CN102787139A - Breeding method capable of improving content of potassium in tobacco leaves - Google Patents

Abstract

The invention provides a breeding method capable of improving the content of potassium in tobacco leaves. According to the invention, total DNA of the high potassium plant pokeweed is introduced into Honghuadajinyuan, a cultivated variety of tobacco, by using a pollen tube pathway method, which results in substantial increase of the content of potassium in later-generation tobacco leaves of Honghuadajinyuan, and therefore, a rich source of variation is provided for breeding and improvement of new varieties of tobacco and innovation of germplasm resources.

Description

A kind of breeding method that improves tobacco tobacco leaf potassium content

Technical field

The present invention relates to a kind of breeding method that improves tobacco tobacco leaf potassium content.

Background technology

According to the great mass of data statistics, potassium is flue-cured tobacco absorbed dose and the highest nutritive element of in-vivo content, also is to influence the critical limitation factor that China's flue cured tobacco quality further improves.And the most of tobacco-growing soils of China can not provide competent potassium to supply with flue-cured tobacco to satisfy the demand that sound tobacco is produced.Therefore, need to drop into a large amount of potash fertilizer in the production.Use chemical fertilizer year after year in a large number, not only increase tobacco grower's production cost, and quicken the deterioration of soil physico-chemical property.Regrettably the average potassium content of China's sound tobacco is still lower, compares at least also low more than 20% (China's sound tobacco about 2%, external sound tobacco is big by 2.5%) with external sound tobacco.Influencing in the flue-cured tobacco body factor of potassium content has manyly, mainly contains: soil and weather; Cultivation step; Kind or genotype etc.Soil and weather are that nature forms, human difficult the change.Cultivation step improves the tobacco leaf potassium content, facts have proved certain limitation for many years, in addition it is also inadvisable to increase the potash fertilizer consumption again.Therefore, the high potassium content new flue-cured tobacco varieties of seed selection will be one of critical path that solves the flue-cured tobacco potassium content.

Yet there are not problems such as affinity, hybrid proterties instability, segregating generation length of distant hybirdization in conventional breeding; Hybridize not affinity phenomenon though somatocyte hybriding technology has overcome to a certain extent, have problems such as regeneration frequency is low, the variation progeny population is little, somaclonal variation, hereditary instability; Can absorb from Hess proposition pollen granule after the research report of foreign DNA; Total DNA leading-in technique is used widely in crop breeding; It has gone beyond the dysgenesia in the conventional breeding; Remedied that cell engineering is loaded down with trivial details, plant regeneration frequency is low, progeny population is little; Be difficult to defectives such as screening, in that at present tobacco genetic background is still clear, the excellent genes source is limited, transgenic research still receives under the situation of trade barrier negative impact the shortcut of the practicable breed improvement of can yet be regarded as.

Utilization has the direct transformation receptor of donor DNA of plants (comprising edge donor dna far away) of goal gene, screening purpose character variation offspring, and the method for cultivating new crop varieties is the practical application on agricultural breeding of plant genetic engineering and molecular biotechnology.Pollen tube passage method is to utilize the pollen tube channel that forms in the flowering of plant fertilization process, and foreign DNA is imported fertilization cell.In recent years, China succeeds on various crop such as cotton, soybean, paddy rice, wheat, tobacco through this technology.

Summary of the invention

The objective of the invention is the deficiency to prior art, a kind of pollen tube passage method is provided, the total genomic dna of high potassium plant Phytolacca acinosa is imported in the big gold dollar of flue-cured tobacco cultivars safflower, in the hope of obtaining the method for high potassium content flue-cured tobacco.

