CN108012929A - A kind of tissue cultures expanding propagation method of pleione bulbocodioides - Google Patents
A kind of tissue cultures expanding propagation method of pleione bulbocodioides Download PDFInfo
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- CN108012929A CN108012929A CN201711451050.1A CN201711451050A CN108012929A CN 108012929 A CN108012929 A CN 108012929A CN 201711451050 A CN201711451050 A CN 201711451050A CN 108012929 A CN108012929 A CN 108012929A
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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
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Abstract
The invention discloses a kind of tissue cultures expanding propagation method of pleione bulbocodioides, including the step of the step of the step of the step of sterilisation step of the selection step of explant, explant, sowing, squamous subculture, culture of rootage, hardening, rooting culture.The present invention utilizes plant tissue culture technique, and it is slow to overcome pleione bulbocodioides tradition division propagation, seed embryo hypoplasia; seed is difficult the problems such as independently developing into intact plant under natural conditions; meet at the same time prolific, reduce the excavation of wild resource, protect wild resource.
Description
Technical field
The present invention relates to the tissue cultures expanding propagation method of a kind of tissue cultivating and seedling technology, more particularly to a kind of pleione bulbocodioides.
Background technology
Pleione bulbocodioides Pleione bulbocodioides (Franch.) Rolfe, orchid family Pleion, ground life or grass of growing nonparasitically upon another plant
This.It is grown in the hayashishita or border stony ground of 1100-3500 meters of height above sea level or on the rock of moss covering, is also common in careless slope slightly
The gravel that the moon covers is on the ground.It is mainly distributed on the Sichuan Province China west and south (in Yuexi, Yanyuan, asbestos, Xichang, wood), WESTERN GUIZHOU extremely
Northern (Kerry, Yin Jiang, Leishan, Kweiyang, Mount Fanjing, Pan county, Anlong, Wangmo County, Nayong), northwestern Yunnan Province to the southeast (Gong Shan,
Wei Xi, Lijing, Dali, Yangbi, Yongning, Mengzi, mountain of papers) and Southeastern Tibet (Cha Walong), Upper Myanmar is also distributed.With root
Stem is used as medicine, and has effects that clearing heat and detoxicating, Sweeling-eliminating medicine powder section, preventing phlegm from forming and stopping coughing, is chiefly used in asthma and cough and arthroncus, venomous snake bite
Treatment etc..Pleione bulbocodioides pattern uniqueness is beautiful, has higher ornamental value, and scape sends from the old pseudobulb base portion of no leaf,
Uprightly, one flower of top tool, flower purple, lavender, pink or near-white sometimes, have purple or dark red color spot on lip, colored
Month phase 4-5, the fruiting period 9-10 months.
The pleione bulbocodioides to circulate currently on the market is mostly wild, and minority is artificial cultivation；Pleione bulbocodioides breeding is mostly division propagation,
Pollen pollination rate is smaller in natural environment, grows that the quantity of fruit pod is also less, and seed embryo hypoplasia can only be in aseptic condition
Under be seeded in culture medium and carry out tissue cultures.
The content of the invention
For overcome the deficiencies in the prior art, the tissue cultures it is an object of the invention to provide a kind of pleione bulbocodioides expand numerous side
Method.Root media using the present invention carry out tissue cultures take root the stage the advantages of be to overcome seed embryo hypoplasia, from
Under right environment the problems such as low reproduction rate；Root growth can be effectively promoted, improves speed and the breeding of pleione bulbocodioides seedling breeding
Quantity, from the stability of maternal source control germplasm, ensures seedling quality, realizes factory's metaplasia of pleione bulbocodioides high quality seed seedling
Production, satisfaction is viewed and admired, medicinal demand, reduces the situation that wild resource spreads unchecked excavation.
