CN105393917B - Clove leaf honeysuckle rapid propagation method - Google Patents
Clove leaf honeysuckle rapid propagation method Download PDFInfo
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- CN105393917B CN105393917B CN201510856683.5A CN201510856683A CN105393917B CN 105393917 B CN105393917 B CN 105393917B CN 201510856683 A CN201510856683 A CN 201510856683A CN 105393917 B CN105393917 B CN 105393917B
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- explant
- sprout
- clove leaf
- blade
- honeysuckle
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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
-
- 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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses clove leaf honeysuckle rapid propagation method, including：(1) explant is prepared：The clove leaf honeysuckle sprout with micro xylem is taken, perula is peeled off using single-edge blade, bud is carried and is no more than the thick xylems of 0.5mm, as explant；(2) sterilize：In super-clean bench, the explant that cutting obtains is moved in the triangular flask for the fresh hydrogenperoxide steam generator for filling 10% with by autoclaved tweezers, instills 35 drop polysorbate60s, concussion 12 minutes, then pours out hydrogenperoxide steam generator, and with aseptic water washing explant, drain away the water, prepare inoculation；(3) it is inoculated with：Explant by sterilization is moved to fill in the blake bottle for starting proliferated culture medium and carries out closing culture；(4) culture of rootage；(5) rooted seedling is subjected to acclimatization and transplantses.Clove leaf honeysuckle can be effectively carried out using this method quickly to breed, and pollution rate is low, inoculation survival rate, inducing clumping bud rate are high.
Description
Technical field
The present invention relates to clove leaf honeysuckle aseptic and rapid propagation method.
Background technology
Clove leaf honeysuckle is Beijing two level endangered plants, and record has 7 plants, but rarely seen one plant in the range of the administrative region of a city of Beijing.
In order to save the endangered plants, its population at individual quantity should be expanded first, but never collect seed for many years, only
A number of individual plant can be obtained by groping the means of tissue-culturing rapid propagation, and utilize the clove leaf honeysuckle quick breeding technology established
Periphery provinces and regions introduces a collection is bred, then together wild environment is arrived in cultivation again with Beijing introduces a collection, realizes that clove leaf is born
The field in winter returns.However, it is extremely difficult to establish clove leaf honeysuckle sterile rapid propagation system.
Thus, effective clove leaf honeysuckle sterile rapid propagation system is established, it is significant.
The content of the invention
The present invention is the following discovery based on inventor and completed：
It is extremely difficult to establish clove leaf honeysuckle sterile rapid propagation system, has perhaps in the first step to explant seeded process
More problems need to solve.The fast numerous the most frequently used explant of breeding, rare or endangered species is stem-segment with single bud, but clove leaf honeysuckle explant
After stem-segment with single bud inoculation, either pollute or be sterilized liquid kill, a variety of thimerosals, more concentration gradients coordinate explant sterilization
Experiment finds that clove leaf honeysuckle stem-segment with single bud contains endophyte, and is endogenetic fungus, result in the failure of explant introducing；
If using blade as explant, although sterile system can be established, inducing clumping bud rate is extremely low, and the probability that makes a variation may
It is larger, it is impossible to the effective way quickly bred as clove leaf honeysuckle.
It is contemplated that at least solves one of technical problem present in prior art.Therefore, one object of the present invention
It is to propose that one kind being capable of effective clove leaf honeysuckle aseptic and rapid propagation method.
