the four seasons orchid seedling that can be transplanted after hardening；Strong sprout
The composition of culture medium is：Miller+0.25mg/L6-BA+0.15mg/LNAA+3mg/L indolebutyric acid+48g/L Moringas leaf juice+
35/L white mulberry vein-juice+22g/L sucrose+7g/L agar, pH 5.6.
Embodiment 3
A kind of tissue culture and rapid propagation method of four seasons orchid, includes disinfection, Fiber differentiation, squamous subculture and the strong sprout training of explant
Support, concretely comprise the following steps：
(1) disinfection of explant：To bloom, four seasons of robust growth, no disease and pests harm it is blue as maternal, cut female parent
Bennet bud on bennet rinses bennet bud, then soaked with the alcohol that volumetric concentration is 75% with flowing water first as explant
2min, then peels off the bract on bennet bud surface, continues the bud that peels a layer from after then soaking 15min in plant disinfectant
Leaf, then continues to soak 10min in the plant disinfectant, with sterile water 5 times, the explant after must sterilizing；The plant
Thimerosal is mixed by the raw material of following parts by weight：0.08 part of glycyrrhizic acid, 1.2 parts of isatis root extract, bupleurum extract 0.8
Part, 8 parts of OP-10,200 parts of water；
(2) Fiber differentiation：By the both ends notch of the explant after the disinfection, explant is seeded in inducing culture
In, light culture 10 days under conditions of being 23-25 DEG C in temperature, then move to temperature be 23-25 DEG C, intensity of illumination be 1500lx's
Interior, daily illumination 14h are cultivated, and incubation time is 42 days；Inducing culture is：MS+0.5mg/L6-BA+0.4mg/
LNAA+0.4mg/L kinetin+55g/L Moringa leaf juice+40g/L white mulberry vein-juice+50mg/L cysteine+25g/L sucrose+8g/L fine jades
Fat, pH 5.5.
(3) squamous subculture：The budlet that Fiber differentiation is sprouted, from after base portion stripping, is inoculated on subculture medium, trains
Support 23-25 DEG C of temperature, light application time 14h/d, intensity of illumination 1500lx；The composition of subculture medium is：Miller+
0.3mg/L6-BA+0.2mg/LNAA+0.2mg/L kinetin+0.08mg/L zeatin+55g/L Moringa leaf juice+40g/L white mulberry vein-juices
+ 25g/L sucrose+8g/L agar, pH 5.6.
(4) strong seedling culture：After 6 generation of shoot proliferation culture, select high more than 2cm, have the seedling of obvious projection on explant
It is transferred in strong seedling culture base and cultivates, 23-25 DEG C of cultivation temperature, light application time 14h/d, intensity of illumination 2500lx, culture is extremely
Radical is 3-5 bars, blade 3-4 pieces, height of seedling 4-8cm, you can hardening, obtains the four seasons orchid seedling that can be transplanted after hardening；Strong sprout
The composition of culture medium is：Miller+0.3mg/L6-BA+0.2mg/LNAA+4mg/L indolebutyric acid+55g/L Moringa leaf juices+40g/
L white mulberry vein-juice+25g/L sucrose+8g/L agar, pH 5.7.
Comparative example 1
Mass concentration is used to carry out disinfection for 1% sodium hypochlorite instead of plant disinfectant, other conditions and 2 phase of embodiment
Together, influence of the disinfection way to induction differentiation rate, explant growth coefficient and rooting rate is investigated.
