CN107278892A - The high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) - Google Patents
The high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) Download PDFInfo
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- CN107278892A CN107278892A CN201710494230.1A CN201710494230A CN107278892A CN 107278892 A CN107278892 A CN 107278892A CN 201710494230 A CN201710494230 A CN 201710494230A CN 107278892 A CN107278892 A CN 107278892A
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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 invention discloses the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora), including explant sterilization, the differentiation of the Fiber differentiation of callus, bud, shoot proliferation culture, two secondary roots and acclimatization and transplantses.Have the beneficial effect that：The time for cultivating purple yu-lan (Magnolia liliflora) seedling using the above method is short, and explant browning rate is low, and dedifferentiation forms callus probability height, and obtained purple yu-lan (Magnolia liliflora) seedling diseases is malicious less, stable hereditary property.The bioactive peptide that amino acid sequence is SCASVCRGKRCGSIFYRGRCYCRCLRC is added in the Fiber differentiation of callus.Above-mentioned bioactive peptide can quickly form colloid in explant incision, polyphenols oozes out in prevention purple yu-lan (Magnolia liliflora) body, above-mentioned active peptides have stronger inoxidizability simultaneously, and polyphenols can be prevented to be oxidized to quinones substance, therefore the content of explant incision quinones substance is very low.
Description
Technical field
The present invention relates to field of plant tissue culture technique, the high-efficiency tissue cultural method of specifically a kind of purple yu-lan (Magnolia liliflora).
Background technology
Purple yu-lan (Magnolia liliflora) (Magnolia liliflora Desr.) also known as the flower bud of lily magnolia, it is Magnoliaceae defoliation small arbor, it is high by 2 ~ 3
M, is distributed in Chinese yunnan, Fujian, Hubei, the ground such as Sichuan, is grown on the area of 300 to 1600 meters of height above sea level, general growth
In hillside edge.Purple yu-lan (Magnolia liliflora) flower is gorgeous pleasant, and fragrance is simple and elegant, and isolated planting or group planting are all very attractive in appearance, is that excellent flower garden, street are green
Change plant, be flowers and the Chinese medicine of Chinese tradition again, but purple yu-lan (Magnolia liliflora) is difficult to transplant and conserved, and has been logged《The world is natural
Protect alliance》Plant Red List, is the flowers and trees being of great rarity.
Now with the development of technology, the method for purple yu-lan (Magnolia liliflora) culture is all being carried out in many areas, wants in this area cultivation kind
Plant purple yu-lan (Magnolia liliflora).Traditional purple yu-lan (Magnolia liliflora) is typically using training methods such as sowing, cuttage, grafting, press strip and division propagations.These cultures
Time-consuming for mode, and breed and be difficult to survive.Some existing purple yu-lan (Magnolia liliflora) tissue cultures modes are the aseptic conditions in closing
Lower progress, required cost is larger, but also there is the easy browning of explant in tissue cultures, the problem of survival rate is not high,
It cannot be used for the amount reproduction of purple yu-lan (Magnolia liliflora).
Prior art such as Authorization Notice No. is the B of CN 103718966 Chinese invention patent, purple yu-lan (Magnolia liliflora) tissue cultures side
Method, the magnolia liliiflora tissue culture method is carried out under the conditions of open, and this method is including to purple yu-lan (Magnolia liliflora) tissue cultures
The antibacterial processing of sterilizing, the selection of explant, the sterilization of explant, the Initial culture of explant, Multiplying culture and the life on ground
The steps such as root culture.In the above method and unresolved purple yu-lan (Magnolia liliflora) explant cultivating process the problem of easy browning.
The content of the invention
It is an object of the invention to provide a kind of explant browning rate is low, dedifferentiation forms the high Qarnet of callus probability
Blue high-efficiency tissue cultural method.
The present invention is directed to the problem of being mentioned in background technology, and the technical scheme taken is：The high-efficiency tissue culture of purple yu-lan (Magnolia liliflora)
Method, including explant sterilization, the differentiation of the Fiber differentiation of callus, bud, shoot proliferation culture, two secondary roots and hardening are moved
Plant.The time for cultivating purple yu-lan (Magnolia liliflora) seedling using the above method is short, and explant browning rate is low, and dedifferentiation forms callus probability height,
Obtained purple yu-lan (Magnolia liliflora) seedling diseases poison less, stable hereditary property, blade area is big, and in bright green, vitality is vigorous, and the root grown is in white
The sturdy type of color, the absorption efficiency to moisture and mineral matter is high.Induction of callus step includes shifting explant lateral bud
To the MS of 0.7 ~ 1.5mg/L6-BA of addition, 0.08 ~ 0.15mg/LIAA, 2.5 ~ 3.5 mg/L and 0.7 ~ 1.8 μ g/L bioactive peptides
The differentiation of induced bud in culture medium.Using above method induction explant dedifferentiation generation callus, explant incision quinone
Class content of material is very low, and browning disappears substantially, and obtained callus cell arrangement is close, quality is softer, purple yu-lan (Magnolia liliflora)
Survival rate be substantially improved.
