Method for detecting purity of GFP transgene cotton crossbreed

Abstract

The invention discloses a method for detecting the purity of a GFP transgene cotton crossbreed, which comprises the following steps: using a GFP homozygote variety as a male parent and a non-GFP variety as a female parent to obtain an F1 crossbreed as a detection object; extracting an F1 generation seed detection sample, soaking the seed in mild water of which the temperature is between 26 and 30DEG C for 20 to 30 hours, then accelerating germination at a temperature of between 28 and 32 DEG C for 20 to 30 hours, removing an external seed coat, and leaving and taking a hypocotyl and an internal seed coat of the same seed as a slice which is placed on the same glass slide respectively; irradiating the slices by blue-violet light of a fluorescence microscope, observing the fluorescence performances of the two slices, and determining that the seed is the F1 crossbreed when only the hypocotyl slice has green fluorescent light but the internal seed coat slice has no green fluorescent light; determining that the seed is a male parent or an F2 generation seed of GFP when both the hypocotyl slice and the internal seed coat slice have the green fluorescent light; and determining that the seed is a female parent or other varieties when both the hypocotyl slice and the internal seed coat slice have no green fluorescent light. The detection method is accurate, quick and economic, and is convenient for application.

Description

Technical field

[0001] The invention belongs to the technical field of biological breeding, and specifically relates to a method for detecting the purity of GFP transgenic cotton hybrids. technical background

[0002] Heterosis is a universal law in biology. The utilization of hybrid vigor is a hot spot in biological breeding. The wide application of the hybrid advantages of rice and corn has played a huge role in solving food security and feed safety. The utilization of cotton hybrid vigor started relatively late. At present, India and China are the countries with the largest application area of ​​cotton hybrids. The highest yield increase of cotton hybrids bred in India can reach more than 50%, and the promotion area accounts for about half of the cotton planting area. my country is a major exporter of traditional textiles. Cotton occupies an important position in my country's textile industry and agricultural production. The annual cotton planting area is about 60 million m...

Examples

Embodiment 1

[0022] A method for detecting the purity of GFP transgenic cotton hybrids, which specifically includes the following steps: 1) F1 hybrids obtained by using GFP homozygous varieties (lines) as male parents and non-GFP varieties (lines) as female parents; 2) F1 extraction Substitute seed test samples, after soaking seeds in warm water at 26°C for 20 hours, germinate at 30°C for 30 hours, remove the outer testa, and save the hypocotyl and inner testa of the same seed as a section and place on the same glass; 3) Illuminate with blue-violet light of a fluorescence microscope, observe the fluorescence performance of the two sections, check with Table 1 to determine whether this seed belongs to F1 hybrids, only the hypocotyl section has green fluorescence and the endocarp section has no green When fluorescent, it can be judged that the seed is an F1 hybrid; when both the hypocotyl slice and the endothelial slice have green fluorescence, the seed can be judged to be the male parent or G...

Embodiment 2

[0027] A method for detecting the purity of GFP transgenic cotton hybrids, specifically including the following steps:

[0028] 1) F1 hybrids obtained by using GFP homozygous varieties (lines) as male parents and non-GFP varieties (lines) as female parents;

[0029] 2) Take the F1 generation seed test sample, soak the seeds in 28℃ warm water for 20, incubate the germination at 28℃ for 30h, remove the outer testa, save the hypocotyl and inner testa of the same seed as a slice respectively On glass

[0030] 3) Use the blue-violet light of a fluorescence microscope to observe the fluorescence performance of the two sections. When only the hypocotyl section has green fluorescence and the endocarp section has no green fluorescence, it can be determined that the seed is an F1 hybrid; when the hypocotyl section and When both endometrium slices have green fluorescence, it can be judged that the seeds are male parent or GFP F2 generation seeds; when the hypocotyl slices and endothelial slice...

