CN102860255A - Magnolia wufengensis variety callus inducing method - Google Patents
Magnolia wufengensis variety callus inducing method Download PDFInfo
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- CN102860255A CN102860255A CN 201110190088 CN201110190088A CN102860255A CN 102860255 A CN102860255 A CN 102860255A CN 201110190088 CN201110190088 CN 201110190088 CN 201110190088 A CN201110190088 A CN 201110190088A CN 102860255 A CN102860255 A CN 102860255A
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Abstract
The invention relates to a magnolia wufengensis tissue culture rapid propagation method and provides a magnolia wufengensis variety callus inducing method. A stem section with buds of a magnolia wufengensis variety is chosen as an optimum explant, an optimum sterilization disinfection system and a culture medium are built, and an optimum primary culture medium and a browning restraining system are chosen, a callus of the magnolia wufengensis variety is induced, a foundation is lain for a vegetative propagation system of the magnolia wufengensis variety, and important significance is achieved in development of applications values of scientific research, greening, appreciation and the like.
Description
1. technical field: the present invention relates to the method that a kind of tissue culture is induced, and relates in particular the method for the callus induction of a kind of safflower yulan mutation.
2. technical background: the mutation of safflower yulan is the new discovery seeds in the Magnoliaceae Magnolia subgenus Yulania, is one of wild monoid of Wufeng County safflower yulan mutation, mainly is distributed in the Tujia Autonomous County of Wufeng domestic, and distributed areas are comparatively narrow.The proterties of safflower yulan mutation seeds is comparatively unique, belongs to seeds with general yulan and compares, and it tree-like tall and big mostly is the high megaphanerophyte of 15-20m, and petal is 9 lobes, inside and outside be redness, color is pure gorgeous, is far to see or closely see all have ornamental value.The discovery of safflower yulan mutation has certain academic significance to problems such as the classification of the fauna of whole Magnoliacea plant and systematic growths, for the research bio-diversity provides evidence, and take high ornamental value as support, has great development and application value aspect road and the urban afforestation.The mutation of safflower yulan is the South-West Hubei endemic tree; quantity is rare; distribution is narrow; at present only find that population faces danger of extinction, in addition in the area in the narrow territory of a slice, Wu Feng Midwest, Hubei; because the locality lacks effective resource management and safeguard measure and people to treasuring the thin of protection of resources consciousness; under the driving of interests, the large tree of safflower yulan of a part is cut down and is resell at a profit, and germ plasm resource runs off serious.Safflower yulan mutation sylvan life seedling is rare under the nature, upgrades difficulty, and percentage of seedgermination and storage rate are all lower.Therefore, not only can carry out the production of batch production grows seedlings to the vegetative research of safflower yulan mutation, improve the reproductive efficiency of safflower yulan mutation, reduce production costs, but also can filter out the clone of merit, satisfy production, view and admire required, significant to the exploitation of safflower yulan mutation scientific research, using value.
3. summary of the invention: the method that the purpose of this invention is to provide a kind of safflower yulan mutation callus induction.Seed, axillalry bud, stem segment with axillary bud and blade take the mutation of safflower yulan is inoculated on the MS medium as explant supplies the examination material respectively, select optimum explant type and draw materials the time, go out again its optimum sterilization system, best base basal culture medium and carbon source take this type as explant selection, optimum just culture base, Browning control system and evoked callus medium.
The safflower yulan mutation callus induction method that the present invention proposes comprises the steps:
(1) selection of optimum explant: seed, axillalry bud, stem segment with axillary bud and the blade that will peel off respectively exosper carry out the outside with clear water and hairbrush and buy clearly, and then with behind 20% the dew medicining liquid dipping 30min, place circulating water to wash 1h, finish preliminary sterilizing works.Again above-mentioned material is placed superclean bench further to disinfect (75% alcohol 30S, 10%NaClO 10min) and shear treatment, the general band embryo part that keeps whole volumes about 1/3 of seed, axillalry bud and stem with bud be otch again, blade is generally taken from the square part of the 1cm*1cm of arteries and veins in the band, and the experiment material after processing is inoculated in MS+1mgL
-16-BA+1mgL
-1Normally cultivate on the NAA minimal medium.