According to an aspect of the present invention, the invention provides a kind of clove leaf honeysuckle rapid propagation method.According to this hair
Bright embodiment, this method comprise the following steps：
(1) explant is prepared：The clove leaf honeysuckle sprout with micro xylem is taken, perula is peeled off using single-edge blade, with
Petiole is quickly removed from base portion afterwards, continues to peel off base portion perula；After perula is fully exfoliated, then extend to branch skin zone,
Branch epidermis around bastem portion is all stripped clean, retains a little light green forming layer；The edge of a knife is slightly tilted, in bastem portion
Under each knife, pay attention to never cutting through xylem, sprout base portion gently chosen with point of a knife, carry bud and be no more than thick wooden of 0.5mm
Portion, as explant；
(2) sterilize：In super-clean bench, 10% is filled with the explant that cutting obtains being moved to by autoclaved tweezers
Fresh hydrogenperoxide steam generator triangular flask in, instill 3-5 drop Tween-60s, concussion 12 minutes, it is molten then to pour out hydrogen peroxide
Liquid, and with aseptic water washing explant, drain away the water, prepare inoculation；
(3) it is inoculated with：The explant by sterilization is moved to from the triangular flask rapidly and fills the training for starting proliferated culture medium
Support in bottle and carry out closing culture, the wherein xylem of sprout base portion will be completely immersed in culture medium；
(4) after the explant in the blake bottle bears Multiple Buds, the Multiple Buds are transferred in root media
Row culture of rootage, to obtain rooted seedling；
(5) rooted seedling is subjected to acclimatization and transplantses.
Quickly bred it is surprisingly found by the inventors that can effectively carry out clove leaf honeysuckle using the method for the present invention, and
And relative to existing clove leaf honeysuckle quick breeding technology, method pollution rate of the invention, the death rate are low, inoculation survival rate,
Inducing clumping bud and rooting rate are high.
In addition, clove leaf honeysuckle according to the above embodiment of the present invention, quickly breeding can also have technology additional as follows
Feature：
According to an embodiment of the invention, the clove leaf honeysuckle sprout is from the annual of wild clove leaf honeysuckle plant
Branch.Thus, clove leaf honeysuckle sprout health easily survives, and grows vigorous, is easy to induce Multiple Buds, is advantageous to numerous enter soon
OK.
According to an embodiment of the invention, the clove leaf honeysuckle sprout is gathered in late August to early September, and prepared
Moisturizing is kept in dark place before explant.Thus, explant easily survives, and is easy to the progress of subsequent step.
According to an embodiment of the invention, before explant is prepared, further comprise entering the clove leaf honeysuckle sprout
The step of row pretreatment.Thereby, it is possible to effectively reduce pollution rate, subsequent inoculations survival rate is improved.
According to an embodiment of the invention, the pretreatment is followed the steps below：By the clove leaf since terminal bud
Running water rinses 6 minutes honeysuckle sprout from top to bottom；Clip under sprout is dipped in into 75% medicinal alcohol 30 seconds, sealed with fritter preservative film
The lower clip of bag；Then whole sprout is put into the solution containing 0.2% dish detergent and soaked 20 minutes；Aseptic water washing 2 times.By
This, subsequent contamination rate is low, and inoculation survival rate is high.
According to an embodiment of the invention, in step (1), when preparing explant：
3 sharp single-edge blades are taken, are soaked 5 minutes in 75% medicinal alcohol；Blade is taken out in calcination on alcolhol burner
10 seconds, the knife back, which is put into small neck, to cool；1 blade is kept to soak, 1 blade is cooling, and 1 blade is in hand outside cutting
Implant, after often doing 5-6 action using blade, more allowing blade replacement.Thereby, it is possible to effectively avoid cross pollution, pollution rate is reduced, is carried
High subsequent inoculations survival rate.
According to an embodiment of the invention, the blade stripping perula for being down to room temperature is taken, in order to avoid sprout of burning.
According to an embodiment of the invention, in step (2), each triangular flask at most 5 explants of sterilization.Thereby, it is possible to
Pollution rate is effectively reduced, improves subsequent inoculations survival rate.
According to an embodiment of the invention, in step (2), when pouring out hydrogenperoxide steam generator, slowly to topple on one side, on one side
Sterilization triangular flask is rotated simultaneously, thimerosal is flowed through each position of bottleneck.Thereby, it is possible to effectively reduce pollution rate.
