CN104604672A - Method for rapidly screening high-nicotine tobacco mutants - Google Patents
Method for rapidly screening high-nicotine tobacco mutants Download PDFInfo
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Abstract
The invention discloses a method for rapidly screening high-nicotine tobacco mutants, and belongs to the biotechnical field. The method comprises the following steps: 1) dibbling tobacco M2-generation seeds onto an aqueous methyl jasmonate solution; 2) 15 days later, screening single plants of which root lengths are significantly suppressed; 3) transferring the single plants screened in the step 2) to an MS culture solution, and culturing for 20 days; 4) transferring the tobacco plants in the step 3) to a provisonal planting plate containing nutrient soil, and 30 days later, transplanting to a field; 5) performing field planting and routine management; 6) determinating nicotine contents at three different time points; 7) selfing and harvesting seeds. According to the method, within 20 days, high-nicotine materials are screened from 4000 China tobacco-100 mutants, and 5 high-nicotine flue-cured tobacco materials are obtained; the highest nicotine content reaches 7.09%, which is the highest nicotine content for current flue-cured tobacco; the efficiency for screening the tobacco mutants is greatly improved, the workload for screening the high-nicotine tobacco is reduced, and the production efficiency is improved.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method of rapid screening high-nicotine tobacco mutant body.
Background technology
Nicotine is the main secondary metabolites of one of tobacco, the main hobby composition in cigarette product, in tobacco seed containing nicotine, tobacco seedling and before pinching nicotine content all very low, after pinching, nicotine content sharply rises, in flue-cured tobacco type, nicotine content generally accounts for 2 ~ 4% of dry weight.
Flue-cured tobacco is the primary raw material of cigarette product, and in recent years, along with tobacco " lowering harm and decreasing coking " implementation, releasing content of coke tar and the smoke hazard index of domestic cigarette progressively reduce, but meanwhile, " fragrance, strength, the concentration " of cigarette also declines to some extent.In order to compensate the deficiency reducing cigarette " fragrance, strength, concentration " after tar, usually mixing in cigarette and joining airing cigarette.But, mix and join airing cigarette and cause again more coarse, the assorted gas of cigarette smoke and excitant is comparatively large, pleasant impression owes totally comfortable, more seriously TSNAs content can be caused to increase, smoke hazard index rising (Yan Keliang, Zeng Xiaoying, Hu Weiyao, Gao Rui, Xu Anchuan, Tang Li. the application of airing tobacco module in Virginian-type cigarette. Chinese tobacco journal, 2012,1:19-25), therefore tobacco enterprise is in the urgent need to improving the nicotine content of flue-cured tobacco, to keep fragrance, strength, the concentration of cigarette under the prerequisite of low tar.In addition, some the novel tobacco goods occurred in the market, as low temperature cigarette, because ignition temperature is low, nicotine release is not enough, also flue-cured tobacco is had to the demand of high-nicotine content.
Although some cultivation steps, as planting density, fertilization type and proportioning, pinch and smear wooden fork period and leaves remained, maturity and bake process temp. and humidity all affect the final nicotine content of flue-cured tobacco, but the cultivation modulator approach that nicotine content increases can be made, " high-nicotine " means " low-quality " usually, as high in protein content, aroma quality is poor, flue gas is coarse, assorted gas is heavy, excitant is large.In generation nineteen ninety, China's cured tobacco production is excessively emphasized " large loose water ", causes tobacco leaf nicotine, protein content too high, have impact on availability.Therefore, need to utilize animal nutrition, start with from inherent hereditary capacity, formulate new high-nicotine kind matter.