(2) the explant optimum selection of time of drawing materials: select sunny calm weather each middle of the month in May, 2009-October, in high noons 12 point-～13 obtain best explant type, be inoculated in above-mentioned (1) medium and normally cultivate.
(3) foundation of optimum sterilization system: get 30 healthy and strong of stem with bud and place circulating water to wash 0.5h, then it is immersed in 15min in 20% " drip and reveal " thimerosal, wash 1h with circulating water again, then in superclean bench, carry out the preliminary sterilization of 30S with 75% alcohol, respectively with NaClO concentration 5%, 10%, 15%, 20% and sterilization time 10min,, 20min,, 30min carries out 12 completely random combined treatment.At last explant is inoculated in above-mentioned (1) MS minimal medium and normally cultivates.
(4) selection of minimal medium and carbon source thereof: will be inoculated into a L through the aseptic explant after the sterilization of (3) step
9(3
4) normally cultivate in the medium experimental design, 3 factors are respectively minimal medium (MS, 1/2MS, WPM), carbon source kind (fructose, sucrose, white granulated sugar), carbon source concentration (15g, 30g, 40g).
(5) Browning control Establishing:
Explant to the mutation of safflower yulan carries out cold treatment and soaks polyvinylpyrrolidine copper (PVP) solution-treated, and concrete grammar is as follows:
1. the notch portion that the spray that will adopt is back wrapped explant with wet husky cloth is cut into the stem section and carries out sterilization treatment after being placed on 4 ℃ of lower refrigeration 2h, 4h.
2. stem with bud is placed on before sterilization in polyvinylpyrrolidine copper (PVP) solution of 1g/LgL-1 and soaks 1h, 2h.
3. the stem with bud after the sterilization cuts in the solution of above-mentioned PVP, inoculation.
Safflower yulan mutation explant is seeded in added dithiothreitol (DTT) (0.5mgL
-1), ascorbic acid Vc (5mgL
-1) and active carbon (2gL
-1) medium on normally cultivate,
By changing condition of culture, control the brown stain of explant, concrete mode is as follows:
1. shading treatment.After secretly cultivating 15 days under 25 ℃, change under the light and cultivate.
2. low temperature shading treatment.Change over to after 15 days under 25 ℃ the full exposure in shading under 15 ℃ of conditions and to cultivate.
(6) the just foundation of culture system
Just normally cultivate on the culture base being inoculated into the MS that has added plant growth regulator (basic element of cell division 6-BA, growth hormone methyl α-naphthyl acetate NAA), adsorbent (AC) and inhibitor (Vc) through the stem with bud after early stage explant processing and the sterilization.
(7) callus induces
To in plant growth regulator (basic element of cell division 6-BA, KT, growth hormone NAA) the evoked callus medium of different content, normally cultivate through the tissue culture plant inoculation of first culture.
Normal condition of cultivating is in the above-mentioned steps: 25 ± 2 ℃ of room temperatures, and relative air humidity about 60%, intensity of illumination is 2000-3000lx, the photoperiod is that light is cultivated 16h, secretly cultivated 8h.
Description of drawings:
Fig. 1 different time is drawn materials on the impact of the brown rate of stem with bud and survival rate
5. embodiment:
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
The selection of embodiment 1. best explants
Safflower yulan mutation embryo, blade, leaf bud and the stem with bud explant of sterilization are inoculated in respectively cultivation (table 1) on the upper MS medium, the survival rate of stem with bud and the rate of sprouting are higher, be respectively 80.3%, 75.6%, the survival rate of blade, embryo, axillalry bud and the rate of sprouting are all lower, be respectively 2%, 3%, 10.6% and 0%, 1.3%, 5.3%.Variance analysis as can be known, stem with bud and other three explants type difference are remarkable, are that safflower yulan mutation tissue is cultivated the best type of explant.
By to stem with bud acquisition time and brown rate relationship analysis as can be known, autumn 9, October gather, the Brown phenomenon weakens, by survival rate more as can be known, the survival rate of drawing materials September will be higher than draws materials about 10% October, therefore, draw materials September and can be used as drawing materials the time of safflower yulan explant the best.
