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Ultralow temperature preservation method and activity identification method for cardiocrinum giganteum var. yunnanense protoplast

A technology of cryopreservation and protoplasts, applied in the field of cell preservation of plant germplasm resources, to achieve the effect of ensuring genetic stability and survival rate

Inactive Publication Date: 2014-01-29
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no research report on the cryopreservation of protoplasts from Lily yunnanensis

Method used

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  • Ultralow temperature preservation method and activity identification method for cardiocrinum giganteum var. yunnanense protoplast
  • Ultralow temperature preservation method and activity identification method for cardiocrinum giganteum var. yunnanense protoplast
  • Ultralow temperature preservation method and activity identification method for cardiocrinum giganteum var. yunnanense protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The cryopreservation method of the protoplasts of Dalilia yunnanensis:

[0028] 1. Callus induction: use the immature embryos germinated from the seeds of Dalilia yunnanensis collected from Ailao Mountain in Yunnan Province as explants, wash the explants with water, soak them in 75% alcohol for 30 s, and then wash them with 0.1% alcohol mercuric chloride for 5 min, rinsed with sterile water for 3 times, and inoculated the sterilized explants on callus induction solid medium: MS+2mg / L KT+1mg / L 2,4-D+3% sucrose +0.7% agar powder, cultured in the dark at 23°C; callus was induced after 40 days. The medium was autoclaved at 121°C for 20 minutes;

[0029] 2. Separation of protoplasts from Dalilia yunnanensis: Take 1g of the above callus on the ultra-clean workbench, cut into about 0.2-0.3cm 2 Put the small pieces in a 10mL centrifuge tube, add the enzyme solution at a ratio of 1:10 between the material and the enzyme solution, and enzymatically hydrolyze it in the dark at 25°...

Embodiment 2

[0040] 1. Callus induction: use the immature embryos germinated from the seeds of Dalilia yunnanensis collected from Ailao Mountain in Yunnan Province as explants, wash the explants with water, soak them in 75% alcohol for 30 s, and then wash them with 0.1% alcohol mercuric chloride for 5 min, rinsed with sterile water for 4 times, and inoculated the sterilized explants on callus induction solid medium: MS+3 mg / L KT+2 mg / L 2,4-D +3 % sucrose + 0.7% agar powder, cultivated in the dark at 27°C; callus was induced after 40 days. The medium was autoclaved at 121°C for 20 minutes;

[0041] 2. Isolation of protoplasts from Dalilia yunnanensis: Take 2g of the above callus on the ultra-clean workbench, cut into about 0.3cm 2 Put the small pieces in a 10mL centrifuge tube, add the enzyme solution at a ratio of 1:15 between the material and the enzyme solution, and dissociate in the dark at 28°C for 12 hours, and occasionally shake it gently a few times during the process. Among them,...

Embodiment 3

[0052] 1. Callus induction: use the immature embryos germinated from the seeds of Dalilia yunnanensis collected from Ailao Mountain in Yunnan Province as explants, wash the explants with water, soak them in 75% alcohol for 30 s, and then wash them with 0.1% alcohol mercuric chloride for 5 min, rinsed with sterile water for 3 times, and inoculated the sterilized explants on callus induction solid medium: MS+2 mg / L KT+2 mg / L 2,4-D +3 % sucrose + 0.7% agar powder, cultured in the dark at 25°C; callus was induced after 40 days. The medium was autoclaved at 121°C for 20 minutes;

[0053] 2. Isolation of protoplasts from Dalilia yunnanensis: Take 1.5g of the above callus on the ultra-clean workbench, cut into about 0.2cm 2 Put the small pieces in a 10mL centrifuge tube, add the enzyme solution at a ratio of 1:10 between the material and the enzyme solution, and enzymatically digest it in the dark at 28°C for 9 hours, and occasionally shake it gently a few times during the process. ...

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Abstract

The invention discloses an ultralow temperature preservation method and an activity identification method for a cardiocrinum giganteum var. yunnanense protoplast. The ultralow temperature preservation method comprises the steps of (1) inducing a callus, (2) separating the cardiocrinum giganteum var. yunnanense protoplast, (3) purifying the cardiocrinum giganteum var. yunnanense protoplast, and (4) freezing, unfreezing, and obtaining the preserved protoplast. The activity identification method comprises the steps of identifying activity, nursing and culturing a protoplast solid, observing division of the protoplast under a microscope, culturing for 2-3 days to see a regenerated cell wall of the protoplast, culturing for 4-6 days to see a firstly-divided cell, and culturing for 30-45 days to see a regenerated cell mass of the protoplast. With the adoption of the ultralow temperature preservation method and the activity identification method, a novel scientific approach is provided for the long-term preservation of cardiocrinum giganteum var. yunnanense which is valuable, rare and endangered and integrates viewing, edible and medicinal values.

Description

technical field [0001] The invention relates to a cryopreservation method and an activity identification method for protoplasts of Dalilia yunnanensis, belonging to the technical field of cell preservation of plant germplasm resources. Background technique [0002] The common ornamental lilies in the market are Liliaceae Lilium plants, and in the large family of Liliaceae plants, there is also a little-known species of Lilium genus ( Cardiocrinum ) rare flowers, this genus got its name because of the hugeness of the plants, which is significantly different from the lily plants. There are only 3 species of Lilium plants in the world, and 2 species are produced in China. They are buckwheat leaf lily ( C. cathayanum ) and Lily ( C. giganteum ), and there is another variety of Dalilia, namely Dalilia yunnanensis ( Cardiocrinum giganteum var .yunnanense ). Lily of Yunnan originates in Ailao Mountain, Gaoligong Mountain and other places in Yunnan Province. It can be used ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N3/00C12Q1/06
Inventor 和凤美朱永平秦晓杰
Owner YUNNAN AGRICULTURAL UNIVERSITY
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