The purpose of the present invention adopts the following technical scheme that realization：A kind of tissue cultures expanding propagation method of pleione bulbocodioides, including,
The selection step of explant：The maternal plant of the 3-4 of health is selected, in pleione bulbocodioides duration of flowering pair mid-April
Pleione bulbocodioides carries out artificial pollination, allows it to grow fruit pod, is taken pains to foster by 4-6 months, and when fruit pod is ripe, i.e., outer kind shell is yellowish green
Color, intact nothing are split, and are then picked to put to refrigerator and are preserved in the environment of 3-7 DEG C 1 week；
The sterilisation step of explant：Fruit pod is taken out, fruit pod is soaked into 10-15min with liquid detergent water or washing powder water, with hair
Brush outwash kind shell surface, and rinse 8-10min with tap water；Clean beaker is put it to, moves to transfer room, in aseptic operating platform
It is upper to be carried out disinfection using medicinal alcohol with mercuric chloride, finally it is put into spare in the sterile disk on superclean bench；
The step of sowing：The fruit pod disinfected is splitted to the endosperm for exposing white filiform with cooling scalpel and tweezers solution,
That is fruit powder；Fruit powder is spread in Initial culture base, lid is sealed after sterilizing bottle cap, goes to culturing room and cultivated, cultivates 30-45
My god, seed starts to sprout；
The step of squamous subculture：The pleione bulbocodioides of sprouting is grown 50-70 days, and pleione bulbocodioides seedling is referred to as when growing to 2-3cm high
Level-one mother's bottle, level-one bottle seedling is gone in subculture medium cultivate 70 days at this time on aseptic operating platform, pleione bulbocodioides seedling grow to
Referred to as two level mother bottle during 5-6cm；
The step of culture of rootage：The two level mother's bottle for growing 70 days is gone in root media on aseptic operating platform and is taken root
Culture 90-120 days；
The step of hardening, rooting culture：Bottle seedling take root after culturing room has cultivated, takes root good, seedling is sturdy, is moved into
Placed 15-20 days in outdoor environment, avoid strong light direct beam, allow it to adapt to external environment；Open bottle cap to place 3-5 days, seedling is fallen
Go out clean culture medium, soak pleione bulbocodioides bottle seedling root 5-10min with the carbendazim for being diluted to 800-1000 times, clean the water that dries in the shade
Point, it is transplanted in matrix and is tamed.
Further, in the selection step of explant, the maternal plant of 3 years of health is selected.
Further, in the sterilisation step of explant, disappeared on aseptic operating platform using medicinal alcohol with mercuric chloride
The concrete operations of poison are as follows：30-60s first is soaked with 75% medicinal alcohol, being stirred continuously during immersion with tweezers makes its disinfection thorough
Bottom, aseptic water washing 3-4 times；4-6min finally is sterilized with 0.2% mercuric chloride, aseptic water washing 4-5 times, is put into superclean bench
On sterile disk in it is spare.
Further, in the step of sowing, the indoor condition of culture of culture is as follows：Temperature is 24 DEG C -26 DEG C, light
It is 2000-2500lx according to intensity, when light application time is 10-12 small/day.
Further, in the step of sowing, the component of the Initial culture base is potassium nitrate 1890-1900mg/L, nitre
Sour ammonium 1640-1650mg/L, calcium chloride dihydrate 440-445mg/L, epsom salt 370-375mg/L, potassium dihydrogen phosphate 160-
170mg/L, potassium iodide 0.8-0.83mg/L, boric acid 6.0-6.2mg/L, four water manganese sulfate 22.3-23.0mg/L, white vitriol
8.6-9.0mg/L, sodium molybdate 0.2-0.25mg/L, cupric sulfate pentahydrate 0.025-0.028mg/L, cobalt chloride 0.028-0.03mg/
L, ferrous sulfate 27.5-29mg/L, ethylenediamine tetra-acetic acid 23-25mg/L, inositol 96-100mg/L, nicotinic acid 0.2-0.3mg/L, salt
Sour pyridoxol 0.25-0.35mg/L, thiamine hydrochloride 0.2-0.3mg/L, glycine 0.8-0.9mg/L, sucrose 2g/L, agar powder
4.8g/L；The pH of culture medium is adjusted to 5.8-6.0 by the Initial culture base with acid-base indicator.
Further, in the step of squamous subculture, the pleione bulbocodioides of the sprouting is 2000-2500lx in intensity of illumination,
Light application time for 10-12 it is small when/day under conditions of grow 50-70 days.
Further, in the step of squamous subculture, the component of the subculture medium is potassium nitrate 1890-1900mg/
L, ammonium nitrate 1640-1650mg/L, calcium chloride dihydrate 440-445mg/L, epsom salt 370-375mg/L, potassium dihydrogen phosphate
160-170mg/L, potassium iodide 0.8-0.83mg/L, boric acid 6.0-6.2mg/L, four water manganese sulfate 22.3-23.0mg/L, seven water sulphur
Sour zinc 8.6-9.0mg/L, sodium molybdate 0.2-0.25mg/L, cupric sulfate pentahydrate 0.025-0.028mg/L, cobalt chloride 0.028-
0.03mg/L, ferrous sulfate 27.5-29mg/L, ethylenediamine tetra-acetic acid 23-25mg/L, inositol 96-100mg/L, nicotinic acid 0.2-
0.3mg/L, puridoxine hydrochloride 0.25-0.35mg/L, thiamine hydrochloride 0.2-0.3mg/L, glycine 0.8-0.9mg/L, also adds
Add the soil of the indolebutyric acid of 0.2mg/L, the 6-benzyladenine of 1.8mg/L, the sucrose of 3g/L, the agar powder of 4.8g/L, 35g/L
The activated carbon powder of fermented bean drink, 0.3g/L；The PH of culture medium is adjusted to 5.8-6.0 by the subculture medium with acid-base indicator.Wherein,
The preparation process of murphy juice is as follows：Take 35g potatoes to pour into 1L sterile waters to crush, form the murphy juice containing mealy potato.