Heritable variation can be produced to the mutagenesis of seed physics and chemistry, from Variants, then select have the germ plasm resource of value to be a breeding methods with application prospect according to demand.But what generally produce due to mutagenesis is all unfavorable proterties, the probability producing merit mutant is very little, therefore from mutagenic progeny screening to meet the material workload of target needs very large.At present, the quick choosing method of high-nicotine mutant material is not also set up, by field period directly measure each tobacco individual plant pinch after nicotine content determine its Nicotine levels, this method needs screened material all to plant, because tobacco plant is tall and big, the mutagenized populations (every part of material needs 25 individual plants) screening one 5000 parts needs the plantation of nearly 70 mu of soils, huge material colony need through sowing, heel in, land for growing field crops Normal culture technique management, to pinch processes such as touching wooden fork, expend very large, and each single-strain blade gather after measure nicotine content also unusual time-intensive, expensive, screening effeciency is very low.
Jasmonates compound comprises jasmonic, methyl jasmonate, is a kind of plant growth regulating substance be extensively present in higher plant body, is also referred to as and hinders inducing hormone, namely plant injured after create a kind of hormone.There are some researches show, after tobbaco, the sharply rising of nicotine content is exactly owing to can produce jasmonate (Pluskota1WE after tobbaco, Qu N, Maitrejean M, Boland W, Baldwin IT.Jasmonates and its mimics differentiallyelicit systemic defence responses in Nicotiana attenuate.Journal of ExperimentalBotany.2007, 58:4071-4082), and to executing methyl jasmonate outside tobacco or carrying out blade injury, key enzyme expression in high-nicotine tobacco bred of nicotine synthesis strengthens, not quite (Dewey R E is then changed at low tobacco bred, Xie J.Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum.Phytochemistry, 2013, 94:10-27, Cane KA, Mayer M, Lidgett AJ, Michael AJ, HamillJD.Molecular analysis of alkaloid metabolism in AABB v.aabb genotype Nicotianatabacum in response to wounding of aerial tissues and methy jasmonate treatmentof cultured roots.Functional Plant Biology.2005,32:305-320), therefore, the material of high-nicotine can be filtered out according to the sensitivity of tobacco to jasmonate.
Summary of the invention
For solving the problem, the invention provides a kind of method of rapid screening high-nicotine tobacco mutant body, high-nicotine material can be obtained fast, significantly reduce the workload of high-nicotine screening mutant, save time and fund, improve productivity effect.
For achieving the above object, the present invention by the following technical solutions:
1) by tobacco M2 for seed program request on the methyl jasmonate aqueous solution;
2) the long remarkable downtrod individual plant of root within 15 days, is screened afterwards;
3) by step 2) cultivate 20 days in the individual plant transfer liquid medium that filters out;
4) by step 3) cigarette strain transfer to and heel in dish containing Nutrition Soil, transplanted land for growing field crops after 30 days;
5) field planting, fertilising;
6) three point in time sampling are divided to measure the nicotine content of individual plant;
7) selfing sowing.
Described step 1) in, methyl jasmonate is commercially available, and the anhydrous alcohol solution with 50% becomes the mother liquor of 100mM/mL, and during use, in the distilled water of 1L sterilizing, add 100 μ L methyl jasmonate mother liquors is 10 μMs/L to final concentration.Sponge thick for 0.2cm is cut into the circle of diameter 9cm, being layered on diameter is in the culture dish of 9cm, sponge is placed onesize filter paper, 121 DEG C of high-temperature sterilizations 20 minutes, 10mL methyl jasmonate solution is poured in culture dish, 30, the even program request seed of each culture dish, has put rear preservative film and has sealed to avoid moisture to evaporate to culture dish.Condition of culture is, temperature 25 DEG C cultivation, and the photoperiod is 16 h light/8 h dark.
Described step 2) in, measure root with common ruler long, select the long remarkable downtrod individual plant of root.
Described step 3) in, liquid nutrient medium is the medium containing MS nutrient solution, and MS is that market is commercially available, takes 4.74gMS powder and joins in 1L distilled water, and 121 DEG C of high-temperature sterilization 20min after heating for dissolving, to avoid bacteria breed.
Described step 5) in, start to pinch when cigarette strain is buddingged, beat 2 ~ 3 tops and bud that do not hack, after pinching, touch weekly wooden fork once.
