CN110278870A - Utilize the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling - Google Patents
Utilize the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling Download PDFInfo
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- CN110278870A CN110278870A CN201910583950.4A CN201910583950A CN110278870A CN 110278870 A CN110278870 A CN 110278870A CN 201910583950 A CN201910583950 A CN 201910583950A CN 110278870 A CN110278870 A CN 110278870A
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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
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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
Abstract
The present invention provides a kind of tissue culture methods with leaf petiole forming seedling through one step culture using Jing Banxia tissue culture tufted seedling, include the following steps: the selection of (1) explant: taking the seed of the tuber of pinellia, after sterile-processed, it carries out vernalization and forms Multiple Buds, select the petiole with leaf as the explant of tissue culture；(2) explant is inoculated in one-step culture base and is cultivated to 4~10cm of height of seedling；The formula of the one-step culture base are as follows: 1/2MS+IBA 0.5~1.2mg/L+NAA, 0.2~0.8mg/L+TDZ, 0.15~0.25mg/L+ sucrose 3%+ agar 0.7%.The present invention as explant and uses special culture medium with leaf petiole using Jing Banxia tissue culture tufted seedling, only need 45 days can sprouting and rooting, and (rooting rate is up to 95% or more to the growth coefficient and seedling quality for having ensured half summer sporelings (of laminaria) of chaste tree；Transplanting survival rate is 100%), to accomplish scale production.
Description
Technical field
The present invention relates to asexual propagation of seedlings technical field, in particular to a kind of band leaf using Jing Banxia tissue culture tufted seedling
The tissue culture method of petiole forming seedling through one step culture.
Background technique
Pinellia Tenore Araeceae Pinellia, is used as medicine with dry tuber, is a kind of important Chinese medicine, and tuber of pinellia stem tuber contains
The chemical components such as volatile oil, nicotine, glutamic acid, sitosterol.Have the function of to drop check spit, be eliminating dampness and eliminating phlegm, dissolving lump and resolving mass etc., be mostly used
In treatment phlegm asthma cough, nausea and vomiting, dizziness etc.." Dragon Lord book on Chinese herbal medicine warp " tuber of pinellia of Gu is referred to as to keep field, water jade, is perennial herb
The excavation of plant, mostly two season of summer and autumn, stem tuber like sand soil at spheroidal or oblateness, be widely used simply, curative effect it is true
The large traditional Chinese medicine cut.Outstanding Jiangling, former Jing Zhou, Qianjiang abound with, and best in quality, are commonly called as " Jing Banxia ".Because its quality is excellent
Good, drug effect is particularly good and becomes the tuber of pinellia and is used as medicine first choice.Make us helpless, the Jing Banxia nowadays supplied in the market is fewer and fewer, existing
The tuber of pinellia of Shi Duoyong is " RHIZOMA TYPHONII FLAGELLIFORMIS ", though there is the title of the tuber of pinellia, " RHIZOMA TYPHONII FLAGELLIFORMIS " is to belong to investigation, do not have in last century the eighties
It receives, the medicinal material of fine, pharmacological property differs widely with Jing Banxia, and medicine valence is as far apart as heaven and earth.Why " the water half investigated and prosecuted once
Can summer " nowadays substitute the tuber of pinellia and propagate its belief on a large scale? since the tuber of pinellia is medicinal and demand for the export is big, cause the increasingly depleted difficulty of wild resource
To meet the requirements, price persistently raises up, and remains high.Though currently, there is the artificial cultivating and growing chaste tree tuber of pinellia that can alleviate market pressure,
But since Jing Banxia growth requires dark and damp environment, in the diastrous weather more time, it will cause the situation of harvest yield deficiency, this
The production of Jing Banxia is caused greatly to limit.In addition, tuber of pinellia continuous cropping plantation easily leads to virus infection, kind property is stepped back, yield and product
Matter decline.Accordingly, it is considered to carry out Jing Banxia seeling industry using plant tissue culture fast breeding technique and carry out artificial growth to be to solve chaste tree
The effective way of tuber of pinellia shortage of resources.