Technical problem to be solved by this invention is to realize through following technical scheme, and its step is following:

A kind of breeding method that improves tobacco tobacco leaf potassium content: utilize the big gold dollar of overall dna importing tobacco bred safflower (Central-South testing station buys from Chinese tobacco) of pollen tube passage method with high potassium plant Phytolacca acinosa and cultivate, specifically may further comprise the steps:

(1) 100 μ l 5M NaCl are newly managed → added to supernatant to the extraction of donor dna: get the freezing blade of the tender tobacco of 0.2g children and go in the inkstone alms bowl → add to change over to after 2 * CTAB extracting solution, 700 μ l grind the 1.5ml centrifuge tube → add isopyknic chloroform in 65 ℃ of insulation 20-30min → cooling back: the centrifugal 10min of primary isoamyl alcohol mixing 10min → 10000r/min → get and the ice-cold ethanol sedimentation DNA of 1ml → choosing DNA precipitates, with 70% ethanol clean dry → be dissolved among the 100 μ lTE; Described 2 * CTAB extract recipe is: 2%CTAB, 1.4M NaCl, 100mM Tris-HCl, 100mM EDTA, pH8.0,2% beta-mercaptoethanol; Described chloroform: the volume ratio of primary isoamyl alcohol is 24: 1;

(2) introduction method: select the open flower in robust growth, stem middle and upper part; Its corolla is taken off; Expose column cap, make chopping block with the left hand thumbnail then and hold column cap, the right hand takes the blade edge to become 45 ℃ of clipped corner 1/4～1/3 column caps with column cap; At last with the DNA extraction drop on column cap, again corolla is put on; 6:00 spent and just decontroled the beginning importing time from morning, and each hour imports once then, and 4:00 spends wilting ending to afternoon.

In the breeding method of described raising tobacco tobacco leaf potassium content, the overall dna of high potassium plant Phytolacca acinosa imported tobacco bred after, screen with tobacco tobacco leaf potassium content 2% the standard of being not less than again.

The tobacco leaf potassium content is measured: during the ripening stage, the whole strain results of representational plant 6 strains are chosen in each processing in the cigarette strain, and the 30min that under 105 ℃, completes after cleaning is then 80 ℃ of oven dry down; Crushing screening, the sample 0.1g that 0.45mm is sieved places the 150mL triangular flask, adds 5% acetate 100mL; Soft rubber ball beyond the Great Wall, oscillation extraction 30min filters with qualitative filter paper on shaking table or vibrator; Discard former milliliters of filtratings, collect the back subsequent filtrate, use the concentration of atomic absorption spectrometry potassium at last.

Positively effect of the present invention is: first with tobacco as acceptor, the total genomic dna of high potassium plant Phytolacca acinosa is imported in the big gold dollar of flue-cured tobacco cultivars safflower, obtained high potassium content flue-cured tobacco; To satisfy the demand that sound tobacco is produced.Use pollen tube channel and import the shape variation that external source plant total genomic dna can cause tobacco receptor; Through screening; Can obtain the kind that the each side shape does very well, and owing to be on autogamous basis, part dna fragmentation or goal gene get into the acceptor gene group; Avoided comprehensive reorganization of donor and acceptor gene, thereby be easy to stablize; Simultaneously, utilize the breeding method of pollen tube channel with plant total genomic dna importing tobacco, simple to operation, also be to solve tobacco source far away crossbreeding and improvement kind at present than one of effective means.

Description of drawings

Fig. 1 measures the mRNA transcriptional level of relevant high potassium gene in high potassium new lines of different tobaccos and the contrast for fluorescence quantitative PCR method.

Embodiment

Following examples are intended to further specify the present invention, and unrestricted the present invention.

Embodiment 1: the inventive method is implemented the performance of the main proterties of back tobacco offspring

Carrying out DNA according to following method imports:

(1) 100 μ l5M NaCl are newly managed → added to supernatant to the extraction of donor dna: get the freezing blade of the tender tobacco of 0.2g children and go in the inkstone alms bowl → add to change over to after 2 * CTAB extracting solution, 700 μ l grind the 1.5ml centrifuge tube → add isopyknic chloroform in 65 ℃ of insulation 20-30min → cooling back: the centrifugal 10min of primary isoamyl alcohol mixing 10min → 10000r/min → get and the ice-cold ethanol sedimentation DNA of 1ml → choosing DNA precipitates, with 70% ethanol clean dry → be dissolved among the 100 μ lTE; Described 2 * CTAB extract recipe is: 2%CTAB, 1.4M NaCl, 100mM Tris-HCl, 100mM EDTA, pH8.0,2% beta-mercaptoethanol; Described chloroform: the volume ratio of primary isoamyl alcohol is 24: 1;