Preferably, the amino acid sequence of bioactive peptide is SCASVCRGKRCGSIFYRGRCYCRCLRC.Above-mentioned activity is short
Peptide can quickly form colloid in explant incision, oozing out for polyphenols in purple yu-lan (Magnolia liliflora) body be prevented, while above-mentioned activity is more
Peptide has stronger inoxidizability, and polyphenols can be prevented to be oxidized to quinones substance, therefore explant incision quinones thing
The content of matter is very low.
Preferably, explant sterilisation step is first with 65 ~ 85% 20 ~ 40s of alcohol-pickled purple yu-lan (Magnolia liliflora) lateral bud, then with sterile
Water is rinsed well, with 0.06 ~ 0.13% HgCl2Sterilize 3 ~ 15min, aseptic water washing 5 ~ 8 times.By the stronger alcohol of permeability
Weak mercuric chloride is used cooperatively with permeability, and Disinfection Effect is good.
Preferably, bud differentiation step is that callus is transferred to the explant sterilized to be inoculated in fructose or grape
Sugar is carbon source, adds in BA0.3 ~ 0.6mg/L and NAA0.008 ~ 0.014mg/L MS culture mediums and cultivates.Callus in the above method
Differentiation rate is high again for tissue, and green projection occurs in 4 ~ 8d explants incision, is further cultured for 15 ~ 25d and Multiple Buds occurs.
Preferably, shoot proliferation incubation step is to separate Multiple Buds with the radix of 2 ~ 4 buds, addition is transferred to 0.32 ~
Cultivated in 0.43mg/LZT, 2.0 ~ 3.2mg/L2,4-D, the MS culture mediums of 5.2 ~ 6.5g/L agar and 23 ~ 34g/L sucrose.It is above-mentioned
Method can obtain substantial amounts of without offspring, and unrooted seedling leaf area is big, and in bright green, vitality is vigorous；Obtained by same explant
Arrive, stabilization characteristics of genetics, the merit of parent can be kept well.
Preferably, two secondary root steps for will be first transferred to without offspring 0.08 ~ 0.14mg/L6-BA of addition and 1.5 ~
In 2.6mg/LNAA MS culture mediums, it is transferred in the 1/2MS culture mediums without any hormone and cultivates after 15 ~ 24d of culture.It is above-mentioned
Method can accelerate rooting rate of the purple yu-lan (Magnolia liliflora) without offspring, and the white sturdy type of root grown, the suction to moisture and mineral matter
Receive efficiency high.
Preferably, acclimatization and transplantses step includes treating that purple yu-lan (Magnolia liliflora) test tube seedling is cultivated in blake bottle to more than 6 leaves
After son and flourishing root system, first opening 1.5 ~ 2.5d of hardening, then adding 0.04 ~ 0.06mg/LIBA, 0.04 ~ 0.06mg/
Soaked in LNAA, the 1/8MS culture mediums of 12 ~ 16g/L sucrose and root is cleaned after 1.5 ~ 2.5d of root, seedling is transferred to equipped with soil sand, and is poured
Have in 1/30 ~ 1/15MS basin, plant ash is added in sandy soil.The cuticula of test tube seedling blade surface has been formed, and is effective against
Low intensive light irradiation and reduction moisture loss, but resisting stress is weak, and the above method can improve purple yu-lan (Magnolia liliflora) seedling straw stiffness, increase
Resist sudden ability by force.
Acclimatization and transplantses step is cultivated including purple yu-lan (Magnolia liliflora) seedling under 25 ~ 30 DEG C of environment, is poured and is moved after a water, 4 ~ 6d every 1 ~ 2d
Enter and cultivated under sunlight.
Compared with prior art, the advantage of the invention is that：
1. the time for cultivating purple yu-lan (Magnolia liliflora) seedling using the above method is short, explant browning rate is low, and dedifferentiation forms callus probability
Height, obtained purple yu-lan (Magnolia liliflora) seedling diseases is malicious less, stable hereditary property, and blade area is big, and in bright green, vitality is vigorous, the root grown
White sturdy type, the absorption efficiency to moisture and mineral matter is high.
