CN106258968A - The iris tissue culture method that a kind of browning rate is low - Google Patents
The iris tissue culture method that a kind of browning rate is low Download PDFInfo
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- CN106258968A CN106258968A CN201610663271.4A CN201610663271A CN106258968A CN 106258968 A CN106258968 A CN 106258968A CN 201610663271 A CN201610663271 A CN 201610663271A CN 106258968 A CN106258968 A CN 106258968A
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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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- 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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Abstract
The present invention relates to field of plant tissue culture technique, be specifically related to the iris tissue culture method that a kind of browning rate is low, comprise the following steps: the making of material selection, material, materials disinfection, pretreatment, inducing culture, enrichment culture, strengthening seedling and rooting and transplanting.Low by browning rate during using this method to make group train, Seedling strain planting percent is high, Seedling strain quality better; strong adaptability after cultivation; by animal nutrition at short notice, meet the demand commercially produced, cultivate iris provide technical support for enterprise scale, industrialization.
Description
Technical field
The present invention relates to field of plant tissue culture technique, be specifically related to the iris tissue culture method that a kind of browning rate is low.
Background technology
Iris is the famous flower of current international flower bed, belongs to the orchid family Phalaenopsis plant, and original producton location is mainly subtropical zone, heat
Band, natural distributed in Oceania, Asia.Iris has viewing period length, color and luster is abundant, pattern is beautiful and fine figure is slim and graceful
Etc. feature, also it is known as " cattleya queen ", is that one is the most universal in orchid, most widely cultivates kind, have
The highest commercial value, is international four one viewing and admiring greatly hot bandwidth.Iris is single stem aerial orchid, and collateral development is very
Few, therefore it is difficult to routine and carries out division propagation, and seldom form seed, seed development is the most relatively difficult, substantially at nature
Under the conditions of sprout difficulty bigger, the Main Means that therefore iris is efficiently bred is exactly tissue culture.
But in iris tissue culture procedures, browning phenomenon seriously annoyings the seeling industry of iris, and brown stain is produced
Thing not only makes Phalaenopsis Explant in Vitro, cell, culture medium brown stain, and suppresses the normal biochemical reactions of Phalaenopsis Explant in Vitro,
The suppression startup of protocorm, growth and the regeneration of iris seedling, drastically influence the further differentiation of Phalaenopsis Explant in Vitro,
Finally even result in the death of Phalaenopsis Explant in Vitro.The browning phenomenon that iris occurs during group training, is iris outer planting
Body is when tissue culture, and Phalaenopsis Explant in Vitro releases some brown materials from its own face to culture medium, make culture medium by
Crossfading into brown, the culture medium of brown can suppress the vigor of Phalaenopsis Explant in Vitro, affects the activity of relevant enzyme, the most in turn to butterfly
The outer implant of phalaenopsis produces poisons, and can affect the further differentiation of Phalaenopsis Explant in Vitro, finally result in iris outer planting time serious
The phenomenon of body Necrosis.
Summary of the invention
It is an object of the invention to provide the iris tissue culture method that a kind of browning rate is low, thus reduce iris in group training
During browning rate, increase planting percent, add work efficiency.
In order to achieve the above object, the technical solution used in the present invention is: the iris tissue culture method that a kind of browning rate is low,
Comprise the following steps:
(1) material selection: choosing trophophase is 45d and the iris bennet that grows fine without pest and disease damage, uses the bennet to be
Outer implant carries out tissue culture；
(2) material makes: bennet is cut into the 3cm sections of band axillalry bud, truncation on joint, cuts sth. askew in 45 ° under joint；
(3) materials disinfection: tentatively cleaned in pond by the iris bennet chosen with soft bristle, removes surface
Silt, then cleans up with liquid detergent, is placed under flowing water flushing 60min, with iris bennet wound of sealing with wax, by butterfly Cymbidium ensifolium (L.) Sw.