According to an embodiment of the invention, in step (2), rinsed 3-5 times with aseptic water washing explant.Thereby, it is possible to
Effectively avoid injuring explant because thimerosal remains.
According to an embodiment of the invention, in step (3), each blake bottle is inoculated with an explant.Thereby, it is possible to effective
Pollution rate is reduced, improves inoculation survival rate.
According to an embodiment of the invention, in step (3), the startup proliferated culture medium is added with 2.5mg/L 6-BA
(6- benzyls aminoadenine) and 0.1mg/L NAA (methyl α-naphthyl acetate) WPM culture mediums (Woody Plant medium).Thus, energy
Enough effectively induction explants bear Multiple Buds.
According to an embodiment of the invention, in step (4), the root media for the addition of 0.1mg/L NAA and
0.3mg/L IBA 1/2MS culture mediums.Thereby, it is possible to effectively induce Multiple Buds to take root, the rooted seedling of stalwartness is obtained.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination accompanying drawings below to embodiment
Substantially and it is readily appreciated that, wherein：
Fig. 1 is shown according to one embodiment of the invention, the signal of the clove leaf honeysuckle sprout of the micro xylem of cutting band
Figure；
Fig. 2 shows that according to one embodiment of the invention xylem is completely immersed in the explant of culture medium in blake bottle；With
And
Fig. 3 is shown according to one embodiment of the invention, and the explant of Multiple Buds is grown in blake bottle.
Embodiment
The solution of the present invention is explained below in conjunction with embodiment.It is it will be understood to those of skill in the art that logical below
It is exemplary to cross the embodiment being described with reference to the drawings, and is merely to illustrate the present invention, and be should not be taken as limiting the scope of the invention.
Unreceipted particular technique or condition in embodiment, according to the technology described by document in the art or condition or according to production
Product specification is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
Embodiment 1：
According to the clove leaf honeysuckle rapid propagation method of the present invention, according to following steps, it is quickly numerous to carry out clove leaf honeysuckle
Grow：
1st, branch is gathered
Explant used in the present invention is the clove leaf honeysuckle sprout with micro xylem, thus, to be wanted according to following
Seek capturing material：
1.1 acquisition time：Late August to early September is optimal, more full compared with winter and spring, branch cortex living cells
It is more, it is easily peeled off.
1.2 acquisition method：After noon 12 points to 2 pm, gather on Centime spiceleaf honeysuckle plant out of office, cut with sharp
Knife clip annotinous branch, separately take a clean small scissors and cut blade from petiole middle and lower part rapidly, retain a bit of petiole,
To protect axillary bud, while pay special attention to not touch off or damage terminal bud, with 5 × 10cm sulfuric acid paper bag protected by bagging.Under branch section
Portion transports laboratory to immediately with sealed plastic bag is put into after pure water infiltration, and transportation makes branch lucifuge.
2nd, clean
After branch transports to laboratory, the clove leaf honeysuckle sprout (see Fig. 1) with micro xylem is taken, at random since terminal bud
Running water is rinsed 6 minutes from top to bottom, and lower clip then is dipped in into 75% medicinal alcohol 30 seconds, cut with fritter preservative film package
Mouthful；Then whole sprout will be put into the solution containing 0.2% dish detergent and soaked 20 minutes；Aseptic water washing 2 times.
3rd, cutting
Take 3 sharp single-edge blades, it is desirable to which cutting edge both ends point of a knife is completely sharp keen；5 points are soaked in 75% medicinal alcohol
Clock；Blade is taken out in calcination on alcolhol burner 10 seconds, the knife back, which is put into small neck, to cool；1 blade is kept to soak, 1 blade
Cooling, 1 blade cutting explant in hand, after often doing 5-6 action using blade, more allowing blade replacement.As shown in figure 1, take
Perula is peeled off with the blade for being down to room temperature；Petiole is quickly then removed from base portion, continues to peel off base portion perula；Perula is completely exfoliated
Afterwards, then extend to branch skin zone, branch epidermis around bastem portion is all stripped clean, retains a little light green and is formed
Layer；The edge of a knife is slightly tilted, and bastem portion each knife up and down, pays attention to never cutting through xylem, sprout base portion is gently chosen with point of a knife, makes pressure
Suppression, which carries, is no more than the thick xylems of 0.5mm, will cut sprout input and fills in the clean beaker of sterilized water, as explant.