The table 1 not growing state of explant of the same race compares (%)
Annotate a: each level of different letter representations is significant difference on P＜0.05 level, and following table is same.
Embodiment 2 explant sterilizations
The best sterilization system (table 2) of safflower yulan mutation stem with bud has been set up in experiment with alcohol and two kinds of disinfectants of NaClO solution.At first with 75% alcohol explant is carried out preliminarily pasteurized, 75% alcohol not only can kill part bacterium and fungi, can also infiltrate the surface of explant, makes the easier explant surface that touches equably of NaClO disinfectant, makes sterilization more thorough.Wherein, selecting concentration and the disinfecting time of suitable NaClO solution is to set up the key of best sterilization system, only have suitable time and concentration guarantee neither to injure bacterium, fungi that vegetable material itself again can the kill plants surface, reach good sterilization effect.As shown in Table 2, processing 4,5 is the survival rate no significant difference between the two, processes producing significant difference with other, and sterilization effect is good.From processing 1 (NaClO 5%-10min) to processing 4 (NaClO 10%-10min), increase along with disinfection concentration or time, survival rate presents the trend that rises gradually, survival rate reaches 83.3%～84%, process the later increase along with disinfection concentration or time of 4 (NaClO10%-10min), survival rate begins to present downward trend, illustrate after this processes to have begun explant is damaged, processing 4 (NaClO 10%-10min) is the critical value of safflower yulan mutation stem with bud explant NaClO sterilization.Therefore, the mode of safflower yulan mutation sterilization the best is: soak 10～20min with the 10%NaClO sterilization behind the 75% alcohol 30S, survival rate can reach 83.3%～84%.
Table 2 disinfecting time and concentration are on the impact of explant
The selection of embodiment 3 minimal mediums and carbon source thereof
This experiment has filtered out the minimal medium of suitable safflower yulan mutation stem with bud cultivation, kind and the concentration of carbon source by the orthogonal design to three kinds of factors.The range analysis result shows (table 3): the kind of minimal medium is to affect the sprout important factor of rate of explant, and its extreme difference value is 15.85, secondly is the concentration of carbon source, and the extreme difference value is 9.92, and the kind of carbon source is sprouted to explant and do not produced considerable influence.The minimal medium that the most suitable safflower yulan mutation starts growth is the MS medium, and sucrose in water ratio white granulated sugar and fructose are more suitable for doing the carbon source of safflower yulan mutation, and carbon source concentration is 30g/LgL
-1Growth to explant is more favourable.
Table 3 minimal medium kind, carbon source kind and concentration are on the impact of the rate of sprouting
The foundation of embodiment 4 Browning control systems
Refrigeration and polyvinylpyrrolidine copper (PVP) all can reduce the brown rate (table 4) of explant to a great extent to the processing of explant, compare with the brown rate of control group 84.6%, the brown stain range of decrease is 10.6%-35.8%, wherein explant cutting process effect in PVP solution is best, only this measure just can make browning rate be reduced to 48.8%, secondly for refrigeration 4h, can make browning rate reduce to 50.7%.
Table 4 different explants is processed the impact on the brown stain effect
Table?3-5?Effects?of?different?explants?treatments?on?the?browning
Dithiothreitol (DTT) (DTT), ascorbic acid (Vc) and active carbon (AC) all can effectively reduce the browning rate (table 5) of safflower yulan mutation explant, compare with control group, the brownization range of decrease is 32.6%-45%, wherein the inhibition of ascorbic acid is best, can make browning rate be reduced to 41.7%, secondly being active carbon, is dithiothreitol (DTT) at last.
The anti-phenol oxide of table 5 is on the impact of brown stain
Table3-6?Effects?of?Oxidation?inhibitor?on?the?browning
Temperature and illumination also are the important environmental factors of brownization of impact in the Plant Tissue Breeding.Experimental result shows (table 6), and shading treatment makes brownization be reduced to 78.1%, and browning rate reduces by 10.8% compared with the control; Shading and low temperature are processed simultaneously, can strengthen the Brown inhibition, make its browning rate be reduced to 64%, and the range of decrease is 24.9%.