Further, in the step of culture of rootage, the component of the root media is potassium nitrate 3790-3800mg/
L, ammonium nitrate 3290-3300mg/L, calcium chloride dihydrate 870-880mg/L, epsom salt 730-740mg/L, potassium dihydrogen phosphate
330-340mg/L, potassium iodide 1.62-1.66mg/L, boric acid 12.0-12.44mg/L, four water manganese sulfate 44.6-46mg/L, seven water
Zinc sulfate 17.2-18mg/L, sodium molybdate 0.4-0.5mg/L, cupric sulfate pentahydrate 0.05-0.06mg/L, cobalt chloride 0.05-
0.06mg/L, ferrous sulfate 55-58mg/L, ethylenediamine tetra-acetic acid 46-50mg/L, inositol 192-200mg/L, nicotinic acid 0.4-
0.6mg/L, puridoxine hydrochloride 0.5-0.7mg/L, thiamine hydrochloride 0.4-0.6mg/L, glycine 1.6-1.8mg/L, is also added
The indolebutyric acid of 2.0mg/L, the 6-benzyladenine of 0.5mg/L, the sucrose of 3g/L, the agar powder of 4.8g/L, the potato of 35g/L
Juice, the bananas juice of 25g/L, the activated carbon powder of 0.3g/L；The PH of culture medium is adjusted to by the root media with acid-base indicator
5.8-6.0.Wherein, the preparation process of murphy juice is as follows：Take 35g potatoes to pour into 1L sterile waters to crush, formed containing mealy potato
Murphy juice.The preparation process of bananas juice is as follows：Take 25g bananas to pour into 1L sterile waters to crush, form the bananas juice containing banana puree.
Further, in the step of hardening, rooting culture, the matrix is by fertile soil, the pine bark crushed, wood fragments
Bits, vermiculite are with mass ratio 3:2:1:1 ratio, which is uniformly mixed, to be made.
Compared with prior art, the beneficial effects of the present invention are：
The present invention uses 1/2MS culture mediums in the Initial culture base of sowing stage, only adds sucrose and agar powder, second-order
The subculture medium and root media that section (squamous subculture), phase III (culture of rootage) use, add one in culture medium
Determine the auxin of concentration；First stage 1/2MS and MS culture medium contrasts, and on the germination and growth of seedling without influence, uses
1/2MS culture mediums can save raw material；Second stage can save raw material using 1/2MS culture mediums, but due to pleione bulbocodioides seedling
Growth time is longer, needs to add certain density auxin in culture medium；Phase III is given birth to due to pleione bulbocodioides seedling
It is longer for a long time, while develop the needs that increase sharply, it is necessary to MS culture mediums and the certain density auxin ability of addition
The nutrition of abundance is provided for its growth；Using plant tissue culture technique, it is slow to overcome pleione bulbocodioides tradition division propagation, seed
Embryonic development is not complete, and seed is difficult the problems such as independently developing into intact plant under natural conditions, while meets prolific, subtracts
The excavation of few wild resource, protects wild resource.Tissue cultures provide suitable humiture, illumination condition for pleione bulbocodioides at the same time,
Sufficient nutritional ingredient, increases light application time, increases the speed of growth of plant, differentiates the root system of prosperity, ensure plant into
Motility rate.
Embodiment
In the following, with reference to embodiment, the present invention is described further, it is necessary to which explanation is, what is do not collided
Under the premise of, new embodiment can be formed between various embodiments described below or between each technical characteristic in any combination.
In the present invention, if not refering in particular to, all parts, percentage are unit of weight, used equipment and raw material etc.
It is commercially available or commonly used in the art.Method in following embodiments, is the normal of this area unless otherwise instructed
Rule method.
