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Efficient sugarcane or sugarcane related species stem tip chromosome flaking method

A technology of chromosome production and related species, applied in the field of cell biology, can solve the problem of difficult to obtain chromosome glass slide specimens of metaphase cells, different root tip thickness, tenderness, and sticky root tips of hydroponic sugarcane and other problems, to achieve the effect of saving film production time and microscopic observation time, clear chromosome structure, and shortening the experimental cycle

Pending Publication Date: 2020-06-05
SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, sugarcane is the most complex aneupolyploid plant, with a large number of chromosomes (range 64-128) and small shape, making it difficult to obtain ideal chromosome slide specimens of metaphase cells
At present, sugarcane root tip meristematic zone tissue is mainly used for production at home and abroad, but there are still some problems in the actual operation process: the hydroponic sugarcane root tip has viscous secretions, which affects the effect of chromosome production
There is no viscous secretion on the root tips of barrel planting or sand culture, but the sediment needs to be cleaned during collection, and the operation is more cumbersome; sugarcane roots are small, with few taproots and many fibrous roots, and the meristem tissue area is very small, generally 1 to 2 roots are used root tip of sugarcane grown in barrel, sand culture or hydroponic culture is greatly affected by nutritional environmental factors, the thickness of the root tip and the degree of tenderness are different, and the division cycle is difficult to grasp. Only a few root tips are in the mitotic stage; it cannot be guaranteed that cells in the middle phase of mitosis can be observed in each slice, and it is necessary to repeatedly collect root tips and duplicate slices for observation, which leads to increased workload; the test and experiment cycle are both Longer, generally it takes about 1 to 2 weeks to identify the number of chromosomes of a material

Method used

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  • Efficient sugarcane or sugarcane related species stem tip chromosome flaking method

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Method for Chromosome Preparation of Sugarcane Shoot Tip

[0063] 1. Using sugarcane as the test material, collect the stem tips of sugarcane in the field in the morning (9:00-11:00) on a sunny day (see figure 2 ) meristem tissue cut into 0.3cm 3 The small blocks are preprocessed.

[0064] 2. Pretreatment with pre-cooled ddH 2 Soak in O sterilized water, pretreat at 4°C for 12h, then fix with Carnot’s fixative solution (1 glacial acetic acid: 3 ethanol, now prepared), 4°C refrigerator for 3 days, and place the meristem tissue in a 70% Store in ethanol at 4°C for long-term use.

[0065] 3. When making slices, take out the stored meristem tissue materials and use ddH 2 Rinse twice with O sterile water, 10 min each time. Then use a mixture of equal volume of 1 mol / L HCl solution and 45% acetic acid aqueous solution at 60°C for 20 min in a water bath for dissociation. with ddH 2 O rinse with sterile water for 10 minutes. Cut off the shoot apex meristem tissue and p...

Embodiment 2

[0094] Chromosome Number Identification of Parent Species and Hybrid Progeny of Sugarcane

[0095] 1. Using sugarcane original parent varieties Valdeira and 57NG208, hybrid parent varieties Mintang 70-611 and RB72-454 and hybrid offspring germplasm F 1 Cliff City 96-48 and BC 2 Yuncane 07-86 was used as the experimental material, and the shoot apical meristematic zone tissues of sugarcane were collected from 9:00 to 11:00 am on a sunny day for pretreatment.

[0096] 2. Put the meristem tissue into a penicillin bottle and pre-treat with pre-cooled ddH 2 Soak in O sterilized water, pretreat at 4°C for 20 h, fix with Carnot’s fixative solution (glacial acetic acid:ethanol = 1:3), and fix in a 4°C refrigerator for 2 to 3 days, and place the meristem tissue at 70 % ethanol at 4°C for long-term storage.

[0097] 3. Take out the stored meristematic tissue material and use ddH 2 Rinse twice with O sterile water, 10 min each time. Then use an equal volume of 1mol / L HCl solution an...

Embodiment 3

[0103] Chromosome Number Identification of Wild Sugarcane Species and Related Genus Plants

[0104] The wild species of sugarcane, cut hand dense and wild species with large stems, are closely related plants of Banmao, Miscanthus, Dianmao and Hebawang. They contain many new resistance genes that are not found in sugarcane. They have strong perennial roots and good clustering It is of great value in the hybrid utilization and genetic improvement of sugarcane. In-depth research on the chromosomal ploidy and cytological genetic behavior of these resources will lay the foundation for the classification, identification, mining and utilization of resources. In the present invention, wild sugarcane species, wild species with large stems and three sugarcane close relatives are used as test materials, and they go to the National Sugarcane Germplasm Resource Garden at 9:00 to 11:00 am on a sunny day to collect stem apex meristems. Tissues are pretreated to investigate whether the techni...

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Abstract

The invention provides an efficient sugarcane or sugarcane related species stem tip chromosome flaking method, and belongs to the technical field of cell biology. The invention provides an efficient sugarcane stem tip chromosome flaking method aiming at solving the problems that sugarcane chromosomes are large in number and small in form and ideal division phase metaphase cells are difficult to obtain through root tip flaking. Chromosome flaking is carried out by utilizing stem tip meristematic region tissues of sugarcane and sugarcane related species in a vigorous growth period, the method has the advantages of convenient material taking, large meristematic area tissue sample size, vigorous division, many metaphase cells and clear and dispersed chromosome structure, and the technical keypoints of material selection, pretreatment, fixation, dissociation dyeing, flaking and chromosome morphology microscopic observation are disclosed. The method is simple and convenient, accurate and reliable in result, good in repeatability, easy to operate and short in experiment period and improves the sugarcane chromosome genome analysis efficiency. Technical support is provided for sugarcane chromosome karyotype research, germplasm resource classification identification and protection utilization.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a high-efficiency method for producing shoot tip chromosomes of sugarcane or sugarcane close relatives. Background technique [0002] Sugarcane is an important sugar crop and energy plant in the world. The genus Saccharum includes 6 species of thin-stemmed wild species, large-stemmed wild species, tropical species, Indian species, Chinese species and succulent spike species (also known as edible spike species) , Banmao, Dianmao and Hebawang in the sugarcane subfamily are all close relatives of sugarcane. Most modern sugarcane varieties contain relatives of 2 to 3 species in Saccharum genus, the genetic basis is relatively narrow, and the species degeneration is serious. In order to breed new and excellent sugarcane varieties, breeders have carried out intra-specific, inter-specific and inter-generic hybridization. It is hoped that the resistance genes of wild sp...

Claims

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Application Information

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IPC IPC(8): G01N1/28
CPCG01N1/2813
Inventor 林秀琴陆鑫蔡青周加芳毛钧吴转娣刘洪博刘新龙李旭娟字秋艳李纯佳徐超华孔春艳
Owner SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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