CN109220792A - A kind of fast numerous method of iris stem apex detoxification regeneration - Google Patents
A kind of fast numerous method of iris stem apex detoxification regeneration Download PDFInfo
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- 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
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Abstract
The invention discloses a kind of fast numerous methods of iris stem apex detoxification regeneration, this method is in combination with multiple stem apex lift-off technology, anti-browning processing, viral inhibitors processing and alternating alternating temperature heat treatment technics, tissue-cultured seedling content of toxins is effectively reduced, improves stem apex survival rate.Multiple stem apex lift-off technology can prevent the virus of plant other parts from spreading to shoot apical meristem, wherein cutting the stem-tip tissue with phyllopodium, the survival rate of tissue-cultured seedling can be improved while effectively reducing toxin content in stem apex；The stem apex browning death rate can be significantly reduced in stem apex preprocessing process, improves stem apex survival rate, and viral inhibitors combine alternately intermittent warming at the same time, can effectively deviate from CyMV and ORSV virus.
Description
Technical field
The present invention relates to a kind of fast numerous methods of iris stem apex detoxification regeneration, belong to tissue culture flower technical field.
Background technique
Iris (Phalaenapsis) is also known as phalaenopsis, belongs to orchid family, Phalaenopsis, because its flower-shape is beautiful, pattern is gorgeous, flower
Phase is long and popular among consumers, is known as the laudatory title of " spending middle queen ".In recent years, international market Flower & Plant trade Industry is rapid, butterfly
Phalaenopsis market is even more to flourish, it has also become China revitalizes high-quality and efficient one of the mainstream kind of the industry of flowers and plants, warp in modern rural area
Ji prospect is boundless.
As iris seedling quick breeding by group culture and international trade are increased, orchid-infecting viruses, which become, hinders butterfly
The technical bottleneck of blue industry development.Due to virus can be operated by daily management in mechanicalness wound, insect vector passed
It broadcasts, and iris commercial seedling mainly uses tissue cultures mode numerous fastly, if virus infection, it is easy to which quickly sprawling causes seriously to damage
It loses.So far, the virus being separated on iris is more than 25 kinds, wherein endangering most heavy, distribution most wide is cymbidium mosaic virus
(Cymbidium mosaic virus, CymMV) and odontoglossum ring spot virus (Odontoglossum ringspot virus,
ORSV).When both viral compound infections, illness, the serious ornamental values and the economic values for influencing orchid can be aggravated.
So far, the main path for obtaining nontoxic seedling includes the methods of stem apex removing, heat treatment and viral inhibitors (Liu
Li Qing etc., 2010).Wherein, Shoot Tip Culture is most widely used, but its detoxification efficiency is often by stem apex size, the stem apex browning death rate
High and condition of culture influence, it is difficult to obtain higher survival rate and virus elimination rate (quiet crystalline substance, 2011).Heat treatment method mainly according to
It can inhibit the duplication of virus and the synthesis of motor protein according to high temperature, virus inactivation caused to inactivate, to cut the tender tip tissue of children
Carry out virus-free culture.However, studies have shown that CymMV and ORSV have certain heat resistance, wherein the mistake poison temperature of CymMV is
60~70 DEG C, the mistake poison temperature of ORSV is 92~94 DEG C, therefore simple heat treatment is unknown to orchid removing CymMV and ORSV effect
Aobvious (Zheng Guohua etc., 2007), and prolonged high-temperature process can also reduce the survival rate of orchid.And HIV suppression agent method is
Using inhibitor such as peacock green, virazole and amino-oligosaccharide etc., reach anti-in plant intracorporal duplication by viral interference
The purpose for viral disease of curing the disease.The concentration of viral inhibitors is the key that detoxification, and the too big stem apex of concentration is dead, and concentration is small can not detoxification.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, the present invention provides a kind of fast numerous side of iris stem apex detoxification regeneration
Method, including multiple stem apex lift-off processing, stem apex anti-browning pretreatment, viral inhibitors combination alternating intermittent warming preculture, stem
Point regeneration culture and quickly breeding etc., can greatly improve stem apex survival rate and virus elimination rate, mention for the production of iris virus-elimination seedlings
For technical support.