A kind of tissue cultures expanding propagation method of pleione bulbocodioides, including,
The selection step of explant：The maternal plant of the 3-4 of health is selected, in pleione bulbocodioides duration of flowering pair mid-April
Pleione bulbocodioides carries out artificial pollination, allows it to grow fruit pod, is taken pains to foster by 4-6 months, and when fruit pod is ripe, i.e., outer kind shell is yellowish green
Color, intact nothing are split, and are then picked to put to refrigerator and are preserved in the environment of 3-7 DEG C 1 week；
The sterilisation step of explant：Fruit pod is taken out, fruit pod is soaked into 10-15min with liquid detergent water or washing powder water, with hair
Brush outwash kind shell surface, and rinse 8-10min with tap water；Clean beaker is put it to, moves to transfer room, in aseptic operating platform
It is upper to be carried out disinfection using medicinal alcohol with mercuric chloride, finally it is put into spare in the sterile disk on superclean bench；
The step of sowing：The fruit pod disinfected is splitted to the endosperm for exposing white filiform with cooling scalpel and tweezers solution,
That is fruit powder；Fruit powder is spread in Initial culture base, lid is sealed after sterilizing bottle cap, goes to culturing room and cultivated, cultivates 30-45
My god, seed starts to sprout；
The step of squamous subculture：The pleione bulbocodioides of sprouting is grown 50-70 days, and pleione bulbocodioides seedling is referred to as when growing to 2-3cm high
Level-one mother's bottle, level-one bottle seedling is gone in subculture medium cultivate 70 days at this time on aseptic operating platform, pleione bulbocodioides seedling grow to
Referred to as two level mother bottle during 5-6cm；
The step of culture of rootage：The two level mother's bottle for growing 70 days is gone in root media on aseptic operating platform and is taken root
Culture 90-120 days；
The step of hardening, rooting culture：Bottle seedling take root after culturing room has cultivated, takes root good, seedling is sturdy, is moved into
Placed 15-20 days in outdoor environment, avoid strong light direct beam, allow it to adapt to external environment；Open bottle cap to place 3-5 days, seedling is fallen
Go out clean culture medium, soak pleione bulbocodioides bottle seedling root 5-10min with the carbendazim for being diluted to 800-1000 times, clean the water that dries in the shade
Point, it is transplanted in matrix and is tamed.
As further embodiment, in the selection step of explant, the maternal plant of 3 years of health is selected.Its
In 3 years raw plant it is optimal can just bloom because pleione bulbocodioides will be grown 2 years or so.
As further embodiment, in the sterilisation step of explant, medicinal alcohol is used on aseptic operating platform
The concrete operations to carry out disinfection with mercuric chloride are as follows：30-60s first is soaked with 75% medicinal alcohol, is constantly stirred with tweezers during immersion
Mixing makes its disinfection thorough, aseptic water washing 3-4 times；4-6min finally is sterilized with 0.2% mercuric chloride, aseptic water washing 4-5 times, puts
It is spare in sterile disk on to superclean bench.Wherein, the relatively other disinfection reagent disinfections of 75% alcohol are more thorough, and the time is more
It is short.A most important link seed disinfection is thorough during with pleione bulbocodioides seed tissue culture propagation, and disinfection thoroughly just can guarantee that seed is broadcast
Kind wild Oryza species do not infect, and in the case where the specified conditions such as temperature, illumination are adapted to, the germination percentage of pleione bulbocodioides seed seedling-raising is more than
85%, ensure that kind is pure.
As further embodiment, in the step of sowing, the indoor condition of culture of culture is as follows：Temperature is
24 DEG C -26 DEG C, intensity of illumination 2000-2500lx, when light application time is 10-12 small/day.
As further embodiment, in the step of sowing, the component of the Initial culture base is potassium nitrate 1890-
1900mg/L, ammonium nitrate 1640-1650mg/L, calcium chloride dihydrate 440-445mg/L, epsom salt 370-375mg/L, phosphoric acid
Potassium dihydrogen 160-170mg/L, potassium iodide 0.8-0.83mg/L, boric acid 6.0-6.2mg/L, four water manganese sulfate 22.3-23.0mg/L,
White vitriol 8.6-9.0mg/L, sodium molybdate 0.2-0.25mg/L, cupric sulfate pentahydrate 0.025-0.028mg/L, cobalt chloride
0.028-0.03mg/L, ferrous sulfate 27.5-29mg/L, ethylenediamine tetra-acetic acid 23-25mg/L, inositol 96-100mg/L, nicotinic acid
0.2-0.3mg/L, puridoxine hydrochloride 0.25-0.35mg/L, thiamine hydrochloride 0.2-0.3mg/L, glycine 0.8-0.9mg/L,
Sucrose 2g/L, agar powder 4.8g/L；The pH of culture medium is adjusted to 5.8-6.0 by the Initial culture base with acid-base indicator.
As further embodiment, in the step of squamous subculture, the pleione bulbocodioides of the sprouting is in intensity of illumination
2000-2500lx, light application time for 10-12 it is small when/day under conditions of grow 50-70 days.
