CN104938340A - Method for conducting rose tissue culturing rapid propagation on explant by using autumn and winter dormant bud - Google Patents

Method for conducting rose tissue culturing rapid propagation on explant by using autumn and winter dormant bud Download PDF

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CN104938340A
CN104938340A CN201510430553.5A CN201510430553A CN104938340A CN 104938340 A CN104938340 A CN 104938340A CN 201510430553 A CN201510430553 A CN 201510430553A CN 104938340 A CN104938340 A CN 104938340A
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bud
autumn
winter
rose
explant
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冯立国
生利霞
王佳
臧姝
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Yangzhou University
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Yangzhou University
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Abstract

The invention discloses a method for conducting rose tissue culturing rapid propagation on an explant by using an autumn and winter dormant bud and belongs to the technical field of ornamental plant seedling asexual reproduction. The method aims to build a rose tissue rapid propagation system with the autumn and winter dormant bud as the explant, and the rose tissue rapid propagation system comprises four stages including disinfection treatment of the explant, primary culturing, multiplication culturing and rooting culturing. The method for conducting the rose tissue culturing rapid propagation on the explant by using the autumn and winter dormant bud has the advantages that the contamination rate is low, the propagation coefficient is high, the cultivating period is short, autumn and winter propagation limit is broken, the problems that a traditional rose propagation method is low in efficiency and long in period are well solved, and the method can be widely applied to actual production.