Stalk is placed on superclean bench with the alcohol disinfecting 30s that the volume fraction crossed through high temperature sterilize is 75%, then with aseptic water washing 5
Secondary, then add 4 with the mercuric chloride solution that mass fraction is 0.1%～5 tween 20 sterilization 12min, rush with sterilized water after sterilization
Wash 6～8 times, be positioned in culture dish standby after blotting surface moisture with sterilized filter paper；
(4) pretreatment: the iris bennet after sterilization is carried out high-temperature process, and temperature is 45 DEG C, and the time is
10min, then under aseptic condition, room temperature places 48h；
(5) inducing culture: by pretreated iris bennet, be seeded in inducing culture and cultivate, first carry out
The light culture of 7d, the light application time then increasing by 2 hours every day carries out light cultivation, until light application time every day is 12h/d,
Intensity of illumination is 1300～1500lx；Front 7～14d use Low-temperature culture, and cultivation temperature is 15～25 DEG C, after Low-temperature culture, adopts
Cultivating with room temperature, cultivation temperature is 25～28 DEG C；Described inducing culture is: MS+0.4～0.8mg/L 6-BA+0.05～
0.08mg/L NAA+10～15g/L sucrose+6～8g/L agar+10～15% coconut juice+290～310mg/L citric acid+1～2g/
L activated carbon, pH value is 5.4～5.5；
(6) enrichment culture: be transferred in proliferated culture medium by the iris bennet after inducing culture, first carries out the dark training of 7d
Supporting, the light application time then increasing by 2 hours every day carries out light cultivation, until light application time every day is 12h/d, and intensity of illumination
It is 1300～1500lx；Front 7～14d use Low-temperature culture, and cultivation temperature is 15～25 DEG C, after Low-temperature culture, use room temperature training
Supporting, cultivation temperature is 25～28 DEG C；Described proliferated culture medium is: MS+1～2mg/L 6-BA+0.1～0.2mg/L NAA+10～
15g/L sucrose+6～8g/L agar+10～15% coconut juice+290～310mg/L citric acid+1～2g/L activated carbon, pH value is 5.4
～5.5；
(7) strengthening seedling and rooting: after enrichment culture, selects have the seedling of obvious projection to proceed to take root in high more than 2cm, outer implant
In culture medium, described root media is: MS+1.4～1.6mg/L 6-BA+0.05～0.08mg/L NAA+10～15g/L sugarcane
Sugar+6～8g/L agar+10～15% coconut juice+8～12% bananas juice+7～10% mashed potatoes, pH value is 5.4～5.5；Cultivate bar
Part: cultivation temperature 25～28 DEG C, light application time is 13h/d, and intensity of illumination is 1300～1500lx；
(8) transplant: when test tube Seedling grows to 4～5cm high, root 2～4, leaf 1～when 2, test tube Seedling is placed on nature light
Lower refining seedling 5～7 days, open bottle cap seedling exercising 1～2 days；Then take out cultivation Seedling, clean the culture medium being attached to root with clear water,
It is careful not to during cleaning damage root, then transplants to sphagna and mixed-matrix that fertile soil mass ratio is 1:2, keep humidity
And ventilation, air humidity is 75～85%, and temperature is 20～25 DEG C.
The iris tissue culture method that a kind of browning rate as above is low, further illustrate into, described inducing culture is:
MS+0.6mg/L 6-BA+0.07mg/L NAA+13g/L sucrose+7g/L agar+13% coconut juice+300mg/L citric acid+1.5g/L
Activated carbon.
The iris tissue culture method that a kind of browning rate as above is low, further illustrate into, described proliferated culture medium is:
MS+1.5mg/L 6-BA+0.15mg/L NAA+13g/L sucrose+7g/L agar+13% coconut juice+300mg/L citric acid+1.5g/L
Activated carbon.
The iris tissue culture method that a kind of browning rate as above is low, further illustrate into, described root media is:
MS+1.5mg/L 6-BA+0.07mg/L NAA+13g/L sucrose+7g/L agar+13% coconut juice+10% bananas juice+9% Rhizoma Solani tuber osi
Mud.