A kind of fast numerous method of iris stem apex detoxification regeneration, includes the following steps:
1) first time stem apex is removed: being chosen the iris tissue-cultured seedling for carrying CyMV and ORSV compound infection, is cut after cleaning
Shoot apical meristem with 2-3 piece phyllopodium is seeded in progress first time virus-free culture on virus-free culture base, obtains a detoxification
Stem apex；
The virus-free culture base are as follows: 1/3MS+6-BA 0.2-1.0mgL-1+NAA 0.05-0.2mg·L-1+ sucrose 12-
15g·L-1+ beef peptone 0.8-1gL-1+ AC (active carbon) 0.8-1.0gL-1；
2) it second of stem apex removing: after a step 1) detoxification stem apex obtained grows 2-3 piece maturation leaflet, cuts
Shoot apical meristem with 2-3 piece phyllopodium is seeded on virus-free culture base described in step 1) and carries out second of virus-free culture,
Obtain secondary detoxification stem apex；
3) stem apex anti-browning pre-processes: after the secondary detoxification stem apex that step 2) obtains grows mature leaflet, cutting band 1-2
The stem-tip tissue of piece phyllopodium is inoculated into dark treatment on anti-browning culture medium, prevents browning dead；
4) viral inhibitors combine alternately alternating temperature preculture: by above-mentioned steps 3) in treated stem apex is transferred to addition disease
On the viral inhibitors culture medium of malicious azoles and amino-oligosaccharide, in combination with alternately alternating temperature heat treatment；
The alternately alternating temperature heat treatment is 33 DEG C of culture 4h, then 20 DEG C of culture 6h, and two kinds of temperature conditions are in the training period
Cycle alternation carries out, and cultivates humidity 50-60%, after dark treatment 12-15 days, illumination 800-1000lux, illumination cultivation time 30-
40 days；
5) above-mentioned steps 4 stem apex regeneration culture: will be passed through) treated stem apex, it is transferred in stem apex regeneration culture medium, obtains
It obtains stem apex and regenerates tissue-cultured seedling；
6) detoxic seedling is quickly bred: being inoculated in increasing after the stem apex regeneration tissue-cultured seedling that step 5) obtains is cut blade and root
It grows culture medium and carries out Multiplying culture, obtain detoxic seedling.
Further, above-mentioned steps 1) described in first time virus-free culture condition of culture be 25-28 DEG C of temperature, humidity
50-60%, after dark treatment 5-7 days, then illumination 800-1000lux, 30-40 days illumination cultivation time.
Further, above-mentioned steps 2) described in second of virus-free culture condition of culture be 25-28 DEG C of temperature, humidity
50-60%, after dark treatment 5-7 days, then illumination 800-1000lux, 30-40 days illumination cultivation time.
Further, above-mentioned steps 3) described in anti-browning culture medium are as follows: 1/3MS+ sucrose 12-15gL-1+ beef protein
Peptone 0.8-1gL-1+ coconut juice 150-200mlL-1+ AC (active carbon) 0.8-1.0gL-1。
Further, above-mentioned steps 3) described in the pretreated condition of culture of stem apex anti-browning be 25-28 DEG C of temperature, it is wet
Spend 50-60%, dark treatment culture 10-15 days.
Further, above-mentioned steps 4) described in viral inhibitors culture medium are as follows: 1/3MS+6-BA 0.2-1.0mg
L-1+NAA 0.05-0.2mgL-1+ sucrose 12-15gL-1+ beef peptone 0.8-1gL-1+ coconut juice 150-200mlL-1+ virazole 45-50mgL-1+ amino-oligosaccharide 45-50mgL-1。
Further, above-mentioned steps 5) described in stem apex regeneration culture medium are as follows: 1/2MS+6-BA 0.2-1.0mgL-1+
NAA 0.05-0.2mg·L-1+ sucrose 12-15gL-1+ beef peptone 0.8-1gL-1+ coconut juice 150-200mlL-1。
Further, above-mentioned steps 5) described in stem apex regeneration culture condition of culture are as follows: 25-28 DEG C of temperature, humidity
50-60%, illumination 800-1000lux, incubation time 30-40 days.
