Method for identifying haploid regenerated by brassica napus microspores simply, conveniently and quickly

A cabbage rape and microspore technology is applied in the application field of plant cytology to achieve the effects of low cost, easy operation and simple process

Inactive Publication Date: 2012-01-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no report on using the number of chloroplasts in leaf stomatal guard cells to identify haploid (n, HP) test-tube plantlets of Brassica napus regenerated from microspores

Method used

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  • Method for identifying haploid regenerated by brassica napus microspores simply, conveniently and quickly
  • Method for identifying haploid regenerated by brassica napus microspores simply, conveniently and quickly
  • Method for identifying haploid regenerated by brassica napus microspores simply, conveniently and quickly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Cut the leaves of sterile seedlings regenerated from Brassica napus microspores in vitro

[0026] Using the method reported by Shi Shuwen et al. (Shi Shuwen et al., Brassica napus and its interspecific and intergenus hybrid microspore embryoid induction, Huazhong Agricultural University Journal, 1993, 12: 544-550) Brassica napus variety Huashuang 4 , Huashuang No. 2, Huayou No. 8, Huashuang No. 1 and Huahuang No. 1 (the above-mentioned varieties are from the rapeseed laboratory of Huazhong Agricultural University, and have been widely promoted and applied in China). Embryoid bodies, the regenerated embryoid bodies were respectively inoculated on the B5 medium with a pH of 6.0 (B5 medium source: Gamborg et al., Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 1968, 50 : 151-158) in a 150ml Erlenmeyer flask, cultivated to complete plantlets with roots, stems and leaves (the culture temperature is 24-26°C, the light is 12h ...

Embodiment 2

[0034] Embodiment 2 (application embodiment: utilize the method for embodiment 1 to identify and discard the microspore haploid plant that does not set seeds)

[0035] Rapeseed varieties Zhongyou 821 (from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, a large-scale promotion variety in China), Huashuang 3 and Huashuang 5 (variety from the rape research laboratory of Huazhong Agricultural University, have been widely promoted and applied in China ) microspores regenerated into embryoid bodies (method reference: Shi Shuwen et al., Induction of microspore embryoid bodies of Brassica napus and its interspecific and intergenus hybrids, Journal of Huazhong Agricultural University, 1993, 12: 544-550). The obtained embryoid bodies were inoculated into Erlenmeyer flasks containing B5 medium (pH 6.0), and cultivated into plantlets (cultivation temperature was 24-26° C., light was 12 hours per day, and light intensity was 1800 lux). Cut off the 5th lea...

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Abstract

The invention belongs to the technical field of plant cytology, and particularly relates to a method for identifying haploid regenerated by brassica napus microspores simply, conveniently and quickly in batch in the early stage in a method of chloroplast counting. The method is characterized by comprising the following steps of: inoculating an embryoid cultured by in-vitro microspores onto a B5 culture medium; when the embryoid is grown into a complete plant, cutting the fifth even leaf sterilely; dyeing the lower epidermis tissue in the middle of the leaf by using iodine-potassium iodide; observing chloroplasts of guard cells of air vents by using an optical microscope; and recording the number of the chloroplasts of 10 air vents on each prepared leaf (namely the same leaf), wherein more than 7 air vents have 6 to 8 chloroplasts, and the plants of the chloroplasts are the haploid. By the method, the problem of long-time subculture of the haploid is solved, and in ten thousands of plants, the cost of reagents, lands and labors can be saved by 19,083 RMB. The method has a simple process, is easy to operate and low in cost and can be used for identifying the ploidy of a large number of plants, and one plant of seedling is identified only for 5 minutes, so the application efficiency of the double haploid (DH) technology is improved.

Description

technical field [0001] The invention belongs to the technical field of plant cytology application, and in particular relates to an early, simple, rapid and large-scale method for identifying haploid plants regenerated from Brassica napus microspores. Background technique [0002] Since the in vitro culture and plant regeneration technology of Brassica napus microspores was successfully established in the late 1980s, double haploid (DH) regenerated from Brassica napus microspores has been widely used to rapidly breed new varieties of Brassica napus and construct genetic Study populations and perform genetic transformation and in vitro mutagenesis. Practice has proved that the Brassica napus DH technology system is an important tool in the research fields of rapeseed genetics, breeding and molecular biology. However, after in vitro microspore culture and double haploid induction, the rate of double haploid plants is more than 30% to 90% in plant populations regenerated from d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 石淑稳王晨吴江生陈琳孙秀丽汪频
Owner HUAZHONG AGRI UNIV
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