A kind of tissue cultivating and seedling method of the aster stem of noble dendrobium
Technical field
The present invention relates to technical field of tissue culture more particularly to a kind of tissue cultivating and seedling methods of the aster stem of noble dendrobium.
Background technology
The aster stem of noble dendrobium (Dendrobium transparens) is orchid family, is mainly distributed on Burma, main point within Chinese territory
Cloth is most from the import of country of Burma in Guangxi;Aster stem of Dendrobium is sagging, slightly meat, cylindrical, not branch, has more piece.Total shape flower
Sequence is multiple, and in the long old stem to fallen leaves, each anthocaulus usually has 2 flowers, flower pattern great achievement purple powder, and film quality is carried out;Petal is closely ellipse
Circle, lip oval are in purple powder, and both sides hold around stamen column and formed horn-like, and horn-like position is dark purple.The aster stem of noble dendrobium provides
Source is rare, pattern it is pure and fresh it is beautiful, have light fragrant, the florescence is up to 2 months, has higher ornamental value.
But there are following defects for the existing aster stem of noble dendrobium:The aster stem of noble dendrobium to circulate on domestic market mainly by from
The import of country of Burma, small part derive from Guangxi, there is no artificial cultivation, maternal less, introduces a collection is rare;Generally pass through division propagation
It is bred with the mode of cuttage, breeding seedling is less, and low reproduction rate, growth cycle is longer, without people under normal habitat
The probability of work pollination result is smaller;Since fruit pod is few, fruit powder is tiny, is not easy the factors such as germination and growth in a natural environment
It restricts so that the aster stem of noble dendrobium seedling breeding with high ornamental value is obstructed.
Invention content
For overcome the deficiencies in the prior art, the purpose of the present invention is to provide a kind of tissue cultivating and seedlings of the aster stem of noble dendrobium
Method can effectively improve the breeding speed of aster stem of noble dendrobium seedling and breeding quantity, from the stabilization of maternal source control germplasm
Property, ensure seedling quality, realize the factorial praluction of aster stem of noble dendrobium high quality seed seedling, meet production requirement, it is dilute to alleviate resource
Scarce present situation.
The purpose of the present invention adopts the following technical scheme that realization:
A kind of tissue cultivating and seedling method of the aster stem of noble dendrobium, including:
Explant selects step:Artificial pollination is carried out in the florescence of aster stem of noble dendrobium maternal plant, after growing fruit pod, is continued
It cultivates, harvesting ripe fruit pod is placed in preserving in refrigerator, obtains spare fruit pod;
Fruit pod sterilisation step:The spare fruit pod is taken, kind shell surface clean is clean, obtain the first clean fruit pod;By institute
It states the first clean fruit pod to move in transfer room, carries out alcohol disinfecting and aseptic water washing, obtain the second clean fruit pod;By described
Two clean fruit pods obtain third cleaning fruit pod with hypochlorite disinfectant and aseptic water washing;The third cleaning fruit pod is risen
Mercury sterilizes and aseptic water washing, obtains the 4th clean fruit pod;
Sow step:The described 4th clean fruit pod is dissected, fruit powder is spread on 1/2MS culture mediums, is transferred in culturing room
Culture, obtains germinating seed;
Subculture step:The germinating seed is placed in culturing room and continues to cultivate, until seedling grows to 2-3cm, is obtained
Take level-one bottle seedling;The level-one bottle seedling is transferred in 1/2MS culture mediums, when growing to 5-6cm, obtains two level bottle seedling;
Culture of rootage step:The two level bottle seedling is transferred in root media and is cultivated, obtains bottle seedling of taking root;
Hardening tames step:The bottle seedling of taking root is placed in culturing room and is cultivated, then moves on in outdoor environment and is tamed and dociled
Change, obtain domesticated seedlings;
Transplant step:The bottle cap of the root media is opened, continues to place the domesticated seedlings 3-5 days, then pour out,
And seedling root is impregnated with carbendazim, it then cleans and dries in the shade, transplant and tamed into matrix.
Further, in the explant selects step, the plant age of aster stem of noble dendrobium maternal plant is 2-4;It is long
Continue to cultivate 3-4 months after going out fruit pod;The ripe fruit pod is placed in 3-7 DEG C of refrigerator and is preserved 1.5-2.5 weeks.
