CN1324952C - Quick breeding method for tissue culture of lemon verbena - Google Patents
Quick breeding method for tissue culture of lemon verbena Download PDFInfo
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- CN1324952C CN1324952C CNB2005100263953A CN200510026395A CN1324952C CN 1324952 C CN1324952 C CN 1324952C CN B2005100263953 A CNB2005100263953 A CN B2005100263953A CN 200510026395 A CN200510026395 A CN 200510026395A CN 1324952 C CN1324952 C CN 1324952C
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Abstract
The present invention relates to a method for lemon verbena's tissue cultivation and fast propagation. The method comprises: adopting lemon verbena's young tender stem segments with knots as explants; during sterilization, using the method of continuously wiping materials, particularly the parts of axillary buds, with a banister brush in sterilization solution to prevent pollution; at the initial stage of inoculation, adopting the methods of cultivation medium transition and dark cultivation to control the browning of the materials; then, cultivating the materials successively in the selected cultivation mediums of an axillary bud induction cultivation medium, a proliferation cultivation medium and a rooting medium, during which the axillary buds can germinate and grow normally, and quickly enter the state of vegetation to produce integrated test-tube plantlets; transplanting the test-tube plantlets into a seedbed of vermiculite and humus soil; one month later, the test-tube plantlets can be transplanted into fields. The method for lemon verbena propagation is free from the influence of external conditions, can be carried out in all the four seasons, can save the occupation of land during seedling cultivation, can reduce production cost, can conserve all the fine characteristics of maternal plants, and can stabilize the inherited characteristics. The method can produce a large amount of fine test-tube plantlets in a short period, and can realize scale and factory production.
Description
Technical field
The present invention relates to a kind of quick breeding method for tissue culture of fragrant plant lemon verbena, more particularly the present invention relates to a kind of axillalry bud induced tissue and cultivate the method for breeding lemon verbena fast.
Background technology
Lemon verbena originates in the ground such as Chile, Argentina and Peru of tropical South America, and also there is cultivation in the Morocco in north African.China has only several strains to be planted in the booth of this experimental group by Japan's introduction.It contains abundant volatile oil, and the essential oil of extraction can be used in the cosmetics, also has very high medical value; Because of its extremely favor of people of distinctive lemon fragrance, be widely used in food, the tea beverage; Yin Qiye look, blade profile are attractive in appearance again, the planting material that also can be used as the flower garden greening, purifies air, and this not only makes the lemon verbena demand heighten, and economic worth also doubles, and therefore lemon verbena is carried out breeding in a large number fast being imminent.
Lemon verbena seminal propagation difficulty, cottage propagation is subjected to all multifactor impacts such as environmental temperature, humidity and soil ph again, and survival rate is low, has directly influenced its popularizing planting, and the cottage propagation coefficient is low again, is difficult to meet the need of market.
Lemon verbena leaf, the close living fur of axil, the sterilization treatment to explant when carrying out tissue culture is quite difficult; Fragrant plant produces secondary metabolites such as essential oil, easily cause material and brownization of medium when tissue culture, and lemon verbena essential oil content height belongs to woody fragrant plant again, than easier brownization of other fragrant plant, makes troubles to tissue culture.
Still do not have both at home and abroad about lemon verbena being carried out the tissue culture report.There is not the ready-made training mode can be for reference, quite important to the selection of its medium.
Summary of the invention
The objective of the invention is at lemon verbena seminal propagation difficulty and cottage propagation survival rate problem on the low side, a kind of quick breeding method for tissue culture of lemon verbena is provided, improve the lemon verbena reproduction coefficient, guarantee the lemon verbena genetic stability.
For realizing this purpose, it is explant that the present invention adopts the young tender stem segment that contains of lemon verbena, take during sterilization in sterilized solution to pollute with the method control of the continuous grooming material of banister brush especially axillalry bud position, brownization of primary stage of inoculation adopting conversion medium and dark cultured method control material, then successively at axillalry bud inducing culture through screening, cultivate in proliferated culture medium and the root media, axillalry bud can also enter vegetative state rapidly by normal germination and growth, obtain complete test-tube plantlet, on the seedbed of vermiculite and humus soil, after one month just implantable land for growing field crops with this test-tube seedling transplanting.
