CN108184667A - A kind of method of Moringa Vitro Quick Reproduction - Google Patents
A kind of method of Moringa Vitro Quick Reproduction Download PDFInfo
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- CN108184667A CN108184667A CN201711493979.0A CN201711493979A CN108184667A CN 108184667 A CN108184667 A CN 108184667A CN 201711493979 A CN201711493979 A CN 201711493979A CN 108184667 A CN108184667 A CN 108184667A
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- moringa
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- multiple buds
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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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
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Environmental Sciences (AREA)
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- Soil Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of methods of Moringa Vitro Quick Reproduction：Explant is inoculated on inducing clumping bud culture medium, is cultivated under 25 ± 1 DEG C, 2000Lx intensities of illumination, daily 12 hours illumination conditions 20~40 days and forms Multiple Buds；Multiple Buds are accessed into proliferated culture medium, 25 ± 1 DEG C, 2000Lx intensities of illumination are cultivated 20 40 days under daily 12 hours illumination conditions, and the bud of access gradually forms new Multiple Buds again；The Multiple Buds that Multiplying culture obtains are cut into the stem section of 3~4cm long, are inoculated in root media, 25 ± 1 DEG C, 2000Lx intensities of illumination, culture to bud is taken root under daily 12 hours illumination conditions, obtains rooted seedling；Using the cotyledonary node of Moringa aseptic seedlings as explant, a large amount of Multiple Buds are formed in a short time, are substantially shorter Moringa sterile propagation system and are established the required time, can effectively solve the supply problem of Moringa high quality seedling.
Description
(1) technical field
The present invention relates to a kind of methods of Moringa Vitro Quick Reproduction, can carry out excellent Moringa seedling by this method
Large-scale production.
(2) background technology
Moringa (Moringa oleifera Lam.) is Moringaceae (Moringaceae) Moringa (Moringa
Adans. a kind of tropical economic tree), about 14 kinds of the whole world, and wherein distribution is relatively wide and utilization and research most
That more is Moringa oleifera, originates in north India Himalayas, drought-enduring, resistance is strong, is adapted to because extensive
Property and abundant nutritive value and one of as plant most popular in recent years.The blade and fruit pod of Moringa contain multiple protein
Matter, vitamin and amino acid have the function of extra-nutrition and Dietotherapy health；Seed contains active flocculation ingredient, can conduct
Water purification agent；Oil ben is then mainly used for the additive of the cosmetics of super quality.The Moringa seedling used in production at present mainly uses seed
Breeding, and sajina seed price is high, budding is irregular, and seedling is in short supply, it is impossible to meet the needs of large area production plantation.State
The content of its ingredient is concentrated mainly on to the research of Moringa outside and is used, and the domestic research to Moringa tissue cultures
Be since in recent years just.Also there are explant differentiation rate, rooting rate are low in tissue culture procedures, transplanting survival rate is not high,
The problems such as easy yellow of test tube seedling is wilted.
(3) invention content
It is an object of the present invention to provide a kind of methods of Moringa Vitro Quick Reproduction, it is possible to reduce comprehensive production cost, it can be real
The mass production of existing Moringa nursery can effectively solve the supply problem of Moringa high quality seedling.
The technical solution adopted by the present invention is：
The present invention provides a kind of method of Moringa Vitro Quick Reproduction, and the method carries out as follows：
(1) explant：It is explant to take the terminal bud of Moringa aseptic seedling or cotyledonary node；
(2) induction of Multiple Buds：Step (1) explant is inoculated on inducing clumping bud culture medium, 25 ± 1 DEG C,
2000Lx intensities of illumination cultivate under daily 12 hours illumination conditions 20~40 days and form Multiple Buds；The inducing clumping bud culture
Base is：MS+0.5~1.0mg/L 6-BA (6- benzyls aminoadenine)；
(3) Multiplying culture of bud：By step (2) Multiple Buds access proliferated culture medium, 25 ± 1 DEG C, 2000Lx intensities of illumination,
It is cultivated 20-40 days under daily 12 hours illumination conditions, the bud of access gradually forms new Multiple Buds again；The bud proliferated culture medium
For：MS+0.5~1.0mg/L 6-BA (preferably 0.5mg/L)；
(4) bud is taken root：The Multiple Buds that step (3) Multiplying culture obtains are cut into the stem section of 3~4cm long, are inoculated in life
In root culture medium, 25 ± 1 DEG C, 2000Lx intensities of illumination are cultivated under daily 12 hours illumination conditions and are taken root to bud, taken root
Seedling；The root media is：1/2MS+0.1~1.0mg/L IBA (indolebutyric acid).