Further, above-mentioned steps 6) described in proliferated culture medium are as follows: 1/2MS+6-BA 1.0-3.0mgL-1+NAA
0.2-0.5mg·L-1+ sucrose 12-15gL-1+ beef peptone 0.8-1gL-1+ coconut juice 150-200mlL-1。
Further, above-mentioned steps 6) described in the condition of culture quickly bred of detoxic seedling are as follows: it is 25-28 DEG C of temperature, wet
After degree 50-60%, dark treatment 5-7d, illumination 1000-1200lux, 40-50 days illumination cultivation time.
Beneficial effects of the present invention:
The present invention is in combination with multiple stem apex lift-off technology, anti-browning processing, viral inhibitors processing and alternating alternating temperature
Tissue-cultured seedling content of toxins is effectively reduced in heat treatment technics, improves stem apex survival rate.Multiple stem apex lift-off technology can prevent plant
The virus of other parts is spread to shoot apical meristem, wherein cutting the stem-tip tissue with phyllopodium, can effectively reduced
Toxin improves the survival rate of tissue-cultured seedling in stem apex while content；And the present inventor studies discovery stem apex preprocessing process can
To significantly reduce the stem apex browning death rate, stem apex survival rate is improved, viral inhibitors combine alternately intermittent warming, energy at the same time
Enough effectively abjection CyMV and ORSV virus.
Specific embodiment
The screening of 1 virus-free culture base key component of embodiment
1 materials and methods
1.1 material
To carry the iris tissue-cultured seedling of CymMV and ORSV compound infection as test material.Cymbidium mosaic virus
(Cymbidium mosaic virus, CymMV) and radula orchid ring spot virus (Odontoglossum ringspot virus,
ORSV) virus detection kit is purchased from An Dezhen Bioisystech Co., Ltd (ADGEN Biotechnology Co., Ltd.).
1.2 method
The sterile working of iris tissue-cultured seedling is carrying out on superclean bench.With dissection under 40 times of cold light source anatomical lens
Needle and scalpel peel off the spire of growing point periphery, expose dimpling, vaulted, shinny growing point.Growing point is cut quickly to be placed on
Virus-free culture base upper surface.Used tool is immersed in sterilized in 75% alcoholic solution after, the calcination and after cooling down on alcolhol burner
It uses.Each culture bottle is inoculated with 3 stem apexs, and each 8 bottles of processing is repeated 2 times.Then dark treatment 2 weeks after inoculation carry out illumination training
It supports, illumination condition 800lux, 22-28 DEG C of temperature.After culture 40 days, survival rate, melting brown rate and the death rate of stem apex are counted.Specifically
Processing is shown in Table 1, table 2, table 3.
The design of 1 MS concentration of table
Remarks: virus-free culture base=basal medium+various concentration MS, basal medium are as follows: 6-BA1.0-3.0mg
L-1+NAA0.2-0.5mg·L-1+ sucrose 25gL-1+ AC (active carbon) 1.0gL-1+ beef peptone 1gL-1。
The design of 2 hormone concentration of table
Remarks: virus-free culture base=basal medium+6-BA+NAA；Basal medium are as follows: 1/2MS+ sucrose 25gL-1+
AC (active carbon) 1.0gL-1+ beef peptone 1gL-1。
The design of 3 sucrose concentration of table
Remarks: virus-free culture base=basal medium+sucrose；Basal medium are as follows: 1/2MS+6-BA1.0-3.0mgL-1
+NAA0.2-0.5mg·L-1+ AC (active carbon) 1.0gL-1+ beef peptone 1gL-1。
1.3 statistical analysis
Test result is all made of Excel2007 and 18.0 statistical software of SPSS is analyzed, with Deng Kenshi duncan's new multiple range method
(Duncan ' s) test is 0.05 horizontal upper comparing difference conspicuousness in P.
