A method for extracting carrot pollen microspores

Abstract

The invention discloses a method for extracting carrot pollen microspores. The method comprises the following steps of: (1) putting a sterilized carrot inflorescence in a mortar with the diameter of 70mm-90mm, adding a B5 fluid medium, and extruding buds by a lapping hammer with the diameter of 25mm-30mm to release the microspores so as to obtain suspension; (2) filtering the suspension obtained in the step (1) by a filter screen with the aperture of 400 meshes and collecting filtrate; and (3) carrying out gradient centrifugation on the filtrate obtained in the step (2) to obtain the microspores according to the following steps of: (a) centrifuging 150g-155g of filtrate for 4-6 min, discarding supernatant and collecting sediments; (b) suspending the sediments obtained in the step (a) by the B5 fluid medium, centrifuging 125g-130g of the product for 4-6 min, discarding supernatant and collecting sediments; and (c) suspending the sediments obtained in the step (b) by the B5 fluid medium, centrifuging 105g-110g of the product for 4-6 min, discarding supernatant and collecting sediments. The method provided by the invention is simply and conveniently operated, and is capable of increasing the extracted microspores, improving the cultivating efficiency, and furthest reducing the influence to the isolated microspore culture caused by impurities such as tissue fragments.

Description

Technical field [0001] The purpose of the present invention is to provide a method for extracting carrot pollen microspores. Background technique [0002] Free microspore culture technology is a fast-developing cell engineering technology, which has been applied in a variety of crops, such as wheat, cabbage, Chinese cabbage, etc. This technology can use the in vitro culture of pollen microspores to quickly obtain homozygous double To create new germplasm for breeding varieties. Carrot free microspore culture technology has been studied (Li Jinrong, Ou Chenggang, Zhuang Feiyun, etc.. Carrot free microspore culture and development process, Acta Horticulture, 2011, 38(8): 1539-1546.), and embryoid plants were obtained , But the number is small, which limits the wide application of this technology in carrot production. One of the main reasons for this problem is that carrot flower organs are small, the flower buds are about 1mm long during the suitable cultivation period, and the d...

AI Technical Summary

Problems solved by technology

Carrot free microspore culture technology has been studied (Li Jinrong, Ou Chenggang, Zhuang Feiyun, etc. Research on carrot free microspore culture and its development process, Acta Horticultural Science, 2011, 38(8):1539-1546.), and embryoid plants were obtained , but the amount is small, which limits the wide application of this technology in carrot production

One of the main reasons for this problem is that the floral organs of carrots are small, the flower buds suitable for cultivation are about 1mm long, and the anther diameter is less than 1mm. It is difficult to fully squeeze the anthers and release the microspores by the method described in the report, so the free microspores obtained The number of spores and the number of induced embryoid bodies are small, and the corresponding number of embryoid bodies finally obtained is also reduced; and during the extraction process, a large amount of somatic tissue impurities will be produced, which will affect the final extraction quality and culture effect

[0003] The existing carrot pollen microspore extraction methods have problems such as small extraction quantity and low extraction efficiency

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