Further, explant is that the African Moringa seed after disinfection is aseptically accessed seed to sprout in step (1)
It is cultivated on hair culture medium, is cultivated under 24 ± 1 DEG C of room temperature of culture, dark, air-proof condition, obtain Moringa aseptic seedling；Described kind
Sub- germination medium is that 0.7% agar of mass concentration, 2% sucrose of mass concentration are added in 1/2MS, and pH value is transferred to 5.8.
Further, African Moringa seed sterilization method is：African Moringa seed full, without deformity and free from insect pests is selected,
Kind of a skin is peelled off, with the alcohol surface sterilization 30s of volumetric concentration 70%, puts the HgCl of mass concentration 0.1%2It impregnates and disappears in aqueous solution
Malicious 12-18min will be kept stirred during disinfection, then with aseptic water washing 4~5 times, the seed after being sterilized.
Further, step (2) the inducing clumping bud culture medium is：MS+0.5mg/L 6-BA.
Further, step (4) described root media is：1/2MS+0.1~0.4mg/L IBA.
MS culture mediums of the present invention form：A great number of elements：1650mg/L NH4NO3、1900mg/L KNO3、440mg/
L CaCl2·2H2O、370mg/L MgSO4·7H2O、170mg/L KH2PO4；Trace element：0.83mg/L KI、6.2mg/L
H3BO3、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、0.25mg/L Na2MoO4·2H2O、0.025mg/L
CuSO4·5H2O、0.025mg/L CoCl2·6H2O、27.8FeSO4·7H2O、37.3mg/L Na2.EDTA·2H2O；It is organic
Object：100mg/L inositols, 0.5mg/L niacin, 0.5mg/L puridoxine hydrochlorides, 0.1mg/L thiamine hydrochlorides, 2.0mg/L glycine,
20 grams/L sucrose, solvent are deionized water.1/2MS culture mediums of the present invention form：A great number of elements reduce half, remaining into
Divide identical with MS culture mediums.
Compared with prior art, the beneficial effects are mainly as follows：(1) using the cotyledonary node of Moringa aseptic seedlings as
Explant forms a large amount of Multiple Buds in a short time, is substantially shorter Moringa sterile propagation system and establishes the required time；
(2) explant sterilization method is improved, with 0.1% HgCl2Solution disinfection seed, makes pollution rate be reduced to 25%；(3) to training
Foster base is improved, and filters out the optimum formula of inducing clumping bud culture medium as MS+0.5~1.0mg/L 6-BA, the increasing of bud
The optimum formula for growing culture medium is MS+0.5~1.0mg/L 6-BA；(4) formula for filtering out best root media is 1/2MS
+0.4mg/L IBA；The above, which is improved, can reduce comprehensive production cost, and improve the stability of its character, it can be achieved that peppery
The mass production of wooden nursery can effectively solve the supply problem of Moringa high quality seedling.
(4) it illustrates
Fig. 1 Moringa seeds cultivate the seedling that 4d, 7d, 10d, 14d are formed respectively on 1/2MS culture mediums；A cultivates 4d's
Moringa seed exposes radicle；B cultivates the Moringa seed of 7d, exposes radicle and plumule；C, cultivates the Moringa seed of 10d, plumule,
Radicle is grown from seed；D cultivates the Moringa seed of 14d, and height of seedling is up to 5 centimetres.
The Multiple Buds that Fig. 2 Moringas terminal bud and cotyledonary node are formed on inducing clumping bud culture medium；After A, terminal bud culture 15d
Base portion forms Multiple Buds；The Multiple Buds formed after B, cotyledonary node culture 8d；C, D, the Multiple Buds formed after cotyledonary node culture 15d.
Fig. 3 buds are when cultivating 20d on root media, the rooted seedling of formation.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1
Material：African Moringa seed, purchased from Beijing Xin Yuan Zhong Ye Co., Ltds.
African Moringa seed full, without deformity and free from insect pests is selected, kind of a skin is peelled off, proceeds as follows respectively：
The sterilization method of 1.1 explant materials：On super-clean bench, with 70% alcohol surface sterilization 30s, mass concentration is put
0.1% HgCl212min is sterilized in aqueous solution, to be kept stirred during disinfection, then with aseptic water washing 4~5 times.Seed
Aseptically seed is accessed on seed germination medium after disinfection, in 24 ± 1 DEG C of room temperature of culture, dark, sealing
Under the conditions of cultivate 4d, 7d, 10d, 14d, shown in the result is shown in Figure 1.Seed germination medium is to add in mass concentration in 1/2MS
0.7% agar, 2% sucrose of mass concentration, pH value are transferred to 5.8.