Description

A kind of autumn and winter sleeping bud that utilizes carries out the method for rose tissue-culturing rapid propagation for explant
Technical field
The present invention relates to a kind of autumn and winter sleeping bud of utilizing and carry out the method for rose tissue-culturing rapid propagation for explant, belong to ornamental plants seedling vegetative propagation technique field.
Background technology
Rose (Rosa rugosa Thunb.) is rose family Rosa machaka, various in style, pattern enriches, give off a strong fragrance, be not only the good plant material of afforestation, and be one of natural perfume plant the most ancient in the world, be the chemical products such as perfume, cosmetics spices source and food, drinks industry important interpolation raw material, or precious traditional Chinese medicine, be integrate view and admire, afforest, medicinal, beauty treatment, cuisines famous flower and tree.The Rosa Damascana extracted by its fresh flower is the famous and precious essential oil in the world, price is extremely expensive, be described as " liquid golden ", be mainly used in high-grade perfume, cosmetics and healthcare industry, have alleviate stress, regulate human endocrine, delay senility, the effect such as beautifying face and moistering lotion.Just because of it is at the extensive use in the fields such as medicinal, edible, daily-use chemical industry, domestic and international market increases day by day to the demand of rose seedling, the huge market demand, has a extensive future.
Traditional rose propagation method breeding cycle is long, and seedling percent is low, and be difficult to realize industrial fast fast-growing and produce, for meeting the need of market, setting up a set of rose rapid propagation system method becomes focus instantly.Utilize rose tissue culture technique can realize the Fast-propagation of rose, comprehensively studies have reported that, multiselect gets blade and tender stem axillalry bud is explant, and the time of drawing materials concentrates on spring and summer plant strain growth season, has no directly to choose autumn and winter sleeping bud tissue and carry out rose related content numerous soon.Therefore, it is that the rose microbody fast breeding technique of explant can break rose breeding autumn and winter restriction with sleeping bud that research is set up a kind of, improves rose reproductive efficiency.
By literature search, at present also without any about utilizing rose sleeping bud to carry out the content published in tissue-culturing rapid propagation.
Summary of the invention
The object of the invention is to break rose breeding autumn and winter restriction, invent a kind of rose sleeping bud autumn and winter that utilizes and carry out microbody method numerous soon.
The present invention is achieved by the following technical solutions: a kind of autumn and winter sleeping bud of utilizing carries out the method for rose tissue-culturing rapid propagation for explant, it is characterized in that comprising following steps:
Disinfecting of A explant: in the annual robust growth of fair weather clip in winter, branch without damage by disease and insect, gently clean down with banister brush, be cut into and be about 4cm, comprise the segment of 3-4 dormancy axillalry bud, with running water 3h after clean gauze parcel, move into aseptic working platform disinfection;
First alcohol-pickled with 75% and at the uniform velocity stir 60s, aseptic water washing 3-5 time, then soak with 3%NaClO and at the uniform velocity stir 10min, aseptic water washing 3-5 time, blot surface moisture with aseptic filter paper after taking-up, be cut into scalpel the single axillary bud stem section being about 1cm for subsequent use;
B Initial culture: Initial culture based formulas is MS+1mg/LBA+0.05-0.1mg/LNAA+30g/L sucrose+7g/L agar, and pH value is 5.8; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; Single axillary bud stem section scalpel after sterilization is peelled off axillalry bud scale (attention is avoided injuring axillalry bud) laterally to access afterwards on Initial culture base, light culture 1 week, Dormant Buds after 20d, cuts sprouting and the sleeping bud grown fine access proliferated culture medium continues to cultivate after 30d;
C Multiplying culture: proliferation culture medium formula is MS+1-2mg/LBA+0.05mg/LNAA+0.6mg/L GA 3+ 30g/L sucrose+7g/L agar, pH value is 5.8; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; A large amount of Multiple Buds can be differentiated be transferred to the sleeping bud 30d on proliferated culture medium by Initial culture base after;
D culture of rootage: prescription of rooting medium is 1/2MS+0.2-0.5mg/LIBA+0.2-0.5mg/LNAA+1-1.5g active carbon+30g/L sucrose+7g/L agar, and pH value is 5.8; Cut the indefinite bud simple bud of robust growth in Multiple Buds, stem height 1-2cm, access root media; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; Start to sprout adventive root after cultivating 2-3 week.
In described step (A), the sterilization method of rose sleeping bud autumn and winter is: 75% alcohol 60s+3%NaClO 10min.
In described step (B), rose sleeping bud autumn and winter Initial culture based formulas is MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar, and pH value is 5.8;
Be inoculated on Initial culture base after in described step (B), rose sleeping bud autumn and winter peels off axillalry bud scale (attention is avoided injuring axillalry bud).
In described step (C), rose sleeping bud autumn and winter proliferation culture medium formula is MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA 3+ 30g/L sucrose+7g/L agar, pH value is 5.8; In described step (D), rose sleeping bud autumn and winter prescription of rooting medium is 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1g active carbon+30g/L sucrose+7g/L agar, and pH value is 5.8.
The present invention successfully passes and chooses rose sleeping bud autumn and winter is explant, and in conjunction with disinfection way, Initial culture, Multiplying culture, a series of tissue culture technique such as culture of rootage, realizes utilizing sleeping bud Fast-propagation rose autumn and winter.
The invention has the advantages that: pollution rate is low, reproduction coefficient is high, and cultivation period is short, breaks breeding restriction autumn and winter, well solves the problem that rose Traditional breeding processes efficiency is low, the cycle is long, can be applicable to actual production.
Embodiment
The inventive method selects rose sleeping bud autumn and winter, by the regulation and control to four key steps such as disinfection way, Initial culture, Multiplying culture, culture of rootage, realizes the Fast-propagation of rose sleeping bud autumn and winter tissue.Specifically comprise following steps:
Disinfecting of A explant: in the annual robust growth of fair weather clip in winter, branch without damage by disease and insect, gently clean down with banister brush, be cut into and be about 4cm, comprise the segment of 3-4 dormancy axillalry bud, with running water 3h after clean gauze parcel, move into aseptic working platform disinfection; First alcohol-pickled with 75% and at the uniform velocity stir 60s, aseptic water washing 3-5 time, then soak with 3%NaClO and at the uniform velocity stir 10min, aseptic water washing 3-5 time, blot surface moisture with aseptic filter paper after taking-up, be cut into scalpel the single axillary bud stem section being about 1cm for subsequent use;
