CN107018906B - A kind of method of green bamboo fruit tissue culture disinfection and callus proliferative induction - Google Patents
A kind of method of green bamboo fruit tissue culture disinfection and callus proliferative induction Download PDFInfo
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- CN107018906B CN107018906B CN201710359419.XA CN201710359419A CN107018906B CN 107018906 B CN107018906 B CN 107018906B CN 201710359419 A CN201710359419 A CN 201710359419A CN 107018906 B CN107018906 B CN 107018906B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention discloses a kind of disinfection of green bamboo fruit tissue culture and callus proliferative induction methods, including acquisition, pretreatment, disinfection, callus induction and callus amplification step.The method of the disinfection of green bamboo fruit tissue culture and callus proliferative induction of the present invention, it is a kind of excellent bamboo plant fruit tissue culture technology, it is high effectively to solve the pollution rate that bamboo plant fruit often occurs during tissue culture, the typical problems such as callus proliferative induction difficulty and low survival rate, significantly improve green bamboo fruit survival rate, callus induction rate and proliferation times, using green bamboo fruit tissue culture survival rate of the present invention up to 88%, Callus induction rate is up to 50%, callus proliferation times are up to 8 times, it lays the foundation to establish and improving mixed raw bamboo tissue culture system, it is widely applied cultivation for green bamboo and reliable guarantee is provided.
Description
Technical field
The invention belongs to technical field of plant asexual propagation, and in particular to a kind of disinfection of green bamboo fruit tissue culture and callus induction
Enrichment procedure.
Background technology
Green bamboo (Pleioblastus distichus) are the excellent of grass family (Poaceae) bitter bamboo category (Pleioblastus)
Goodly by class ornamental plant, leaf is emerald green, and extremely resistance to trimming, and the application demand in afforestation is increasing at present, warp
Ji value is also higher and higher, and [Zhang Chunxia, Luo Renxiang, Ding Xingcui wait the tissue cultures of green bamboo and quickly breed [J] plant physiologies
Learn communication, 2010,46 (5)：477-478].
Green bamboo originate in Japan, in the 1980s introduce Nanjing, bloom for the first time in March, 2015, May fruit maturation
[woods tree swallow, model is graceful, Jiang Mingyun, with waiting 3 kinds (kinds) big by bamboo flower organ morphology feature and emendation [J] Nanjing forestry
Learn journal (natural science edition), 2017,41 (1)：189-193].
Bamboo plant reinforces germplasm since blooming cycle is long, the fertile rate of seed is low, Post flowering Large Scale Death phenomenon
The preservation of resource and the protection of genetic diversity are the bases of bamboo class genetic breeding.Bamboo Tissue culture fast breeding technique is quick with it
Efficiently, the advantages that easy to operation and transport, important work is played for the preservation and vast propagation of Fine species for planting germ plasm resource
With [Zhang Min, Lu Yishan, Jiang Zeping, wait Bamboo Tissue culture studies present situations and the Jiangsu application prospect [J] forestry science and technology, and 2007,
34(5)：40-45].
Research about bamboo plant fruit embryo is more and more, but fruit disinfection and callus induction increase the bottleneck pole planted
The earth limits the research of bamboo plant tissue culture.Tissue culture experiment display, green bamboo are a kind of mixed raw bamboos of the one kind being easier to tissue culture, therefore
Establish its embryo system has very important research significance for mixing for raw bamboo class.Therefore by inexpensive, easy to operate
Seed disinfection method and efficient callus proliferation formula carry out the embryo selection that tissue culture experiment is undoubtedly a kind of cost effective
Mode.
Invention content
Goal of the invention：For the deficiencies in the prior art, the object of the present invention is to provide a kind of green bamboo fruit tissue cultures
Disinfection and callus proliferative induction method overcome the problems such as tissue culture pollution rate is high, and callus proliferative induction is difficult, have at low cost, behaviour
Make the advantages that easy.
Technical solution：In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is：
The method of a kind of disinfection of green bamboo fruit tissue culture and callus proliferative induction, includes the following steps：
1) mid-May acquires green bamboo ripening fruits in fine when, will acquire during green bamboo fruit maturation
Fruit handled immediately；
2) the inside and outside bran piece of fruit is peelled off, cleans fruit surface with liquid detergent, flowing water rinses, then is impregnated with carbendazim solution
Fruit finally rinses under flowing water, for use；
3) on aseptic operating platform, fruit is impregnated with alcohol, aseptic water washing for several times, then with mercuric chloride impregnates, sterile water punching
It washes and sterilizes for several times；
4) it will be cultivated in the material access callus inducing medium after disinfection, inducing culture group becomes：MS is cultivated
Base+2,4-D 1.5~3mg/L+KT, 0.3~1mg/L+30g/L sucrose+2.5g/L gellan gums；
5) callus is subjected to Multiplying culture in proliferated culture medium, proliferated culture medium group becomes：MS culture mediums+2,4-D
1~3mg/L+6-BA, 0.5~1.5mg/L+30g/L sucrose+2.5g/L gellan gums.