(2) introduction method: select the open flower in robust growth, stem middle and upper part; Its corolla is taken off; Expose column cap, make chopping block with the left hand thumbnail then and hold column cap, the right hand takes the blade edge to become 45 ℃ of clipped corner 1/4～1/3 column caps with column cap; At last with the DNA extraction drop on column cap, again corolla is put on; 6:00 spent and just decontroled the beginning importing time from morning, and each hour imports once then, and 4:00 spends wilting ending to afternoon.

1. the performance of tobacco offspring Other Main Agronomic Characters

With the Phytolacca acinosa is donor, and the big gold dollar of flue-cured tobacco cultivars safflower is an acceptor, and preparation imports combination, and 20 flowers of each combined treatment obtain D after the maturation ₀1082 (D ₀For importing contemporary seed).Plantation D in 2000 ₀, totally 212 strains develop into normal plant.Ripening stage (transplanting back 65d) gets middle leaf respectively and measures the tobacco leaf potassium content, and the result sees table 1.Can know that from table 1 after the total genomic dna of donor plant pokeweed imported flue-cured tobacco, the tobacco leaf potassium content had produced big luffing.15% cigarette strain tobacco leaf potassium content is higher than 2.5%, 1 strain tobacco leaf potassium content is wherein arranged up to 5.06%.It is thus clear that foreign DNA imports to be handled, and has significantly changed acceptor tobacco leaf potassium content, proves the validity that imports.

Table 1 D ₀For potassium content in tobacco leaf (middle leaf)

During results, the tobacco leaf potassium content is higher than single receipts of 2.5% cigarette strain and singly takes off, and obtains D ₁For seed, through plantation in continuous 9 years, being higher than 2.5% with the tobacco leaf potassium content was main selective pressure, considers plant type and disease resistance simultaneously, with the D that selects ₉Three strain systems in generation carry out main proterties examination, and the result sees table 2.Can know the D after the importing by table 2 ₉Other Main Agronomic Characters for strain system is compared with acceptor (the big gold dollar of safflower), does not all have plant height, the number of sheets, pitch and breeding time much variations.And aspect leaf area (long * wide), stem girth and three proterties of output, different strain performances is different.D ₉-05 and D ₉-06 leaf area is maximum, compares with acceptor to reach utmost point level of signification difference; D ₉-05 and D ₉-12 output is the highest, compares with acceptor to reach level of signification difference; Aspect stem girth, compare D with acceptor ₉-06 and D ₉-12 two strains reach utmost point level of signification difference.

Table 2 D ₉For Other Main Agronomic Characters basic inquiry table

Annotate: ^*Be illustrated in LSR _0.05There were significant differences for level, ^*Be illustrated in LSR _0.01There were significant differences for level.

2. the performance of tobacco offspring tobacco leaf potassium content

The height of potassium content in tobacco leaf is as one of important indicator of quality evalution.Test-results shows that the potassium content in tobacco leaf of new lines is all compared the bigger raising (table 3) of photograph (the big gold dollar of safflower); Compare with contrast, with D ₉-05 and D ₉-06 amplification is maximum, wherein D ₉-05 middle leaf potassium content mean number surpasses 1.9%, and summation surpasses 5.7%; D ₉-06 bottom leaf potassium content mean number surpasses 2.17%, and summation surpasses 6.51%.

Three the new lines potassium content in tobacco leaf of table 3 and the significance of difference of comparing

Annotate: ^*Be illustrated in LSR _0.05There were significant differences for level, ^*Be illustrated in LSR _0.01There were significant differences for level.During multiple comparisons, same area compares.