B Initial culture: Initial culture based formulas is MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar, and pH value is 5.8; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; Single axillary bud stem section scalpel after sterilization is peelled off axillalry bud scale (attention is avoided injuring axillalry bud) laterally to access afterwards on Initial culture base, light culture 1 week, Dormant Buds after 20d, cuts sprouting and the sleeping bud grown fine access proliferated culture medium continues to cultivate after 30d;
C Multiplying culture: proliferation culture medium formula is MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA 3+ 30g/L sucrose+7g/L agar, pH value is 5.8; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; A large amount of Multiple Buds can be differentiated be transferred to the sleeping bud 30d on proliferated culture medium by Initial culture base after;
D culture of rootage: prescription of rooting medium is 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1g active carbon ++ 30g/L sucrose+7g/L agar, pH value is 5.8; Cut the indefinite bud simple bud of robust growth in Multiple Buds, stem height 1-2cm, access root media; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; Start to sprout adventive root after cultivating 2-3 week.
The research of the explant process related in the present invention's following instance and sterilization, Initial culture, Multiplying culture, culture of rootage technology absolutely proves technical scheme of the present invention, to prove the authenticity of this achievement in research, correctness and operability.
A. explant the best disinfects the research of mode
Rose variety " purple branch rose " selected by examination material, by the explant fetched with after method pretreatment in above-mentioned A, on aseptic working platform with 3 kinds of disinfection way to the disinfection of single dormancy leaf stem section, be inoculated in after peelling off scale on Initial culture base and cultivate, often process 20, repeat 3 times, after condition of culture same B, 20d, add up pollution rate, melting brown rate and starting rate.
Test finds the prolongation along with disinfecting time, and pollution rate reduces gradually, and melting brown rate raises gradually, and starting rate declines gradually; Wherein process 2 pollution rates comparatively to process 1 and significantly significantly decline, fall is greater than 30%, and starting rate still can remain on more than 90%; Process 3 pollution rates and be less than 10% than process 2 fall, starting rate is but low to moderate 77.8%; Consider, determine that 75% alcohol 60s+3%NaClO 10min is that purple branch rose winter dormancy bud tissue cultures the best disinfects mode.
The different disinfection way of table 1 is on the impact of explant
Process Disinfection way Pollution rate % Melting brown rate % Starting rate %
1 75% alcohol 60s+3%NaClO 5min 50 0 100
2 75% alcohol 60s+3%NaClO 10min 17 6.25 93.75
3 75% alcohol 60s+3%NaClO 15min 9.8 22.2 77.8
B. the research of best Initial culture base
Plant growth regulator is the requisite material of the purple branch rose winter dormancy bud regeneration of induction, for determining best concentration combination, arranges 4 process, often process inoculation 20 explants, repeat 3 times, after 20d, add up starting rate, observe and record its growing state.
Test finds that BA plays a crucial role to the regeneration of induction purple branch rose winter dormancy bud, and the raising of BA concentration is conducive to the sprouting of sleeping bud; When BA concentration is identical, 0.05mg/LNAA is higher than 0.1mg/LNAA starting rate.Process 3 has callus to generate with process 4 sleeping buds while sprouting into indefinite bud, is conducive to further Multiplying culture, and process 3 starting rates are the highest, therefore determine that MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar is best Initial culture base.
The impact that table 2 variable concentrations plant growth regulator is sprouted explant indefinite bud
Process BA mg/L NAA mg/L Starting rate % Upgrowth situation
1 0.5 0.05 83.8 Have no callus, the indefinite bud blade latitude of emulsion is large
2 0.5 0.1 78.4 Have no callus, indefinite bud growing way is poor
3 1 0.05 93 Have callus to generate, indefinite bud growing way is good, and color is green
4 1 0.1 86 Have callus to generate, indefinite bud grows fine
C. the research of optimum multiplication medium
For realizing the Fast-propagation of purple branch rose, Multiplying culture need be carried out to the indefinite bud that Initial culture is sprouted, BA, NAA and GA are set 34 process of three plant growth Auto-regulator variable concentrations combinations, cut by the sleeping bud explant that best Initial culture base is sprouted and be transferred on it, often process 20, repeat 3 times, observed and recorded upgrowth situation, adds up growth coefficient after 30d.
Test finds to add GA 3contribute to the propagation of indefinite bud, growth coefficient is with GA 3concentration raises and presents ascendant trend; Increase BA concentration, adventitious bud proliferation coefficient significantly rises, and clump bud is tillered many, and growing way is good, therefore determines that process 4 that growth coefficient is the highest is filled a prescription MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA 3+ 30g/L sucrose+7g/L agar is optimum multiplication medium.
Table 3 variable concentrations plant growth regulator is on the impact of explant adventitious bud proliferation
Process BA mg/L NAA mg/L GA 3mg/L Growth coefficient Upgrowth situation
1 1 0.05 0.3 2.0 Clump bud is few, most of mounted blade
2 2 0.05 0.3 3.0 Clump bud is more, and indefinite bud color is light green
3 1 0.05 0.6 2.8 Less from bud, grow fine
4 2 0.05 0.6 4.4 Clump bud is many, and growing way is vigorous, and color is green
D. the research of best root media
With 1/2MS medium for minimal medium, add NAA, IBA, the active carbon of variable concentrations, cut the indefinite bud simple bud access root media of robust growth, stem height 1-2cm, totally 4 process, each process 20 indefinite buds, repeat 3 times, observe and record condition of rooting, after 30d, adding up rooting rate.
Test finds except process 4, other 3 process rooting rates all reach more than 90%, process 2 rooting rates the highest, but observe adventitious bud rooting situation and growing way discovery, process 1 adventive root short and thick, plant part growing way is fast, and process 2 adventive root elongated, plant part growing way is slow, partial blade turns yellow, process 3 adventive root short and thick, aerial growth is slow, and plant is short and small.More than comprehensive, determine that 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1g active carbon+30g/L sucrose+7g/L agar is best root media.
Table 4 variable concentrations plant growth regulator is on the impact of adventitious bud rooting
Process NAA mg/L IBA mg/L Active carbon g Rooting rate %
1 0.2 0.2 1 90.9
2 0.2 0.5 1.5 95.4
3 0.5 0.2 1.5 90.9
4 0.5 0.5 1 86.3
It is low that the present invention has pollution rate, and reproduction coefficient is high, and cultivation period is short, breaks the advantages such as breeding restriction autumn and winter, well solve the problem that rose Traditional breeding processes efficiency is low, the cycle is long, can be widely used in actual production.
The present invention utilizes rose sleeping bud autumn and winter to be explant, is realized the Fast-propagation of rose by tissue culture technique.The present invention, to the reproduction coefficient improving rose, shortens cultivation period, breaks breeding restriction autumn and winter, realizes the large-scale production of rose seedling significant.