Although the success of Jing Banxia artificial growth, expanding plantation, there are still obstacles: first, propagation method is still confined to block
Stem breeding and bulbil breeding, seminal propagation are seldom used in actual production or are hardly used；Second, pest control techniques with
Production mismatches, and virosis, rot disease, epidemic disease etc. are larger to Influence of production；Third, stem tuber is planted, planting cost is high；Fourth,
Large-scale planting area is less.And the foundation of tissue culturing system, it plays an important role, takes off for the control of Jing Banxia continuous cropping diseases
The plantation of malicious seedling can greatly reduce the incidence of its disease, improve the quality and yield of " Jing Banxia ", and keep its excellent
Quality makes " Jing Banxia " to realize large area plantation, in response to the market demand, allowing people's living standard to be improved.
Documents 1: application No. is " CN201410023655.0 ", a kind of entitled " tissue training of tuber of pinellia forming seedling through one step culture
The method of supporting and new tuber of pinellia culture medium " discloses a kind of method for tissue culture of tuber of pinellia forming seedling through one step culture, with 6-BA and NAA hormone
Combination is explant to strain bud, stem tuber, blade and petiole, carries out forming seedling through one step culture induction, stem tuber differentiation efficiency highest, every piece more
Injured tissue differentiates 12-14 seedling, planting percent 95%.Stem tuber seedling number of days 35-40d, blade and petiole need 2-3 months.
However the case haves the shortcomings that slow, Organ Differentiation low efficiency of callus proliferation speed etc. is significant insufficient.In addition, the case is in explant
There are more limiting factor, stem tuber and strain buds in body selection is not thorough as explant in the presence of disinfection, the high problem of pollution rate；And
Seedling overlong time, it is difficult to meet the needs of market.
Thus, how to develop it is a kind of using Jing Banxia tissue culture tufted seedling with leaf petiole as the side of explant forming seedling through one step culture
Method, and improve its rise in value efficiency and differentiation efficiency and become technical problem urgently to be resolved to meet the market demand.
Summary of the invention
It is an object of the invention to overcome the defect of the prior art, a kind of band using Jing Banxia tissue culture tufted seedling is provided
The tissue culture method of leaf petiole forming seedling through one step culture as explant and uses special training with leaf petiole using Jing Banxia tissue culture tufted seedling
Base is supported, under the culture medium, the first long root system of explant after absorbing nutrition, then sprouts, can fully absorb the battalion in culture medium in this way
Support substance, thus the seedling time is short, only need 45 days can sprouting and rooting, and ensured the growth coefficient (up to 22 of half summer sporelings (of laminaria) of chaste tree
More than) and seedling quality (rooting rate is up to 95% or more；Transplanting survival rate is 100%), to accomplish scale production, meet life
Needs in production.
The present invention is implemented as follows:
The present invention provides a kind of tissue culture method with leaf petiole forming seedling through one step culture using Jing Banxia tissue culture tufted seedling, specific to wrap
Include following steps:
The selection of step 1, explant: taking the seed of the tuber of pinellia, after sterile-processed, carry out vernalization and forms Multiple Buds, selection
Explant of the petiole with leaf as tissue culture；
Explant is inoculated in one-step culture base and cultivates to 4~10cm of height of seedling by step 2；One step at
The formula of seedling culture medium are as follows: 0.15~0.25mg/L+ of 1/2MS+IBA 0.5~1.2mg/L+NAA, 0.2~0.8mg/L+TDZ
Sucrose 3%+ agar 0.7%.