Further, in the fruit pod sterilisation step, spare fruit pod liquid detergent water or washing powder water are impregnated
15-20min cleans down kind of a shell surface, and rinse 10-15min with tap water with hairbrush, obtains the first clean fruit pod;By described in
First clean fruit pod is put into clean beaker, is moved to transfer room, is impregnated 30-60s with 75% medicinal alcohol on aseptic operating platform,
It is stirred continuously during immersion with tweezers, ensures that disinfection is thorough, then with aseptic water washing 3-4 time, obtain the second cleaning fruit pod;It will
A concentration of 2% hypochlorite disinfectant shell 1-2min of the second clean fruit pod, then with aseptic water washing 3-4 times, obtains
To third cleaning fruit pod;The third cleaning fruit pod is sterilized into 6-8min with 0.1% mercuric chloride, then with aseptic water washing 4-5
It is secondary, obtain the 4th clean fruit pod.
Further, in the sowing step, institute is dissected with the scalpel and tweezers of 280-290 DEG C of high-temperature sterilization of process
The 4th clean fruit pod is stated, after fruit powder is spread to 1/2MS culture mediums, and covers the culture medium bottle cap by disinfection;The culturing room
Temperature for 24-26 DEG C, intensity of illumination 2000-2500Lx, light application time is 10-12 hour/day, culture 30-35 days.
Further, in the sowing step, 2% sucrose, the fine jade of 4.8g/L are added in the 1/2MS culture mediums
Cosmetics, and the pH of culture medium is adjusted to 5.8-6.0.
Further, in the subculture step, the germinating seed is placed in intensity of illumination as 2000-2500Lx
Culturing room in continue to cultivate, light application time is 10-12 hour/day, is grown 50-60 days, grows to 2-3cm up to seedling, acquisition
Level-one bottle seedling;The level-one bottle seedling is transferred in 1/2MS culture mediums in an aseptic environment and is cultivated 50-60 days, grows to 5-6cm
When, obtain two level bottle seedling.
Further, in the subculture step, in the 1/2MS culture mediums add 0.8mg/L methyl α-naphthyl acetate,
The 6 benzyladenine 6-BA of 1.5mg/L, 3% sucrose, the agar powder of 4.8g/L, the murphy juice of 35g/L and 0.3g/L activity
Carbon dust, and the pH of culture medium is adjusted to 5.8-6.0.
Further, in the culture of rootage step, the two level bottle seedling is transferred to training of taking root in an aseptic environment
It supports and is cultivated 85-95 days in base, obtain bottle seedling of taking root;The root media is MS culture mediums, is added in the MS culture mediums
The methyl α-naphthyl acetate of 2.0mg/L, the 6 benzyladenine 6-BA of 1.0mg/L, 3% sucrose, the agar powder of 4.8g/L, 35g/L potato
The activated carbon powder of juice, the bananas juice of 25g/L and 0.3g/L, and the pH of culture medium is adjusted to 5.8-6.0.
Further, in the hardening tames step, the bottle seedling of taking root is placed in culturing room and is cultivated 85-95 days,
Then it is moved into outdoor environment and places 15-30 days, obtain domesticated seedlings.
Further, in the transplant step, carbendazim is diluted with 800-1000 times of water, soak is obtained and impregnates
Seedling root 15-20min, is then cleaned and is dried in the shade with clear water.
Compared with prior art, the beneficial effects of the present invention are:
(1) tissue cultivating and seedling method of the aster stem of noble dendrobium provided by the present invention can effectively improve aster stem of noble dendrobium kind
The breeding speed of seedling and breeding quantity, from the stability of maternal source control germplasm, ensure seedling quality, realize that the aster stem of noble dendrobium is excellent
The factorial praluction of matter seed seedling, meets production requirement, alleviates the present situation of scarcity of resources.
(2) tissue cultivating and seedling method of the aster stem of noble dendrobium provided by the present invention, most important link kind during breeding
Son disinfection is thorough, and disinfection thoroughly just can guarantee that seed sowing wild Oryza species do not infect, and be suitble in specified conditions such as temperature, illumination
In the case of, the germination percentage of aster stem of noble dendrobium seed seedling-raising is more than 80%, ensures that kind is pure.In sowing and subculture step,
Culture medium uses 1/2MS culture mediums, by being compared with MS culture mediums, on the germination and growth of seedling without influence, is cultivated using 1/2MS
Base can save raw material, and every bottle of culture medium can save the cost of 1.0-1.5 members in production.