2 results and analysis
Influence of the 2.1 MS concentration to iris shoot tip meristem
Influence of the MS concentration to iris shoot tip meristem is shown in Table 5.MS concentration to iris stem apex melting brown rate, the death rate,
Survival rate and growing way feature have significant impact.With the reduction of MS concentration, the stem apex death rate is substantially reduced, survival rate significantly increases
Add, wherein M3 handles stem apex leaf color jade green, and growing way health is most suitable for shoot tip meristem culture, and MS concentration is too high or too low equal
Cause shoot tip meristem jaundice browning, vitrifying obvious.
Influence of the 4 MS concentration of table to iris shoot tip meristem
Note: p < 0.05. same letter indicates that difference is not significant, and different letters indicate significant difference.
Influence of 2.2 hormone combinations to iris shoot tip meristem
Hormone combination has significant impact (to be shown in Table iris stem apex melting brown rate, the death rate, survival rate and growing way feature
6).With the reduction of 6-BA concentration, the stem apex death rate is substantially reduced, survival rate dramatically increases, and wherein A3 handles stem apex survival rate
Highest, stem apex bud is small, and growing way is weak, though and A5 processing survival rate is slightly lower compared with A3 processing, but stem apex growth potential is healthy and strong, is most suitable training
Support base.In addition, the 6-BA of high concentration causes shoot tip meristem jaundice browning, vitrifying serious, and there is variation phenomenon.
Influence of 5 hormone combination of table to iris shoot tip meristem
Note: p < 0.05. same letter indicates that difference is not significant, and different letters indicate significant difference.
Influence of 2.3 sucrose concentrations to iris shoot tip meristem
Sucrose concentration has significant impact (to be shown in Table iris stem apex melting brown rate, the death rate, survival rate and growing way feature
7).With the raising of sucrose concentration, the stem apex death rate is obviously increased, survival rate significantly reduces, and wherein T2 is handled, and stem apex growing way is strong
Health, leaf color jade green are most suitable for shoot tip meristem culture.Sucrose concentration it is too high or too it is low cause shoot tip meristem jaundice it is brown
Change, vitrifying are obvious.
Influence of 6 sucrose concentration of table to iris shoot tip meristem
Note: p < 0.05. same letter indicates that difference is not significant, and different letters indicate significant difference.
3. conclusion
According to the interpretation of result of table 4-6, the virus-free culture base of optimization is 1/3MS+6-BA 1.0mgL-1+NAA
0.2mg·L-1+ sucrose 15gL-1+ beef peptone 1gL-1+ AC (active carbon) 1.0gL-1。
2 stem apex of embodiment is repeatedly removed, viral inhibitors and alternating intermittent warming influence survival rate and detoxification efficiency
1. materials and methods
1.1 material
To carry the iris tissue-cultured seedling of CymMV and ORSV compound infection as test material.Cymbidium mosaic virus
(Cymbidium mosaic virus, CymMV) and radula orchid ring spot virus (Odontoglossum ringspot virus,
ORSV) virus detection kit is purchased from An Dezhen Bioisystech Co., Ltd (ADGEN Biotechnology Co., Ltd.).
1.2 method
The sterile working of iris tissue-cultured seedling is carrying out on superclean bench.With dissection under 40 times of cold light source anatomical lens
Needle and scalpel peel off the spire of growing point periphery, expose dimpling, vaulted, shinny growing point.Growing point is cut quickly to be placed on
Virus-free culture culture medium upper surface.Used tool is immersed in sterilized in 75% alcoholic solution after, the calcination and cold on alcolhol burner
But it uses afterwards.
1.2.1 influence of the stem apex removing number to stem apex survival rate and detoxification efficiency
3 experimental groups are designed, the stem apex of the experimental material of test group 1 is removed 1 time, the stem apex of the experimental material of test group 2
The stem apex of removing 2 times, the experimental material of test group 3 is removed 3 times, counts survival rate, the detoxification of the stem apex of each test group respectively
Rate and the mitogenetic seedling growth characteristics of stem apex.
