Tissue culture method utilizing Dianthus spp. cotyledon and application of tissue culture method in genetic transformation of Dianthus spp.

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a tissue culture method utilizing Dianthus spp. cotyledon and application of the tissue culture method in genetic transformation of Dianthus spp.. The method has the advantages of being high in propagation coefficient, high in propagation speed, good in rooting effect, simple, convenient and the like; and compared with a method for tissue culture rapid propagation by utilizing leaves to obtain regenerated plants, the method has the propagation coefficient as high as 32-35, has high propagation speed and shortens the culture period. A rapid propagation production technology is provided for the Dianthus spp., and industrialized production is carried out; and meanwhile, genetic transformation is carried out by the regeneration method, so that genetic transformation can be rapidly completed, transgenic plants are obtained, and a powerful technical platform is constructed for further carrying out agrobacterium tumefaciens-mediated Chinese dianthus transgenosis research.

Description

Technical field [0001] The present invention belongs to the field of plant gene engineering, and in particular, the present invention relates to a tissue culture method using stone bamboo leaves and its application in the genetic conversion of stone bamboo. Background technique [0002] Dianthus chinensis is a two-year herb and plants originated in China. It is widely used in fresh cut flowers, flower beds, garden greening, and rich floral advantages to improve economic value. In recent years, the group culture and fast-growing technology have made a lot of results, however, the regenerative rate is low, and the regeneration seedlings are more slender, and the industrialized group will quickly cultivate the difficulty of production. In the prior art, the graduation thesis of Chen Xu (2006) introduced the use of blades to establish Chinese stone bamboo to establish China Shizhu, which: the extent of plant blades in pre-culture groups (MS base medium) 3-4d from + 2, 4-D1.0mg / L + ...

AI Technical Summary

Problems solved by technology

In recent years, the tissue culture and rapid propagation techniques of Dianthus have achieved a lot of results. However, the regeneration rate is low, and the regenerated seedlings are relatively slender. It is difficult to cultivate production seedlings by industrial tissue culture and rapid propagation.

In the prior art, in the graduation thesis of Chen Xu (2006), it has been introduced to utilize leaves as explants to establish the genetic transformation system of Dianthus chinensis. +2,4-D1.0mg / L+6-BA 0.1mg / L) for 3-4d, and then transferred to differentiation medium (MS basal medium+NAA 0.3mg / L+6-BA 1.0mg / L), its regeneration rate is 68%, but it is greatly affected by the state of the leaves; in the patent "A Method for Preparing Dianthus High-frequency Regeneration Plants" (200910046388.8) published in 2009, the method for obtaining Dianthus regeneration plants is by means of protoplasts However, the method of obtaining protoplasts is complicated, and cellulase and pectinase are needed in the operation, which is costly; Zhang Jingjing (2011) reported in detail in her graduation thesis that firstly using leaves to induce callus, Furthermore, the genetic transformation system for differentiation and budding has low transformation efficiency. At the same time, direct regeneration of leaves is introduced, and the regeneration rate is 56%. The optimal differentiation medium is consistent with that reported by Chen Xu.

Shi Heping et al. (2013) reported in the article that Dianthuschinensis (Dianthuschinensi s) utilizes hairy root induction and its plant regeneration method, and its induction plant regeneration medium is: MS basal medium+NA A 0.02mg / L+6-BA 1.0mg / L, adventitious buds can be formed in 30 days, but this method needs to induce the generation of sufficient hairy roots first, then dedifferentiate them to obtain callus, and finally differentiate into new plants, complicated process

[0003] There are many ways to obtain regenerated plants. However, it is not difficult to find that the genetic transformation of Dianthus chinensis mediated by Agrobacterium is greatly affected by the state of the leaves of tissue culture seedlings. At the same time, another method has been reported, by preparing protoplasts The method of inducing regeneration is costly and inefficient

However, in other species, such as Su Xiaoyu (2007) found in carnations, the regeneration rate of regenerated plants was different when explants inoculated with different parts, and the genetic transformation efficiency was also greatly improved when Agrobacterium was transferred to different explants. difference

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