The invention has the beneficial effects as follows: low by browning rate during using this method to make group train, Seedling strain planting percent is high, Seedling
Strain quality better, strong adaptability after cultivation, by animal nutrition at short notice, meet the demand commercially produced, for
Enterprise scale, industrialization are cultivated iris and are provided technical support.
Accompanying drawing explanation
Fig. 1 is schematic flow sheet of the present invention.
Detailed description of the invention
Below in conjunction with the accompanying drawings the specific embodiment of the invention is further elaborated.
As it is shown in figure 1, the iris tissue culture method that a kind of browning rate of present invention offer is low, comprise the following steps: material
Choose, material making, materials disinfection, pretreatment, inducing culture, enrichment culture, strengthening seedling and rooting and transplanting, pass through this method
Iris browning rate during group training can be reduced, increase planting percent,
Below each step in method is elaborated.
(1) material selection: choosing trophophase is 45d and the iris bennet that grows fine without pest and disease damage, uses the bennet to be
Outer implant carries out tissue culture；Use the iris bennet grown fine without pest and disease damage can reduce the sickness rate of Seedling strain, it is ensured that
Do not disturbed by other factors during group training, increased outer implant survival rate.Select the iris bennet pair that trophophase is different
During group training, browning phenomenon also can have a certain impact, and table 1 is in the iris bennet tissue culture procedures of different growing stages
Brown stain impact:
Brown stain impact in the iris bennet tissue culture procedures of table 1 different growing stages
Cultivating in table 1, the bennet of different growing stages is placed on identical culture medium, outside condition of culture is homogeneous
Causing, trophophase is that the iris bennet browning rate of 30d is on the low side compared with other groups as can be seen from Table 1, and 45d takes second place, along with butterfly
The increase of Cymbidium ensifolium (L.) Sw. stalk trophophase, browning rate increases the most accordingly, and the browning rate growth with it of iris bennet tissue culture be described
Phase close relation, but owing to the iris bennet tissue Maturity that trophophase is 30d is the highest, outside making after tissue culture
Implant upgrowth situation is poor, so combined factors considers in terms of browning rate and upgrowth situation two, selecting trophophase is 45d and nothing
The iris bennet that pest and disease damage grows fine is as outer implant.
(2) material makes: bennet is cut into the 3cm sections of band axillalry bud, truncation on joint, cuts sth. askew in 45 ° under joint；
The brownization performance in tissue culture of the sections of table 2 different length
Being placed in identical culture medium by the bennet sections of different length in table 2 and cultivate, outside condition of culture is equal
Unanimously, inoculation number is consistent, and outer implant carries out the cutting of different length when carrying out tissue culture, a length of
The sections browning rate of 3cm is minimum, increases over time, and brown stain rate of rise is the slowest, and two groups that length is less, browning rate
And rate of rise is bigger than normal compared with 3cm length one group, this phenomenon illustrates that iris bennet cuts as outer implant to be made
Being difficult to the least, the outer implant cut is the least, and tangent plane is the biggest with the ratio of volume, and the injury to Phalaenopsis Explant in Vitro is the biggest,
Thus causing browning degree the biggest, cutting is too small simultaneously, also easily causes dead bud phenomenon.And along with cutting the increase of length,
Browning rate also increases, and due to the increase of sections, the Phalaenopsis Explant in Vitro made is difficult to sterilizing, and bacterial bearing rate is high, too increases iris
Browning rate in tissue culture procedures.Therefore bennet is cut into the 3cm sections of band axillalry bud, can effectively reduce browning rate.Under joint in
Cut sth. askew for 45 °, outer implant can be inserted in downwards in culture medium, increase tangent plane simultaneously, assimilation effect can be improved.
(3) materials disinfection: tentatively cleaned in pond by the iris bennet chosen with soft bristle, removes surface
Silt, then cleans up with liquid detergent, is placed under flowing water flushing 60min, with iris bennet wound of sealing with wax, by butterfly Cymbidium ensifolium (L.) Sw.