In step 2), 0.5h is rinsed under flowing water, then fruit 0.5h is impregnated with 0.5% carbendazim solution, finally in stream
It is underwater to rinse 2h, for use.
In step 3), on aseptic operating platform, fruit 30s, aseptic water washing 4 times are impregnated with 70% alcohol, then use
0.1% mercuric chloride impregnates 7min, aseptic water washing 4 times.
In step 4), inducing culture group becomes：1.5~2mg/L+KT0.3 of MS culture medium+2,4-D~0.5mg/L+
30g/L sucrose+2.5g/L gellan gums.
In step 4), inducing culture group becomes：MS culture medium+2,4-D 1.5mg/L+KT 0.5mg/L+30g/L sucrose
+ 2.5g/L gellan gums.
In step 5), proliferated culture medium group becomes：1~3mg/L+6-BA0.5 of MS culture medium+2,4-D~1.5mg/L+
30g/L sucrose+2.5g/L gellan gums.
In step 5), proliferated culture medium group becomes：MS culture medium+2,4-D 1mg/L+6-BA 1mg/L+30g/L sucrose+
2.5g/L gellan gum.
The green bamboo fruit tissue culture disinfection of the present invention and callus proliferative induction method, for bamboo plant fruit tissue culture pollution rate
High, the problems such as callus proliferative induction is difficult, explores the disinfection of green bamboo fruit tissue culture and callus proliferative induction method.This method master
If including acquisition → pretreatment → disinfection → callus induction → callus proliferation.According to said method carry out green bamboo fruit tissue culture, pollution
Rate is relatively low, and survival rate is high, and Callus induction rate is high, and proliferation is fast.
Advantageous effect：Compared with prior art, the method for green bamboo fruit tissue culture of the invention disinfection and callus proliferative induction,
It is a kind of excellent bamboo plant fruit tissue culture technology, effectively solves the pollution that bamboo plant fruit often occurs during tissue culture
Rate is high, and the typical problems such as callus proliferative induction difficulty and low survival rate significantly improve green bamboo fruit survival rate, callus induction
Rate and proliferation times, using green bamboo fruit tissue culture survival rate of the present invention up to 88%, Callus induction rate is up to 50%, callus proliferation times
Up to 8 times, laying the foundation for raw bamboo tissue culture system is mixed to establish and improving, being widely applied cultivation for green bamboo provides reliable guarantee.
Description of the drawings
Fig. 1 is the green bamboo fruit figure with bran piece；
Fig. 2 is the green bamboo fruit figure of bran piece；
Fig. 3 is after callus induces 1 week, to expose radicle figure；
Fig. 4 is after callus induces 2 weeks, to form callus figure；
Fig. 5 is the callus figure after callus induces 1.5 months；
Fig. 6 is the callus figure after callus induces 4 months；
Fig. 7 is the callus figure after callus is proliferated 2 months；
Fig. 8 is the callus figure after callus is proliferated 4 months.
Specific implementation mode
With reference to specific embodiment, the present invention is described further.
1 green bamboo fruit explant of embodiment selects
With the process of green bamboo fruit growth, when fine, the green bamboo of robust growth and no disease and pests harm are acquired
Immature fruit, ripening fruits, take back.Partial mature fruit is dried in the shade, is stored in 4 DEG C of refrigerators.
The present embodiment sets 4 processing altogether.The green bamboo immature fruit of fresh acquisition, ripening fruits, 4 DEG C are preserved 2 months
And after 4 DEG C of ripening fruits disinfections for preserving 6 months, it is inoculated in initial culture medium that (initial culture medium is：MS culture mediums+2,
4-D 2mg/mL+KT 1mg/mL+30g/L sucrose+2.5g/L gellan gums).30 explants of each processing inoculation, are repeated 3 times,
Pollution rate is counted after 15d, and survival rate is counted after 25d.
The results are shown in Table 1, from the point of view of pollution rate, although handle 1 result pollution rate it is minimum, be 5.3%, it goes out
More rate is 0.This illustrates that immature green bamboo fruit is not suitable for callus induction.In processing 3 and 4, pollution rate is higher and healing rate is low,
Even 0, illustrate to increase with the time of seed storage, pollution rate improves, and healing rate reduces.It can be seen that fresh acquisition
Green bamboo fruit is the best explant to form callus.