4th, sterilize
In super-clean bench, 10% is filled with sprout (explant that i.e. cutting obtains) being moved to by autoclaved tweezers
Fresh hydrogenperoxide steam generator triangular flask in (every bottle at most sterilization 5 explants), instill few drops of (3-5 drops) Tween-60s, shake
Shake, concussion 12 minutes, pour out hydrogenperoxide steam generator, when pouring out hydrogenperoxide steam generator will on one side slowly topple over, on one side at the same rotate
Triangular flask is sterilized, thimerosal is flowed through each position of bottleneck, then with aseptic water washing explant 3-5 times, is drained away the water, is prepared
Inoculation.
5th, it is inoculated with
Left hand is horizontal to hold disinfection bottle and medium bottle, keeps two bottles and desktop horizontal parallel, right hand aseptic nipper will pass through
The explant of sterilization is moved to fill in the blake bottle for starting proliferated culture medium from sterilization triangular flask and cultivated, and is shortened as far as possible outer
Implant rests on bottle outer in-flight time, and the xylem of sprout base portion is completely immersed in into culture medium (see Fig. 2), each medium bottle
An explant is only inoculated with, tweezers are placed on support, closed the lid in medium bottle rapidly by the right hand.
Wherein, it is WPM+6BA 2.5mg/L+NAA 0.1mg/L to start proliferated culture medium.
As shown in figure 3, after blake bottle closing culture, explant has grown Multiple Buds.
6th, take root and transplant
The Multiple Buds of above-mentioned acquisition are transferred is taken root in root media 1/2MS+0.1mg/L NAA+0.3mg/L IBA
Cultivate, after rooted seedling to be obtained, carried out acclimatization and transplantses.
The present invention is to breed clove leaf honeysuckle using tissue culture method first, both at home and abroad beyond example.Also, sent out through statistics
Existing, when quickly being bred using the method progress clove leaf honeysuckle of the present invention, for Contamination rate control within 20%, the death rate 16% is left
The right side, inoculation survival rate 64%, significant effect are better than existing traditional clove leaf honeysuckle reproduction technique.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
Claims (8)
1. a kind of clove leaf honeysuckle rapid propagation method, it is characterised in that comprise the following steps：
(1) explant is prepared：Take the clove leaf honeysuckle sprout with micro xylem, using single-edge blade peel off perula, then from
Base portion quickly removes petiole, continues to peel off base portion perula；After perula is fully exfoliated, then extend to branch skin zone, by bud
Branch epidermis is all stripped clean around base portion, retains a little light green forming layer；The edge of a knife is slightly tilted, and bastem portion is each up and down
One knife, pay attention to never cutting through xylem, sprout base portion is gently chosen with point of a knife, carry bud and be no more than the thick xylems of 0.5mm, make
For explant；
(2) sterilize：In super-clean bench, with the explant that cutting obtains moved to by autoclaved tweezers fill 10% it is new
In the triangular flask of fresh hydrogenperoxide steam generator, 3-5 drop Tween-60s are instilled, shakes 12 minutes, then pours out hydrogenperoxide steam generator, and
With aseptic water washing explant, drain away the water, prepare inoculation；
(3) it is inoculated with：The explant by sterilization is moved to from the triangular flask rapidly and fills the blake bottle for starting proliferated culture medium
In carry out closing culture, the wherein xylem of sprout base portion will be completely immersed in culture medium,
Each blake bottle is inoculated with an explant, and the startup proliferated culture medium is added with 2.5mg/L 6-BA and 0.1mg/L
NAA WPM culture mediums；
(4) after the explant in the blake bottle bears Multiple Buds, the Multiple Buds is transferred in root media and given birth to
Root culture, to obtain rooted seedling,
The root media is the 1/2MS culture mediums that with the addition of 0.1mg/L NAA and 0.3mg/L IBA；
(5) rooted seedling is subjected to acclimatization and transplantses.