Comparative example 2
Moringa juice and white mulberry vein-juice are replaced using banana puree, other conditions are same as Example 2, i.e. the composition of inducing culture
For：MS+0.4mg/L6-BA+0.3mg/LNAA+0.3mg/L kinetin+100g/L banana puree+45mg/L cysteine+22/L sugarcanes
Sugar+7g/L agar, pH 5.5；The composition of subculture medium is：Miller+0.25mg/L6-BA+0.15mg/LNAA+
0.15mg/L kinetin+0.06mg/L zeatin+100g/L banana puree+22g/L sucrose+7g/L agar, pH 5.6；Strong sprout is trained
Support base composition be：Miller+0.25mg/L6-BA+0.15mg/LNAA+3mg/L indolebutyric acid+100g/L banana purees+22g/L
Sucrose+7g/L agar, pH 5.6；Using the identical method culture four seasons orchid seedling of embodiment 2, in incubation, statistics, which induces, divides
Rate, 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：MS+ activated carbons
1g/L+6-BA 0.4mg/L+NAA0.3mg/L+ agar 6g/L+ sucrose 20g/L；The composition of subculture medium is：1/2MS+ activity
Charcoal 1g/L+6-BA 0.25mg/L+NAA0.15mg/L+ agar 6g/L+ sucrose 20g/L；The composition of strong seedling culture base is：1/2MS+
Activated carbon 1g/L+6-BA 0.25mg/L+NAA0.15mg/L+ agar 6g/L+ sucrose 20g/L.Using the identical method of embodiment 2
Four seasons orchid seedling is cultivated, 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 four seasons 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 | 3 | 3 | 4 | 18 | 4 | 5 |
From the results shown in Table 1, plant disinfectant using the present invention, the microbiological contamination situation ratio of four seasons orchid explant are adopted
It is slightly good with conventional hypochlorous acid disinfection.
2nd, the growing state during tissue culture：
After the explant Fiber differentiation 50 days of four seasons orchid, the differentiation and proliferation situation of each group explant is counted, will induction training
Foster protocorm squamous subculture is after 80 days, statistical average inductivity and average rooting rate；Continue strong seedling culture to count after 65 days
Average root long, the results are shown in Table 2 for each group.
Table 2
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Average stem length (cm) | 3.5 | 3.9 | 3.6 | 3.0 | 2.8 | 2.3 |
| Value-added coefficient | 8.5 | 10.5 | 8.9 | 6.0 | 4.5 | 4.2 |
| Average inductivity (%) | 72.5 | 75.5 | 69.4 | 62.5 | 52.8 | 50.8 |
| Average rooting rate (%) | 93.0 | 95.0 | 93.0 | 82.0 | 68.2 | 62.2 |
| Average root long | 2.5 | 2.5 | 2.4 | 2.2 | 1.98 | 1.95 |
From the results shown in Table 2, the average stem length of embodiment 1-3, growth coefficient, average inductivity, average life
Root rate, average root long are better than comparative example 1, illustrate the change due to disinfection way, mode using the present invention, does not injure explant
Body, during tissue culture, the growing state in each stage is all relatively preferable.Relative to comparative example 2, the average stem length of embodiment 1-3,
Growth coefficient be averaged inductivity, average rooting rate, average root long it is obvious preferably, illustrate to compare addition banana puree, use is peppery
Wooden leaf juice and white mulberry vein-juice are engaged as nutritional agents, the composition of culture medium is more suitable for the growth of four seasons orchid explant.Relative to
Comparative example 3, for embodiment 3 because the culture medium used is different, the growing state in each stage is also obvious preferable.Planting experiment shows
Tissue-cultured seedling in embodiment 1-3 plants survival rate up to more than 95%.
Described above is the detailed description for the present invention preferably possible embodiments, but embodiment is not limited to this hair
Bright patent claim, equal change or the modification change completed under the technical spirit suggested by all present invention, should all belong to
Cover the scope of the claims in the present invention.