The different explants of table 1 select the influence to explant culture
Therefore, should be during mid-May, green bamboo fruit maturation according to the above results, acquisition is emerald green in fine when
Bamboo ripening fruits pre-processes the fruit of fresh acquisition immediately.
2 green bamboo fruit explant of embodiment sterilizes
By the green bamboo fruit of fresh acquisition, inside and outside bran piece (such as Fig. 1,2) is peelled off, about half spoonful of liquid detergent is added, cleans fruit
0.5h is rinsed in surface under flowing water；It is impregnated with 0.5% carbendazim solution, rinses 2h under flowing water, bring aseptic operating platform into
On, fruit 30s, aseptic water washing 4 times are impregnated with 70% alcohol, then impregnated with 0.1% mercuric chloride, aseptic water washing 4 times.
The present embodiment sets 6 processing altogether.Disinfection way is：0.5% carbendazim disinfection 0, the mercuric chloride of 0.5h and 0.1% disappear
Poison 5,7,9min combinations, set 6 kinds of processing altogether.Fruit after disinfection is inoculated in initial culture medium that (initial culture medium is：MS is cultivated
Base+2,4-D 2mg/mL+KT 1mg/mL+30g/L sucrose+2.5g/L gellan gums).30 explants of each processing inoculation, repeat
3 times, pollution rate is counted after 15d, survival rate is counted after 25d.
The results are shown in Table 2, and with the growth of 0.1% mercuric chloride processing time, pollution rate reduces, and survival rate increases, but
It is processing 3, although pollution rate is minimum, up to 12.6%, its healing rate reduces.Because disinfecting time is too long to the embryo shape of fruit
At injury.In addition, using 0.5% carbendazim 0.5h than without using pollution rate be substantially reduced, survival rate increase.Thus may be used
See, processing 2 is best sterilization method.
Influence of the different disinfectant processing different times of table 2 to explant culture
Therefore, the best sterilization method of green bamboo fruit is：Liquid detergent cleans, and flowing water rinses 0.5h, and 0.5% carbendazim is molten
Liquid impregnates 0.5h, and flowing water rinses 2h, and 70% alcohol impregnates 30s, aseptic water washing 4 times, and 0.1% mercuric chloride impregnates 7min, nothing
Bacterium water rinses 4 times.
3 green bamboo fruit callus of embodiment induces
The present embodiment selects MS for minimal medium, is produced using growth regulator 2.4-D and KT combination induction green bamboo fruit
Raw callus carries out callus induction, each processing inoculation by test material of the processed fruit of best sterilization method of embodiment 2
30 explants, are repeated 3 times, and are cultivated in 25 ± 2 DEG C, illumination 3000lx, 14h/d, are observed 1 time per 7d, count callus
Inductivity, observe and record the upgrowth situation of callus.
The results are shown in Table 3, and the best hormone prescription of green bamboo fruit callus induction is processing 4, i.e.,：MS+2,4-D
1.5mg/L+KT 0.5mg/L+30g/L sucrose+2.5g/L gellan gums.Result data is compared it is found that handle in 1-9, is gone out
More rate is not low, illustrates that these processing can induce to form callus.Wherein, 4 healing rate highests are handled, for the induction training of best callus
Support base.After callus induces 1 week, expose radicle, as shown in Figure 3；Callus induces 2 weeks, forms callus, as shown in Figure 4；Callus induces
Callus after 1.5 months, as shown in Figure 5；Callus induce 4 months after callus figure, as shown in Figure 6.
3 hormon of table is with the experimental result for comparing the induction of green bamboo fruit callus
| Processing | 2,4-D | KT | Healing rate |
| 1 | 1.5mg/L | 0.3mg/L | 63% |
| 2 | 2mg/L | 0.3mg/L | 62.4% |
| 3 | 3mg/L | 0.3mg/L | 61.6% |
| 4 | 1.5mg/L | 0.5mg/L | 68.9% |
| 5 | 2mg/L | 0.5mg/L | 62.4% |
| 6 | 3mg/L | 0.5mg/L | 58.7% |
| 7 | 1.5mg/L | 1mg/L | 52% |
| 8 | 2mg/L | 1mg/L | 50.9% |
| 9 | 3mg/L | 1mg/L | 45.1% |
4 green bamboo fruit callus of embodiment is proliferated
The callus induced is transferred on callus proliferated culture medium, callus proliferated culture medium selects MS for basic culture
Base is combined using growth regulator 2.4-D and 6-BA, 30 explants of each processing inoculation, in 25 ± 2 DEG C, illumination 3000lx,
It is cultivated in 14h/d, each moon is observed 1 time, is counted the proliferation rate of callus, is observed and recorded the upgrowth situation of callus.
