A method for establishing a lily embryogenic callus regeneration system with pistil as explant

A technology of embryogenic callus and explants, applied in plant regeneration, botany equipment and methods, horticultural methods, etc., can solve the problems of strict disinfection requirements for tissue culture, affecting the improvement of lily genetic engineering, and destroying rare and endangered species. Achieve the effect of good embryogenicity of callus, high embryogenicity, and tender explants

Inactive Publication Date: 2016-08-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the embryogenic callus regeneration system of lily is also the decisive basis of the genetic transformation system of lily, which in turn affects the genetic engineering improvement of lily.
[0003] At present, the establishment of lily callus regeneration system mostly uses the scales of field bulbs as explants. The disadvantage is that the bulbs have been in the soil environment for a long time and carry many germs, which makes the tissue culture disinfection strict and the pollution rate is high.
In addition, when it is necessary to establish a laboratory in vitro system of wild lily germplasm, bulbs are used as explants, and bulbs can only be dug after flowering. There are two outstanding shortcomings: first, the determination of the location and species of wild lily. The identification largely depends on the eye-catching floral organs and their characteristics. After the flowering period, all or part of the aboveground parts of wild lilies have withered. Confusion; secondly, digging bulbs is equivalent to the complete collection of wild resources, and it is even more difficult to restore damage to rare and endangered species

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Establishment of lily embryogenic callus regeneration system with oriental lily 'Sorbonne' pistil as explant

[0049] (1) At the flower bud stage, take 1.7cm-long young lily flower buds with 1cm-long pedicels, refrigerate them in a sealed bag in a refrigerator at 4 degrees Celsius, and take them out after a week of refrigeration for processing.

[0050] (2) Soak the flower buds in tap water with detergent for 30 minutes, rinse them under running water for 75 minutes, clean them, and use 1% w / v sodium hypochlorite solution with 0.1% v / v Tween on the ultra-clean workbench , disinfect the surface for 8 minutes, and then rinse with sterile water 3 times. The pistil is stripped from the flower bud, the stigma is excised, and inoculated on the petri dish filled with callus induction medium made in step 2, wherein the inoculation of the pistil is based on the surface of the contact medium; The base was cultured for 8 days at 25 degrees Celsius in the dark, and callu...

Embodiment 2

[0056] Example 2 Establishment of lily embryogenic callus regeneration system with oriental lily 'Sorbonne' pistil as explant

[0057] (1) At the flower bud stage, take 2cm-long young lily flower buds with 1.5cm-long pedicels, seal them in a bag and refrigerate them in a refrigerator at 4 degrees Celsius for a week before proceeding to follow-up operations.

[0058] (2) Soak the flower buds in tap water added with detergent for 18 minutes, rinse them under running water for 30 minutes, and wash them with 1% (w / v) Disinfect the surface with sodium hypochlorite solution for 12 minutes, and then rinse with sterile water for 3 times. The pistil is stripped from the flower bud, the stigma is excised, and inoculated on the petri dish filled with callus induction medium made in step 2, wherein the inoculation of the pistil is based on the surface of the contact medium; The base was cultured for 13 days at 25 degrees Celsius in the dark, and callus clumps appeared, and the contamina...

Embodiment 3

[0064] Example 3 Establishment of lily embryogenic callus regeneration system with oriental lily 'Sorbonne' pistil as explants

[0065](1) At the flower bud stage, young lily flower buds with a length of 1 cm and a pedicel with a length of 1.3 cm were taken, sealed in a bag and refrigerated at 4 degrees Celsius. After a week of refrigeration, the explants were taken out for processing.

[0066] (2) Soak the flower buds in tap water added with detergent for 5 minutes, rinse them under running water for 120 minutes, and wash them with 1% (w / v) Tween added with 0.1% (v / v) v) Disinfect the surface with sodium hypochlorite solution for 15 minutes, and then rinse it with sterile water for 3 times. The pistil is stripped from the flower bud, the stigma is excised, and inoculated on the petri dish filled with callus induction medium made in step 2, wherein the inoculation of the pistil is based on the surface of the contact medium; The base was cultured for 16 days at 25 degrees Cels...

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Abstract

The invention relates to the technical field of quick reproduction of plants, and aims to provide a method for establishing a lily embryogenic callus regeneration system by using pistils as explants. The method comprises the following steps: material acquisition, callus-induced culture medium preparation, explant inoculation and callus induction, small plant regeneration, successive transfer culture of small plants, and transplanting of small plants. The method has the characteristics of low pollution rate, high callus inductivity, large callus conglomeration, favorable callus embryogenic property, high regeneration capacity and high reproducibility; especially, as pistils in the young bud are used as the explants, the pollution rate can be lowered to 0, the callus inductivity of up to 100% can be obtained, the callus conglomeration diameter can be approximate to 1cm after culture for 130 days, the callus conglomeration reproducibility is 100%, the embryogenic property is high, and each pistil can obtain 10-40 small plants on the whole.

Description

technical field [0001] The invention relates to the technical field of rapid plant propagation, in particular to a method for establishing a lily embryogenic callus regeneration system using pistil as an explant. Background technique [0002] Lilium embryogenic callus regeneration system has the highest proliferation rate and multiplication rate compared with other regeneration systems such as bud regeneration system, which is of great significance for the preservation of excellent germplasm. In addition, the embryogenic callus regeneration system of lily is also the decisive basis of the genetic transformation system of lily, which in turn affects the genetic engineering improvement of lily. [0003] At present, most of the lily callus regeneration systems are established with the scales of field bulbs as explants. The disadvantage is that the bulbs have been in the soil environment for a long time and carry many germs, so the requirements for tissue culture disinfection ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00
Inventor 张琳夏宜平张夏菲马怡迪杜方吴昀
Owner ZHEJIANG UNIV
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