CN110393147A - The method for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root - Google Patents
The method for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root Download PDFInfo
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- CN110393147A CN110393147A CN201910723876.1A CN201910723876A CN110393147A CN 110393147 A CN110393147 A CN 110393147A CN 201910723876 A CN201910723876 A CN 201910723876A CN 110393147 A CN110393147 A CN 110393147A
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- tobacco
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- mist
- nicotine
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
Abstract
The method for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root, tobacco cloud and mist 87 is mutated two generation seeds and cloud and mist 87 compares seed and distinguishes program request, when cloud and mist 87 compares seed and grows two panels true leaf, cloud and mist 87 is mutated two generation materials and 87 control material seedling of cloud and mist extracts clean ratio to root system, it filters out the mutant material KH65 with more flourishing root system and cultivates transplanting, according to upper after cigarette strain is mature, in, the nicotine content of lower tobacco leaf sampling and measuring single plant KH65, it blooms to KH65 cigarette strain, it is selfed sowing, it is sowed again in second year, the root condition of mutant material is identified again, the nicotine content of single plant Ripening Mutant material is measured by sampling again after cigarette strain is mature, it grows to and blooms to Ripening Mutant cigarette strain, it is selfed sowing, obtain high-nicotine tobacco mutant body.The present invention is by the root system performance of screening tobacco EMS mutant material, and quickly identification obtains the tobacco mutant material of high-nicotine content.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of method of quickly screening high-nicotine tobacco mutant body.
Background technique
Alkaloid is a kind of secondary metabolites mostly important in tobacco.Alkaloid in tobacco mainly includes nicotine
(Nicotine, nicotine), nornicotine (Nornicotine), anabasine (Anatibine) and anabasine
(Anabasine).Nicotine (Nicotine) is mostly important in cultivation tobacco (Nicotiana tabacum) while being also to contain
Measure highest secondary metabolite, nicotine is most economic value in tobacco as a kind of alkaloid known to the public the biggest
Part, the number of content can directly determine quality of tobacco.Alkaloid accounts for about dry in known tobacco breds all at present
The 2%~4% of weight, wherein nicotine can account for the 90%-95% of nicotiana alkaloids total amount.
Nicotine can respond biotic and abiotic stress, be the important means that tobacco resists stress from outside.But nicotine
For too high levels to cigarette strain itself also toxic effect, therefore when not being forced, cigarette strain nicotine content is lower, works as cigarette
Grass is by insect bite, and when germ is infected, cigarette strain can rapidly generate a large amount of nicotine and can provide the growth and development of itself
Protective effect.During production of cigarettes, nicotine content is extremely important to the quality of cigarette, and how much content can determine cigarette
The harmony of chemical component.The too low cigarette of nicotine content does not have enough satisfactions.
With the rise of tobacco industry reducing tar and reducing harm strategy, while tar reduction the nicotine content in cigarette can dropped together
It is low, and this reduction to nicotine content will cause consumer's pleasant feeling deficiency.Therefore raising nicotine appropriate contains in tobacco
Amount is to solve the problems, such as such means.
The nicotine content of tobacco can be improved using the method for topping in agricultural production at present, but this raising is by product
The intrinsic nicotine content limitation of kind.Therefore the kind for cultivating high-nicotine is the preferred embodiment for solving the problems, such as this.
Traditional breeding system is mainly to pass through to find new resource, is then returned main breed with new resources repeatedly
It hands over, final new varieties of the acquisition with purpose character of selfing.But this breeding mode usually requires to take a substantial amount of time, together
Shi Bixu has suitable high-nicotine resource.Conventional breeding means would generally encounter breeding resources and have the chain superfluous of unfavorable character
Remaining, this is by the quality of strong influence tobacco bred.Another problem that tobacco breeding faces simultaneously is exactly the heredity back of tobacco
Scape is narrow, may can not find suitable breeding resources.
The method that the prior art obtains high-nicotine material also by the EMS mutant for screening resistance to jasmonic, but it is led
What is obtained is the mutant material on resistance to jasmonic path, since these mutant materials are all the materials on hormone path, because
This can have many development phenotypes.
Do not allow to plant transgene tobacco at present in view of Chinese, therefore new high-nicotine is formulated using the method for EMS mutagenesis
Kind be effective method.It but is to pass through the measurement nicotine content of one plant of strain of conventional means in character in tobacco in view of nicotine
The problems such as needing to expend a large amount of manpower and material resources, and being likely present measuring error or inaccuracy.Therefore one is chosen
The tobacco that the method for simple possible obtains high-nicotine content is a necessary thing.
Performance Liquid Chromatography Analysis for Nicotine in Tobacco is to generate in tobacco root, therefore healthy and strong root system will promote the synthesis of nicotine.Relatively
In the data of nicotine, root data are easier to obtain, and do not need to put into a large amount of analysis detection cost.Root healthy and strong simultaneously
System is also conducive to the growth and development of cigarette strain.