Test group 1 chooses the iris tissue-cultured seedling for carrying CyMV and ORSV compound infection, seedling age 60d, in superclean bench
On, by root culture based on being cleaned in sterile water, it is placed in stainless steel disc, dries up moisture, cut stem apex under body formula anatomical lens
The shoot apical meristem of >=0.5mm 2 phyllopodium of band, is seeded in 1/3MS+6-BA 1.0mgL-1+NAA 0.2mg·L-1+ sugarcane
Sugared 15gL-1+ beef peptone 1gL-1+ AC (active carbon) 1.0gL-1Virus-free culture base on, 3 stem apexs of every bottle of inoculation,
8 bottles of each test group, is repeated 2 times, and 25-28 DEG C of cultivation temperature, humidity 60%, after dark treatment 7 days, illumination 800lux, when culture
Between 30 days, after being handled using anti-browning transfer stem apex regeneration culture medium.
The test stem apex of test group 2 obtains a detoxification stem apex after the test operation of same test group 1, long to the stem apex
Secondary stem apex removing is carried out after 1-2 piece maturation vanelets out, cuts mitogenetic group of stem apex of 2 phyllopodium of band of a detoxification stem apex
It knits, is seeded in 1/3MS+6-BA 1.0mgL-1+NAA 0.2mg·L-1+ sucrose 15gL-1+ beef peptone 1gL-1+AC
(active carbon) 1.0gL-1Virus-free culture base on, 3 stem apexs of every bottle of inoculation, 8 bottles of each test group is repeated 2 times, cultivation temperature
25-28 DEG C, humidity 60%, after dark treatment 7 days, illumination 800lux, incubation time 30 days, stem of transferring after being handled using anti-browning
Sharp regeneration culture medium.
The stem apex of test group 3 obtains secondary detoxification stem apex after the test operation of same test group 2, grows 1- to the stem apex
Stem apex removing three times is carried out after 2 mature vanelets, cuts the shoot apical meristem of 2 phyllopodium of band of secondary detoxification stem apex,
It is seeded in 1/3MS+6-BA 1.0mgL-1+NAA 0.2mg·L-1+ sucrose 15gL-1+ beef peptone 1gL-1+ AC is (living
Property charcoal) 1.0gL-1Virus-free culture base on, 3 stem apexs of every bottle of inoculation, 8 bottles of each test group is repeated 2 times, cultivation temperature 25-
28 DEG C, humidity 60%, after dark treatment 7 days, illumination 800lux, incubation time 30 days, stem apex of transferring after being handled using anti-browning
Regeneration culture medium.
1.2.2 stem apex anti-browning pre-processes the influence to iris stem apex survival rate
Design 2 experimental groups, the detoxification stem apex of test group 1 is pre-processed by anti-browning, the detoxification stem apex of test group 2 without
Anti-browning pretreatment, counts different tests group stem apex melting brown rate and survival rate respectively.
The detoxification stem apex obtained in 1.2.1 is seeded in anti-browning culture medium 1/3MS+ sucrose 15gL by test group 1-1+ ox
Meat peptone 1gL-1+ coconut juice 200mlL-1+ AC (active carbon) 1.0gL-1, 25-28 DEG C of cultivation temperature, humidity 60%, secretly
Switching stem apex regeneration culture medium after processing 15d.
Test group 2 directly transfers the detoxification stem apex obtained in 1.2.1 stem apex regeneration culture medium.