Method of the present invention specifically comprises the steps:
1, explant sterilization: choose lemon verbena trunk base portion children shoot, remove blade, be placed in the liquid detergent water and embathe, putting into 70% alcohol again after running water flushing stirred 15～20 seconds, take out to change over to immediately in the smart solution of saturated bleaching that contains Tween-80 and soaked 9～10 minutes, constantly use banister brush grooming axillalry bud position therebetween, take out with behind the aseptic water washing 3～4 times and water is blotted with the blotting paper of sterilizing, then the young tender stem segment that contains that cuts contiguous terminal bud is made explant, be seeded in rapidly on the MS medium, put into the dark place and cultivate, material was transferred on the new MS medium in second day;
2, axillalry bud is induced: will be on the MS medium no longer the explant of brownization change inducing culture MS+6-BA0.5～1.0mg/L+NAA0.1mg/L+ agar 7.0g/L+ sucrose 30g/L over to, pH5.8, cultivation temperature is 25 ± 2 ℃, periodicity of illumination 12h.d
-1, luminous intensity is 1200lux;
3, shoot proliferation: cut single bud in the inducing culture, remove 2/3 blade, cut the stem section that contains joint and be inoculated into proliferated culture medium MS+6-BA0.25～0.5mg/L+NAA0.1mg/L+ agar 7.0g/L+ sucrose 30g/L, pH5.8, cultivation temperature is 25 ± 2 ℃, periodicity of illumination 14h.d
-1, luminous intensity is 2000lux;
4, root induction: length is that the bud of the stalwartness of 1.5～2.0cm is inoculated into root media 1/2MS+IBA0.5mg/L+ agar 6.0g/L+ sucrose 25g/L in the clip enrichment culture, pH5.8, and cultivation temperature is 20 ± 2 ℃, periodicity of illumination 10h.d
-1, luminous intensity is 1000lux;
5, acclimatization and transplants: the seedling of selecting long 2～5cm, height of seedling 3～5cm of root, open blake bottle, add a little sterile water hardening 5-7 days, careful then water is washed the root agar medium off, transplants on the seedbed that vermiculite that high-temperature sterilization crosses and humus soil (ratio 1: 1) are housed, water sufficient water, last cover continues to cultivate in the greenhouse with film, and early stage, humidity was controlled at about 70%, 20 ± 2 ℃ of temperature, periodicity of illumination 10h.d
-1, intensity of illumination 1000lux, the later stage is recovered temp. and humidity under the nature gradually.Move into the land for growing field crops after one month.
The present invention breeds lemon verbena compared with prior art, has substantial progress:
1, improved reproductive efficiency: lemon verbena seminal propagation difficulty, not only reproduction coefficient is low to adopt cottage method, the labor cost height, complex management, and also survival rate is low.The present invention gets the tender stem segments that lemon verbena contains joint, carries out the quick breeding that axillalry bud is induced with tissue culture method, is not subjected to the influence of external condition, the four seasons all can carry out, and not only save the occupation of land of growing seedlings, and reduce production costs, and whole merits of parent have been preserved, stabilization characteristics of genetics.This method can form a large amount of good test-tube plantlets in a short time, carries out scale, batch production production.
2, controlled pollution effectively: lemon verbena is because of the densely covered glandular hairs in its surface, objectively exist the contradiction that needs long-time sterilization and don't resistance to kill, existing sterilization method with the medicining liquid dipping explant is easy to kill and wound material, is difficult to guarantee the quantity of effective explant.The present invention is strict control disinfecting time when sterilizing operation, take in thimerosal with the continuous grooming material of the banister brush especially method at axillalry bud position, Disinfection Effect is obviously improved, and pollution rate is controlled at below 8.94%, has also guaranteed enough effective explant quantity.
3, suppressed medium and brownization of explant: fragrant plant produces secondary metabolites such as essential oil, explant is inoculated into directly by existing cultured method easily causes material and brownization of medium in the inducing culture, and lemon verbena essential oil content height, belong to woody fragrant plant again, than easier brownization that causes material and medium of other fragrant plant.It is explant that the present invention selects for use lemon verbena trunk base portion newly to extract the tender stem segments that contiguous terminal bud contains joint on the branch out, putting into the dark place after inoculation cultivates, and material is transferred on the new medium in second day, extremely brownization is not transferred on the inducing culture again, controlled brownization of material and medium effectively.