As further embodiment, in the step of squamous subculture, the component of the subculture medium is potassium nitrate
1890-1900mg/L, ammonium nitrate 1640-1650mg/L, calcium chloride dihydrate 440-445mg/L, epsom salt 370-375mg/
L, potassium dihydrogen phosphate 160-170mg/L, potassium iodide 0.8-0.83mg/L, boric acid 6.0-6.2mg/L, four water manganese sulfate 22.3-
23.0mg/L, white vitriol 8.6-9.0mg/L, sodium molybdate 0.2-0.25mg/L, cupric sulfate pentahydrate 0.025-0.028mg/L,
Cobalt chloride 0.028-0.03mg/L, ferrous sulfate 27.5-29mg/L, ethylenediamine tetra-acetic acid 23-25mg/L, inositol 96-100mg/
L, nicotinic acid 0.2-0.3mg/L, puridoxine hydrochloride 0.25-0.35mg/L, thiamine hydrochloride 0.2-0.3mg/L, glycine 0.8-
0.9mg/L, also adds the indolebutyric acid of 0.2mg/L, the 6-benzyladenine of 1.8mg/L, the sucrose of 3g/L, the agar of 4.8g/L
Powder, the murphy juice of 35g/L, the activated carbon powder of 0.3g/L；The PH of culture medium is adjusted to by the subculture medium with acid-base indicator
5.8-6.0。
As further embodiment, in the step of culture of rootage, the component of the root media is potassium nitrate
3790-3800mg/L, ammonium nitrate 3290-3300mg/L, calcium chloride dihydrate 870-880mg/L, epsom salt 730-740mg/
L, potassium dihydrogen phosphate 330-340mg/L, potassium iodide 1.62-1.66mg/L, boric acid 12.0-12.44mg/L, four water manganese sulfates
44.6-46mg/L, white vitriol 17.2-18mg/L, sodium molybdate 0.4-0.5mg/L, cupric sulfate pentahydrate 0.05-0.06mg/L,
Cobalt chloride 0.05-0.06mg/L, ferrous sulfate 55-58mg/L, ethylenediamine tetra-acetic acid 46-50mg/L, inositol 192-200mg/L,
Nicotinic acid 0.4-0.6mg/L, puridoxine hydrochloride 0.5-0.7mg/L, thiamine hydrochloride 0.4-0.6mg/L, glycine 1.6-1.8mg/
L, also add the indolebutyric acid of 2.0mg/L, the 6-benzyladenine of 0.5mg/L, the sucrose of 3g/L, 4.8g/L agar powder,
The murphy juice of 35g/L, the bananas juice of 25g/L, the activated carbon powder of 0.3g/L；The root media will be cultivated with acid-base indicator
The PH of base is adjusted to 5.8-6.0.
As further embodiment, in the step of hardening, rooting culture, the matrix by fertile soil, crush
Pine bark, wood fragments bits, vermiculite are with mass ratio 3:2:1:1 ratio, which is uniformly mixed, to be made.
It is specific embodiment of the present invention below, used raw material, equipment etc. remove special limit in the following embodiments
It can be obtained outside fixed by buying pattern.
Embodiment 1-3The Initial culture base of pleione bulbocodioides
The proportioning according to the form below 1 weighs raw material respectively, is prepared in accordance with the following steps, difference is what is added
Raw material proportioning is different, prepares pleione bulbocodioides Initial culture base product, specifically refers to table 1：
Table 1：The raw material proportioning table of embodiment 1-3
Embodiment 4-6Pleione bulbocodioides subculture medium
The proportioning according to the form below 2 weighs raw material respectively, is prepared in accordance with the following steps, difference is what is added
Raw material proportioning is different, prepares pleione bulbocodioides subculture medium product, specifically refers to table 2：
Table 2：The raw material proportioning table of embodiment 4-6
Embodiment 7-9Pleione bulbocodioides root media
The proportioning according to the form below 3 weighs raw material respectively, is prepared in accordance with the following steps, difference is what is added
Raw material proportioning is different, prepares pleione bulbocodioides root media product, specifically refers to table 3：
Table 3：The raw material proportioning table of embodiment 7-9
A kind of tissue cultures expanding propagation method of 10 pleione bulbocodioides of embodiment
The tissue cultures expanding propagation method of the pleione bulbocodioides, including,
The selection step of explant：Select health the maternal plant of 3 years, mid-April pleione bulbocodioides duration of flowering to only
Garlic is blue to carry out artificial pollination, allows it to grow fruit pod, is taken pains to foster by 4-6 months, and when fruit pod is ripe, i.e., outer kind shell is yellowish green
Color, intact nothing are split, and are then picked to put to refrigerator and are preserved in the environment of 3-7 DEG C 1 week；
The sterilisation step of explant：Fruit pod is taken out, fruit pod is soaked into 10-15min with liquid detergent water or washing powder water, with hair
Brush outwash kind shell surface, and rinse 8-10min with tap water；Clean beaker is put it to, moves to transfer room, in aseptic operating platform
Upper first to soak 30-60s with 75% medicinal alcohol, being stirred continuously during immersion with tweezers makes its disinfection thorough, aseptic water washing 3-4
It is secondary；4-6min finally is sterilized with 0.2% mercuric chloride, aseptic water washing 4-5 times, is put into standby in the sterile disk on superclean bench
With.Wherein, the relatively other disinfection reagent disinfections of 75% alcohol are more thorough, and the time is shorter.