Embodiment 2: the inventive method is implemented the performance of back tobacco offspring quality of tobacco

1. tobacco leaf visual appearance performance

Tobacco leaf visual appearance evaluation result shows (table 4): big gold dollar of safflower and D ₉-05 and D ₉-12 article tie up to and show basically identical on the ripening degree, and D ₉-06 strain maturation is later; What oil content was maximum is the big gold dollar of safflower, secondly is D ₉-06 strain; Big gold dollar of safflower and D ₉-05, D ₉-06 and D ₉-12 structure major parts are loose; D ₉-05, D ₉-06 and D ₉-12 all are better than K327 on plumpness, uniformity coefficient, color and luster.This explanation, new lines be better than the big gold dollar of contrast safflower slightly, but difference are not remarkable on the tobacco leaf visual appearance.

The tobacco leaf visual appearance evaluation of the different strains of table 4 relatively

Annotate: the B in the position, C, X represent upper tobacco leaf, middle part tobacco leaf and lower tobacco leaf (down together) respectively.

2. tobacco leaf chemical composition performance

Get the big gold dollar blade of different strain and Flos Carthami respectively, baking back tobacco leaf mixing cigarette appearance is carried out the routine analysis (table 5) of chemical ingredients: 1. in lower blade, and D ₉-06 and D ₉The total sugar content of-12 strains is lower than the contrast big gold dollar of safflower and D slightly ₉The big gold dollar of a little higher than contrast safflower of-05 strain, but all do not reach level of signification; D ₉-05, D ₉-06 and D ₉The reducing sugar of-12 new lines all is higher than the contrast big gold dollar of safflower and total nitrogen and nicotine and is lower than slightly and contrasts the big gold dollar of safflower, but does not all reach level of signification; Reducing sugar/nicotine, K ₂O and Cl ^-On all be higher than the contrast big gold dollar of safflower and at K ₂O/Cl ^-On all be lower than contrast safflower big gold dollar.2. in the blade of middle part, the total reducing sugar of new lines, reducing sugar, reducing sugar/nicotine, K ₂O and K ₂O/Cl ^-All be higher than contrast safflower big gold dollar, wherein on the total reducing sugar with the difference of contrast not significantly, reaching level of signification, on reducing sugar/nicotine, removing and D with the difference of contrast on the reducing sugar with the difference of contrast ₉The significantly not outer same D of-12 strain differences ₉-05 and D ₉-06 strain is remarkable level, at K ₂O is last to be removed and BD ₉-06 strain difference reaches the outer same D of level of signification ₉-12 and D ₉-05 strain difference not significantly, at K ₂O/Cl ^-Go up same D ₉-12, D ₉-05 and D ₉-06 strain reaches utmost point level of signification; Total nitrogen and Cl ^-All be lower than the big gold dollar of contrast safflower, contrast and D ₉-06 strain difference reaches level of signification and and D ₉-12 and D ₉-05 strain difference is not remarkable; Has only D ₉The nicotine of-12 strains is higher than the big gold dollar of contrast safflower, D ₉-05 and D ₉The nicotine of-06 strain is lower than the contrast big gold dollar of safflower and contrast and D ₉-06 strain has reached level of signification; 3. in upper blade, the total reducing sugar of new lines, reducing sugar and Cl ^-All be lower than the big gold dollar of contrast safflower, wherein remove and D impinging upon on the total reducing sugar ₉The significantly not outer same D of-12 strain differences ₉-05 and D ₉-06 strain is remarkable level, on reducing sugar difference all significantly and at Cl ^-Last difference all reaches utmost point level of signification; At K ₂O and K ₂O/Cl ^-On all be higher than contrast safflower big gold dollar, wherein contrast and D ₉-06 strain difference all significantly and and D ₉-12 and D ₉-05 strain difference all reaches level of signification; D ₉-05 and D ₉The total nitrogen of-06 strain and nicotine all are higher than the contrast big gold dollar of safflower and difference reaches utmost point level of signification, and D ₉-12 strains be lower than not level of signification of the contrast big gold dollar of safflower and difference; D ₉It is not remarkable that the reducing sugar/nicotine of-12 strains is higher than the contrast big gold dollar of safflower and difference, and D ₉-05 and D ₉Be lower than the contrast big gold dollar of safflower and the difference of-06 strain reach level of signification.