Embodiment 3

[0032] A method for detecting the purity of GFP transgenic cotton hybrids, specifically including the following steps:

[0033] 1) F1 hybrids obtained by using GFP homozygous varieties (lines) as male parents and non-GFP varieties (lines) as female parents;

[0034] 2) Take the F1 generation seed test sample, soak the seeds in 30℃ warm water for 24 hours, incubate the germination at 30℃ for 24 hours, remove the outer testa, and save the hypocotyl and inner testa of the same seed as a section and place them in the same On glass

[0035] 3) Use the blue-violet light of a fluorescence microscope to observe the fluorescence performance of the two sections. When only the hypocotyl section has green fluorescence and the endocarp section has no green fluorescence, it can be determined that the seed is an F1 hybrid; when the hypocotyl section and When both endometrium slices have green fluorescence, it can be judged that the seeds are male parent or GFP F2 generation seeds; when the hypocot...

Technical field

[0001] The invention belongs to the technical field of biological breeding, and specifically relates to a method for detecting the purity of GFP transgenic cotton hybrids. technical background

[0002] Heterosis is a universal law in biology. The utilization of hybrid vigor is a hot spot in biological breeding. The wide application of the hybrid advantages of rice and corn has played a huge role in solving food security and feed safety. The utilization of cotton hybrid vigor started relatively late. At present, India and China are the countries with the largest application area of ​​cotton hybrids. The highest yield increase of cotton hybrids bred in India can reach more than 50%, and the promotion area accounts for about half of the cotton planting area. my country is a major exporter of traditional textiles. Cotton occupies an important position in my country's textile industry and agricultural production. The annual cotton planting area is about 60 million m...

Examples

Embodiment 1

[0022] A method for detecting the purity of GFP transgenic cotton hybrids, which specifically includes the following steps: 1) F1 hybrids obtained by using GFP homozygous varieties (lines) as male parents and non-GFP varieties (lines) as female parents; 2) F1 extraction Substitute seed test samples, after soaking seeds in warm water at 26°C for 20 hours, germinate at 30°C for 30 hours, remove the outer testa, and save the hypocotyl and inner testa of the same seed as a section and place on the same glass; 3) Illuminate with blue-violet light of a fluorescence microscope, observe the fluorescence performance of the two sections, check with Table 1 to determine whether this seed belongs to F1 hybrids, only the hypocotyl section has green fluorescence and the endocarp section has no green When fluorescent, it can be judged that the seed is an F1 hybrid; when both the hypocotyl slice and the endothelial slice have green fluorescence, the seed can be judged to be the male parent or G...

Embodiment 2

[0027] A method for detecting the purity of GFP transgenic cotton hybrids, specifically including the following steps:

[0028] 1) F1 hybrids obtained by using GFP homozygous varieties (lines) as male parents and non-GFP varieties (lines) as female parents;

[0029] 2) Take the F1 generation seed test sample, soak the seeds in 28℃ warm water for 20, incubate the germination at 28℃ for 30h, remove the outer testa, save the hypocotyl and inner testa of the same seed as a slice respectively On glass

[0030] 3) Use the blue-violet light of a fluorescence microscope to observe the fluorescence performance of the two sections. When only the hypocotyl section has green fluorescence and the endocarp section has no green fluorescence, it can be determined that the seed is an F1 hybrid; when the hypocotyl section and When both endometrium slices have green fluorescence, it can be judged that the seeds are male parent or GFP F2 generation seeds; when the hypocotyl slices and endothelial slice...

Embodiment 3

[0032] A method for detecting the purity of GFP transgenic cotton hybrids, specifically including the following steps:

[0033] 1) F1 hybrids obtained by using GFP homozygous varieties (lines) as male parents and non-GFP varieties (lines) as female parents;

[0034] 2) Take the F1 generation seed test sample, soak the seeds in 30℃ warm water for 24 hours, incubate the germination at 30℃ for 24 hours, remove the outer testa, and save the hypocotyl and inner testa of the same seed as a section and place them in the same On glass

[0035] 3) Use the blue-violet light of a fluorescence microscope to observe the fluorescence performance of the two sections. When only the hypocotyl section has green fluorescence and the endocarp section has no green fluorescence, it can be determined that the seed is an F1 hybrid; when the hypocotyl section and When both endometrium slices have green fluorescence, it can be judged that the seeds are male parent or GFP F2 generation seeds; when the hypocot...

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