The step of sowing：The fruit pod disinfected is splitted to the endosperm for exposing white filiform with cooling scalpel and tweezers solution,
That is fruit powder；Fruit powder is spread in the Initial culture base of embodiment 2, seals lid after sterilizing bottle cap, go to culturing room is in temperature
24 DEG C -26 DEG C, intensity of illumination 2000-2500lx, light application time for 10-12 it is small when/day under conditions of cultivated, cultivate
30-45 days, seed started to sprout；
The step of squamous subculture：The pleione bulbocodioides of sprouting is 2000-2500lx in intensity of illumination, and light application time is small for 10-12
When/day under conditions of grow 50-70 days, referred to as level-one mother bottle when pleione bulbocodioides seedling grows to 2-3cm high, at this time by level-one bottle seedling
Go in the subculture medium of embodiment 5 and cultivate 70 days on aseptic operating platform, pleione bulbocodioides seedling length to referred to as two level during 5-6cm
Female bottle；
The step of culture of rootage：The two level mother's bottle for growing 70 days is gone to the training of taking root of embodiment 8 on aseptic operating platform
Support culture of rootage 90-120 days in base；
The step of hardening, rooting culture：Bottle seedling take root after culturing room has cultivated, takes root good, seedling is sturdy, is moved into
Placed 15-20 days in outdoor environment, avoid strong light direct beam, allow it to adapt to external environment；Open bottle cap to place 3-5 days, seedling is fallen
Go out clean culture medium, soak pleione bulbocodioides bottle seedling root 5-10min with the carbendazim for being diluted to 800-1000 times, clean the water that dries in the shade
Point, it is transplanted in matrix and is tamed.The matrix is by fertile soil, the pine bark crushed, wood fragments bits, vermiculite with mass ratio
3:2:1:1 ratio, which is uniformly mixed, to be made.
The present invention utilizes plant tissue culture technique, overcomes that pleione bulbocodioides tradition division propagation is slow, and seed embryonic development is not
Entirely, seed is difficult the problems such as independently developing into intact plant under natural conditions, while meets prolific, reduces wild money
The excavation in source, protects wild resource.Tissue cultures provide suitable humiture, illumination condition, sufficient battalion for pleione bulbocodioides at the same time
Form point, increase light application time, increase the speed of growth of plant, differentiate the root system of prosperity, ensure the survival rate of plant.
1/2MS culture mediums are used in the Initial culture base of sowing stage, only addition sucrose and agar powder, second stage (after
Be commissioned to train foster), the phase III (culture of rootage) use subculture medium and root media, a certain concentration is added in culture medium
Auxin；First stage 1/2MS and MS culture medium contrasts, and on the germination and growth of seedling without influence, is trained using 1/2MS
Foster base can save raw material；Second stage can save raw material using 1/2MS culture mediums, but due to the pleione bulbocodioides seedling early growth time
It is longer, need to add certain density auxin in culture medium；Phase III due to the pleione bulbocodioides seedling early growth time compared with
It is long, while develop needs that increase sharply, it is necessary to which that with MS culture mediums and to add certain density auxin could be its growth
Sufficient nutrition is provided.
A most important link seed disinfection is thorough during with pleione bulbocodioides seed tissue culture propagation, and disinfection thoroughly just can guarantee that kind
Son sowing wild Oryza species do not infect, in the case where the specified conditions such as temperature, illumination are adapted to, the germination percentage of pleione bulbocodioides seed seedling-raising
More than 85%, ensure that kind is pure.
The above embodiment is only the preferred embodiment of the present invention, it is impossible to the scope of protection of the invention is limited with this,
The change and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed scope.