1.2.3 the influence of viral inhibitors and temperature to stem apex survival rate and detoxification efficiency
Test material is handled by the operating procedure filtered out in 1.2.1 and 1.2.2, obtains detoxification stem apex, it will
It is divided into 5 test groups, 8 bottles of each test group, and 3 stem apexs of every bottle of inoculation are repeated 2 times.Test group 1 is viral inhibitors culture
Virazole 50mgL is added in base-1, cultivation temperature be 35 DEG C, test group 2 be addition virazole 50mgL-1, not special designing
Cultivation temperature (i.e. at room temperature), test group 3 does not add virazole, cultivation temperature is 35 DEG C, and test group 4 is not added virazole, handed over
It is 33 DEG C (4h)/20 DEG C (6h) for intermittent warming, test group 5 adds virazole 50mgL-1, alternately intermittent warming be 33 DEG C
(4h)/20 DEG C (6h), is seeded in 1/3MS+6-BA 1.0mgL for the stem apex of each test group-1+NAA 0.2mg·L-1+ sugarcane
Sugared 15gL-1+ beef peptone 1gL-1+ coconut juice 200mlL-1+ virazole 50mgL-1+ amino-oligosaccharide 50mgL-1,
Humidity 60% is cultivated, after dark treatment 15 days, illumination 800lux, the illumination cultivation time 30 days.
1.3 statistical analysis
Test result is all made of Excel2007 and 18.0 statistical software of SPSS is analyzed, with Deng Kenshi duncan's new multiple range method
(Duncan ' s) test is 0.05 horizontal upper comparing difference conspicuousness in P.
2 results and analysis
2.1 stem apexs remove influence of the number to stem apex survival rate and detoxification efficiency
Stem apex remove number to iris stem apex melting brown rate, the death rate, survival rate and growing way feature have significant impact (see
Table 7).With the increase of removing number, the stem apex death rate is obviously increased, wherein secondary stem apex lift-off processing stem apex leaf color is yellowish green,
It grows fine and virus elimination rate is higher, be most suitable for shoot tip meristem culture.
7 stem apex of table removes influence of the number to stem apex survival rate and detoxification efficiency
Note: p < 0.05. same letter indicates that difference is not significant, and different letters indicate significant difference.
2.2 stem apexs pre-process the influence to iris stem apex survival rate
Stem apex pretreatment has significant impact (being shown in Table 8) to iris stem apex melting brown rate, survival rate.Directly switching stem apex at
Motility rate is substantially reduced, melting brown rate dramatically increases, and it is up to 92% that wherein stem apex, which pre-processes 15 days stem apex survival rates,.
8 stem apex of table pre-processes the influence to stem apex survival rate
The influence of 2.3 viral inhibitors and temperature to stem apex survival rate and detoxification efficiency
Viral inhibitors and temperature to iris stem apex survival rate, detoxification efficiency and growing way feature have significant impact (see
Table 9).It can be found that 35 DEG C of high temperature are lethal temperature, the death rate 100%, individual virazole to iris from 5 processing
Processing and individual 33 DEG C/20 DEG C intermittent warmings cannot deviate from virus, and only after the intermittent warming of+33 DEG C of virazole/20 DEG C,
The removal efficiency that the removal efficiency of CyMV is 100%, ORSV is 60%, and the mitogenetic seedling growing way of stem apex is substantially healthy, is able to satisfy subsequent expansion
Numerous demand.Therefore ,+33 DEG C of virazole/20 DEG C intermittent warming is the most suitable poison-removing method of stem apex.
The influence of 9 viral inhibitors of table and temperature to stem apex survival rate and detoxification efficiency
Remarks: "-" stem apex is dead, no record.
3. conclusion
Secondary stem apex lift-off processing is most suitable for shoot tip meristem culture；Stem apex survival rate is most after stem apex pre-processes for stem apex
A height of 92%, significantly improve stem apex survival rate；Virazole is added in viral inhibitors culture medium, 33 DEG C/20 DEG C intermittent warmings are
The most suitable poison-removing method of stem apex, virus elimination rate improve.
The fast numerous operating method of 3 iris stem apex detoxification of embodiment regeneration
1. material
To carry 1031 tissue-cultured seedling of iris of CymMV and ORSV compound infection as test material.Cymbidium mosaic virus
(Cymbidium mosaic virus, CymMV) and radula orchid ring spot virus (Odontoglossum ringspot virus,
ORSV) virus detection kit is purchased from An Dezhen Bioisystech Co., Ltd (ADGEN Biotechnology Co., Ltd.).