2. it is SCASVCRGKRCGSIFYRGRCYCRCLRC that amino acid sequence is added in the Fiber differentiation of callus
Bioactive peptide.Above-mentioned bioactive peptide can quickly form colloid in explant incision, prevent polyphenols in purple yu-lan (Magnolia liliflora) body
Ooze out, while above-mentioned active peptides have stronger inoxidizability, polyphenols can be prevented to be oxidized to quinones substance, because
The content of this explant incision quinones substance is very low.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora), including explant sterilization, the differentiation of the Fiber differentiation of callus, bud, subculture increasing
Grow culture, two secondary roots and acclimatization and transplantses.The time for cultivating purple yu-lan (Magnolia liliflora) seedling using the above method is short, and explant browning rate is low, takes off
Callus probability height is differentiated to form, obtained purple yu-lan (Magnolia liliflora) seedling diseases is malicious less, stable hereditary property, blade area is big, in bright green,
Vitality is vigorous, the white sturdy type of the root grown, and the absorption efficiency to moisture and mineral matter is high.Induction of callus is walked
It is rapid to include explant lateral bud being transferred to addition 1.1mg/L6-BA, 0.13mg/LIAA, 2.8 mg/L and 1.2 μ g/L bioactive peptides
MS culture mediums in induced bud differentiation.Using above method induction explant dedifferentiation generation callus, explant otch
Place's quinones substance content is very low, and browning disappears substantially, and obtained callus cell arrangement is close, quality is softer, purple
The survival rate of yulan is substantially improved.
The amino acid sequence of bioactive peptide is SCASVCRGKRCGSIFYRGRCYCRCLRC.Above-mentioned bioactive peptide can be outside
Implant incision quickly forms colloid, prevents oozing out for polyphenols in purple yu-lan (Magnolia liliflora) body, at the same above-mentioned active peptides have compared with
Strong inoxidizability, can prevent polyphenols to be oxidized to quinones substance, therefore the content of explant incision quinones substance
It is very low.
Explant sterilisation step is first with 80% alcohol-pickled purple yu-lan (Magnolia liliflora) lateral bud 20s, then, use clean with aseptic water washing
0.1% HgCl2Sterilize 6min, aseptic water washing 6 times.The stronger alcohol of permeability and the weak mercuric chloride of permeability are used cooperatively,
Disinfection Effect is good.
Bud differentiation step is that callus is transferred to the explant sterilized to be inoculated in using fructose or glucose as carbon source,
Add in BA0.4mg/L and NAA0.011mg/L MS culture mediums and cultivate.Differentiation rate is high again for callus in the above method, outside 7d
There is green projection in implant incision, is further cultured for 22d and Multiple Buds occurs.
Shoot proliferation incubation step is separates Multiple Buds with the radix of 3 buds, and addition is transferred to 0.39mg/LZT, 2.8mg/
Cultivated in the MS culture mediums of L2,4-D, 6.1g/L agar and 29g/L sucrose.The above method can obtain substantial amounts of without offspring, unrooted
Seedling leaf area is big, and in bright green, vitality is vigorous；Obtained, stabilization characteristics of genetics, can kept well by same explant
The merit of parent.
Two secondary root steps are the MS culture mediums for adding 0.12mg/L6-BA and 2.2mg/LNAA will to be first transferred to without offspring
In, it is transferred in the 1/2MS culture mediums without any hormone and cultivates after culture 22d.The above method can accelerate purple yu-lan (Magnolia liliflora) without offspring
Rooting rate, and the white sturdy type of root grown, the absorption efficiency to moisture and mineral matter is high.
Acclimatization and transplantses step includes treating that purple yu-lan (Magnolia liliflora) test tube seedling is cultivated in blake bottle to more than 6 leaves and prosperity
After root system, first opening hardening 2d, then in the 1/8MS culture mediums of addition 0.05mg/LIBA, 0.05mg/LNAA, 14g/L sucrose
Soak and root is cleaned after root 2d, seedling is transferred to equipped with soil sand, and pour in 1/20MS basin, plant ash is added in sandy soil.Test tube seedling
The cuticula of blade surface has been formed, and is effective against low intensive light irradiation and is reduced moisture loss, but resisting stress is weak,
The above method can improve purple yu-lan (Magnolia liliflora) seedling straw stiffness, and enhancing resists sudden ability.
Acclimatization and transplantses step is cultivated including purple yu-lan (Magnolia liliflora) seedling under 28 DEG C of environment, is poured and is moved into after a water, 4 ~ 6d every 1 ~ 2d
Cultivated under sunlight.