The invention has the following advantages:
A kind of tissue culture method with leaf petiole forming seedling through one step culture using Jing Banxia tissue culture tufted seedling provided by the invention, through this
The discovery of inventor's a large amount of Making Innovation Experiments using the formula 0.5~1.2mg/L+NAA of culture medium 1/2MS+IBA 0.2~
0.8mg/L+TDZ 0.15~0.25mg/L+ sucrose 3%+ agar 0.7%, selecting petiole only to need for explant 45 days can be quick
Seedling, and ensured the growth coefficient and seedling quality of half summer sporelings (of laminaria) of chaste tree.10 days or so, incision formation was evident that callus group
Knit (Fig. 1)；20 days or so, it is seen that callus obviously expands, and generates mitogenetic (Fig. 2) a large amount of；30 days or so, it is seen that
Apparent bud point differentiation (Fig. 3)；After 45 days, callus differentiation and seedling emergence, and it is long to 4-10cm (Fig. 4), root system is vigorous, seedling ratio
Relatively strong, withered blade is initial explant blade；Specifically:
(1) under the culture medium of the formula, the first long root system of explant after absorbing nutrition, then sprouts, can fully absorb in this way
Nutriment in culture medium, thus the seedling time is short；Petiole is selected in documents 1 to need 2-3 months when explant；And
The present invention equally select petiole be explant only need 45 days can sprouting and rooting, and ensured the growth coefficient and kind of half summer sporelings (of laminaria) of chaste tree
Seedling quality, accomplishes scale production, and meets the needs in production.
(2) growth coefficient is up to, and callus propagation coefficient reaches 22 or more；Rooting rate is up to 99% or more；Transplanting is deposited
Motility rate is 100%.
(3) 1/2 MS is used compared with comparative example 1, in method of the invention, nutritional need is relatively low.
Detailed description of the invention
Fig. 1 is the picture of 10 days or so the callus formed in embodiment 1；
Fig. 2 is 20 days or so in embodiment 1, and callus obviously expands, and generates a large amount of mitogenetic picture；
Fig. 3 is the picture that bud point is obviously broken up 30 days or so in embodiment 1；
Fig. 4 is the picture of callus differentiation and seedling emergence after 45 days in embodiment 1.
Specific embodiment
Embodiment 1
(1) Seeds preprocess: annual 6-7 month or 10-11 month, mature Jing Banxia seed, fruit except going are collected
Skin, 35 degree water-bath 3-4 days.
(2) it vernalization and selecting explant: on superclean bench, taking sterile petri dish, culture dish is two layers of aseptic filter paper,
It being soaked with sterile water, the seed after disinfection is placed on culture dish, seed disinfection mode is that 0.1% potassium permanganate sterilizes 25min,
Then 75% ethanol disinfection 5min, last 0.1% mercuric chloride sterilize 10min, aseptic water washing 4-5 times, and each 3min or so is kept away
Light, 25 DEG C of vernalization select the petiole with leaf as the explant of tissue culture when plumular axis length 2-3cm.
(3) one-step culture: explant being inoculated in one-step culture base and is placed in illumination/dark=12h/12h,
It is cultivated in 25 DEG C of culturing room, the one-step culture based formulas are as follows:
1/2MS+IBA 1mg/L+NAA 0.5mg/L+TDZ 0.2mg/L+ sucrose 3%+ agar 0.7%.
As shown in Figure 1,10 days or so, incision formation is evident that callus.
As shown in Fig. 2, 20 days or so, it is seen that callus obviously expands, and generates mitogenetic a large amount of.
As shown in figure 3,30 days or so, it is seen that apparent bud point differentiation.
As shown in figure 4, after 45 days, callus differentiation and seedling emergence, and it is long to 4-10cm.
(4) acclimatization and transplants: the root system of the tissue-cultured seedling is rinsed well, and cultivation on turfy soil in cultivating.
Embodiment 2
The formula of one-step culture base described in the embodiment are as follows: 1/2MS+IBA 1mg/L+NAA0.5mg/L+TDZ
0.15mg/L+ sucrose 3%+ agar 0.7%；Other operating procedures are identical as the embodiment 1.
Embodiment 3
The formula of one-step culture base described in the embodiment are as follows: 1/2MS+IBA 1mg/L+NAA0.5mg/L+TDZ
0.25mg/L+ sucrose 3%+ agar 0.7%；Other operating procedures are identical as the embodiment 1.
Embodiment 4
The formula of one-step culture base described in the embodiment are as follows: 1/2MS+IBA 0.5mg/L+NAA0.5mg/L+TDZ
0.2mg/L+ sucrose 3%+ agar 0.7%；Other operating procedures are identical as the embodiment 1.