Claims (6)

1. utilize autumn and winter sleeping bud to carry out a method for rose tissue-culturing rapid propagation for explant, it is characterized in that comprising following steps:
(A) the disinfecting of explant: the annual robust growth of fair weather clip, branch without damage by disease and insect after autumn and winter fallen leaves, gently clean down with banister brush, be cut into the segment comprising 3-4 dormancy axillalry bud, after running water after clean gauze parcel, move into aseptic working platform disinfection;
First alcohol-pickled with 75% and at the uniform velocity stir 60s, aseptic water washing 3-5 time, then soak with 3%NaClO and at the uniform velocity stir 5-10min, aseptic water washing 3-5 time, blot surface moisture with aseptic filter paper after taking-up, the single axillary bud stem section be cut into scalpel is for subsequent use;
(B) Initial culture: Initial culture based formulas is MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar, and pH value is 5.8; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; Laterally access on Initial culture base after single axillary bud stem section scalpel after sterilization being peelled off axillalry bud scale, light culture 1 week, Dormant Buds after 20d, cut sprouting after 30d and the sleeping bud grown fine access proliferated culture medium continues to cultivate;
(C) Multiplying culture: proliferation culture medium formula is MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA 3+ 30g/L sucrose+7g/L agar, pH value is 5.8; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; A large amount of Multiple Buds can be differentiated be transferred to the sleeping bud 30d on proliferated culture medium by Initial culture base after;
(D) culture of rootage: prescription of rooting medium is 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1-1.5g active carbon+30g/L sucrose+7g/L agar, and pH value is 5.8; Cut the indefinite bud simple bud of robust growth in Multiple Buds, stem height 1-2cm, access root media; Condition of culture is intensity of illumination 2000-3000Lx, light irradiation time 14h/d, temperature 25 ± 2 DEG C; Start to sprout adventive root after cultivating 2-3 week.
2. the autumn and winter sleeping bud that utilizes according to claim 1 carries out the method for rose tissue-culturing rapid propagation for explant, it is characterized in that the sterilization method of rose sleeping bud autumn and winter in described step (A) is: 75% alcohol 60s+3%NaClO 10min.
3. a kind of autumn and winter sleeping bud of utilizing according to claim 1 carries out the method for rose tissue-culturing rapid propagation for explant, it is characterized in that in described step (B), rose sleeping bud autumn and winter Initial culture based formulas is MS+1mg/LBA+0.05mg/LNAA+30g/L sucrose+7g/L agar, pH value is 5.8.
4. the autumn and winter sleeping bud that utilizes according to claim 1 carries out the method for rose tissue-culturing rapid propagation for explant, it is characterized in that being inoculated on Initial culture base after in described step (B), rose sleeping bud autumn and winter peels off axillalry bud scale (attention is avoided injuring axillalry bud).
5. the autumn and winter sleeping bud that utilizes according to claim 1 carries out the method for rose tissue-culturing rapid propagation for explant, it is characterized in that in described step (C), rose sleeping bud autumn and winter proliferation culture medium formula is MS+2mg/LBA+0.05mg/LNAA+0.6mg/L GA 3+ 30g/L sucrose+7g/L agar, pH value is 5.8.
6. a kind of autumn and winter sleeping bud of utilizing according to claim 1 carries out the method for rose tissue-culturing rapid propagation for explant, it is characterized in that in described step (D), rose sleeping bud autumn and winter prescription of rooting medium is 1/2MS+0.2mg/LIBA+0.2mg/LNAA+1g active carbon+30g/L sucrose+7g/L agar, pH value is 5.8.
CN201510430553.5A 2015-07-21 2015-07-21 Method for conducting rose tissue culturing rapid propagation on explant by using autumn and winter dormant bud Pending CN104938340A (en)

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CN111887155A (en) * 2020-06-29 2020-11-06 南京锦江园林景观有限公司 Method for improving efficient and rapid seedling formation of rosa plants
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