Summary of the invention
During solving production of cigarettes, the problem of nicotine content deficiency, the object of the present invention is to provide one kind to pass through sieve
The method for selecting tobacco root to be quickly obtained high-nicotine tobacco mutant body intends the root system performance by screening tobacco EMS mutant material,
Quickly identification obtains the tobacco mutant material of high-nicotine content, the workload of high-nicotine mutant screening, section is greatly decreased
It saves time and fund, improves productivity effect.
In order to achieve the above objectives, the invention adopts the following technical scheme:
The method for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root, steps are as follows:
1) tobacco cloud and mist 87 is mutated two generation seeds and the control seed of cloud and mist 87 is put be sowed on floating seedling tray respectively；
2) when growing two panels true leaf to the control seed of cloud and mist 87, cloud and mist 87 is mutated two generation materials and is compareed with cloud and mist 87
Material seedling extracts clean ratio to root condition, filters out the mutant material KH65 with more flourishing root system；
3) the mutant material KH65 single plant that step 2) filters out is transferred in small flower and is cultivated 20 days, then transplant to
Big flowerpot or big Tanaka；
4) it is sampled after cigarette strain is mature, the nicotine content of single plant KH65 is measured by sampling according to upper, middle and lower portion tobacco leaf；
5) sowing is selfed to blooming to KH65 cigarette strain collateral generation；
6) the KH65 cigarette strain seed that harvest obtains is sowed again in second year, by KH65 cigarette strain seed and the control kind of cloud and mist 87
Point is sowed on floating seedling tray respectively；
7) when growing two panels true leaf to the control seed of cloud and mist 87, the root condition of mutant material is identified again,
KH65 material and 87 control material seedling of cloud and mist are extracted into clean ratio to root condition, filter out with more flourishing root system at
Ripe mutant material；
8) the Ripening Mutant material single plant that step 7) filters out is transferred in small flower and is cultivated 20 days, then transplant to
Big flowerpot or big Tanaka；
9) it is sampled after cigarette strain is mature, single plant Ripening Mutant material is measured by sampling according to upper, middle and lower portion tobacco leaf
Nicotine content；
10) it grows to and blooms to Ripening Mutant cigarette strain, be selfed sowing, screening obtains high-nicotine tobacco mutant body.
Above-mentioned steps 2) and step 7), root length is measured using ruler, root system phenotype is observed on negatoscope.
Compared with prior art, the invention has the following advantages:
(1) present invention obtains the healthy and strong tobacco mutant material of root system by directly screening and obtains high-nicotine material, not only may be used
The problem of to avoid that can only obtain mutant material on jasmonic path, can also obtain the more flourishing mutant material of root system simultaneously,
Play the effect killed two birds with one stone.
(2) present invention greatly enhances the efficiency of screening tobacco mutant body, the screening operation of high-nicotine tobacco is alleviated
Amount, improves productivity effect.
(3) present invention measures the nicotine content of cigarette strain upper, middle and lower portion blade simultaneously, and the nicotine result of measurement is more accurate.
(4) the method for the present invention can be used for the high-nicotine mutant material screening of other flue-cured tobacco cultivars or for the high cigarette of tobacco
Alkali breed improvement and genetic research, have a wide range of application.
Detailed description of the invention
Fig. 1 is the root system comparison that screening obtains that tobacco EMS mutant material KH65 seedling compares with cloud and mist 87；
Fig. 2 is that high-nicotine mutant KH65 compares upper middle lower part blade nicotine content with cloud and mist 87；
Fig. 3 is the root system comparison diagram that mature EMS mutant material KH65 cigarette strain is compareed with cloud and mist 87；
Fig. 4 is that EMS mutant material KH65 cigarette strain and 87 cigarette strain surface area of root systern of cloud and mist compare histogram；
Fig. 5 is EMS mutant material KH65 cigarette strain and 87 cigarette strain growing way comparison diagram of cloud and mist.
Specific embodiment
The present invention is described further with reference to the accompanying drawings and examples.
The method for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root, steps are as follows:
1. program request mutant seeds
2000 parts of mutation two generations (M2) seeds are taken to carry out the screening of high-nicotine flue-cured tobacco material from 87 mutant library of tobacco cloud and mist,
Every part of seed takes 5, while choosing cloud and mist 87 and compareing seed, and program request is cultivated in floating seedling tray respectively.
2. mutant material bodies root system screens
It is when growing two panels true leaf to 87 control material of cloud and mist, EMS mutant material and 87 control material of cloud and mist is young
It is thin to extract, root length is then measured using ruler, root system phenotype, comparison mutant material and cloud and mist 87 are observed on negatoscope
Root condition is shown in Fig. 1.A mutant material KH65 is identified in 2000 parts of materials, compared with check variety cloud and mist 87, tool
There is more flourishing root system.