Stalk is placed on superclean bench with the alcohol disinfecting 30s that the volume fraction crossed through high temperature sterilize is 75%, then with aseptic water washing 5
Secondary, then add 4 with the mercuric chloride solution that mass fraction is 0.1%～5 tween 20 sterilization 12min, rush with sterilized water after sterilization
Wash 6～8 times, be positioned in culture dish standby after blotting surface moisture with sterilized filter paper；
The outer implant after table 3 different disinfecting time process browning phenomenon in tissue culture
Bennet sections different for disinfecting time in table 3 is placed in identical culture medium and cultivates, outside cultivation bar
Part is all consistent, and inoculation number is consistent, the rate value that one group of browning rate of the 12min that sterilizes as can be seen from Table 3 and browning rate increase
Minimum, other groups compare all that some is bigger than normal therewith, thus the mercuric chloride solution using mass fraction to be 0.1% adds 4～5 tell
Temperature-20 sterilization 12min can the generation of effective Restrain browning phenomenon.
(4) pretreatment: the iris bennet after sterilization is carried out high-temperature process, and temperature is 45 DEG C, and the time is
10min, then under aseptic condition, room temperature places 48h；The high-temperature process using the short time can make its interior polyphenol oxidase lose
Deactivation, thus reach the purpose of BPH resistant rice variety in iris tissue culture procedures.
(5) inducing culture: by pretreated iris bennet, be seeded in inducing culture and cultivate, first carry out
The light culture of 7d, the light application time then increasing by 2 hours every day carries out light cultivation, until light application time every day is 12h/d,
Intensity of illumination is 1300～1500lx, and front 7d when the most just starting is light culture, and then 8d uses the illumination cultivation of 2 hours,
9d uses the illumination cultivation of 4 hours, and 10d uses the illumination cultivation of 6 hours, and 11d uses the illumination cultivation of 8 hours, the
12d uses the illumination cultivation of 10 hours, and 13d uses the illumination cultivation of 12 hours, and 14d still uses the illumination of 12 hours to train
Support, the most all use the illumination cultivation of 12h/d；Browning rate can be reduced, if during light culture by the light culture of a period of time
Between too high, the Seedling strain after cultivation can be made again to turn to be yellow, be difficult to growth, so use 7d light culture.By stepped increase light
According to the time, outer implant can be made gradually to adapt to environment, thus alleviate the harm of external implant.
Front 7～14d use Low-temperature culture, and cultivation temperature is 15～25 DEG C, after Low-temperature culture, use room temperature to cultivate, cultivate
Temperature is 25～28 DEG C, uses the Low-temperature culture of a period of time, it is possible to reduce the activity of polyphenol oxidase, Restrain browning phenomenon
Produce；Described inducing culture is: MS+0.4～0.8mg/L 6-BA+0.05～0.08mg/L NAA+10～15g/L sucrose+6
～8g/L agar+10～15% coconut juice+290～310mg/L citric acid+1～2g/L activated carbon, pH value is 5.4～5.5, and table 4 is
The various embodiments of inducing culture:
While citric acid is as antioxidant, also regulating the pH value of culture medium, activated carbon can effectively press down as adsorbent
The generation of brown stain processed, agar consumption is the biggest, and culture medium hardness is the biggest, is unfavorable for the absorption of nutrient, and agar consumption is the least, training
Foster base is difficult to solidification, add coconut juice can the generation of effective Restrain browning, described MS basal medium is prior art, does not does
It is specifically described.As preferably, described inducing culture is: MS+0.6mg/L 6-BA+0.07mg/L NAA+13g/L sucrose+7g/
L agar+13% coconut juice+300mg/L citric acid+1.5g/L activated carbon, is composition and the consumption of culture medium 1 in table 4.
