CN109275564A - A kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts the method for building up of rapid propagation system - Google Patents
A kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts the method for building up of rapid propagation system Download PDFInfo
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- CN109275564A CN109275564A CN201811213424.0A CN201811213424A CN109275564A CN 109275564 A CN109275564 A CN 109275564A CN 201811213424 A CN201811213424 A CN 201811213424A CN 109275564 A CN109275564 A CN 109275564A
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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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- Developmental Biology & Embryology (AREA)
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Abstract
The invention discloses the method for building up that a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts rapid propagation system, disinfection and inoculation including explant blade, leaf inserts three steps of Multiplying culture and Rooting and hardening-off culture, basal medium is used in incubation, and the specific culture medium of additional MS nutrient solution and hormone, first by the blade as explant by wiping, concussion washing, oblique cutting enters in basal medium downward at leaf bright end after flushing and sterile deionized water are rinsed, it is formed from petiole base addition Multiplying culture liquid evoking adventive bud, minimal medium is transferred to after the adventitious bud clump of formation is finally divided into single plant, from simple bud root, addition Rooting and hardening-off culture liquid carries out strong plantlets and rootage.The present invention inserts rapid propagation cultivation by the leaf that tissue culture technique realizes Qinling Mountains purpleflower stonecrop herb; growth coefficient is high; the feature that natural propagation mode can be overcome slower; the consistent high quality seedling of a large amount of stabilization characteristics of genetics is obtained in a short time, and there is positive effect to Qinling Mountains purpleflower stonecrop herb fast breeding and plasm resource protection.
Description
Technical field
The present invention relates to field of plant tissue culture technique, specially a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts rapid propagation system
Method for building up.
Background technique
Qinling Mountains purpleflower stonecrop herb (Petrocosmea qinlingensis W.T.Wang) is Gesneriaceae (Gesneriaceae)
Petrocosmea (Petrocosmea) plant is the distinctive herbaceos perennial in Qinling area, the month at florescence 8-9, is born in mountainous region rock
Shi Shang, is listed in national II grade of Top-rated protected wild plants, and be decided to be endangered species by 650 meters of height above sea level.Qinling Mountains purpleflower stonecrop herb is stone butterfly
Butterfly belongs to the type of distribution northernmost, and Natural Populations quantity is few, and multiple field investigation does not find its wild population, and researcher was once
Think that this kind has become extinct.Shaanxi Province's investigating of wild plant resources in 2015 has rediscovered this kind, and distributed areas are very narrow,
It is only limitted near the Cha Dianzhen of Shaanxi Province Mian County, is born on lime stone precipices dark and damp, that moss accumulation is more more.
Purpleflower stonecrop herb seed sterility in the Qinling Mountains under natural conditions, vegetative propagation speed are again very slow.Habitat is unique and modes of reproduction limits
System is that this kind causes the major reason being on the point of.The quick breeding that Qinling Mountains purpleflower stonecrop herb is carried out using plant tissue culture technique, can be short
It is obtained in time largely with the consistent high quality seedling of maternal plant genetic background, the feature for overcoming natural propagation mode slower, still,
The report quickly bred in the purpleflower stonecrop herb test tube of the Qinling Mountains is established not yet before this, more without successful example.It is achieved that the Qinling Mountains
The quick breeding of purpleflower stonecrop herb returns the field of Qinling Mountains purpleflower stonecrop herb to have great importance with plasm resource protection.
Summary of the invention
The purpose of the present invention is to provide one kind using band handle blade as explant material, inserts by blade disinfection, inoculation, leaf
Proliferation and strong plantlets and rootage and etc. successfully realize Qinling Mountains purpleflower stonecrop herb leaf insert rapid propagation cultivation, can overcome natural propagation mode compared with
Slow feature obtains the consistent high quality seedling of a large amount of stabilization characteristics of genetics in a short time, and technology is easily grasped, and growth coefficient is high
Qinling Mountains purpleflower stonecrop herb test tube internal lobe insert rapid propagation system.
To achieve the above object, present invention provide the technical scheme that
A kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts the method for building up of rapid propagation system, comprising the following steps:
(1) disinfection and inoculation of blade: the band handle blade that health is won from the purpleflower stonecrop herb maternal plant of the Qinling Mountains is explant material,
Explant material is cleaned and is sterilized, then oblique cutting enters basic culture downward by explant material sterile working inferior lobe pommel
In base, basal medium sterilizes at 121.3 DEG C 60min after adding water to be mixed by the perlite, vermiculite and peat of certain volume ratio
It is made；
(2) leaf inserts Multiplying culture: oblique cutting enters the petiole base of the explant material of basal medium to training from step (1)
Support and add Multiplying culture liquid in base, the formation of evoking adventive bud, Multiplying culture liquid be MS nutrient solution+IBA, IBA concentration be 100~
600mg/L, pH value 5.8；
(3) Rooting and hardening-off culture: being divided into single plant for adventitious bud clump obtained in step (2), selects sprout greater than 2cm and band
There is the single plant of 3~5 leaves to be transferred to basal medium, the progress of Rooting and hardening-off culture liquid is added into basal medium from simple bud base portion
Strong plantlets and rootage, it is 200mg/L, pH value 5.8 that wherein Rooting and hardening-off culture liquid, which is MS nutrient solution+IBA, IBA concentration,.