The induction of 1.2 Multiple Buds：After step 1.1 dark culturing 10 days, the seed sprouted is carried out to 12h dark successively,
12h illumination (2000Lx) is cultivated, and is then taken the terminal bud and cotyledonary node of 14d age Moringa aseptic seedlings, is inoculated in inducing clumping bud respectively
On culture medium, after being cultivated 20 days, 25 days, 30 days under 25 ± 1 DEG C of temperature, 2000Lx illumination conditions, the generation of Multiple Buds is observed
Situation, as a result as shown in Figure 2.Inducing clumping bud culture medium is：MS+0.5mg/L 6-BA are the best cultures for inducing Multiple Buds；
The Multiplying culture of 1.3 buds：After inducing clumping bud culture 30d, the preferable Multiple Buds of growth selection are cut from cotyledonary node
Under be about the regenerated buds of 2~3cm, access proliferated culture medium, 25 ± 1 DEG C, cultivate under 2000Lx illumination conditions 20 days, 25 days, 30
My god, the bud of access gradually forms new Multiple Buds again, and general 30 days are 1 shoot proliferation period, and each subculture cycle is by 1 bud
It can be proliferated to form 6-7 bud, the bud proliferated culture medium is：MS+0.5mg/L 6-BA, the most suitable culture for Moringa bud proliferation
Base.
1.4 buds are taken root：High about 3-4 centimetres of the bud that Multiplying culture obtains from base portion is cut, accesses root media
In, culture medium is 1/2MS+0.1mg/L IBA, 25 ± 1 DEG C, culture 20d is taken root to bud under 2000Lx illumination conditions；As a result see figure
Shown in 3.
1.5 conclusion
African Moringa test tube seedling is cultivated 20 days in root media, and the root system of 1.2 centimeter lengths is grown in incision.
Embodiment 2
By explant in 1 step 1.1 of embodiment in the HgCl of mass concentration 0.1%2Disinfecting time changes respectively in aqueous solution
For 12min, 15min, 18min, other operations the results are shown in Table 1 with embodiment 1.
1 mercuric chloride processing time of table is to Moringa seed sprouting, the influence of seedling development
It can be seen in table 1 that 0.1%HgCl2When 15min is sterilized in solution, the germination rate highest of Moringa seed reaches
66.7%.
Embodiment 3
The terminal bud of 14d age Moringa aseptic seedlings will be taken in 1 step 1.2 of embodiment, be inoculated in the clump of addition various concentration 6-BA
The 6-BA concentration of inducing culture of sprouting is changed to 0.25mgL respectively-1、0.5mg·L-1、1.0mg·L-1, other operations are the same as real
Example 1 is applied, the results are shown in Table 2.
The influence that 2 various concentration 6-BA of table is proliferated Moringa terminal bud
As seen from Table 2, when adding 0.5mg/L 6-BA in culture medium, the Multiple Buds number that Moringa terminal bud generates is most, training
It supports the average bud number that 30 days the latter terminal buds generate and reaches 6.7.
Embodiment 4
The cotyledonary node of 14d age Moringa aseptic seedlings will be taken in 1 step 1.2 of embodiment, be inoculated in addition various concentration 6-BA's
The 6-BA concentration of inducing clumping bud culture medium is changed to 0.25mgL respectively-1、0.5mg·L-1、1.0mg·L-1, other operations are together
Embodiment 1, the results are shown in Table 3.
3 various concentration 6-BA of table forms Moringa cotyledonary node the influence of Multiple Buds
As seen from Table 3, when adding 0.5mg/L 6-BA in culture medium, the Multiple Buds number that Moringa cotyledonary node generates is most,
The average bud number that 30 days the latter terminal buds of culture generate reaches 16.
Embodiment 5
It is material to choose African Moringa seed, and operating procedure such as embodiment 1, each culture medium is also in the same manner as in Example 1, only
It is to add 0.1mg/L, 0.2mg/L, 0.4mg/L, 0.8mg/L IBA respectively in step (4), root media, 25 ± 1 DEG C,
Cultivated under 2000Lx illumination conditions 20 days to bud take root when, other operations are with embodiment 1, record rooting rate, mean elements peace
Equal root long, the results are shown in Table 4.
