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Method for preparing high-frequency regeneration plant of pink

A technology for regenerating plants and dianthus, applied in the field of plant biology

Inactive Publication Date: 2010-08-25
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the method of using the protoplast regeneration method to regenerate Dianthus plants at a high frequency

Method used

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  • Method for preparing high-frequency regeneration plant of pink
  • Method for preparing high-frequency regeneration plant of pink

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1. obtain the protoplast of carnation and Chinese carnation

[0082] Carnation (Dianthus caryophyllus, species is Tuareg, red flower) and Chinese carnation (Dianthus chinensis, species is D. Versicolor Fish ex Link, also known as Dianthus xingan) after amplification and propagation of test tube seedlings as starting materials, taken The newly proliferated top three pairs of stretched leaves are wilted for a short time, and the lower epidermis is gently torn off with tweezers or cut into thin strips with a width of about 1-1.5mm with a scalpel, and placed in CPW salt with 0.7mol / L mannitol The plasmolysis solution (pH 5.6) was separated for 1-1.5h, and then replaced with a filter-sterilized enzymatic hydrolysis solution [the composition of the enzyme solution was 2% cellulase (Cellulase Onozuka R-10), 0.5% isolated enzyme (Macerozyme R- 10), 0.5mol / L mannitol was dissolved in CPW salt solution]. Put it in a 24°C incubator for 12-14 hours in the dark for enzym...

Embodiment 2

[0085] Example 2. Protoplast culture of regenerative cells

[0086] Place an appropriate amount of protoplasts into six sterile glass petri dishes with a diameter of 6 cm, and inject 2 ml of liquid culture solution into each petri dish, so that the density of protoplasts is 2.5×10 5 cells / ml; seal with tape (Parafilm), and carry out liquid shallow layer dark culture (24°C), the medium is modified KM8p (KM8p+0.5mg / L 2,4-D+0.5mg / L NAA), culture 4 -After 5 days, part of the protoplasts began to grow walls, most of the cells were oval, and the regenerative cells could be seen to divide for the first time after 7 days of culture.

[0087] At this time, the division frequency of regenerative cells of Carnation was 6.7%, and the division frequency of regenerative cells of Chinese Dianthus was 5.8%. When cultured for 10 days, the division frequency of regenerative cells of Carnation reached 24.6%, and that of Chinese Dianthus reached 21.4%.

Embodiment 3

[0088] Example 3. Cultivate regenerated plants

[0089] During the cultivation process, 0.3-0.5ml of fresh sterile culture medium should be added every 10 days to maintain normal osmotic pressure and make the dividing cells continue to divide. When a small cell mass of 8-32 cells is formed, it should be added in time 0.5ml liquid culture medium with reduced osmotic pressure (MS culture fluid+1mg / L2,4-D+0.2mg / L NAA containing 1% (weight ratio) glucose and 2% (weight ratio) sucrose) 1-2 times, Thereafter, the regenerative cells of the protoplast can develop towards the direction of embryogenic cells.

[0090] When cultured to 95-100 days, you can see a large number of milky white, compact structure of small calli visible to the naked eye, use a glass pipette to suck them together to add 0.5mg / L BA and 0.2mg / L NAA, 0.2% Gelrite solidified On the MS proliferation medium, after 5-6 weeks of culture, when the callus grows to a diameter of about 3-4mm, the callus piece of this size ...

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Abstract

The invention relates to a method for preparing a high-frequency regeneration plant of pink, which comprises: 1, obtaining the protoplast of the pink; 2, subjecting the protoplast obtained by the step 1 to liquid shallow culture and enrichment culture to allow the protoplast to grow into regenerative cells and further into calluses; 3, subjecting the calluses obtained by the step 2 to bud initiation culture to grow seedlings on the calluses; and 4, subjecting a seedling obtained by the step 3 to rooting culture to grow the seedling into pink plant. The method of the invention adopts a protoplast culture technique and overcomes the technical drawback that it is difficult to obtain a regeneration plant of the pink from the protoplast of the pink in the prior art.

Description

technical field [0001] The invention belongs to the field of plant biology, and more specifically, the invention relates to a method for preparing high-frequency regeneration plants of carnation and Chinese carnation. Background technique [0002] Caryophyllales are dicotyledonous plants belonging to the order Cayophyllales. Widely distributed around the world, mainly in temperate or warm temperate regions of the northern hemisphere. Dianthus is a genus of Caryophyllaceae, native to Europe and Asia, and a small part extends southward to Africa. [0003] Carnation (Dianthus caryophyllus) is one of the four major cut flowers in the world. It belongs to the genus Caryophyllaceae and is a perennial root herb. Plant height 30-60cm, stem clustered, smooth, slightly white powder, with enlarged nodes on the stem. Opposite leaves, linear-lanceolate, entire, base clasping, with white powder and grayish green, with 3-5 obvious veins. The flowers are mostly solitary at the top of th...

Claims

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Application Information

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IPC IPC(8): A01H4/00C12N5/04
Inventor 朱木兰卫志明
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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