Method for simply and rapidly identifying transgenic seeds and estimating copy numbers

Abstract

The invention provides a method for simply and rapidly identifying transgenic seeds and estimating copy numbers. According to the invention, the red fluorescent protein (FP) gene (driven by callus / seed coat-specific promoters) is closely connected in series with the target gene and then introduced into the rice male sterile mutant ms26 by means of Agrobacterium tumefaciens-mediated transformation. The transgenic plants are self-crossed to generate the following two types of seeds: non-fluorescent seeds free of transgenic components and fluorescent seeds containing transgenic components. The two types of seeds can be directly separated with the naked eye or with the aid of a fluorescence microscopy, and the separation method is simple and has high accuracy up to 100%. Further, according to the proportions of the fluorescent seeds and the non-fluorescent seeds, the copy numbers of the exogenous gene in the transgenic plant line can be estimated. Since the method provided by the invention does not need the molecular detection of the germinated seeds and young seedlings, the identified seeds can be conveniently applied to the late-stage scientific researches or production.

Description

technical field [0001] The invention belongs to the field of biological technology and the technical field of plant breeding. The invention provides a simple and effective method for quickly identifying transgenic seeds and non-transgenic seeds. The method does not need to germinate the seeds, and can conveniently distinguish the transgenic and non-transgenic seeds for scientific research or production. Background technique [0002] Transgenic technology has shown great application potential in crop quality improvement and resistance enhancement. However, the transgenic T0 generation is often transgenic heterozygous, and its selfing produces seeds of three genotypes of 1:2:1, a quarter of which are homozygous non-transgenic seeds, a quarter of which are homozygous transgenic seeds, and two One-third of the seeds were heterozygous transgenic seeds. In traditional transformation methods, transgenic seeds and non-transgenic seeds cannot be identified at the seed stage, so eac...

AI Technical Summary

Problems solved by technology

There are many problems with this method. First, different stages of seedling development have different degrees of resistance to herbicides and antibiotics. It is difficult to control the dosage of screening agents. If it is too high, the transgenic seedlings may be killed, and if it is too low, it may kill non- Transgenic seedlings mixed in

Second, the potential risks of herbicides and antibiotics to the environment and food safety have received more and more attention. Therefore, people have tried to use safer screening markers during the transformation process, such as key enzymes in the carbohydrate metabolism process such as PMI, xylA, etc., but it is difficult to screen transgenic plants with such screening markers in the field

Currently, there is no fast, stable, and secure way to do this

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