3. continuing to cultivate
The KH65 of acquisition and 87 control material of cloud and mist are transplanted in small flower and continue culture 20 days.Then it will grow up
KH65 and the transplanting of cloud and mist 87 to big flowerpot or big Tanaka are cultivated.
4. nicotine content measures
About 60 days after transplanting mature cigarette strains are sampled, are taken respectively according to upper, middle and lower portion tobacco leaf in identical leaf position
Then the blade of KH65 and cloud and mist 87 carry out nicotine content measurement.Tobacco-containing material in establishing criteria YC/T 160-2002 detection
Nicotine content.As a result see Fig. 2, show nicotine content in the upper middle lower part tobacco leaf in KH65 mutant material improve 30% with
On.
Nicotine content measuring method is as follows:
Total alkaloid (in terms of nicotine) and p-aminophenyl sulphur with 5% acetic acid aqueous solution extracted tobacco sample, in extract liquor
Acid and cyanogen chloride reaction, cyanogen chloride react generation by potassium cyanide and toluene-sodium-sulfonchloramide online.Reaction product colorimeter is measured in 460nm.
Key instrument equipment: Continuous Flow Analysis instrument (U.S. API) (German SEALAA3) (French ALLIANCE).
Configure reagent:
35 solution of Brij (polyethoxy laurel ether): in water plus 5 drip 22%Brij35, stir evenly.
Buffer solution A: weighing 2.35g sodium chloride (NaCl), 7.60g Boratex (Na2B4O3·10H2O), dissolved with water,
Then it is transferred in 1L volumetric flask, 1mL Brij 35 is added, is diluted to 1L with distilled water.It is filtered using preceding with qualitative filter paper.
Buffer solution B: claim 26g disodium hydrogen phosphate (Na2HPO4), 10.4g citric acid [COH (COOH) (CH2COOH)2·
H2O], 7g p-aminobenzene sulfonic acid (NH2C6H4SO3H), dissolved, be then transferred in 1L volumetric flask with water, 1mL Brij 35 is added, used
Distilled water is diluted to 1L.It is filtered using preceding with qualitative filter paper.
Toluene-sodium-sulfonchloramide solution (the chloro- 4- methyl benzenesulfonamide sodium salt of N-) [CH3C6H4SO2N(Na)Cl·3H2O]: take 8.65g chloramines
T, it is soluble in water, it is then transferred in the volumetric flask of 500mL, is settled to scale with water.It is filtered using preceding with qualitative filter paper.
0.22mol/L NaOH buffer: NaOH 8.8g, Na2HPO426.0g C6H8O7·H2O (Citric Acid Mono)
10.4g is dissolved with water and is settled to 1000mL.
P-aminobenzene sulfonic acid buffer: C is weighed6H7NO3S (p-aminobenzene sulfonic acid) 7g, Na2HPO426.0g C6H8O7·H2O
(Citric Acid Mono) 10.4g, is dissolved with water and is settled to 1000mL.
Toluene-sodium-sulfonchloramide: weighing toluene-sodium-sulfonchloramide 1.2g, is settled to 100mL with pure water dissolution, is saved with brown reagent bottle.
Potassium cyanide: KCN 0.4g is settled to 100mL with pure water dissolution.
Na2CO3Solution: 10gNa2CO3, distilled water dissolves and is settled to 1000mL.
Analytical procedure: it weighs 0.3g cigarette sample and (is accurate to 0.0001g) in 150mL triangular flask or plastic bottle；50mL is added
5% acetic acid solution covers plug；The oscillation extraction 30min on common shaking table, revolving speed control 170r/min, are filtered with filter paper
Machine.(concentration range that such as sample liquid concentration exceeds working stamndard liquid, then should dilute)
As a result calculating and statement:
The content of total alkaloid in terms of butt, is obtained by following formula:
In formula:
The Instrument observation value of C --- sample liquid total alkaloid, unit mg/mL；
The volume of V --- extract liquor, unit mL；
The quality of m --- sample, unit mg；
The moisture content of W --- sample, unit %.
As a result average value to measure twice is accurate to 0.01% as measurement result.
5. being selfed sowing
To KH65 cigarette strain collateral generation to blooming, be selfed bagging, reserve seed for planting.