The different strain main chemical compositions of table 5 relatively

3. smoke panel test quality performance of tobacco leaf

Each kind (being) is smoked panel test total points with D ₉The middle leaf of-06 strain the highest (53.56) is with D ₉The bottom leaf of-12 strains minimum (44.71).All in all, the big gold dollar of the quality of smokeing panel test safflower is put up the best performance D ₉-06 strain is slightly inferior, D ₉-12 strains are the poorest, but difference not significantly (table 5).

The tobacco leaf of the different strains of table 5 is smoked panel test quality evalution relatively

Annotate: in the fragrance type, Luzhou-flavor (N), middle odor type (Z), in dense partially (ZN), dense partially in (NZ).

In the assorted gas type, withered and burnt smell (K), living blue or green gas (S), xylon gas (M), the smell of soil (T), pollen gas (H)

Embodiment 3: the inventive method is implemented back and the expression of potassium genes involved in the tobacco offspring

The mRNA transcriptional level that the employing real time fluorescence quantifying PCR method has been measured relevant high potassium gene in high potassium new lines of different tobaccos and the contrast changes (Fig. 1).The result shows: in different new lines, with control material ratio, D ₉-05, D ₉-06 and D ₉High potassium expression of gene amount is the most obvious in-12 new lines, and the new lines potassium content that this this test of explanation obtains is higher; On this basis, in the different genes relevant with potassium, Ntpk1 gene, Cbl9 gene and the Gork gene expression amount in three new lines is all than higher; The Ntkc1 gene is at D ₉Expression amount in-05 new lines is very high, but at D ₉-06 and D ₉Lower in-12 new lines; The Tork1 gene is at D ₉Expression amount in-12 new lines is very high, at D ₉Expression amount in-06 new lines is than higher, but at D ₉Lower in-05 new lines; The Kat1 gene is at D ₉Expression amount in-12 new lines is very high, at D ₉-05 and D ₉In-06 new lines than higher.

Claims (2)

1. a breeding method that improves tobacco tobacco leaf potassium content is characterized in that, utilizes in the big gold dollar of overall dna importing tobacco bred safflower of pollen tube passage method with high potassium plant Phytolacca acinosa and cultivates, and specifically may further comprise the steps:

(1) 100 μ l 5M NaCl are newly managed → added to supernatant to the extraction of donor dna: get the freezing blade of the tender tobacco of 0.2g children and go in the inkstone alms bowl → add to change over to after 2 * CTAB extracting solution, 700 μ l grind the 1.5ml centrifuge tube → add isopyknic chloroform in 65 ℃ of insulation 20-30min → cooling back: the centrifugal 10min of primary isoamyl alcohol mixing 10min → 10000r/min → get and the ice-cold ethanol sedimentation DNA of 1ml → choosing DNA precipitates, with 70% ethanol clean dry → be dissolved among the 100 μ lTE; Described 2 * CTAB extract recipe is: 2%CTAB, 1.4M NaCl, 100mM Tris-HCl, 100mM EDTA, pH8.0,2% beta-mercaptoethanol; Described chloroform: the volume ratio of primary isoamyl alcohol is 24: 1;

(2) introduction method: select the open flower in robust growth, stem middle and upper part; Its corolla is taken off; Expose column cap, make chopping block with the left hand thumbnail then and hold column cap, the right hand takes the blade edge to become 45 ℃ of clipped corner 1/4～1/3 column caps with column cap; At last with the DNA extraction drop on column cap, again corolla is put on; 6:00 spent and just decontroled the beginning importing time from morning, and each hour imports once then, and 4:00 spends wilting ending to afternoon.

2. the breeding method of raising according to claim 1 tobacco tobacco leaf potassium content is characterized in that, the overall dna of high potassium plant Phytolacca acinosa is imported tobacco bred after, screen with tobacco tobacco leaf potassium content 2.5% the standard of being not less than again.

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