Method for establishing lily embryogenic callus regeneration system by using stamens as explants

An embryogenic callus and explant technology, which is applied in the fields of plant regeneration, botanical equipment and methods, horticultural methods, etc., can solve the problems of strict requirements for tissue culture disinfection, affecting the improvement of lily genetic engineering, and destruction of rare and endangered species, etc. To achieve the effect of good callus embryos, high embryogenicity and tender explants

Active Publication Date: 2014-05-21
HANGZHOU LANDSCAPING
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

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' stamens as explants

[0049] (1) Take 0.5m-long young lily flower buds at the flower bud stage, put them in sealed bags and refrigerate them 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. Use tweezers and a scalpel to separate the flower buds, and inoculate the filaments with anthers on a petri dish filled with callus induction medium, whichever is in contact with the surface of the medium; inoculate the medium inoculated with explants at 25 degrees Celsius, completely The callus began to appear after 40 day...

Embodiment 2

[0056] Example 2 Establishment of lily embryogenic callus regeneration system with oriental lily ‘Sorbonne’ stamens as explants

[0057] (1) At the flower bud stage, take 1.5cm-long young lily flower buds, 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 10 minutes, and then rinse with sterile water for 3 times. Use tweezers and a scalpel to separate the flower buds, and inoculate the filaments with anthers on a petri dish filled with callus induction medium, whichever is in contact with the surface of the medium; inoculate the medium inoculated with explants at 25 degrees Celsius, completely Callus began to appear after 25 days of cultivation under dark conditions, and the con...

Embodiment 3

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

[0065] (1) At the flower bud stage, young lily flower buds with a length of 2 cm were taken, sealed in a bag and refrigerated at 4 degrees Celsius. After refrigerated for a week, 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. Use tweezers and a scalpel to separate the flower buds, and inoculate the filaments with anthers on a petri dish filled with callus induction medium, whichever is in contact with the surface of the medium; inoculate the medium inoculated with explants at 25 degrees Celsius, completely The callus began to appear after 16 days...

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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 stamens 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 antheral filaments in the young bud are used as the explants, the pollution rate can be lowered to 0, the embryogenic callus inductivity of up to 15% can be obtained, the callus conglomeration reproducibility is 100%, the embryogenic property is high, and 6 stamens contained in each bud can obtain 60-120 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 with stamens as explants. 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 are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 张琳夏宜平沈柏春魏素芬
Owner HANGZHOU LANDSCAPING
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