Preferably, in step (1) and (3) basal medium preparation process are as follows:
Perlite, vermiculite and peat are measured first, in accordance with the volume ratio of 2:1:1 and water is added to be mixed, after the saturation that absorbs water respectively
Be fitted into the culture bottle of 240mL, be packed into volume be culture bottle volume 1/3, then cover ventilating cover, finally by culture bottle in
Sterilize 60min at 121.3 DEG C, obtains basal medium.
Preferably, sucrose, pH value 5.8 is not added in MS nutrient solution in step (1) and (3).
Preferably, the cleaning of step (1) Explants and disinfecting process are as follows:
It first using the dust and impurity on banister brush wiping removal surface, is then placed in 250mL wide-mouth bottle, instills 2 drops
Tap water is added after dish washing liquid, uses tap water to rinse 30~40min after 10~15min of concussion washing, is finally gone using sterilizing
Ionized water rinses 7~8 times, each 1min.
Preferably, the IBA for the MS nutrient solution+1mL that the additional amount of Multiplying culture liquid is 1mL in step (2), adventitious bud clump
Forming the time is 4~8 weeks.
Preferably, the IBA for the MS nutrient solution+0.5mL that the additional amount of Rooting and hardening-off culture liquid is 1mL in step (3), culture
Become within 3~6 weeks the strong sprout of well developed root system, part single plant is normally bloomed in culture bottle after culture 8 weeks.
Preferably, the condition of culture in step (1) (2) (3) is 25+2 DEG C of cultivation temperature, intensity of illumination 60-100umol
m-2·S-1, light application time 16hd-1, humidity 60-70%.
Compared with prior art, beneficial effects of the present invention are as follows:
1, the present invention inserts proliferation and strong plantlets and rootage by blade disinfection, inoculation, leaf using band handle blade as explant material
And etc. by tissue culture technique successfully realize Qinling Mountains purpleflower stonecrop herb leaf insert rapid propagation cultivation, establish the high Qinling Mountains of growth coefficient
Purpleflower stonecrop herb test tube internal lobe inserts rapid propagation system.
2, the feature that method provided by the invention can overcome Qinling Mountains purpleflower stonecrop herb natural propagation mode slower, in a short time
The consistent high quality seedling of a large amount of stabilization characteristics of genetics is obtained, planting percent is high, and technology is easily grasped.
Specific embodiment
Following specific embodiments are a further detailed description of the invention, and are not limitations of the present invention.
Embodiment 1
(1) preparation of culture medium: culture medium used in the purpleflower stonecrop herb incubation of the Qinling Mountains, including basis culture are prepared first
The specific culture medium of base and additional MS nutrient solution and hormone, the wherein process for preparation of basal medium are as follows: according to the volume of 2:1:1
Than measuring perlite, vermiculite and peat and water being added to be mixed, in the culture bottle for being respectively charged into 240mL after the saturation that absorbs water, it is packed into body
Product is the 1/3 of culture bottle volume, then covers ventilating cover, and finally culture bottle sterilizes 60min at 121.3 DEG C；Multiplying culture
Liquid is that the concentration of MS nutrient solution+IBA, IBA are 100mg/L；Rooting and hardening-off culture liquid is that the concentration of MS nutrient solution+IBA, IBA are
200mg/L；Sucrose, pH value 5.8 is not added in MS nutrient solution used in Multiplying culture liquid and Rooting and hardening-off culture liquid.
(2) disinfection and inoculation of blade: it is explant material that health care belt handle blade is won from the purpleflower stonecrop herb maternal plant of the Qinling Mountains, will
It first wipes removal surface dirt and impurity with banister brush, is then placed in 250mL wide-mouth bottle and instills 2 drop dish washing liquids, be added
Tap water shakes washing 10min, then rinses 30min using tap water, finally using sterile deionized water flushing 8 times, every time
1min；Band handle blade petiole after cleaning and sterilizing is passed down through sterile working process oblique cutting to enter in basal medium.