Description of the drawings
Fig. 1 is the aster stem of noble dendrobium of the present invention in sowing stage, the sprouting figure of seed;
Fig. 2 is vegetative map of the aster stem of noble dendrobium in the squamous subculture stage of the present invention;
Fig. 3 is take root figure of the aster stem of noble dendrobium in the culture of rootage stage of the present invention;
Fig. 4 is that the aster stem of noble dendrobium of the present invention tames the hardening figure in stage in hardening;
Fig. 5 is cultivation figure of the aster stem of noble dendrobium in the transplanting stage of the present invention.
Specific embodiment
In the following, with reference to attached drawing and specific embodiment, the present invention is described further, it should be noted that not
Under the premise of conflicting, new implementation can be formed between various embodiments described below or between each technical characteristic in any combination
Example.
A kind of tissue cultivating and seedling method of the aster stem of noble dendrobium, including:
Explant selects step:Artificial pollination is carried out in the florescence of aster stem of noble dendrobium maternal plant, after growing fruit pod, is continued
It cultivates, harvesting ripe fruit pod is placed in preserving in refrigerator, obtains spare fruit pod;
Fruit pod sterilisation step:Spare fruit pod is taken, kind shell surface clean is clean, obtain the first clean fruit pod;It is clean by first
Net fruit pod is moved in transfer room, carries out alcohol disinfecting and aseptic water washing, obtains the second clean fruit pod;Second clean fruit pod is used
Hypochlorite disinfectant and aseptic water washing obtain third cleaning fruit pod;Third cleaning fruit pod with mercuric chloride is sterilized and sterile water rushes
It washes, obtains the 4th clean fruit pod;
Sow step:The 4th clean fruit pod is dissected, fruit powder is spread on 1/2MS culture mediums, is transferred in culturing room and cultivates,
Obtain germinating seed;
Subculture step:Germinating seed is placed in culturing room and continues to cultivate, until seedling grows to 2-3cm, obtains one
Grade bottle seedling;Level-one bottle seedling is transferred in 1/2MS culture mediums, when growing to 5-6cm, obtains two level bottle seedling;
Culture of rootage step:Two level bottle seedling is transferred in root media and is cultivated, obtains bottle seedling of taking root;
Hardening tames step:The bottle seedling that will take root, which is placed in culturing room, cultivates, and then moves on in outdoor environment and is tamed, is obtained
Take domesticated seedlings;
Transplant step:The bottle cap of root media is opened, continues to place domesticated seedlings 3-5 days, then pour out, and with more bacterium
Seedling root is impregnated in spirit, is then cleaned and is dried in the shade, transplants and tamed into matrix.
As further embodiment, in step is sowed, 2% sucrose, 4.8g/L are added in 1/2MS culture mediums
Agar powder, and the pH of culture medium is adjusted to 5.8-6.0;In subculture step, 0.8mg/ is added in 1/2MS culture mediums
The methyl α-naphthyl acetate of L, the 6 benzyladenine 6-BA of 1.5mg/L, 3% sucrose, the agar powder of 4.8g/L, 35g/L murphy juice and
The activated carbon powder of 0.3g/L, and the pH of culture medium is adjusted to 5.8-6.0;In culture of rootage step, root media is trained for MS
Base is supported, the methyl α-naphthyl acetate of 2.0mg/L, 6 benzyladenine 6-BA, 3% sucrose, the 4.8g/ of 1.0mg/L are added in MS culture mediums
The agar powder of L, the murphy juice of 35g/L, the bananas juice of 25g/L and 0.3g/L activated carbon powder, and the pH of culture medium is adjusted to
5.8-6.0。
At seeding time, culture medium uses 1/2MS culture mediums, only adds sucrose and agar powder, and when squamous subculture all uses 1/
2MS culture mediums add certain density auxin in culture medium, and by being compared with MS culture mediums, seedling is sprouted
Length occurs without influence, raw material can be saved using 1/2MS culture mediums, every bottle of culture medium can save 1.0-1.5 members in production
Cost.
As further embodiment, in hardening tames step, domesticated seedlings are grown in culturing room 60 days or so can be with
It moves it in the greenhouse that temperature is 22-28 DEG C, humidity is 45%-75% and carries out hardening 30 days, culturing room can be saved in this way
The power consumption of fluorescent lamp and air-conditioning can monthly save the cost of 1200-1500 members or so;Then it is moved into outdoor environment.