Described step 6) in, three time points are respectively, when buddingging, pinch latter 15 days, pinch latter 45 days.Get middle leaf (from bottom leaf number the 9th leaf) when buddingging and measure nicotine content, pinch and within latter 15 days, get upper leaf (top first leaf), pinch and within latter 45 days, get middle leaf (from bottom leaf number the 10th leaf), blade toasts 1 day through 60 DEG C after fetching, remove cigarette muscle, clay into power with sample milling machine, cross 600 mesh sieves, take 1g powder, measure nicotine content, nicotine content assay method adopts ultraviolet spectrophotometry to carry out, method is with reference to the carrying out (Li Weili of Li Weili etc., Ma Yinhai, Duan Yaojun, Xu Jicang. the free nicotine in determined by ultraviolet spectrophotometry tobacco. Agriculture of Anhui science, 2012, 40:3600-3601).Each sample in triplicate.
Compared with prior art, the present invention has following beneficial effect:
1. the present invention completed the high-nicotine material of 4000 parts of Zhongyan-100 mutant in 20 days, and obtain 5 parts of high-nicotine flue-cured tobacco materials, the highest nicotine content reaches 7.09%, for the nicotine content that current Virginia tobacco is the highest, the present invention greatly improves the efficiency of screening tobacco mutant body, alleviate the screening operation amount of high-nicotine tobacco, improve productivity effect.
2. the screening of the high-nicotine material in the present invention can be carried out in culture dish, easy to operate, saves time and expense.
3. nicotine content of the present invention measure respectively before pinching (when buddingging), pinch latter 15 days, pinch and carried out 3 sub-sampling replications in latter 45 days, ensure the accuracy of high-nicotine screening in early stage.
4. the present invention also may be used for other flue-cured tobacco cultivars high-nicotine mutant material screening or for the breed improvement of tobacco high-nicotine and genetic research.
Accompanying drawing explanation
Fig. 1 is the 11MZE273654-3 and the root long comparison diagram of control material (Zhongyan-100) on clear water and 10 μMs of methyl jasmonates that filter out;
Fig. 2 a is the nicotine content block diagram before high-nicotine mutant is pinched;
Fig. 2 b is the nicotine content block diagram that high-nicotine mutant is pinched latter 15 days;
Fig. 2 c is the nicotine content block diagram that high-nicotine mutant is pinched latter 45 days.
In Fig. 1, a is that the root of Zhongyan-100 on clear water is long; B is that the root of 11MZE273654-3 on clear water is long; C is that Zhongyan-100 root on 10 μMs of methyl jasmonates is long; D is that 11MZE273654-3 root on 10 μMs of methyl jasmonates is long.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described further.
1. program request mutant seeds
From tobacco mutant body storehouse, get 4000 parts of sudden change two generations (M2) seeds carry out high-nicotine flue-cured tobacco material screening, every part of seed gets 30, and program request is cultivated in containing the culture dish of 10 μMs of methyl jasmonates, does clear water contrast simultaneously.In addition, except program request mutant, every day again the Zhongyan-100 seed that do not suddenly change of program request 3 parts as parallel control.
2. methyl jasmonate sensitive mutant screening
Tobacco seed program request is in culture dish, 5 days start to show money or valuables one carries unintentionally, within 7 days, root starts growth, mutant and Zhongyan-100 (contrast) is measured respectively long containing the root on 10 μMs of methyl jasmonates and clear water when 15 days, as shown in table 1, in table 1, the root length of mutant in clear water contrast is the mean value of 30 individual plants, and Zhongyan-100 is the mean value of 90 individual plants.
The root measurement data of table 1 mutant and Zhongyan-100
As shown in Figure 1, because the growth of methyl jasmonate to root has inhibitory action, after the methyl jasmonate of interpolation 10 μMs cultivates 15 days, the root of Zhongyan-100 is long average between 2.11cm-2.24cm, but be numbered 11MZE273654, 11MZE274507, there are 5 individual plants more responsive to the performance of methyl jasmonate in the single strain of 11MZE275288, its root is long only has about 1cm, select 11MZE273654-3, 11MZE273654-12, 11MZE274507-27, 11MZE275288-5, 11MZE275288-25, 11MZE273654-3 and the root of control material (Zhongyan-100) on clear water and 10 μMs of methyl jasmonates long.