(6) enrichment culture: be transferred in proliferated culture medium by the iris bennet after inducing culture, first carries out the dark training of 7d
Supporting, the light application time then increasing by 2 hours every day carries out light cultivation, until light application time every day is 12h/d, and intensity of illumination
It is 1300～1500lx；Front 7～14d use Low-temperature culture, cultivation temperature is 15～25 DEG C, in like manner with inducing culture step in one
Cause, first light culture, by stepped increase light application time, after Low-temperature culture, use room temperature to cultivate, cultivation temperature be 25～
28℃；Described proliferated culture medium is: MS+1～2mg/L 6-BA+0.1～0.2mg/L NAA+10～15g/L sucrose+6～8g/L
Agar+10～15% coconut juice+290～310mg/L citric acid+1～2g/L activated carbon, pH value is 5.4～5.5；Table 5 is propagation training
The various embodiments of foster base:
As preferably, described proliferated culture medium is: MS+1.5mg/L 6-BA+0.15mg/L NAA+13g/L sucrose+7g/L
Agar+13% coconut juice+300mg/L citric acid+1.5g/L activated carbon, is composition and the consumption of culture medium 7 in table 5.
(7) strengthening seedling and rooting: after enrichment culture, selects have the seedling of obvious projection to proceed to take root in high more than 2cm, outer implant
In culture medium, described root media is: MS+1.4～1.6mg/L 6-BA+0.05～0.08mg/L NAA+10～15g/L sugarcane
Sugar+6～8g/L agar+10～15% coconut juice+8～12% bananas juice+7～10% mashed potatoes, pH value is 5.4～5.5；Cultivate bar
Part: cultivation temperature 25～28 DEG C, light application time is 13h/d, and intensity of illumination is 1300～1500lx；Table 6 is root media
Various embodiments:
As preferably, described root media is: MS+1.5mg/L 6-BA+0.07mg/L NAA+13g/L sucrose+7g/L
Agar+13% coconut juice+10% bananas juice+9% mashed potatoes, is composition and the consumption of culture medium 13 in table 6.
(8) transplant: when test tube Seedling grows to 4～5cm high, root 2～4, leaf 1～when 2, test tube Seedling is placed on nature light
Lower refining seedling 5～7 days, open bottle cap seedling exercising 1～2 days；Then take out cultivation Seedling, clean the culture medium being attached to root with clear water,
It is careful not to during cleaning damage root, then transplants to sphagna and mixed-matrix that fertile soil mass ratio is 1:2, keep humidity
And ventilation, air humidity is 75～85%, and temperature is 20～25 DEG C.It is low by browning rate during using this method to make group train,
Seedling strain planting percent is high, Seedling strain quality better, and strong adaptability after cultivation by animal nutrition at short notice, meets business
The demand that metaplasia is produced, cultivates iris provide technical support for enterprise scale, industrialization.
The present invention is not limited to examples detailed above, in claims of the present invention limited range, and art technology
Various deformation or amendment that personnel can make without creative work are all protected by this patent.
Claims (4)
1. the iris tissue culture method that a browning rate is low, it is characterised in that comprise the following steps:
(1) material selection: choosing trophophase is 45d and the iris bennet that grows fine without pest and disease damage, and employing bennet is outer planting
Body carries out tissue culture；
(2) material makes: bennet is cut into the 3cm sections of band axillalry bud, truncation on joint, cuts sth. askew in 45 ° under joint；
(3) materials disinfection: tentatively cleaned in pond by the iris bennet chosen with soft bristle, removes the mud on surface
Sand, then cleans up with liquid detergent, is placed under flowing water flushing 60min, with iris bennet wound of sealing with wax, by iris bennet
It is placed on superclean bench with the alcohol disinfecting 30s that the volume fraction crossed through high temperature sterilize is 75%, then with aseptic water washing 5
Secondary, then add 4 with the mercuric chloride solution that mass fraction is 0.1%～5 tween 20 sterilization 12min, rush with sterilized water after sterilization