Embodiment 5
The formula of one-step culture base described in the embodiment are as follows: 1/2MS+IBA 1.2mg/L+NAA0.5mg/L+TDZ
0.2mg/L+ sucrose 3%+ agar 0.7%；Other operating procedures are identical as the embodiment 1.
Embodiment 6
The formula of one-step culture base described in the embodiment are as follows: 1/2MS+IBA 1mg/L+NAA0.2mg/L+TDZ
0.2mg/L+ sucrose 3%+ agar 0.7%；Other operating procedures are identical as the embodiment 1.
Embodiment 7
The formula of one-step culture base described in the embodiment are as follows: 1/2MS+IBA 1mg/L+NAA0.8mg/L+TDZ
0.2mg/L+ sucrose 3%+ agar 0.7%；Other operating procedures are identical as the embodiment 1.
Comparative example 1
The formula of culture medium used in the comparative example are as follows:
1/2MS+IBA 1mg/L+NAA 0.5mg/L+TDZ 0.2mg/L+ sucrose 3%+ agar 0.7%；Other operation steps
It is rapid identical as the embodiment 1.
Comparative example 2
The formula of culture medium used in the comparative example are as follows:
1/2MS+IBA 1.5mg/L+NAA 0.5mg/L+TDZ 0.2mg/L+ sucrose 3%+ agar 0.7%；Other operations
Step is identical as the embodiment 1.
Comparative example 3
The formula of culture medium used in the comparative example are as follows:
1/2MS+IBA 1mg/L+NAA0.5mg/L+TDZ 0.1mg/L+ sucrose 3%+ agar 0.7%；Other operating procedures
It is identical as the embodiment 1.
Experimental example 1
It while is at least repeated 3 times in above embodiments 1- embodiment 7 and comparative example 1- comparative example 3, sees respectively
Examine and calculate callus quality, the callus of Jing Banxia in above embodiments 1- embodiment 7 and comparative example 1- comparative example 3
Hyperblastosis multiple, Multiple Buds quantity (i.e. a bud, which taps into culture medium it, can grow how many a buds), rooting rate, Yi Jicheng
Motility rate simultaneously counts as follows:
Table 1
| Group | Callus quality | Callus proliferation times | Rooting rate | Survival rate | Seedling-growing time |
| Embodiment 1 | It is greyish white loose | 28 times | 99% | 100% | 45 days |
| Embodiment 2 | It is greyish white loose | 23 times | 98% | 99% | 46 days |
| Embodiment 3 | It is greyish white loose | 22 times | 97% | 98% | 47 days |
| Embodiment 4 | It is greyish white loose | 26 times | 95% | 97% | 48 days |
| Embodiment 5 | It is greyish white loose | 27 times | 96% | 99% | 49 days |
| Embodiment 6 | It is greyish white loose | 25 times | 94% | 99% | 47 days |
| Embodiment 7 | It is greyish white loose | 24 times | 95% | 99% | 47 days |
| Comparative example 1 | It is yellowish loose | 8 times | 25% | 24% | 190 days |
| Comparative example 2 | It is yellowish loose | 5 times | 12% | 16% | 144 days |
| Comparative example 3 | White dense | 4 times | 14% | 23% | 89 days |
In comparative example 1, callus is first generated, root is then differentiated, until there is the differentiation of bud at six month, week
Phase is longer.And the basal medium in comparative example 1 is MS culture medium, nutritional need is high.
In comparative example 2, compared with Example 1, the concentration of IBA is only changed to 1.5mg/L, the root system of formation is very fast, more
When injured tissue is unconspicuous, just differentiate root, although forming root, callus is also further formed, but growth rate compared with
Embodiment 1 is substantially reduced.Thus the raising for guessing IBA concentration, can quickly make incision differentiate root, but callus is obvious
Less than normal, the quantity of root system is also less, this concentration may be higher, to produce inhibiting effect.
In comparative example 3, compared with Example 1, the concentration of TDZ is only changed to 0.1mg/L, the speed that callus is formed with
And the equal embodiment 1 of size is low, the callus of formation is smaller, and root system generates less.