As a result such as table 4 shows, the best hormone prescription of green bamboo fruit callus proliferation is processing 4：MS+2,4-D1mg/L+6-BA
1mg/L+30g/L sucrose+2.5g/L gellan gums.Result data is compared it is found that with 2, the increase of 4-D concentration is proliferated
Multiple reduces, and with the increase of 6-BA concentration, proliferation times first increase to be reduced afterwards.4 callus proliferation times highests are handled, are best
Callus proliferated culture medium.Callus figure of the callus proliferation after 2 months, as shown in Figure 7；Callus figure of the callus proliferation after 4 months, such as schemes
Shown in 8.
4 hormon of table is with the experimental result for comparing callus proliferation
| Processing | 2,4-D | 6-BA | Callus increment multiple after 4 months |
| 1 | 1mg/L | 0.5mg/L | 7.0 again |
| 2 | 2mg/L | 0.5mg/L | 6.6 again |
| 3 | 3mg/L | 0.5mg/L | 5.4 again |
| 4 | 1mg/L | 1mg/L | 8.1 again |
| 5 | 2mg/L | 1mg/L | 7.3 again |
| 6 | 3mg/L | 1mg/L | 6.5 again |
| 7 | 1mg/L | 1.5mg/L | 7.5 again |
| 8 | 2mg/L | 1.5mg/L | 6.1 again |
| 9 | 3mg/L | 1.5mg/L | 5.4 again |
In conclusion entire culture formed the good callus of a large amount of growth conditions after 8 months.
Claims (6)
1. a kind of method of green bamboo fruit tissue culture disinfection and callus proliferative induction, which is characterized in that include the following steps：
1) mid-May acquires green bamboo ripening fruits in fine when, by the fruit of acquisition during green bamboo fruit maturation
It is real to be handled immediately；
2) the inside and outside bran piece of fruit to be peelled off, fruit surface is cleaned with liquid detergent, flowing water rinses, then impregnates fruit with carbendazim solution,
It is finally rinsed under flowing water, for use；
3) on aseptic operating platform, fruit, aseptic water washing are impregnated with alcohol, then impregnated with mercuric chloride, aseptic water washing disinfection；
4) it will be cultivated in the material access callus inducing medium after disinfection, inducing culture group becomes：MS culture mediums+2,
4-D 1.5~3mg/L+KT, 0.3~1mg/L+30g/L sucrose+2.5g/L gellan gums；
5) callus is subjected to Multiplying culture in proliferated culture medium, proliferated culture medium group becomes：MS culture mediums+2,4-D 1~
3mg/L+6-BA 0.5~1.5mg/L+30g/L sucrose+2.5g/L gellan gums.
2. the method for green bamboo fruit tissue culture disinfection according to claim 1 and callus proliferative induction, which is characterized in that step
2) in, 0.5h is rinsed under flowing water, then impregnate fruit 0.5h with 0.5% carbendazim solution, finally rinse 2h under flowing water, wait for
With.
3. the method for green bamboo fruit tissue culture disinfection according to claim 1 and callus proliferative induction, which is characterized in that step
3) in, on aseptic operating platform, fruit 30s, aseptic water washing 4 times are impregnated with 70% alcohol, then impregnated with 0.1% mercuric chloride
7min, aseptic water washing 4 times.
4. the method for green bamboo fruit tissue culture disinfection according to claim 1 and callus proliferative induction, which is characterized in that step
4) in, inducing culture group becomes：MS culture medium+2,4-D 1.5~2mg/L+KT, 0.3~0.5mg/L+30g/L sucrose+
2.5g/L gellan gum.
5. the method for green bamboo fruit tissue culture disinfection according to claim 1 or 4 and callus proliferative induction, which is characterized in that
In step 4), inducing culture group becomes：MS culture medium+2,4-D 1.5mg/L+KT 0.5mg/L+30g/L sucrose+2.5g/L
Gellan gum.
6. the method for green bamboo fruit tissue culture disinfection according to claim 1 and callus proliferative induction, which is characterized in that step
5) in, proliferated culture medium group becomes：MS culture medium+2,4-D 1mg/L+6-BA 1mg/L+30g/L sucrose+2.5g/L gellan gums.
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