2. according to the method for claim 1, it is characterised in that the clove leaf honeysuckle sprout is born from wild clove leaf
The annotinous branch of winter plant,
The clove leaf honeysuckle sprout is gathered in late August to early September, and moisturizing is kept in dark place before explant is prepared.
3. according to the method for claim 1, it is characterised in that before explant is prepared, further comprise to the fourth
The step of spiceleaf honeysuckle sprout is pre-processed.
4. according to the method for claim 3, it is characterised in that follow the steps below the pretreatment：
By the clove leaf honeysuckle sprout, running water rinses 6 minutes from top to bottom since terminal bud；Clip under sprout is dipped in 75%
Medicinal alcohol 30 seconds, with clip under fritter preservative film package；Then whole sprout is put into the solution containing 0.2% dish detergent and soaked
Bubble 20 minutes；Aseptic water washing 2 times.
5. according to the method for claim 1, it is characterised in that in step (1), when preparing explant：
3 sharp single-edge blades are taken, are soaked 5 minutes in 75% medicinal alcohol；Blade is taken out in calcination on alcolhol burner 10 seconds,
The knife back, which is put into small neck, to cool；1 blade is kept to soak, 1 blade is cooling, 1 blade cutting explant in hand,
After often doing 5-6 action using blade, more allowing blade replacement.
6. according to the method for claim 5, it is characterised in that take the blade stripping perula for being down to room temperature.
7. according to the method for claim 1, it is characterised in that in step (2), the at most sterilization 5 of each triangular flask is outer
Implant.
8. according to the method for claim 1, it is characterised in that in step (2), when pouring out hydrogenperoxide steam generator, one
Side slowly topples over, on one side while rotates sterilization triangular flask, thimerosal is flowed through each position of bottleneck,
In step (2), with aseptic water washing explant 3-5 times.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510856683.5A CN105393917B (en) | 2015-11-30 | 2015-11-30 | Clove leaf honeysuckle rapid propagation method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510856683.5A CN105393917B (en) | 2015-11-30 | 2015-11-30 | Clove leaf honeysuckle rapid propagation method |
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| CN105393917A CN105393917A (en) | 2016-03-16 |
| CN105393917B true CN105393917B (en) | 2017-11-24 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106171971B (en) * | 2016-06-27 | 2018-05-08 | 中国科学院植物研究所 | A kind of method of clove leaf honeysuckle tissue-culturing rapid propagation |
| CN106212289A (en) * | 2016-08-26 | 2016-12-14 | 北京林业大学 | A kind of method setting up oil Paeonia suffruticosa bulbil efficient aseptic culture system |
| CN106386489A (en) * | 2016-09-12 | 2017-02-15 | 北华大学 | Tissue culture and rapid propagation method of lonicera ruprechtiana |
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| CN102217542B (en) * | 2011-05-23 | 2012-09-05 | 北京农业职业学院 | Method for propagating red-leaf lonicera maackii quickly |
| CN102630563B (en) * | 2012-03-28 | 2013-06-26 | 常熟市滨江农业科技有限公司 | Method for raising seedlings of Lonicera sempervirens |
| CN103535285B (en) * | 2013-11-04 | 2015-10-14 | 中国科学院昆明植物研究所 | The Fast-propagation of honeysuckle seedling and in-vitro conservation method |
| CN104756869B (en) * | 2015-04-09 | 2016-04-20 | 广西壮族自治区林业科学研究院 | The preparation of masson pine fine individual plant aseptic explant and initial bud inducement method |
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