Claims (8)
1. a kind of tissue culture and rapid propagation method of four seasons orchid, includes disinfection, Fiber differentiation, squamous subculture and the strong seedling culture of explant,
It is characterized in that concretely comprise the following steps：
(1) disinfection of explant：To bloom, four seasons of robust growth, no disease and pests harm it is blue as maternal, cut maternal bennet
On bennet bud as explant, rinse bennet bud with flowing water first, then 1min- is soaked with the alcohol that volumetric concentration is 75%
2min, then peels off the bract on bennet bud surface, continues to peel off one after then soaking 8min-15min in plant disinfectant
Layer bract, then continues to soak 5min-10min in the plant disinfectant, with sterile water 3-5 times, the explant after must sterilizing
Body；The plant disinfectant is mixed by the raw material of following parts by weight：0.05-0.08 parts of glycyrrhizic acid, isatis root extract
0.8-1.2 parts, 0.5-0.8 parts of bupleurum extract, 6-8 parts of OP-10,150-200 parts of water；
(2) Fiber differentiation：By the both ends notch of the explant after the disinfection, explant is seeded in inducing culture,
Light culture 10 days, then moves to the interior that temperature is 23-25 DEG C, intensity of illumination is 1500lx under conditions of temperature is 23-25 DEG C,
Daily illumination 10-14h is cultivated, and incubation time is 40-45 days；
(3) squamous subculture：The budlet that Fiber differentiation is sprouted, from after base portion stripping, is inoculated on subculture medium, culture temperature
23-25 DEG C, light application time 12-14h/d, intensity of illumination 1500lx of degree；
(4) strong seedling culture：After shoot proliferation culture 4-6 generations, high more than 2cm, the seedling for having obvious projection on explant is selected to turn
Enter and cultivated in strong seedling culture base, 23-25 DEG C of cultivation temperature, light application time 12-14h/d, intensity of illumination 2500lx, culture is extremely
Radical is 3-5 bars, blade 3-4 pieces, height of seedling 4-8cm, you can hardening, obtains the four seasons orchid seedling that can be transplanted after hardening.
2. the tissue culture and rapid propagation method of four seasons orchid according to claim 1, it is characterised in that：The inducing culture is：MS+
0.3-0.5mg/L6-BA+0.2-0.4mg/LNAA+0.2-0.4mg/L kinetin+40-55g/L Moringa leaf juice+30-40g/L mulberries
Leaf juice+40-50mg/L cysteine+20-25g/L sucrose+6-8g/L agar, pH 5.4-5.5.
3. the tissue culture and rapid propagation method of four seasons orchid according to claim 1, it is characterised in that：The composition of the subculture medium
For：Miller+0.2-0.3mg/L6-BA+0.1-0.2mg/LNAA+0.1-0.2mg/L kinetin+0.05-0.08mg/L corns
Element+40-55g/L Moringa leaf juice+30-40g/L white mulberry vein-juice+20-25g/L sucrose+6-8g/L agar, pH 5.5-5.6.
4. the tissue culture and rapid propagation method of four seasons orchid according to claim 1, it is characterised in that：The composition of the strong seedling culture base
For：Miller+0.2-0.3mg/L6-BA+0.1-0.2mg/LNAA+2-4mg/L indolebutyric acid+40-55g/L Moringa leaf juices+30-
40g/L white mulberry vein-juice+20-25g/L sucrose+6-8g/L agar, pH 5.6-5.7.
5. the tissue culture and rapid propagation method of four seasons orchid according to claim 1, it is characterised in that：The bupleurum extract is by bavin
Decocted 2 times, each 1.5-2h with the 8-10 times of water measured recklessly, merge extracting solution, filtering and concentrating is obtained to dry.
6. the tissue culture and rapid propagation method of four seasons orchid according to claim 1, it is characterised in that：The isatis root extract be by
Radix Isatidis is decocted 2 times, each 1.5-2h with the 8-10 times of water measured, and merges extracting solution, and filtering and concentrating is obtained to dry.
7. the tissue culture and rapid propagation method of four seasons orchid according to claim 1, it is characterised in that：The preparation side of the Moringa leaf juice
Method is：First clean and blade and then fully smash to pieces, then with filtered through gauze, the filtrate be white mulberry vein-juice.
8. the tissue culture and rapid propagation method of four seasons orchid according to claim 1, it is characterised in that：The white mulberry vein-juice is by fresh mulberry
Leaf is fully smashed to pieces after cleaning, then with filtered through gauze, the filtrate is white mulberry vein-juice.
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Citations (2)
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| CN106069772A (en) * | 2016-07-12 | 2016-11-09 | 成都东山兰韵农业有限公司 | A kind of sword-leaved cymbidium tissue culture quick propagation culturing method |
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|---|---|---|---|---|
| CN106069772A (en) * | 2016-07-12 | 2016-11-09 | 成都东山兰韵农业有限公司 | A kind of sword-leaved cymbidium tissue culture quick propagation culturing method |
| CN106386899A (en) * | 2016-09-07 | 2017-02-15 | 马鞍山纽泽科技服务有限公司 | Environment-friendly disinfector for fruit and vegetable |
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