Claims (9)
- A kind of 1. tissue cultures expanding propagation method of pleione bulbocodioides, it is characterised in that including,The selection step of explant：Select health 3-4 maternal plant, mid-April pleione bulbocodioides duration of flowering to only garlic Orchid carries out artificial pollination, allows it to grow fruit pod, is taken pains to foster by 4-6 months, when fruit pod is ripe, i.e., outer kind shell yellow green, Intact nothing is split, and is then picked to put to refrigerator and is preserved in the environment of 3-7 DEG C 1 week；The sterilisation step of explant：Fruit pod is taken out, fruit pod is soaked into 10-15min with liquid detergent water or washing powder water, with hairbrush brush Net kind shell surface, and rinse 8-10min with tap water；Clean beaker is put it to, moves to transfer room, is adopted on aseptic operating platform Carried out disinfection, be finally put into spare in the sterile disk on superclean bench with medicinal alcohol and mercuric chloride；The step of sowing：The fruit pod disinfected is splitted to the endosperm for exposing white filiform, i.e. fruit with cooling scalpel and tweezers solution Powder；Fruit powder is spread in Initial culture base, lid is sealed after sterilizing bottle cap, goes to culturing room and cultivated, is cultivated 30-45 days, Seed starts to sprout；The step of squamous subculture：The pleione bulbocodioides of sprouting is grown 50-70 days, referred to as level-one when pleione bulbocodioides seedling grows to 2-3cm high Female bottle, level-one bottle seedling is gone in subculture medium cultivate 70 days at this time on aseptic operating platform, pleione bulbocodioides seedling length to 5-6cm When referred to as two level mother bottle；The step of culture of rootage：The two level mother's bottle for growing 70 days is gone into culture of rootage in root media on aseptic operating platform 90-120 days；The step of hardening, rooting culture：Bottle seedling take root after culturing room has cultivated, takes root good, seedling is sturdy, is moved into outdoor Placed 15-20 days in environment, avoid strong light direct beam, allow it to adapt to external environment；Open bottle cap to place 3-5 days, seedling is poured out and is washed Net culture medium, soaks pleione bulbocodioides bottle seedling root 5-10min with the carbendazim for being diluted to 800-1000 times, cleans the moisture that dries in the shade, will It is transplanted in matrix and is tamed.
- 2. the tissue cultures expanding propagation method of pleione bulbocodioides as claimed in claim 1, it is characterised in that in the selection step of explant In, select the healthy maternal plant of 3 years.
- 3. the tissue cultures expanding propagation method of pleione bulbocodioides as claimed in claim 1, it is characterised in that in the sterilisation step of explant In, it is as follows using medicinal alcohol and concrete operations that mercuric chloride carries out disinfection on aseptic operating platform：First soaked with 75% medicinal alcohol 30-60s is steeped, being stirred continuously during immersion with tweezers makes its disinfection thorough, aseptic water washing 3-4 times；Finally with 0.2% mercuric chloride 4-6min is sterilized, aseptic water washing 4-5 times, is put into spare in the sterile disk on superclean bench.
- 4. the tissue cultures expanding propagation method of pleione bulbocodioides as claimed in claim 1, it is characterised in that in the step of sowing, institute It is as follows to state the indoor condition of culture of culture：Temperature is 24 DEG C -26 DEG C, intensity of illumination 2000-2500lx, light application time 10- 12 it is small when/day.
- 5. the tissue cultures expanding propagation method of pleione bulbocodioides as claimed in claim 1, it is characterised in that in the step of sowing, institute The component for stating Initial culture base is potassium nitrate 1890-1900mg/L, ammonium nitrate 1640-1650mg/L, calcium chloride dihydrate 440- 445mg/L, epsom salt 370-375mg/L, potassium dihydrogen phosphate 160-170mg/L, potassium iodide 0.8-0.83mg/L, boric acid 6.0-6.2mg/L, four water manganese sulfate 22.3-23.0mg/L, white vitriol 8.6-9.0mg/L, sodium molybdate 0.2-0.25mg/L, Cupric sulfate pentahydrate 0.025-0.028mg/L, cobalt chloride 0.028-0.03mg/L, ferrous sulfate 27.5-29mg/L, ethylenediamine tetrem Sour 23-25mg/L, inositol 96-100mg/L, nicotinic acid 0.2-0.3mg/L, puridoxine hydrochloride 0.25-0.35mg/L, thiamine hydrochloride 0.2-0.3mg/L, glycine 0.8-0.9mg/L, sucrose 2g/L, agar powder 4.8g/L；The Initial culture base is indicated with soda acid The pH of culture medium is adjusted to 5.8-6.0 by agent.
- 6. the tissue cultures expanding propagation method of pleione bulbocodioides as claimed in claim 1, it is characterised in that the squamous subculture the step of In, the pleione bulbocodioides of the sprouting is 2000-2500lx in intensity of illumination, light application time for 10-12 it is small when/day under conditions of grow 50-70 days.
- 7. the tissue cultures expanding propagation method of pleione bulbocodioides as claimed in claim 1, it is characterised in that the squamous subculture the step of In, the component of the subculture medium is potassium nitrate 1890-1900mg/L, ammonium nitrate 1640-1650mg/L, calcium chloride dihydrate 440-445mg/L, epsom salt 370-375mg/L, potassium dihydrogen phosphate 160-170mg/L, potassium iodide 0.8-0.83mg/L, boron Sour 6.0-6.2mg/L, four water manganese sulfate 22.3-23.0mg/L, white vitriol 8.6-9.0mg/L, sodium molybdate 0.2-0.25mg/ L, cupric sulfate pentahydrate 0.025-0.028mg/L, cobalt chloride 0.028-0.03mg/L, ferrous sulfate 27.5-29mg/L, ethylenediamine tetraacetic Acetic acid 23-25mg/L, inositol 96-100mg/L, nicotinic acid 0.2-0.3mg/L, puridoxine hydrochloride 0.25-0.35mg/L, thiamine hydrochloride Plain 0.2-0.3mg/L, glycine 0.8-0.9mg/L, indolebutyric acid, the 6- benzyl glands of 1.8mg/L for also adding 0.2mg/L are fast Purine, the sucrose of 3g/L, the agar powder of 4.8g/L, the murphy juice of 35g/L, the activated carbon powder of 0.3g/L；The subculture medium is used The PH of culture medium is adjusted to 5.8-6.0 by acid-base indicator.
