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Extraction method of protoplast of barnyard grass

An extraction method and protoplast technology, which are applied in the agricultural field to achieve the effects of high yield, large quantity and good integrity

Inactive Publication Date: 2016-12-21
HUNAN AGRI BIOTECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the biological research on barnyardgrass is carried out relatively late, and the preparation of its protoplasts has not been reported. Therefore, the preparation of barnyardgrass protoplasts by an efficient and economical method can lay a certain technology for somatic cell hybridization and genetic engineering of barnyardgrass. Base

Method used

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  • Extraction method of protoplast of barnyard grass
  • Extraction method of protoplast of barnyard grass

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1. Select barnyardgrass seeds that are full and of the same maturity, and pass the weight concentration of 2% KNO 3 After soaking the seeds overnight, they were broadcast live in a culture pot (28 cm in diameter, 17 cm in height), and the culture medium was paddy field soil;

[0029] 2. When the seedlings obtained in step (1) grow to the 4-leaf stage, use a blade to cut off the relatively young leaves on the main stem, wash them with distilled water three times, and set aside;

[0030] 3. Dry the leaves obtained in step (2) with filter paper, cut the leaves into filaments of about 1.0mm lengthwise along the main veins of the leaves with a blade, and quickly and gently transfer the materials to 10mL of enzymatic hydrolysis solution Put the test material in a centrifuge tube, and completely immerse the test material in the enzymatic hydrolysis solution, shake the enzymatic hydrolysis at 25°C in the dark, and enzymatic hydrolysis for 6 hours, with a rotation speed of 60r / m...

Embodiment 2

[0036] 1. Select barnyardgrass seeds that are full and of the same maturity, and pass the weight concentration of 2% KNO 3 After soaking the seeds overnight, they were broadcast live in a culture pot (28 cm in diameter, 17 cm in height), and the culture medium was paddy field soil;

[0037] 2. When the seedlings obtained in step (1) grow to the 4-leaf stage, use a blade to cut off the relatively young leaves on the main stem, wash them with distilled water three times, and set aside;

[0038] 3. Dry the leaves obtained in step (2) with filter paper, cut the leaves into filaments of about 1.0mm lengthwise along the main veins of the leaves with a blade, and quickly and gently transfer the materials to 10mL of enzymatic hydrolysis solution Put the test material in a centrifuge tube, and completely immerse the test material in the enzymatic hydrolysis solution, shake the enzymatic hydrolysis for 8 hours at 25°C in the dark, and the rotation speed is 60r / min;

[0039]Preparation ...

Embodiment 3

[0044] 1. Choose plump, consistent barnyardgrass seeds, and live-seed them in cultivation pots (diameter 28cm, height 17cm) after soaking overnight with weight concentration 2% KNO3, and the cultivation substrate is paddy field soil;

[0045] 2. When the seedlings obtained in step (1) grow to the 4-leaf stage, cut off the tender main stem with a blade, rinse with distilled water three times, and set aside;

[0046] 3. Dry the main stem obtained in step (2) with filter paper, cross-cut the main stem with a blade to cut it into filaments of about 1.0mm, and quickly and gently transfer the material to a 10mL centrifuge filled with enzymatic hydrolysis solution. Put the test material in the tube, and completely immerse the test material in the enzymatic hydrolysis solution, shake the enzymatic hydrolysis for 3 hours at 25°C in the dark, and the rotation speed is 60r / min;

[0047] Preparation of enzymolysis solution: using water as solvent, mix cellulase R-10 with a volume concentr...

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Abstract

The invention discloses an extraction method of protoplast of barnyard grass. The extraction method includes the following steps: selecting barnyard grass seeds having same fullness and maturity, and directly sowing the seeds in a culture medium; cutting main stems and tender leaves on the main stems after 3-5 leaf stage of seedlings; wiping the main stems and tender leaves, cross-cutting the main stems and vertical-shredding the leaves along the main veins of the leaves into fine strips being 0.5-1.5 mm, and quickly and gently moving the fine strips into a 10 ml centrifugal tube containing an enzymolysis liquid until the fine strips, as a sample, are completely immersed in the enzymolysis liquid; performing vibrating enzymolysis for 3-6 h at 25 DEG C in a dark condition; and after enzymolysis, performing centrifugation and removing a supernatant, and collecting protoplast; and adding a washing liquid for re-suspension, performing centrifugation and removing a supernatant again to obtain a solution, i.e. a pure protoplast. The separation materials of the method are easy to obtain and the method has simple extraction operations. The method is large in number of separated protoplast, is high in yield, has good integrality and lays fundament for protoplast transfection and fusion.

Description

technical field [0001] The invention relates to the field of agricultural technology, in particular to a method for extracting barnyard grass biomass. Background technique [0002] Plant protoplast refers to a single cell coated with a cell membrane after the cell wall is removed. It can be applied to the fusion between distant parents, easy to introduce foreign genes, and is also used to separate various organelles (nuclei, chloroplasts, mitochondria, vacuoles, etc.) etc.), and can provide technical support for research on material transport and information exchange between cells. Barnyardgrass [Echinochloa crusgalli (L.) Beauv] is an annual grass plant, and is one of the main malignant weeds in global agricultural production. Stronger than rice etc. C 3 Plants seriously endanger the security of world food production. The development of herbicides mostly takes barnyardgrass as the primary target. However, due to the large-scale application of chemical herbicides in rece...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04C12N2509/00
Inventor 彭琼柏连阳罗为桂李祖任邬腊梅王立峰杨浩娜
Owner HUNAN AGRI BIOTECH RES CENT
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