Efficient genetic transformation method for embryogenic callus of cotyledon embryo of rubber tree

Abstract

The invention belongs to the field of biotech, and relates to an efficient genetic transformation method for embryogenic callus of cotyledon embryo of rubber tree. The method comprises the following steps: conducting the tissue culture of the anther or the internal integument of rubber trees, using the cotyledon embryo as explant and carrying out induction and genetic transformation; then, inducing the transformed embryogenic callus into embryo and inducing the selected mature embryos into sprouts. The invention provides a simple-flow, extra-high efficiency, low-cost transformation method which overcomes the serious defects like low frequency of embryo production rate in rubber tree genetic transformation system and the long induction time of the embryogenic callus. Meanwhile, the invention also compensates the defect that the obtainment of transformed rubber tree acceptor materials is prone to natural conditions limitations and greatly shortens the transformation circle, thus making it a high-efficient genetic transformation method for rubber free. The invention is of great scientific as well as using value, and also provides a new technical route for the formation of transformation systems for rubber tree.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a plant genetic transformation method, in particular to a rubber tree high-efficiency genetic transformation method using sliced ​​embryogenic callus of somatic cotyledon embryos of rubber tree as transformation acceptor material. Background technique [0002] Hevea brasiliensis Muell.Arg is the main source of natural rubber, one of the four major industrial raw materials, and is also an energy plant. The chemical composition of natural rubber, rubber hydrocarbons, has been bio-improved and can be used as a substitute for fossil energy. Rubber trees are perennial, cross-pollinated plants, and the conventional breeding cycle is long, generally 30 years. At the same time, the genes within the rubber tree species are narrow, and it is difficult to make a major breakthrough in cross-breeding. Therefore, genetic engineering breeding has become the core content of rubber tree biotech...

AI Technical Summary

Problems solved by technology

Later, in 1994-1998, Arokiaraj and others successfully transferred β-glucuronidase (GUS) and neomycin phosphotransferase gene (NPTII) into rubber (GL 1 clone ), it is still expressed normally in the third-generation clones of proliferation culture, showing a high degree of stability, and this is an important feature that transgenic plants need to possess. The genetic transformation system of rubber trees has been initially established. Callus as the transformation recipient, and the use of NPT II as the screening gene for transformation have become the basic method for the transformation of Hevea brasiliensis, but the transformation efficiency is extremely low

In 2000, Montoro of the French International Agricultural Research and Development Center used the subcultured fragile inner integumentary callus and NPT II to screen genes to study the role of calcium in the transformation of rubber tree (PB 260 clone) Agrobacterium, but there was no transgenic plants

In 2006, Montoro et al. carried out suspension culture through the fragile callus in the integument of the rubber tree, and used the Agrobacterium-mediated method to transfer the GUS gene and the NPT II screening gene into the rubber tree (PB260clone) to obtain transgenic plants, but the transformation process There are a large number of vitrified seedlings produced, and the related tissue culture technology is not yet perfect

In 2006, Chen Xiongting et al. bombarded rubber anther dedifferentiated callus with gene gun to carry out transformation experiments, introduced GUS gene, NPT II gene, and GAI dwarf gene into Brasilia brasiliensis (Haiken 2 clone), and used gene gun to transform rubber tree callus. It was explored and preliminarily determined that the GAI gene has been integrated into the rubber genome, but after the rubber callus was bombarded by the gene gun, the degree of browning was relatively serious, and the embryoid body regeneration rate and seedling emergence rate were relatively low

At present, the main problems in the cultivation of anthers and inner integuments are: the explants are limited by the flowering and fruiting time of rubber, the embryo formation rate of dedifferentiated callus is low, and it is difficult to preserve for a long time, so the plant regeneration frequency is low. The production cost is high, and it is difficult to meet the large demand for tissue culture seedlings in production and the needs of biotechnology experiments such as establishing an efficient genetic transformation system by relying on somatic embryonic plant stem section micropropagation technology in the later stage