3. continue to cultivate
Above individual plant and Zhongyan-100 contrast are transferred in the culture dish containing 10mL MS nutrient solution and continue to cultivate, after 20 days, tobacco grew into for four leaf phases, and growth is comparatively healthy and strong.
4. heel in
Mutant and contrast cigarette strain are carefully transferred to and heels in dish containing Nutrition Soil, after 30 days, transplanted land for growing field crops.
5. field planting, fertilising
Test is carried out on Mao Zhuan farm, Zhucheng, spacing in the rows 0.5 meter, line-spacing 1.2 meters, often row 25 strain, test the consumption fertilising executing pure N 7kg (mass ratio of N, P, K is 1:1:1) according to every mu, wherein base manure respectively accounts for 50% with topdressing, and the 30d after transplanting that topdresses uses, and other control measures are carried out according to land for growing field crops conventional cultivation.
6. nicotine content measures
As shown in Figure 2 a, transplant latter 60 days tobaccos to budding, get middle part blade to dry, measure nicotine content, can find out that the nicotine content of blade in the middle part of Zhongyan-100 when not pinching is being 0.42%, and 11MZE273654-3,11MZE273654-12,11MZE274507-27,11MZE275288-5,11MZE275288-25 individual plant nicotine content filtered out by methyl jasmonate is 1.09% ~ 1.35%, higher than contrast 1 ~ 2 times.
Pinch after buddingging, after 15 days, Zhongyan-100 and these individual plant nicotine contents filtered out all obviously raise, and as shown in Figure 2 b, this creates owing to pinching to hinder inducing hormone-jasmonic, jasmonic can stimulate the nicotine of tobacco root to synthesize, as can be seen from Fig. 2 b, at this moment the nicotine content of Zhongyan-100 adds nearly 3 times, reaches 2.05%, and the nicotine content of several individual plants of our screening also sharply rises, the nicotine content 4.84% of 11MZE273654-12.
As shown in Figure 2 c, continue conventional field management, pinch 45 days time measure middle part nicotine content of tobacco leaves, the nicotine content of Zhongyan-100 is 3.81%, and the nicotine content of the several materials filtered out the highest reach 7.09%, the nicotine content of other several individual plants is also high more than 0.5 ~ 1 times than contrast.
Measure nicotine content by three different times, the nicotine content that the high-nicotine mutant material selected by screen for seeds is shown is significantly higher than contrast, shows that this individual plant is high-nicotine mutant material.
7. selfing sowing
No longer touch wooden fork after 45 days, treat collateral generation to blooming, selfing bagging, to reserve seed for planting.
Claims (10)
1. a method for rapid screening high-nicotine tobacco mutant body, is characterized in that: comprise the following steps:
(1) by tobacco M2 for seed program request on the methyl jasmonate aqueous solution;
Within (2) 15 days, screen the long remarkable downtrod individual plant of root afterwards;
(3) individual plant that step (2) filters out is moved on in liquid nutrient medium and cultivate 20 days;
(4) the cigarette strain of step (3) is moved on to and heel in dish containing Nutrition Soil, after 30 days, transplanted land for growing field crops;
(5) field planting, fertilising;
(6) nicotine content is measured three different time sections;
(7) selfing sowing.
2. the method for rapid screening high-nicotine tobacco mutant body as claimed in claim 1, it is characterized in that: in described step (1), the methyl jasmonate concentration used is 10 μMs, and the methyl jasmonate aqueous solution of interpolation is 10mL.
3. the method for rapid screening high-nicotine tobacco mutant body as claimed in claim 1, is characterized in that: in described step (1), and culture dish, sponge and filter paper are all through 121 DEG C of high-temperature sterilization process 20 minutes, and sealing avoids moisture to evaporate.