4, filter out effective culture medium prescription: still do not have relevant report to the lemon verbena tissue culture so far both at home and abroad, selecting medium for use is a link of most critical.The present invention carries out axillalry bud with MS medium supplemented 6-BA1.0mg/L and NAA0.1mg/L and induces, the normal germination and growth of explant axillalry bud: carry out enrichment culture with MS medium supplemented 6-BA0.50mg/L and NAA0.1mg/L, enter vegetative state rapidly, switching in 20 days 1 time, growth coefficient is more than 3 times; Carry out root induction with additional IBA0.5mg/L among the 1/2MS, rooting rate is up to 99%, and the bar number of taking root is many, just can obtain a large amount of tissue cultivating seedling in a short time.
Embodiment
Below by specific embodiment technical scheme of the present invention is further described.
1. explant sterilization is removed the lemon verbena children shoot of blade, in liquid detergent water, embathed 20 minutes, the back was washed 2 hours with running water, drain, putting into 70% alcohol stirred 15 seconds, taking-up changes in the smart solution of saturated bleaching that contains Tween-80 soaked 10 minutes, therebetween with the continuous grooming material of banister brush especially axillalry bud position, on clean bench, take out then, use aseptic water washing 3～4 times, with sterilization blotting paper water is blotted again, cut the young tender stem segment that contains of terminal bud lower end, be seeded in rapidly on the MS medium, put into the dark place and cultivate, and changed in the new MS medium in second day.
2. axillalry bud is induced will not have the stem section of pollution, brownization to change medium MS+6-BA1.0mg/L+NAA0.1mg/L+ agar 7.0g/L+ sucrose 30g/L, pH5.8 over to.25 ± 2 ℃ of cultivation temperature, periodicity of illumination 12h.d
-1, luminous intensity 1200lux.
3. shoot proliferation cuts single bud in the inducing culture, removes 2/3 blade, cuts to contain stem segment and be inoculated into medium MS+6-BA0.5mg/L+NAA0.1mg/L+ agar 7.0g/L+ sucrose 30g/L, pH5.8.25 ± 2 ℃ of cultivation temperature, periodicity of illumination 14h.d
-1, luminous intensity is 2000lux.
4. length is that the bud of the stalwartness of 1.5～2.0cm is inoculated into medium 1/2MS+IBA0.5mg/L+ agar 6.0g/L+ sucrose 25g/L, pH5.8 in the root induction clip enrichment culture.Cultivation temperature is 20 ± 2 ℃, photoperiod 10h.d
-1, luminous intensity is 1000lux.
5. acclimatization and transplants is selected the seedling of the long 2～5cm of root, height of seedling 3～5cm stalwartness, opens blake bottle, adds a little sterile water, takes exercise 5～7 days in culturing room, makes it adapt to external environment condition.Careful then water is washed the root agar medium off, transplants on the seedbed that vermiculite that high-temperature sterilization crosses and humus soil (ratio 1: 1) are housed, and waters sufficient water, and last cover is with film, continuation cultivation in the greenhouse.Early stage, controlled humidity was about 70%, and temperature is at 20 ± 2 ℃, photoperiod 10h.d
-1, intensity of illumination 1000lux, the later stage, seedling began to grow new root after 20 days progressively near the temperature under the nature, humidity, illumination condition.After 30 days, seedling is moved into the land for growing field crops of doing furrow, fertilising in advance, water sufficient water, the cover shade net.Remove shade net after one week, by general nursery bed management, the statistics survival rate reaches 89% after two weeks.