Embodiment 2：
The high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora), comprises the following steps：
1）Explant is sterilized：First with 75% alcohol-pickled purple yu-lan (Magnolia liliflora) lateral bud 30s, then it is clean with aseptic water washing, with 0.1%
HgCl2Sterilize 7min, aseptic water washing 6 times；
2）Induction of callus：Explant lateral bud is transferred into addition 1mg/L6-BA, 0.1mg/LIAA and 1.5 μ g/L to live
The differentiation of induced bud, in 25 DEG C, 18d is cultivated under the conditions of illumination 2000lx in the MS culture mediums of property small peptide.The amino of bioactive peptide
Acid sequence is SCASVCRGKRCGSIFYRGRCYCRCLRC；
3）Bud breaks up：Callus is transferred to the explant sterilized to be inoculated in using fructose or glucose as carbon source, added
Cultivated in BA0.5mg/L and NAA0.01mg/L MS culture mediums.The notch after culture 6d under the conditions of 25 DEG C, illumination 2000lx
Separate existing green raised, be further cultured for 20d and Multiple Buds occur, be further cultured for 5d Multiple Buds and grow to 2 ~ 4cm；
4）Shoot proliferation culture：Multiple Buds are separated with the radix of 3 buds, is transferred to and adds 0.4mg/LZT, 2.5mg/L2,4-D,
In the MS culture mediums of 6g/L agar and 30g/L sucrose, and 10d is cultivated under the conditions of 25 DEG C, illumination 2000lx；
5）Two secondary roots：To first it be transferred to without offspring in addition 0.1mg/L6-BA and 2mg/LNAA MS culture mediums, light culture
It is transferred to after 20d in the 1/2MS culture mediums without any hormone and continues light culture 14d；
6）Acclimatization and transplantses：Cultivated after purple yu-lan (Magnolia liliflora) test tube seedling in blake bottle to after with more than 6 leaves and flourishing root system, first
Open hardening 2d, then washed in the 1/8MS culture mediums of addition 0.05mg/LIBA, 0.05mg/LNAA, 14g/L sucrose after leaching root 2d
Net root, seedling is transferred to equipped with soil sand, and is poured in 1/20MS basin, and plant ash is added in sandy soil.Acclimatization and transplantses step includes
Purple yu-lan (Magnolia liliflora) seedling is cultivated under 27 DEG C of environment, is poured to move into after a water, 4 ~ 6d under sunlight every 1 ~ 2d and is cultivated.
Embodiment 3：
The high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora), comprises the following steps：
1）Explant is sterilized：First with 70% alcohol-pickled purple yu-lan (Magnolia liliflora) lateral bud 40s, then it is clean with aseptic water washing, with 0.1%
HgCl2Sterilize 7min, aseptic water washing 7 times；
2）Induction of callus：Explant lateral bud is transferred to addition 0.9mg/L6-BA, 0.12mg/LIAA and 1.1 μ g/L
The differentiation of induced bud, 19d is cultivated under the conditions of 25 DEG C, illumination 2500lx in the MS culture mediums of bioactive peptide.The ammonia of bioactive peptide
Base acid sequence is SCASVCRGKRCGSIFYRGRCYCRCLRC；
3）Bud breaks up：Callus is transferred to the explant sterilized to be inoculated in using fructose or glucose as carbon source, added
Cultivated in BA0.48mg/L and NAA0.009mg/L MS culture mediums.The otch after culture 6d under the conditions of 25 DEG C, illumination 2500lx
There is green projection in part, is further cultured for 23d and Multiple Buds occurs, is further cultured for 5d Multiple Buds and grows to 2 ~ 4cm；
4）Shoot proliferation culture：Multiple Buds are separated with the radix of 3 buds, is transferred to and adds 0.38mg/LZT, 2.2mg/L2,4-D,
In the MS culture mediums of 6g/L agar and 30g/L sucrose, and 10d is cultivated under the conditions of 25 DEG C, illumination 2500lx；
5）Two secondary roots：To first it be transferred to without offspring in addition 0.1mg/L6-BA and 2mg/LNAA MS culture mediums, light culture
It is transferred to after 20d in the 1/2MS culture mediums without any hormone and continues light culture 14d；
6）Acclimatization and transplantses：Cultivated after purple yu-lan (Magnolia liliflora) test tube seedling in blake bottle to after with more than 6 leaves and flourishing root system, first
Open hardening 2d, then in the 1/8MS culture mediums of addition 0.047mg/LIBA, 0.047mg/LNAA, 14g/L sucrose after leaching root 2d
Root is cleaned, seedling is transferred to equipped with soil sand, and is poured in 1/20MS basin, plant ash is added in sandy soil.Acclimatization and transplantses step bag
Include purple yu-lan (Magnolia liliflora) seedling to cultivate under 27 DEG C of environment, pour to move into after a water, 4 ~ 6d under sunlight every 1 ~ 2d and cultivate.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail embodiment described above, it should be understood that it is described above only
For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should be included in the scope of the protection.