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:
A kind of tissue cultivating and seedling method of the aster stem of noble dendrobium, including:
Explant selects step:Plant age is selected as 3 years aster stem of noble dendrobium maternal plants, in aster stem of noble dendrobium maternal plant
Florescence (mid-June) carries out artificial pollination, after growing fruit pod, continues to cultivate 3 months, (outer kind of shell is yellowish green for harvesting ripe fruit pod
Color, intact nothing are split) it is placed in preserving 2 weeks in 5 DEG C of refrigerator;
Fruit pod sterilisation step:Spare fruit pod is taken, 15min is impregnated with liquid detergent water, cleans down kind of a shell surface with hairbrush, be used in combination
Tap water rinses 12min, obtains the first clean fruit pod;First clean fruit pod is put into clean beaker, transfer room is moved to, in nothing
50s is impregnated with 75% medicinal alcohol on bacterium operation console, is stirred continuously during immersion with tweezers, ensures that disinfection is thorough, then with nothing
Bacterium water rinses 4 times, obtains the second clean fruit pod;By the second clean fruit pod with a concentration of 2% hypochlorite disinfectant shell 2min,
Then it uses aseptic water washing 3 times, obtains third cleaning fruit pod;Third cleaning fruit pod is sterilized into 7min with 0.1% mercuric chloride, then
With aseptic water washing 5 times, the 4th clean fruit pod is obtained;
Sow step:With the 4th clean fruit pod of scalpel and tweezers dissection by 280-290 DEG C of high-temperature sterilization;By yellow
After fruit powder is spread to 1/2MS culture mediums, and the culture medium bottle cap by disinfection is covered, 2% sugarcane is added in 1/2MS culture mediums
The agar powder of sugar, 4.8g/L, and the pH of culture medium is adjusted to 5.8-6.0, it is then transferred in culturing room and cultivates, the temperature of culturing room
It is 25 DEG C, intensity of illumination 2300Lx to spend, and light application time is 11 hours/day, cultivates 32 days, obtains germinating seed;
Subculture step:Germinating seed is placed in the culturing room that intensity of illumination is 2300Lx and continues to cultivate, during illumination
Between for 11 hours/day, grow 55 days, until seedling grows to 2-3cm, obtain level-one bottle seedling;In an aseptic environment by level-one bottle seedling
It is transferred in 1/2MS culture mediums, and 6 benzyladenines of the methyl α-naphthyl acetate of 0.8mg/L, 1.5mg/L is added in 1/2MS culture mediums
6-BA, 3% sucrose, the agar powder of 4.8g/L, the murphy juice of 35g/L and 0.3g/L activated carbon powder, and by the pH of culture medium
It is adjusted to 5.8-6.0;Culture 55 days when growing to 5-6cm, obtains two level bottle seedling;
Culture of rootage step:Two level bottle seedling is transferred in root media in an aseptic environment and is cultivated 90 days, obtains life
Root bottle seedling;Root media is MS culture mediums, and the methyl α-naphthyl acetate of 2.0mg/L, the 6 benzyl glands of 1.0mg/L are added in MS culture mediums
Purine 6-BA, 3% sucrose, the agar powder of 4.8g/L, the murphy juice of 35g/L, the bananas juice of 25g/L and 0.3g/L activated carbon
Powder, and the pH of culture medium is adjusted to 5.8-6.0;
Hardening tames step:Domesticated seedlings are grown in culturing room can move it to temperature for 60 days or so as 22-28 DEG C, humidity
To carry out hardening in the greenhouse of 45%-75% 30 days, take root good, seedling is sturdy, is then moved into outdoor environment and (avoids strong light
Direct projection) in place 20 days, allow its adapt to external environment, obtain domesticated seedlings;
Transplant step:The bottle cap of root media is opened, continues to place domesticated seedlings 4 days, then pour out, and with 900 times
Water dilution carbendazim, obtain soak simultaneously impregnate seedling root 17min, then cleaned and dried in the shade with clear water, transplanted into matrix
It is tamed.
The result of the test of embodiment 1:In step is sowed, the germination percentage of aster stem of noble dendrobium seed seedling-raising is 90%, also, purple
The garden stem of noble dendrobium is when fruit pod is germinateed (see Fig. 1) and in squamous subculture (see Fig. 2), culture of rootage (see Fig. 3), hardening domestication (see figure
4) when and transplanting is (see Fig. 5), the growing way of the aster stem of noble dendrobium is good.
