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Method for early-stage screening of high-nicotine and low-nicotine tobacco converter plants

A technology of transformed strains and nicotine, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems that the identification results are affected by environmental factors, it is difficult to ensure the size of the target group, and the efficiency and timeliness are not enough, so as to reduce the Average nicotine conversion rate, easy to promote, and the effect of increasing the ratio

Inactive Publication Date: 2015-09-30
HUBEI TOBACCO SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is reliable and effective, it has the following disadvantages: 1. The sampling period is still late at the group tree stage after transplanting, and the field was wasted after removing the transformed strains, and it is difficult to ensure the size of the target group; 2. It needs chemical induction methods Yes; 3. The identification results are affected by environmental factors: such as drug concentration, modulation temperature and time, factors affecting chemical detection, etc.
Therefore, although there is a good reference, it is still not efficient and timely enough to meet the actual needs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Early screening of burley tobacco high and low nicotine transformants. The specific steps are as follows:

[0025] A. On the ultra-clean workbench, 10 copies of burley tobacco varieties used for screening of high and low nicotine transformants (including 2 control varieties: B37 high nicotine transformants with a conversion rate of 85%; B37 low smoke) Alkali-converted materials with a conversion rate of 3.1%) were put into the marked 2ml sterile centrifuge tube, poured into 70% ethanol for surface disinfection for 60 seconds, rinsed with sterile water once for 3 minutes, and pipetted The sterile water is sucked by the gun, and then saturated calcium hypochlorite (Ca(ClO) with a concentration of 10%) 2 ) Disinfect the solution for 8 minutes, turn the cover up and down several times to fully disinfect, rinse with sterile water 3 times, 3 minutes each time to remove residual Ca(ClO) 2 Solution, after sucking the sterile water with a pipette, use the tip of the tip t...

example 2

[0034] Example 2. Early screening of high and low nicotine transformants of Maryland tobacco. The specific steps are as follows:

[0035] A. On the ultra-clean workbench, 6 Maryland tobacco varieties used for screening of high and low nicotine transformants (including 2 control varieties: Md609 high nicotine transforming materials, with a conversion rate of 90%; Md609 low nicotine transformants) Transform the materials, the conversion rate is 2.7%) respectively into the marked 2ml sterile centrifuge tube, pour 75% ethanol for surface disinfection for 55 seconds, rinse with sterile water once for 5 minutes, use a pipette Aspirate the sterile water, and then use 10% saturated calcium hypochlorite (Ca(ClO) 2 ) Disinfect the solution for 10 minutes, turn the cover up and down several times to fully disinfect, rinse with sterile water twice, 5 minutes each time to remove residual Ca(ClO) 2 Solution, after sucking the sterile water with a pipette, use the tip of the tip to suck the deto...

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Abstract

The invention relates to a method for early-stage screening of high-nicotine and low-nicotine tobacco converter plants. The method mainly comprises the steps of taking aseptic seedling leaves from indoor tobacco seed sprout, adopting a CTAB small-sample method to extract tobacco genome DNA, using the DNA as a template, using a nicotine converted specific primer to perform PCR amplification, adopting 2% agarose gel electrophoresis to separate and show a PCR amplification product, observing, reading and counting molecular marker band spectrums in a gel-imaging system and comparing the band spectrums with a contrast characteristic band spectrum to screen high-nicotine and low-nicotine conversion materials. The method is not limited by environment conditions and seasonal influence, does not need artificial chemical inducted tobacco conversion, high-nicotine and low-nicotine converter plant screening is performed before sowing, and time cost and economic cost are greatly saved. In addition, the method is accurate, efficient, simple and convenient to operate and easy to centralize and popularize, can be used for screening of high-nicotine and low-nicotine conversion materials of different types of tobaccos and can be also used for quick purification and identification performed according to specific nicotine conversion characters.

Description

Technical field [0001] The present invention relates to a method for early screening of tobacco high and low nicotine transformants, in particular to a method for early screening of tobacco high and low nicotine transformants by using nicotine-specific primers for PCR amplification and based on characteristic band profiles, belonging to tobacco The field of planting technology. Background technique [0002] Studies have shown that common tobacco mainly includes four alkaloids: nicotine, nornicotine, neonicotine and pseudoequiine. Ordinary tobacco belongs to nicotine accumulation type. Nicotine accounts for more than 90% of the total alkaloid content. Nornicotine is the second largest alkaloid, but generally only accounts for 2-5% of the total alkaloid content. The other two alkaloids The content is extremely low. Nornicotine is also called desmethylnicotine, which is mainly formed by the demethylation of nicotine under the action of demethylase. Although the content of nornico...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q1/686C12Q2600/13C12Q2531/113
Inventor 蔡长春程玲冯吉
Owner HUBEI TOBACCO SCI RES INST
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