(3) leaf insert Multiplying culture: the petiole base that oblique cutting enters basal medium from step (2) added into culture medium by
The Multiplying culture liquid that the pH for the IBA composition that the MS nutrient solution of 1mL and the concentration of 1mL are 100mg/L is 5.8, then in 25+2's
Cultivation temperature, 60~100umolm-2·S-1Intensity of illumination and 60~70% humidity under carry out 16hd-1Illumination
To the formation of evoking adventive bud, culture initially forms adventitious bud for 4 weeks, and adventitious bud first occurs at petiole base, cultivates the 5th week
The growth coefficient of each blade is 3.67, and the maximum growth coefficient of single blade is 7, cultivates being averaged to the 8th week each blade
Growth coefficient is 22.67, and wherein the maximum growth coefficient of single blade is up to 36.
(4) Rooting and hardening-off culture: the adventitious bud that step (3) is obtained is selected sprout greater than 2cm and is had from single plant is divided into
The single plant of 3~5 leaves is transferred to basal medium, and MS nutrient solution and 0.5mL by 1mL are added from simple bud base portion into culture medium
The Rooting and hardening-off culture liquid that the pH of IBA that concentration is 200mg/L composition is 5.8, then in 25+2 DEG C cultivation temperature, 60~
100umol·m-2·S-1Intensity of illumination and 60~70% humidity under carry out 16hd-1Illumination, to take root
Strong sprout, culture start to take root on the 2nd week, become the strong sprout of well developed root system after the 6th week, after the 8th week part single plant in culture bottle just
Normally opened flower.
Embodiment 2
It is identical as the basic step of embodiment 1, the difference is that Multiplying culture liquid used in step (3) is sought by the MS of 1mL
The IBA that nutrient solution and 1mL concentration are 200mg/L is formed, pH 5.8.
It is added after the Multiplying culture liquid cultivates under the conditions of same as Example 1, initially forms adventitious bud within the 4th week, it is indefinite
Bud can be formed in face of blade and the back side, and majority occurs in the nearly petiole base of blade, and minority occurs to cultivate the 5th week in leaf margin
The growth coefficient of each blade is 11.67, and the maximum growth coefficient of single blade is 16, cultivates putting down to the 8th week each blade
Equal growth coefficient is 51, and wherein the maximum growth coefficient of single blade is up to 68.
Embodiment 3
It is identical as the basic step of embodiment 1, the difference is that Multiplying culture liquid used in step (3) is sought by the MS of 1mL
The IBA that nutrient solution and 1mL concentration are 400mg/L is formed, pH 5.8.
It is added after the Multiplying culture liquid cultivates under the conditions of same as Example 1, the 4th week starts in the nearly petiole base of blade
Adventitious bud is formed, while petiole base forms a small amount of white Hairy root, the growth coefficient of the 5th week each blade of culture is
9.33, the maximum growth coefficient of single blade is 12, and cultivating to the average coefficient of proliferation of the 8th week each blade is 33.67, wherein
The maximum growth coefficient of single blade is up to 37.
Embodiment 4
It is identical as the basic step of embodiment 1, the difference is that Multiplying culture liquid used in step (3) is sought by the MS of 1mL
The IBA that nutrient solution and 1mL concentration are 600mg/L is formed, pH 5.8.
It is added after the Multiplying culture liquid cultivates under the conditions of same as Example 1, the 3rd week petiole base forms a large amount of obvious
White Hairy root, culture initially forms adventitious bud on the 4th week, and adventitious bud majority occurs in the nearly petiole base of blade, in front and
The back side can be formed, and the growth coefficient of the 5th week each blade of culture is 4.33, and single blade maximum growth coefficient is 6, and culture is extremely
The average coefficient of proliferation of 8th week each blade is 16.33, and wherein the maximum growth coefficient of single blade is up to 21.
Comparative example
It is identical as the basic step of embodiment 2, the difference is that the MS that Multiplying culture liquid used in step (3) is 1mL is sought
(IBA+TDZ+NAA) of nutrient solution and 1mL, wherein the concentration of IBA is 200mg/L, and TDZ concentration is 2.0mg/L, and NAA concentration is
0.2mg/L, pH 5.8.