6. sowing again
It by the cigarette strain seed of the KH65 of harvest upper one year, sows again, by KH65 cigarette strain seed and the control kind of cloud and mist 87
Point is sowed on floating seedling tray respectively；
7. identifying again
When growing two panels true leaf to the control seed of cloud and mist 87, the root condition of mutant material is identified again, it will
KH65 material and 87 control material seedling of cloud and mist extract clean ratio and (measure root length to root condition using ruler, seeing piece
Root system phenotype is observed on lamp), filter out the Ripening Mutant material with more flourishing root system.It is mature EMS prominent shown in Fig. 3
Become the root system comparison diagram that material KH65 cigarette strain is compareed with cloud and mist 87, Fig. 4 is mature EMS mutant material KH65 cigarette strain and cloud and mist 87
Cigarette strain surface area of root systern compares histogram, by Fig. 3, Fig. 4 as it can be seen that the root system of mutant material KH65 is obviously than control cloud and mist 87
Well developed root system.By root Area computing, the surface area of root systern of KH65 mutant is about 3 times of cloud and mist 87.
8. cultivating again
The Ripening Mutant material single plant filtered out is transferred in small flower and is cultivated 20 days, then transplant to big flowerpot or
The big Tanaka of person.KH65 mutant plants and 87 control material of cloud and mist are planted into cylindrical glass bottle simultaneously, compare two plant
Growing way, Fig. 5 is EMS mutant material KH65 cigarette strain and 87 cigarette strain growing way comparison diagram of cloud and mist, it can be found that 87 plant of cloud and mist is long
It is higher, but internode is away from larger, and then growing way is more healthy and strong for KH65 mutant plants.
9. sample detection again
It is sampled after cigarette strain is mature, the cigarette of single plant Ripening Mutant material is measured by sampling according to upper, middle and lower portion tobacco leaf
Alkali content；
10. obtaining high-nicotine tobacco mutant body
To Ripening Mutant cigarette strain collateral generation to blooming, it is selfed sowing, screening obtains high-nicotine tobacco mutant body.
The method of the present invention successfully obtains well developed root system and the higher tobacco of nicotine content by the screening to mutant
Mutant material.
Claims (2)
1. the method for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root, which is characterized in that steps are as follows:
1) tobacco cloud and mist 87 is mutated two generation seeds and the control seed of cloud and mist 87 is put be sowed on floating seedling tray respectively；
2) when growing two panels true leaf to the control seed of cloud and mist 87, cloud and mist 87 is mutated two generation materials and 87 control material of cloud and mist
Seedling extracts clean ratio to root condition, filters out the mutant material KH65 with more flourishing root system；
3) the mutant material KH65 single plant that step 2) filters out is transferred in small flower and is cultivated 20 days, then transplanted to great Hua
Basin or big Tanaka；
4) it is sampled after cigarette strain is mature, the nicotine content of single plant KH65 is measured by sampling according to upper, middle and lower portion tobacco leaf；
5) it grows to and blooms to KH65 cigarette strain, be selfed sowing；
6) the KH65 cigarette strain seed that harvest obtains is sowed again in second year, and KH65 cigarette strain seed and cloud and mist 87 are compareed seed point
Other point is sowed on floating seedling tray；
7) when growing two panels true leaf to the control seed of cloud and mist 87, the root condition of mutant material is identified again, it will
KH65 material and 87 control material seedling of cloud and mist extract clean ratio to root condition, filter out the maturation with more flourishing root system
Mutant material；
8) the Ripening Mutant material single plant that step 7) filters out is transferred in small flower and is cultivated 20 days, then transplanted to great Hua
Basin or big Tanaka；
9) it is sampled after cigarette strain is mature, the nicotine of single plant Ripening Mutant material is measured by sampling according to upper, middle and lower portion tobacco leaf
Content；
10) it grows to and blooms to Ripening Mutant cigarette strain, be selfed sowing, screening obtains high-nicotine tobacco mutant body.
2. the method according to claim 1 for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root, special
Sign is, above-mentioned steps 2) and step 7), root length is measured using ruler, root system phenotype is observed on negatoscope.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112335543A (en) * | 2020-11-13 | 2021-02-09 | 云南省烟草农业科学研究院 | Method for obtaining developed root system tobacco mutant by screening EMS mutagenesis group |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104604672A (en) * | 2015-01-12 | 2015-05-13 | 中国农业科学院烟草研究所 | Method for rapidly screening high-nicotine tobacco mutants |
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2019
- 2019-08-07 CN CN201910723876.1A patent/CN110393147A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104604672A (en) * | 2015-01-12 | 2015-05-13 | 中国农业科学院烟草研究所 | Method for rapidly screening high-nicotine tobacco mutants |
Non-Patent Citations (3)
| Title |
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| 烤烟科研组: "烤烟营养器官与产质关系的研究", 《贵州农学院学报》 * |
| 马新明等: "烟草根系生长发育与地上部相关性的研究", 《中国烟草学报》 * |
| 高迟銮等: "切根对烤烟根系生长及化学成分的影响", 《河南农业大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112335543A (en) * | 2020-11-13 | 2021-02-09 | 云南省烟草农业科学研究院 | Method for obtaining developed root system tobacco mutant by screening EMS mutagenesis group |
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Application publication date: 20191101 |