2. method
(1) the iris tissue-cultured seedling for carrying CyMV and ORSV compound infection, seedling age 60d, super repeatedly stem apex removing: are chosen
On net workbench, by root culture based on being cleaned in sterile water, it is placed in stainless steel disc, moisture is dried up, under body formula anatomical lens
The shoot apical meristem for cutting 2 phyllopodium of band is seeded in 1/3MS+6-BA 1.0mgL-1+NAA 0.2mg·L-1+ sucrose
15g·L-1+ beef peptone 1gL-1+ AC (active carbon) 1.0gL-1Minimal medium on, it is 25-28 DEG C of cultivation temperature, wet
Degree 60%, after dark treatment 7d, illumination 800lux incubation time 30 days, obtains a detoxification stem apex.
Secondary stem apex removing is carried out after a detoxification stem apex grows 2-3 piece maturation leaflet, cuts the stem of 2 phyllopodium of band
Second of virus-free culture is carried out on sharp separate living tissue inoculation virus-free culture base, obtains secondary detoxification stem apex.
(2) stem apex anti-browning pre-processes: after secondary detoxification stem apex grows 1-2 piece maturation leaflet, cutting 1 phyllopodium of band
Stem-tip tissue be inoculated into anti-browning pretreatment culture medium on.Anti-browning pre-processes culture medium are as follows: is seeded in 1/3MS+ sucrose
15g·L-1+ beef peptone 1gL-1+ coconut juice 200mlL-1+ AC (active carbon) 1.0gL-1, 25-28 DEG C of cultivation temperature, wet
Degree 60%, dark treatment 15d prevents Necrosis.
(3) viral inhibitors combine alternately alternating temperature preculture: processed detoxification stem apex is transferred to viral inhibitors training
It supports on base, viral inhibitors culture medium 1/3MS+6-BA 1.0mgL-1+NAA 0.2mg·L-1+ sucrose 15gL-1+ beef egg
White peptone 1gL-1+ coconut juice 200mlL-1+ virazole 50mgL-1+ amino-oligosaccharide 50mgL-1, alternately temperature match curing conditions are 33
DEG C processing 4h, 20 DEG C of processing 6h, two kinds of temperature conditions cycle alternation in the training period, humidity 60%, after dark treatment 15d, illumination
800lux, incubation time 30 days.
(4) stem apex regeneration culture: the stem apex after culture is transferred in regeneration culture medium, regeneration culture medium 1/2MS+
6-BA 1.0mg·L-1+NAA 0.2mg·L-1+ sucrose 15gL-1+ beef peptone 1gL-1+ coconut juice 200mlL-1, training
25-28 DEG C of temperature, humidity 60% are supported, illumination 800lux incubation time 30 days, obtains stem apex and regenerates tissue-cultured seedling.
(5) detoxic seedling is quickly bred: stem apex regeneration tissue-cultured seedling being cut blade and root, is inoculated in proliferated culture medium: 1/
2MS+6-BA 1.0mg·L-1+NAA 0.2mg·L-1+ sugar 15gL-1+ beef peptone 1gL-1+ coconut juice 150-200ml
L-1, 25-28 DEG C of cultivation temperature, humidity 60%, after dark treatment 7d, illumination 1200lux incubation time 40 days, can be obtained detoxification
Seedling.
3. conclusion
This method combines at multiple stem apex lift-off technology, anti-browning processing, viral inhibitors processing and alternating alternating temperature heat
Tissue-cultured seedling content of toxins is effectively reduced in reason technology, improves stem apex survival rate.