Claims (1)
1, a kind of quick breeding method for tissue culture of lemon verbena is characterized in that comprising the steps:
1) explant sterilization: the young shoot of choosing lemon verbena trunk base portion, remove blade, be placed in the liquid detergent water and embathe, putting into 70% alcohol after running water flushing stirred 15～20 seconds, take out to change over to immediately in the smart solution of saturated bleaching that contains Tween-80 and soaked 9～10 minutes, constantly use banister brush grooming axillalry bud position during this time, take out with behind the aseptic water washing 3～4 times and water is blotted with the blotting paper of sterilizing, then the young tender stem segment that contains that cuts contiguous terminal bud is made explant, be seeded in rapidly on the MS medium, put into the dark place and cultivate, material was transferred on the new MS medium in second day;
2) axillalry bud is induced: will be on the MS medium no longer the explant of brownization change in the inducing culture, inducing culture consist of MS+6-BA0.5～1.0mg/L+NAA0.1mg/L+ agar 7.0g/L+ sucrose 30g/L, pH5.8, cultivation temperature is 25 ± 2 ℃, periodicity of illumination 12h.d
-1, luminous intensity is 1200lux;
3) shoot proliferation: cut single bud in the inducing culture, after removing 2/3 blade, cutting the stem section that contains joint is inoculated in the proliferated culture medium, proliferated culture medium consist of MS+6-BA0.25～0.5mg/L+NAA0.1mg/L+ agar 7.0g/L+ sucrose 30g/L, pH5.8, cultivation temperature is 25 ± 2 ℃, periodicity of illumination 14h.d
-1, luminous intensity is 2000lux;
4) root induction: length is that the bud of the stalwartness of 1.5～2.0cm is inoculated in the root media in the clip enrichment culture, root media consist of 1/2MS+IBA0.5mg/L+ agar 6.0g/L+ sucrose 25g/L, pH5.8, cultivation temperature is 20 ± 2 ℃, periodicity of illumination 10h.d
-1, luminous intensity is 1000lux;
5) acclimatization and transplants: the seedling of selecting long 2～5cm, height of seedling 3～5cm of root, open blake bottle, in bottle, added the sterile water hardening 5-7 days, and washed the root agar medium then off, transplant on the vermiculite of sterilizing and the humus soil ratio of sterilizing are 1: 1 seedbed, water sufficient water, cover continues to cultivate in the greenhouse with film on the seedbed, and humidity is controlled at 70%, 20 ± 2 ℃ of temperature, periodicity of illumination 10h.d
-1, intensity of illumination 1000lux recovers temp. and humidity under the nature gradually, moves into the land for growing field crops after one month.
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| CNB2005100263953A CN1324952C (en) | 2005-06-02 | 2005-06-02 | Quick breeding method for tissue culture of lemon verbena |
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| CNB2005100263953A CN1324952C (en) | 2005-06-02 | 2005-06-02 | Quick breeding method for tissue culture of lemon verbena |
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| CN1324952C true CN1324952C (en) | 2007-07-11 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101053313B (en) * | 2007-05-31 | 2010-04-21 | 上海交通大学 | Method for tissue culture of Japanese premna |
| CN102668988A (en) * | 2012-06-08 | 2012-09-19 | 江苏九久环境科技有限公司 | Fast seedling breeding method for tissue cultivation of callicarpa bodinieri and method for transplanting rooting seedlings of callicarpa bodinieri |
| CN104472233B (en) * | 2014-12-15 | 2017-08-04 | 成都苗夫现代苗木科技有限公司 | A kind of cuttage breeding method of willow leaf Verbena officinalis |
| CN108077071B (en) * | 2016-11-21 | 2020-04-24 | 四川七彩林科股份有限公司 | Culture medium for culturing vitex agnus-castus tissue and rapid propagation method |
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Non-Patent Citations (4)
| Title |
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| 园艺植物组织培养中的褐化现象及抗褐化研究进展 周俊辉，周家容，曾浩森，等，园艺学报，第27卷 2000 * |
| 柠檬马鞭草在不同生长期精油含有率及组分变化的研究 沈校军，姚雷，黄健，上海交通大学学报（农业科学版），第22卷第1期 2004 * |
| 柠檬马鞭草在不同生长期精油含有率及组分变化的研究 沈校军，姚雷，黄健，上海交通大学学报（农业科学版），第22卷第1期 2004;园艺植物组织培养中的褐化现象及抗褐化研究进展 周俊辉，周家容，曾浩森，等，园艺学报，第27卷 2000;植物组织培养过程中的污染原因及控制措施 赵佐敏，艾勇，贵州农业科学，第32卷第1期 2004 * |
| 植物组织培养过程中的污染原因及控制措施 赵佐敏，艾勇，贵州农业科学，第32卷第1期 2004 * |
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