By upper table 1 it is found that the Jing Banxia Multiple Buds breeding coefficient obtained using cultural method of the present invention reaches 22
More than, Multiple Buds are healthy and strong, are inoculated into callus differential medium and are easy to take root after accelerating culture medium, rooting rate is reachable
99% or more, transplanting survival rate reaches as high as 100%, and callus proliferation speed is fast, high-quality；Can be in short 45 days
Complete tuber of pinellia plant can be formed, the proliferation number and seedling quality of half summer sporelings (of laminaria) of chaste tree is ensured, accomplishes scale production, meet production
On needs.It is also not any in relation to report at present using the band leaf petiole of Jing Banxia tissue culture tufted seedling as explant
Road can achieve the effect that of the invention.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of tissue culture method with leaf petiole forming seedling through one step culture using Jing Banxia tissue culture tufted seedling, which is characterized in that specific packet
Include following steps:
The selection of step 1, explant: taking the seed of the tuber of pinellia, after sterile-processed, carry out vernalization and forms Multiple Buds, selection band leaf
Explant of the petiole as tissue culture；
Explant is inoculated in one-step culture base and cultivates to 4~10cm of height of seedling by step 2；The forming seedling through one step culture training
Support the formula of base are as follows: 1/2MS+IBA 0.5~1.2mg/L+NAA, 0.2~0.8mg/L+TDZ, 0.15~0.25mg/L+ sucrose
3%+ agar 0.7%.
2. the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling is utilized as described in claim 1, it is special
Sign is, the final concentration of 1mg/L of the IBA.
3. the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling is utilized as described in claim 1, it is special
Sign is, the final concentration of 0.5mg/L of the NAA.
4. the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling is utilized as described in claim 1, it is special
Sign is, the final concentration of 0.2mg/L of the TDZ.
5. the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling is utilized as described in claim 1, it is special
Sign is, the specific steps sterilized in the step 1 are as follows: potassium permanganate sterilizes 25min, and then ethanol disinfection 5min, finally rises
Mercury sterilizes 10min, aseptic water washing 4-5 times.
6. the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling is utilized as claimed in claim 5, it is special
Sign is that the concentration of the potassium permanganate is 0.1%, and the concentration of alcohol is 75%, and the concentration of mercuric chloride is 0.1%.
7. the tissue culture method with leaf petiole forming seedling through one step culture of Jing Banxia tissue culture tufted seedling is utilized as described in claim 1, it is special
Sign is, in the step 1 the step of vernalization are as follows: on superclean bench, takes sterile petri dish, culture dish is two layers of sterile filter
Paper is soaked with sterile water, and the seed after disinfection is placed on culture dish and is protected from light, vernalization under room temperature.
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Cited By (4)
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| CN112106665A (en) * | 2020-10-28 | 2020-12-22 | 中国科学院昆明植物研究所 | Culture medium for one-step seedling formation of amorphophallus rivieri and application and tissue culture method |
| CN113973701A (en) * | 2021-10-25 | 2022-01-28 | 南京博方生物科技有限公司 | Method for producing pinellia ternata bulbil by water culture method |
| CN115176706A (en) * | 2022-08-04 | 2022-10-14 | 云南中医药大学 | One-step seedling formation efficient breeding method for pinellia ternata leaves |
| CN115380821A (en) * | 2022-07-28 | 2022-11-25 | 甘肃中医药大学 | Rapid propagation method of pinellia ternata artificial seed stems |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112106665A (en) * | 2020-10-28 | 2020-12-22 | 中国科学院昆明植物研究所 | Culture medium for one-step seedling formation of amorphophallus rivieri and application and tissue culture method |
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| CN115380821A (en) * | 2022-07-28 | 2022-11-25 | 甘肃中医药大学 | Rapid propagation method of pinellia ternata artificial seed stems |
| CN115380821B (en) * | 2022-07-28 | 2023-08-11 | 甘肃中医药大学 | Rapid propagation method of pinellia tuber artificial seed stems |
| CN115176706A (en) * | 2022-08-04 | 2022-10-14 | 云南中医药大学 | One-step seedling formation efficient breeding method for pinellia ternata leaves |
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Application publication date: 20190927 |