Claims (10)
1. a kind of fast numerous method of iris stem apex detoxification regeneration, which comprises the steps of:
1) first time stem apex is removed: choosing the iris tissue-cultured seedling for carrying CyMV and ORSV compound infection, band 2-3 is cut after cleaning
The shoot apical meristem of piece phyllopodium is seeded in progress first time virus-free culture on virus-free culture base, obtains a detoxification stem apex；
The virus-free culture base are as follows: 1/3MS+6-BA 0.2-1.0mgL-1+NAA 0.05-0.2mg·L-1+ sucrose 12-15g
L-1+ beef peptone 0.8-1gL-1+ active carbon 0.8-1.0gL-1；
2) second of stem apex removing: after a step 1) detoxification stem apex obtained grows 2-3 piece maturation leaflet, band 2- is cut
The shoot apical meristem of 3 phyllopodium is seeded on virus-free culture base described in step 1) and carries out second of virus-free culture, obtains
Secondary detoxification stem apex；
3) stem apex anti-browning pre-processes: after the secondary detoxification stem apex that step 2) obtains grows mature leaflet, cutting band 1-2 piece leaf
The stem-tip tissue of former base is inoculated into dark treatment on anti-browning culture medium, prevents browning dead；
4) viral inhibitors combine alternately alternating temperature preculture: by above-mentioned steps 3) in treated that stem apex is transferred to addition virazole
On the viral inhibitors culture medium of amino-oligosaccharide, in combination with alternately alternating temperature heat treatment；
The alternately alternating temperature heat treatment is 33 DEG C of culture 4h, then 20 DEG C of culture 6h, two kinds of temperature conditions recycle in the training period
Alternately, humidity 50-60% is cultivated, after dark treatment 12-15 days, illumination 800-1000lux, 30-40 days illumination cultivation time；
5) stem apex regeneration culture: will pass through above-mentioned steps 4) treated stem apex, it is transferred in stem apex regeneration culture medium, acquisition stem
Point regeneration tissue-cultured seedling；
6) detoxic seedling is quickly bred: being inoculated in proliferation training after the stem apex regeneration tissue-cultured seedling that step 5) obtains is cut blade and root
It supports base and carries out Multiplying culture, obtain detoxic seedling.
2. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 1)
The condition of culture of first time virus-free culture is 25-28 DEG C of temperature, humidity 50-60%, after dark treatment 5-7 days, then illumination 800-
1000lux, 30-40 days illumination cultivation time.
3. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 2)
The condition of culture of second of virus-free culture is 25-28 DEG C of temperature, humidity 50-60%, after dark treatment 5-7 days, then illumination 800-
1000lux, 30-40 days illumination cultivation time.
4. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 3)
Anti-browning culture medium are as follows: 1/3MS+ sucrose 12-15gL-1+ beef peptone 0.8-1gL-1+ coconut juice 150-200mlL-1+
Active carbon 0.8-1.0gL-1。
5. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 3)
The pretreated condition of culture of stem apex anti-browning be 25-28 DEG C of temperature, humidity 50-60%, dark treatment culture 10-15 days.
6. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 4)
Viral inhibitors culture medium are as follows: 1/3MS+6-BA 0.2-1.0mgL-1+NAA 0.05-0.2mg·L-1+ sucrose 12-15gL-1+ beef peptone 0.8-1gL-1+ coconut juice 150-200mlL-1+ virazole 45-50mgL-1+ amino-oligosaccharide 45-
50mg·L-1。
7. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 5)
Stem apex regeneration culture medium are as follows: 1/2MS+6-BA 0.2-1.0mgL-1+NAA 0.05-0.2mg·L-1+ sucrose 12-15gL-1+
Beef peptone 0.8-1gL-1+ coconut juice 150-200mlL-1。
8. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 5)
The condition of culture of stem apex regeneration culture are as follows: 25-28 DEG C of temperature, humidity 50-60%, illumination 800-1000lux, incubation time 30-
40 days.
9. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 6)
Proliferated culture medium are as follows: 1/2MS+6-BA 1.0-3.0mgL-1+NAA 0.2-0.5mg·L-1+ sucrose 12-15gL-1+ beef
Peptone 0.8-1gL-1+ coconut juice 150-200mlL-1。
10. the fast numerous method of iris stem apex detoxification regeneration as described in claim 1, which is characterized in that in the step 6)
The condition of culture quickly bred of detoxic seedling are as follows: after 25-28 DEG C of temperature, humidity 50-60%, dark treatment 5-7d, illumination 1000-
1200lux, 40-50 days illumination cultivation time.
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