Table 6 low temperature and shading treatment are on the impact of brownization control
Talbe?3-7?Effects?of?low?temperature?and?shading?treatment?on?browning
Embodiment 5 is culture just
Stem with bud after the sterilization is inoculated on the first culture base of additional 6-BA, NAA, Vc and AC various combination of MS and cultivates, about 10 days, leaf bud expands rapidly and germinates new leaves, and 90% explant can be finished and start growth.Added behind the plant growth regulator survival rate of explant be significantly improved (table 7), compare significant difference with contrast (processing 1), variance analysis as can be known, hormone kind and concentration make a significant impact the first culture of safflower yulan mutation explant, and the survival rate of processing 5,6,7,9,10 is higher, can reach more than 85%, with processing 2,3,4,8 significant differences, no significant difference between five kinds of processing, the processing that wherein survival rate is the highest appears at No. 9, MS+2.0mgL
-16-BA+1.5mgL
-1NAA, survival rate can reach 88.3%, is higher than contrast 77.7%.
Added anti-browning agent Vc and adsorbent A C in medium after, compare for No. 1 with control group, brown rate significantly decreases, and wherein effect is reasonable is to process 9:Vc5mgL
-1+ AC3mgL
-1With processing 10:Vc6mgL
-1+ AC2mgL
-1, be respectively 25.1%, 32.7%, contrast on year-on-year basis the 54%-61% that descended.The anti-browning effect of anti-browning agent Vc is better than adsorbent A C, and the former brown rate is about 50%, and the latter's brown rate is 70%-80%.It is best that both are combined with effect, and brown rate is between 25%-40%.
Analysis-by-synthesis as can be known, be fit to safflower yulan mutation stem with bud just the medium of culture be: MS+6-BA2.0mg.L
-1+ NAA 1.5mg.L
-1+ VC 5mg.L
-1L+AC 3mg.L
-1
The foundation of the first culture of table 7 and Browning control system
Embodiment 6 callus inductions
First culture aseptic seedling is transferred in the inducing culture of the additional not growth regulator of the same race of MS (table 8), produce a small amount of white callus after 10 days around the axillalry bud of explant and bottom the stem section, this callus can not continue differentiation, through brownization is dead voluntarily about a week.After 20 days, produce a large amount of green callus in explant stem section bottom, there is granular kick on the surface, continues to cultivate, and after 30 days, As time goes on such callus has browning to begin to occur.As shown in Table 8, plant growth regulator concentration and kind produce significant difference to the healing rate of test-tube plantlet, process 7,8,9 healing rates higher, each is processed and produces significant difference in P＜0.05 level with other, illustrates that the concentration of the plant cell growth element NAA that is fit to safflower yulan mutation stem with bud explant callus induction is 1.5-2.5mgL
-1, the concentration of basic element of cell division 6-BA is 8-12mgL
-1, the concentration of KT is 3.5-5mgL
-1, what wherein healing rate was the highest number is 9:MS+6-BA12mgL for processing
-1+ KT5mgL
-1Healing rate is 45.4%.Experiment shows, the healing rate effect of the 6-BA basic element of cell division is far below kinetin KT, and concentration is lower than 2mgL
-16-BA when processing, the healing rate of test-tube plantlet is lower than 5.0%, kinetin KT can significantly increase the callus induction rate of safflower yulan mutation stem with bud, working concentration is 1mgL separately
-1Can make the healing rate of explant reach 12.8% during KT, and use 1mgL separately
-1The healing rate of 6-BA (0.93%) only 0.93%.Analyze as can be known, the callus induction rate that both are combined with (processing 5-9) is all high than single use one plant growth regulators inductivity HORMONE TREATMENT (processing 1-4) inductivity, illustrate the basic element of cell division of the same race not to be combined with effect better.The concentration that is fit to the plant cell growth element NAA of safflower yulan stem with bud explant callus induction is 1.5-2.5mgL
-1
Analysis-by-synthesis, the medium that is fit to safflower yulan mutation stem with bud callus induction is: MS+6-BA 12mg.L-1+KT 5mg.L-1+NAA 2.5mg.L-1
Table 8 growth regulator is induced callus
The above only is optimal way of the present invention; should be pointed out that the common laborer for the art, under the principle prerequisite that does not break away from the present technique invention; can also make some suitable improvements and modifications, these improvements and modifications also should be considered as in the protection domain of the present invention.