It is added after the Multiplying culture liquid cultivates under the conditions of same as Example 2, initially forms adventitious bud within the 4th week, it is indefinite
Bud majority occurs be formed at the nearly petiole end of blade in front and back, and the growth coefficient of the 5th week each blade of culture is
5.67, single blade maximum growth coefficient is 6, and cultivating to the average coefficient of proliferation of the 8th week each blade is 28.33, wherein single
The maximum growth coefficient of a blade is up to 32.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (7)
1. the method for building up that a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts rapid propagation system, which comprises the following steps:
(1) disinfection and inoculation of blade: the band handle blade that health is won from the purpleflower stonecrop herb maternal plant of the Qinling Mountains is explant material, externally
Implant material is cleaned and is sterilized, and then by explant material sterile working inferior lobe pommel, oblique cutting enters basal medium downward
In, the basal medium sterilizes at 121.3 DEG C after adding water to be mixed by the perlite, vermiculite and peat of certain volume ratio
60min is made；
(2) leaf inserts Multiplying culture: oblique cutting enters the petiole base of the explant material of basal medium to culture medium from step (1)
Middle addition Multiplying culture liquid, the formation of evoking adventive bud, the Multiplying culture liquid are MS nutrient solution+IBA, and the IBA concentration is
100~600mg/L, pH value 5.8；
(3) Rooting and hardening-off culture: being divided into single plant for adventitious bud clump obtained in step (2), selects sprout greater than 2cm and with 3
The single plant of~5 leaves is transferred to basal medium, and Rooting and hardening-off culture liquid is added into basal medium from simple bud base portion and is given birth to
Root strong sprout, the Rooting and hardening-off culture liquid are MS nutrient solution+IBA, and the IBA concentration is 200mg/L, pH value 5.8.
2. a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe according to claim 1 inserts the method for building up of rapid propagation system, feature exists
In the preparation process of basal medium in the step (1) and (3) are as follows:
Perlite, vermiculite and peat are measured first, in accordance with the volume ratio of 2:1:1 and water is added to be mixed, and are respectively charged into after the saturation that absorbs water
In the culture bottle of 240mL, it is packed into 1/3 that volume is culture bottle volume, ventilating cover is then covered, finally by culture bottle in 121.3
Sterilize 60min at DEG C, obtains basal medium.
3. a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe according to claim 1 inserts the method for building up of rapid propagation system, feature exists
In sucrose, pH value 5.8 is not added in the MS nutrient solution in the step (2) and (3).
4. a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe according to claim 1 inserts the method for building up of rapid propagation system, feature exists
In the cleaning of step (1) Explants and disinfecting process are as follows:
It first using the dust and impurity on banister brush wiping removal surface, is then placed in 250mL wide-mouth bottle, 2 drop of instillation is washed clean
Tap water is added after essence, uses tap water to rinse 30~40min after 10~15min of concussion washing, finally using sterilizing deionization
Water rinses 7~8 times, each 1min.
5. a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe according to claim 1 inserts the method for building up of rapid propagation system, feature exists
In the additional amount of Multiplying culture liquid is the IBA of the MS nutrient solution+1mL of 1mL, the formation time of adventitious bud clump in the step (2)
It is 4~8 weeks.
6. a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe according to claim 1 inserts the method for building up of rapid propagation system, feature exists
In, in the step (3) additional amount of Rooting and hardening-off culture liquid be 1mL MS nutrient solution+0.5mL IBA, culture 3~6 weeks at
For the strong sprout of well developed root system, part single plant is normally bloomed in culture bottle after culture 8 weeks.
7. a kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe according to claim 1 inserts the method for building up of rapid propagation system, feature exists
In the condition of culture in the step (1) (2) (3) is 25+2 DEG C of cultivation temperature, 60~100umolm of intensity of illumination-2·S-1, light application time 16hd-1, humidity 60~70%.
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| CN201811213424.0A CN109275564A (en) | 2018-10-18 | 2018-10-18 | A kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts the method for building up of rapid propagation system |
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| CN201811213424.0A CN109275564A (en) | 2018-10-18 | 2018-10-18 | A kind of Qinling Mountains purpleflower stonecrop herb test tube internal lobe inserts the method for building up of rapid propagation system |
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|---|---|---|---|---|
| CN1860858A (en) * | 2006-02-20 | 2006-11-15 | 云南师范大学 | Method for producing test-tube carmus for high efficiency commercialized prodn. of devilstrongue |
| CN105145354A (en) * | 2015-09-08 | 2015-12-16 | 浙江省农业科学院 | Method for aseptically seeding ebony and establishing leaf-cutting rapid propagation system |
| CN105309312A (en) * | 2015-11-09 | 2016-02-10 | 广西壮族自治区药用植物园 | Petrocosmea sinensis tissue culture and rapid propagation method |
-
2018
- 2018-10-18 CN CN201811213424.0A patent/CN109275564A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1860858A (en) * | 2006-02-20 | 2006-11-15 | 云南师范大学 | Method for producing test-tube carmus for high efficiency commercialized prodn. of devilstrongue |
| CN105145354A (en) * | 2015-09-08 | 2015-12-16 | 浙江省农业科学院 | Method for aseptically seeding ebony and establishing leaf-cutting rapid propagation system |
| CN105309312A (en) * | 2015-11-09 | 2016-02-10 | 广西壮族自治区药用植物园 | Petrocosmea sinensis tissue culture and rapid propagation method |
Non-Patent Citations (1)
| Title |
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| 蒋景龙: "珍稀濒危植物秦岭石蝴蝶的繁育研究现状", 《分子植物育种》 * |
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