Wash 6～8 times, be positioned in culture dish standby after blotting surface moisture with sterilized filter paper；
(4) pretreatment: the iris bennet after sterilization is carried out high-temperature process, and temperature is 45 DEG C, and the time is 10min, so
Under rear aseptic condition, room temperature places 48h；
(5) inducing culture: by pretreated iris bennet, be seeded in inducing culture and cultivate, first carries out 7d's
Light culture, the light application time then increasing by 2 hours every day carries out light cultivation, until light application time every day is 12h/d, illumination
Intensity is 1300～1500lx；Front 7～14d use Low-temperature culture, and cultivation temperature is 15～25 DEG C, after Low-temperature culture, often use
Temperature is cultivated, and cultivation temperature is 25～28 DEG C；Described inducing culture is: MS+0.4～0.8mg/L 6-BA+0.05～0.08mg/
L NAA+10～15g/L sucrose+6～8g/L agar+10～15% coconut juice+290～310mg/L citric acid+1～2g/L activity
Charcoal, pH value is 5.4～5.5；
(6) enrichment culture: the iris bennet after inducing culture is transferred in proliferated culture medium, first carries out the light culture of 7d,
Then the light application time increasing by 2 hours every day carries out light cultivation, until light application time every day is 12h/d, intensity of illumination is
1300～1500lx；Front 7～14d use Low-temperature culture, and cultivation temperature is 15～25 DEG C, after Low-temperature culture, use room temperature to cultivate,
Cultivation temperature is 25～28 DEG C；Described proliferated culture medium is: MS+1～2mg/L 6-BA+0.1～0.2mg/L NAA+10～
15g/L sucrose+6～8g/L agar+10～15% coconut juice+290～310mg/L citric acid+1～2g/L activated carbon, pH value is 5.4
～5.5；
(7) strengthening seedling and rooting: after enrichment culture, selects have the seedling of obvious projection to proceed to root culture in high more than 2cm, outer implant
In base, described root media is: MS+1.4～1.6mg/L 6-BA+0.05～0.08mg/LNAA+10～15g/L sucrose+6
～8g/L agar+10～15% coconut juice+8～12% bananas juice+7～10% mashed potatoes, pH value is 5.4～5.5；Condition of culture:
Cultivation temperature 25～28 DEG C, light application time is 13h/d, and intensity of illumination is 1300～1500lx；
(8) transplant: when test tube Seedling grows to 4～5cm high, root 2～4, leaf 1～when 2, test tube Seedling is placed under nature light refining
Seedling 5～7 days, open bottle cap seedling exercising 1～2 days；Then take out cultivation Seedling, clean the culture medium being attached to root with clear water, clean
Time be careful not to damage root, then transplant to sphagna and mixed-matrix that fertile soil mass ratio is 1:2, keep humidity and logical
Wind, air humidity is 75～85%, and temperature is 20～25 DEG C.
The iris tissue culture method that a kind of browning rate the most according to claim 1 is low, it is characterised in that: described inducing culture
Base is: MS+0.6mg/L 6-BA+0.07mg/L NAA+13g/L sucrose+7g/L agar+13% coconut juice+300mg/L citric acid+
1.5g/L activated carbon.
The iris tissue culture method that a kind of browning rate the most according to claim 1 is low, it is characterised in that: described enrichment culture
Base is: MS+1.5mg/L 6-BA+0.15mg/L NAA+13g/L sucrose+7g/L agar+13% coconut juice+300mg/L citric acid+
1.5g/L activated carbon.
The iris tissue culture method that a kind of browning rate the most according to claim 1 is low, it is characterised in that: described root culture
Base is: MS+1.5mg/L 6-BA+0.07mg/L NAA+13g/L sucrose+7g/L agar+13% coconut juice+10% bananas juice+9%
Mashed potatoes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111264394A (en) * | 2020-04-07 | 2020-06-12 | 无锡向山兰园科技有限公司 | Tissue culture method of cymbidium hybridum |
| CN113973713A (en) * | 2021-10-28 | 2022-01-28 | 北京市昌平职业学校 | Butterfly orchid tissue culture method |
-
2016
- 2016-08-12 CN CN201610663271.4A patent/CN106258968A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111264394A (en) * | 2020-04-07 | 2020-06-12 | 无锡向山兰园科技有限公司 | Tissue culture method of cymbidium hybridum |
| CN113973713A (en) * | 2021-10-28 | 2022-01-28 | 北京市昌平职业学校 | Butterfly orchid tissue culture method |
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