Influences of the 4 various concentration IBA of table to Moringa rooting of vitro seedling
| IBA concentration (mg/L) | Rooting rate (%) | Mean elements | Average root long (cm) |
| 0.1 | 63.0 | 4.3 | 1.20 |
| 0.2 | 70.0 | 3.7 | 1.17 |
| 0.4 | 77.0 | 4.5 | 1.08 |
| 0.8 | 54.0 | 6.2 | 0.86 |
As seen from Table 4, when adding 0.4mg/L IBA in root media, the rooting rate highest of Moringa test tube seedling reaches
77%, mean elements 4.5, a length of 1.08cm of average root.
Claims (6)
- A kind of 1. method of Moringa Vitro Quick Reproduction, it is characterised in that the method carries out as follows：(1) explant：It is explant to take the terminal bud of Moringa aseptic seedling or cotyledonary node；(2) induction of Multiple Buds：Step (1) explant is inoculated on inducing clumping bud culture medium, in 25 ± 1 DEG C, 2000Lx Intensity of illumination cultivates under daily 12 hours illumination conditions 20~40 days and forms Multiple Buds；The inducing clumping bud culture medium is：MS + 0.5~1.0mg/L 6-BA；(3) Multiplying culture of bud：By step (2) Multiple Buds access proliferated culture medium, 25 ± 1 DEG C, 2000Lx intensities of illumination, daily It is cultivated 20-40 days under 12 hours illumination conditions, the bud of access gradually forms new Multiple Buds again；The bud proliferated culture medium is： MS+0.5~1.0mg/L 6-BA；(4) bud is taken root：The Multiple Buds that step (3) Multiplying culture obtains are cut into the stem section of 3~4cm long, are inoculated in training of taking root It supports in base, 25 ± 1 DEG C, 2000Lx intensities of illumination, culture to bud is taken root under daily 12 hours illumination conditions, obtains rooted seedling；Institute Stating root media is：1/2MS+0.1~1.0mg/L IBA.
- 2. the method for Moringa Vitro Quick Reproduction as described in claim 1, it is characterised in that explant is will to sterilize in step (1) African Moringa seed afterwards, which is aseptically accessed on seed germination medium, to be cultivated, black in 24 ± 1 DEG C of room temperature of culture Secretly, it is cultivated under air-proof condition, obtains Moringa aseptic seedling；The seed germination medium is that mass concentration 0.7% is added in 1/2MS Agar, 2% sucrose of mass concentration, pH value are transferred to 5.8.
- 3. the method for Moringa Vitro Quick Reproduction as claimed in claim 2, it is characterised in that African Moringa seed sterilization method is： African Moringa seed full, without deformity and free from insect pests is selected, kind of a skin is peelled off, with the alcohol surface sterilization of volumetric concentration 70% 30s puts the HgCl of mass concentration 0.1%2Soaking disinfection 12-18min in aqueous solution will be kept stirred, Ran Houyong during disinfection Aseptic water washing 4~5 times, the seed after being sterilized.
- 4. the method for Moringa Vitro Quick Reproduction as described in claim 1, it is characterised in that step (2) the inducing clumping bud training Foster base is：MS+0.5mg/L 6-BA.
- 5. the method for Moringa Vitro Quick Reproduction as described in claim 1, it is characterised in that step (3) the bud proliferated culture medium For：MS+0.5mg/L 6-BA.
- 6. the method for Moringa Vitro Quick Reproduction as described in claim 1, it is characterised in that step (4) described root media For：1/2MS+0.1~0.4mg/L IBA.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115136890A (en) * | 2022-06-13 | 2022-10-04 | 华南农业大学 | Efficient regeneration method of moringa oleifera plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016164224A1 (en) * | 2013-11-01 | 2016-10-13 | Rutgers, The State University Of New Jersey | Extracts from plants of the moringaceae family and methods of making |
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2017
- 2017-12-31 CN CN201711493979.0A patent/CN108184667A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016164224A1 (en) * | 2013-11-01 | 2016-10-13 | Rutgers, The State University Of New Jersey | Extracts from plants of the moringaceae family and methods of making |
Non-Patent Citations (2)
| Title |
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| 杨俊俊等: "辣木快速繁殖体系", 《上海交通大学学报（农业科学版）》 * |
| 陈涵斌: "辣木快速繁殖体系建立", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115136890A (en) * | 2022-06-13 | 2022-10-04 | 华南农业大学 | Efficient regeneration method of moringa oleifera plants |
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