4. the method for rapid screening high-nicotine tobacco mutant body as claimed in claim 1, is characterized in that: in described step (3), containing MS nutrient solution in described liquid nutrient medium.
5. the method for rapid screening high-nicotine tobacco mutant body as claimed in claim 1, is characterized in that: in described step (3), do not add sucrose in liquid nutrient medium, and carries out 121 DEG C of high-temperature sterilization process 20min to described liquid nutrient medium.
6. the method for the rapid screening high-nicotine tobacco mutant body as described in right 1, is characterized in that: in described step (5), and field planting is according to spacing in the rows 0.5 meter, and line-spacing 1.2 meters, often row 25 strain is carried out.
7. the method for the rapid screening high-nicotine tobacco mutant body as described in right 1, is characterized in that: in the fertilizer adopted in described step (5), the mass ratio of N, P, K is 1:1:1.
8. the method for the rapid screening high-nicotine tobacco mutant body as described in right 1, is characterized in that: in described step (5), fertilising adopts base manure and topdresses, and base manure respectively accounts for 50% with topdressing, and the 30d after transplanting that topdresses uses.
9. the method for the rapid screening high-nicotine tobacco mutant body as described in right 1, is characterized in that: in described step (6), and three time periods are when buddingging respectively, pinch latter 15 days and pinch latter 45 days.
10. the method for the rapid screening high-nicotine tobacco mutant body as described in right 1, is characterized in that: in described step (7), and selfing sowing is no longer touch wooden fork after 45 days, treat collateral generation to blooming, selfing bagging, to reserve seed for planting.
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Cited By (7)
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| CN106879458A (en) * | 2015-12-16 | 2017-06-23 | 中国农业科学院烟草研究所 | A kind of method of EMS mutagenesis tobacco seed |
| CN107981413A (en) * | 2018-01-02 | 2018-05-04 | 四川三联新材料有限公司 | A kind of flavouring for being used to heat the cigarette that do not burn mends fragrant blank pipe material and preparation method thereof |
| CN107981412A (en) * | 2018-01-02 | 2018-05-04 | 四川三联新材料有限公司 | A kind of flavouring for being used to heat the cigarette that do not burn mends fragrant gel phase-change material and preparation method thereof |
| CN108175126A (en) * | 2018-01-02 | 2018-06-19 | 四川三联新材料有限公司 | A kind of perfume material for the fragrant section of cigarette production of not burning for the heating of cigar style and preparation method thereof |
| CN108175127A (en) * | 2018-01-02 | 2018-06-19 | 四川三联新材料有限公司 | A kind of mode of heating smoking material and preparation method thereof |
| CN110393147A (en) * | 2019-08-07 | 2019-11-01 | 云南省烟草农业科学研究院 | The method for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root |
| CN111448983A (en) * | 2020-01-21 | 2020-07-28 | 河南中烟工业有限责任公司 | Method for screening high-nicotine tobacco variety mutant |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106879458A (en) * | 2015-12-16 | 2017-06-23 | 中国农业科学院烟草研究所 | A kind of method of EMS mutagenesis tobacco seed |
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| CN108175126A (en) * | 2018-01-02 | 2018-06-19 | 四川三联新材料有限公司 | A kind of perfume material for the fragrant section of cigarette production of not burning for the heating of cigar style and preparation method thereof |
| CN108175127A (en) * | 2018-01-02 | 2018-06-19 | 四川三联新材料有限公司 | A kind of mode of heating smoking material and preparation method thereof |
| CN110393147A (en) * | 2019-08-07 | 2019-11-01 | 云南省烟草农业科学研究院 | The method for being quickly obtained high-nicotine tobacco mutant body by screening tobacco root |
| CN111448983A (en) * | 2020-01-21 | 2020-07-28 | 河南中烟工业有限责任公司 | Method for screening high-nicotine tobacco variety mutant |
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