SEQUENCE LISTING
<110>Pujiang County Mei Ze bio tech ltd
<120>The high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora)
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 27
<212> PRT
<213>It is artificial synthesized
<400> 1
Ser Cys Ala Ser Val Cys Arg Gly Lys Arg Cys Gly Ser Ile Phe Tyr
1 5 10 15
Arg Gly Arg Cys Tyr Cys Arg Cys Leu Arg Cys
20 25
Claims (8)
1. the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora), including explant sterilization, the differentiation of the Fiber differentiation of callus, bud, subculture
Multiplying culture, two secondary roots and acclimatization and transplantses, it is characterised in that：Described induction of callus step is included explant
Lateral bud is transferred to the MS cultures of 0.7 ~ 1.5mg/L6-BA of addition, 0.08 ~ 0.15mg/LIAA and 0.7 ~ 1.8 μ g/L bioactive peptides
The differentiation of induced bud in base.
2. the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) according to claim 1, it is characterised in that：Described bioactive peptide
Amino acid sequence is SCASVCRGKRCGSIFYRGRCYCRCLRC.
3. the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) according to claim 1, it is characterised in that：Described explant sterilization
Step for first use 65 ~ 85% 20 ~ 40s of alcohol-pickled purple yu-lan (Magnolia liliflora) lateral bud, then with aseptic water washing totally, with 0.06 ~ 0.13%
HgCl2Sterilize 3 ~ 15min, aseptic water washing 5 ~ 8 times.
4. the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) according to claim 1, it is characterised in that：Described bud differentiation step
It is inoculated in for callus to be transferred to the explant sterilized using fructose or glucose as carbon source, adds BA0.3 ~ 0.6mg/L
Cultivated with NAA0.008 ~ 0.014mg/L MS culture mediums.
5. the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) according to claim 1, it is characterised in that：Described shoot proliferation training
It is to separate Multiple Buds with the radix of 2 ~ 4 buds to support step, adds and is transferred to 0.32 ~ 0.43mg/LZT, 2.0 ~ 3.2mg/L2,4-D,
Cultivated in the MS culture mediums of 5.2 ~ 6.5g/L agar and 23 ~ 34g/L sucrose.
6. the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) according to claim 1, it is characterised in that：Described two secondary roots step
Rapid is first to be transferred to without offspring in addition 0.08 ~ 0.14mg/L6-BA and 1.5 ~ 2.6mg/LNAA MS culture mediums, culture 15 ~
It is transferred in the 1/2MS culture mediums without any hormone and cultivates after 24d.
7. the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) according to claim 1, it is characterised in that：Described acclimatization and transplantses step
Suddenly include cultivating in blake bottle to after with more than 6 leaves and flourishing root system after purple yu-lan (Magnolia liliflora) test tube seedling, first opening hardening
1.5 ~ 2.5d, then in the 1/8MS culture mediums for adding 0.04 ~ 0.06mg/LIBA, 0.04 ~ 0.06mg/LNAA, 12 ~ 16g/L sucrose
Root is cleaned after 1.5 ~ 2.5d of middle leaching root, seedling is transferred to equipped with soil sand, and is poured in 1/30 ~ 1/15MS basin, is added in sandy soil
Plant ash.
8. the high-efficiency tissue cultural method of purple yu-lan (Magnolia liliflora) according to claim 1, it is characterised in that：Described acclimatization and transplantses step
Suddenly cultivated including purple yu-lan (Magnolia liliflora) seedling under 25 ~ 30 DEG C of environment, pour to move into after a water, 4 ~ 6d under sunlight every 1 ~ 2d and cultivate.
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| CN111557197A (en) * | 2020-03-09 | 2020-08-21 | 霸州市禹德农业科技有限公司 | Magnolia liliiflora division planting method |
| CN114158481A (en) * | 2021-12-27 | 2022-03-11 | 浙江宜格企业管理集团有限公司 | Preparation method of magnolia callus culture |
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| CN111557197A (en) * | 2020-03-09 | 2020-08-21 | 霸州市禹德农业科技有限公司 | Magnolia liliiflora division planting method |
| CN114158481A (en) * | 2021-12-27 | 2022-03-11 | 浙江宜格企业管理集团有限公司 | Preparation method of magnolia callus culture |
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Application publication date: 20171024 |