- 8. the tissue cultures expanding propagation method of pleione bulbocodioides as claimed in claim 1, it is characterised in that the culture of rootage the step of In, the component of the root media is potassium nitrate 3790-3800mg/L, ammonium nitrate 3290-3300mg/L, calcium chloride dihydrate 870-880mg/L, epsom salt 730-740mg/L, potassium dihydrogen phosphate 330-340mg/L, potassium iodide 1.62-1.66mg/L, Boric acid 12.0-12.44mg/L, four water manganese sulfate 44.6-46mg/L, white vitriol 17.2-18mg/L, sodium molybdate 0.4- 0.5mg/L, cupric sulfate pentahydrate 0.05-0.06mg/L, cobalt chloride 0.05-0.06mg/L, ferrous sulfate 55-58mg/L, ethylenediamine Tetraacethyl 46-50mg/L, inositol 192-200mg/L, nicotinic acid 0.4-0.6mg/L, puridoxine hydrochloride 0.5-0.7mg/L, hydrochloric acid sulphur Amine element 0.4-0.6mg/L, glycine 1.6-1.8mg/L, indolebutyric acid, the 6- benzyl glands of 0.5mg/L for also adding 2.0mg/L are fast Purine, the sucrose of 3g/L, the agar powder of 4.8g/L, the murphy juice of 35g/L, the bananas juice of 25g/L, the activated carbon powder of 0.3g/L；Institute State root media and the PH of culture medium is adjusted to 5.8-6.0 with acid-base indicator.
- 9. the tissue cultures expanding propagation method of pleione bulbocodioides as claimed in claim 1, it is characterised in that in hardening, rooting culture In step, the matrix is by fertile soil, the pine bark crushed, wood fragments bits, vermiculite with mass ratio 3:2:1:1 ratio is uniformly mixed It is made.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108739376A (en) * | 2018-04-28 | 2018-11-06 | 中国科学院武汉植物园 | Turn round the blue rapid propagation method of valve U.S. hat |
| CN110972956A (en) * | 2020-01-02 | 2020-04-10 | 昭通芸生农业科技开发有限公司 | Pleione seed tissue culture method |
| CN114223545A (en) * | 2022-01-12 | 2022-03-25 | 中国科学院昆明植物研究所 | Culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds and application and cultivation method thereof |
| CN114431145A (en) * | 2022-02-15 | 2022-05-06 | 杭州市农业科学研究院 | Pleione tissue rapid propagation method based on somatic embryo approach |
| CN115093276A (en) * | 2022-05-13 | 2022-09-23 | 和田博正农业科技有限公司 | Preparation and application method of broad-spectrum rooting agent |
| CN115997685A (en) * | 2023-01-18 | 2023-04-25 | 云南山里红生物科技有限公司 | Tissue culture and rapid propagation method of cymbidium sinense |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739376A (en) * | 2018-04-28 | 2018-11-06 | 中国科学院武汉植物园 | Turn round the blue rapid propagation method of valve U.S. hat |
| CN110972956A (en) * | 2020-01-02 | 2020-04-10 | 昭通芸生农业科技开发有限公司 | Pleione seed tissue culture method |
| CN114223545A (en) * | 2022-01-12 | 2022-03-25 | 中国科学院昆明植物研究所 | Culture medium for improving germination rate of Pleione bulbocodioides distant hybrid seeds and application and cultivation method thereof |
| CN114431145A (en) * | 2022-02-15 | 2022-05-06 | 杭州市农业科学研究院 | Pleione tissue rapid propagation method based on somatic embryo approach |
| CN114431145B (en) * | 2022-02-15 | 2023-01-03 | 杭州市农业科学研究院 | Pleione tissue rapid propagation method based on somatic embryo approach |
| CN115093276A (en) * | 2022-05-13 | 2022-09-23 | 和田博正农业科技有限公司 | Preparation and application method of broad-spectrum rooting agent |
| CN115997685A (en) * | 2023-01-18 | 2023-04-25 | 云南山里红生物科技有限公司 | Tissue culture and rapid propagation method of cymbidium sinense |
| CN115997685B (en) * | 2023-01-18 | 2023-10-20 | 云南山里红生物科技有限公司 | Tissue culture and rapid propagation method of cymbidium sinense |
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