Claims (8)
1. the method for a safflower yulan mutation callus induction, it is characterized in that may further comprise the steps: seed, axillalry bud, stem segment with axillary bud and the blade take the mutation of safflower yulan is inoculated on the MS medium as explant supplies the examination material respectively, select optimum explant type and draw materials the time, go out again its optimum sterilization system, best base basal culture medium and carbon source take this type as explant selection, optimum just culture base, Browning control system and evoked callus medium.
2. callus induction method according to claim 1 is characterized in that, the best explant of described safflower yulan mutation callus induction is stem with bud, and the optimum time of drawing materials is September.
3. callus induction method according to claim 1, it is characterized in that, the optimum sterilization system of described safflower yulan mutation is take stem with bud as explant, with aseptic water washing behind 75% alcohol-pickled explant 30s 3-4 time, then soak 10min again with aseptic water washing 4 times with 10%NaClO liquid.
4. callus induction method according to claim 1 is characterized in that, described safflower yulan mutation best base basal culture medium is the MS medium, and optimum carbon source is 30g.L
-1Sucrose.
5. callus induction method according to claim 1 is characterized in that, below three kinds of methods all can effectively suppress brownization of safflower yulan mutation:
(1) before inoculation, branch is immersed in cutting or refrigeration branch 4h in the PVP solution;
(2) in medium, add 5mg.L
-1Ascorbic acid or 2g.L
-1Active carbon;
(3) after shading under 15 ℃ of conditions is cultivated 15 days, change under 25 ℃ the full exposure and cultivate.
6. callus induction method according to claim 1 is characterized in that, described safflower yulan mutation tissue is cultivated optimum just culture base and is: MS+6-BA 2.0mg.L
-1+ NAA 1.5mg.L
-1+ VC 5mg.L
-1L+AC 3mg.L
-1
7. callus induction method according to claim 1 is characterized in that, described safflower yulan mutation tissue is cultivated optimum callus inducing medium and is: MS+6-BA 12mg.L
-1+ KT 5mg.L
-1+ NAA 2.5mg.L
-16-BA is combined with KT two Plants conditioning agents and induces successful to be higher than independent use.
8. callus induction method according to claim 1 is characterized in that, medium all adds 7g.L
-1Agar, PH are 5.8-6.0, and 121 ℃ of 20min of autoclaving, condition of culture are 25 ± 2 ℃ of room temperatures, relative air humidity about 60%, and intensity of illumination is 2000-3000lx, the photoperiod is that light is cultivated 16h and the dark 8h of cultivation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103238520A (en) * | 2013-05-20 | 2013-08-14 | 宜宾云辰乔木园林有限责任公司 | Method for carrying out tissue culture and seedling growing of michelia champaca |
| CN105660408A (en) * | 2016-02-26 | 2016-06-15 | 邓珂 | Michelia alba callus induction method |
| CN106962094A (en) * | 2017-02-27 | 2017-07-21 | 焦杰彪 | A kind of Magnolia wufengensis seeding growing seedlings method |
| CN107509635A (en) * | 2017-10-11 | 2017-12-26 | 陈金水 | A kind of in vitro tissue culture and rapid propagation method of Chinese anise |
| 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 |
-
2011
- 2011-07-08 CN CN 201110190088 patent/CN102860255A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103238520A (en) * | 2013-05-20 | 2013-08-14 | 宜宾云辰乔木园林有限责任公司 | Method for carrying out tissue culture and seedling growing of michelia champaca |
| CN103238520B (en) * | 2013-05-20 | 2015-03-18 | 宜宾云辰乔木园林有限责任公司 | Method for carrying out tissue culture and seedling growing of michelia champaca |
| CN105660408A (en) * | 2016-02-26 | 2016-06-15 | 邓珂 | Michelia alba callus induction method |
| CN106962094A (en) * | 2017-02-27 | 2017-07-21 | 焦杰彪 | A kind of Magnolia wufengensis seeding growing seedlings method |
| CN107509635A (en) * | 2017-10-11 | 2017-12-26 | 陈金水 | A kind of in vitro tissue culture and rapid propagation method of Chinese anise |
| 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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