Embodiment 2:
Embodiment 2 and embodiment 1 the difference lies in:
In explant selection step:Plant age is selected after growing fruit pod, to continue to train for 4 years aster stem of noble dendrobium maternal plants
It educates 4 months, harvesting ripe fruit pod is placed in 7 DEG C of refrigerator and preserves 2.5 weeks;
In fruit pod sterilisation step:Spare fruit pod is taken, 20min is impregnated with washing powder water, kind of a shell surface is cleaned down with hairbrush, and
15min is rinsed with tap water, obtains the first clean fruit pod;First clean fruit pod is put into clean beaker, moves to transfer room,
30s is impregnated with 75% medicinal alcohol on aseptic operating platform, is stirred continuously during immersion with tweezers, ensures that disinfection is thorough, then uses
Aseptic water washing 3 times obtains the second clean fruit pod;By the second clean fruit pod with a concentration of 2% hypochlorite disinfectant's shell
1min then with aseptic water washing 4 times, obtains third cleaning fruit pod;Third cleaning fruit pod is sterilized with 0.1% mercuric chloride
6min then with aseptic water washing 4 times, obtains the 4th clean fruit pod;
It sows in step:The temperature of culturing room is 24 DEG C, intensity of illumination 2500Lx, and light application time is 12 hours/day, is trained
It supports 30 days;
In subculture step:Germinating seed is placed in the culturing room that intensity of illumination is 2000Lx and continues to cultivate, illumination
Time is 12 hours/day, is grown 50 days;
In culture of rootage step:Two level bottle seedling is transferred in root media and is cultivated 95 days;
In hardening domestication step:The bottle seedling that will take root is placed in culturing room and cultivates 85 days, is then moved into outdoor environment
It places 30 days;
In transplant step:The bottle cap of root media is opened, continues to place domesticated seedlings 5 days, then pour out, and with 800
Water dilution carbendazim again, obtains soak and simultaneously impregnates seedling root 15min;
It is other identical with specific embodiment 1.
The result of the test of embodiment 2:In step is sowed, the germination percentage of aster stem of noble dendrobium seed seedling-raising is 89%, also, purple
For the garden stem of noble dendrobium when fruit pod is germinateed and in squamous subculture, culture of rootage, hardening domestication and transplanting, the growing way of the aster stem of noble dendrobium is equal
Well, the schematic diagram of each growth phase is similar to Example 1, and details are not described herein.
Embodiment 3:
Embodiment 3 and embodiment 1 the difference lies in:
In explant selection step:Plant age is selected after growing fruit pod, to continue to train for 2 years aster stem of noble dendrobium maternal plants
It educates 3.5 months, harvesting ripe fruit pod is placed in 3 DEG C of refrigerator and preserves 1.5 weeks;
In fruit pod sterilisation step:Spare fruit pod is taken, 15min is impregnated with washing powder water, kind of a shell surface is cleaned down with hairbrush, and
10min is rinsed with tap water, obtains the first clean fruit pod;First clean fruit pod is put into clean beaker, moves to transfer room,
60s is impregnated with 75% medicinal alcohol on aseptic operating platform, is stirred continuously during immersion with tweezers, ensures that disinfection is thorough, then uses
Aseptic water washing 3 times obtains the second clean fruit pod;By the second clean fruit pod with a concentration of 2% hypochlorite disinfectant's shell
1.5min then with aseptic water washing 4 times, obtains third cleaning fruit pod;Third cleaning fruit pod is sterilized with 0.1% mercuric chloride
8min then with aseptic water washing 4 times, obtains the 4th clean fruit pod;
It sows in step:The temperature of culturing room is 26 DEG C, intensity of illumination 2000Lx, and light application time is 10 hours/day, is trained
It supports 35 days;
In subculture step:Germinating seed is placed in the culturing room that intensity of illumination is 2500Lx and continues to cultivate, illumination
Time is 10 hours/day, is grown 60 days;
In culture of rootage step:Two level bottle seedling is transferred in root media and is cultivated 85 days;
In hardening domestication step:The bottle seedling that will take root is placed in culturing room and cultivates 95 days, is then moved into outdoor environment
It places 15 days;
In transplant step:The bottle cap of root media is opened, continues to place domesticated seedlings 3 days, then pour out, and with 1000
Water dilution carbendazim again, obtains soak and simultaneously impregnates seedling root 20min;
It is other identical with specific embodiment 1.
3 result of the test of embodiment:In step is sowed, the germination percentage of aster stem of noble dendrobium seed seedling-raising is 88%, also, aster
For the stem of noble dendrobium when fruit pod is germinateed and in squamous subculture, culture of rootage, hardening domestication and transplanting, the growing way of the aster stem of noble dendrobium is good
Good, the schematic diagram of each growth phase is similar to Example 1, and details are not described herein.
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 variation 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 range.