Highly effective genetic conversion method for gum tree embryonic callus

Abstract

The invention discloses a highly-efficient genetic transformation method of the embryogenic callus of rubber trees, which is characterized in that anters of rubber trees or inner integuments which are used as explant are proceeded tissue culture, the culture is induced to produce embryogenic callus of rubber trees, then the embryogenic callus is proceeded successive transfer enrichment culture, and the embryogenic callus after enrichment is utilized as acceptor material to proceed genetic transformation. The successive transfer enrichment culture is proceeded to resistant embryogenic calli which is achieved after the transformation, and the resistant embryogenic calli after the successive transfer enrichment culture is induced the culture to produce embryo which further becomes plants in the process of embryoid induction. The invention has the advantages of simple art procedure, excessively high transformation efficiency and low cost, overcomes the serious shortcomings of extremely low percentage of immature embryos of the existing genetic transformation system of rubber trees, and the like, resolves the disadvantages that the achievement of the materials of transformation acceptor of rubber trees is easy to be restricted by natural conditions, such as seasons, and greatly reduces the transformation period, thereby the invention is an effective genetic transformation method of rubber trees and has great scientific value and application value.

Description

technical field [0001] The invention relates to a plant genetic transformation method, in particular to a rubber tree high-efficiency genetic transformation method using subcultured embryogenic callus as a transformation recipient material. Background technique [0002] In 1991, Arokiaraj from the Malaysian Rubber Research Institute injected the stem of 2-week-old isolated rubber seedlings with a hypodermic syringe into the carcinogenic Agrobacterium 541 / 71 for inoculation, and formed a tumor after 3 weeks, and the diameter of the tumor could reach 2cm after 8 weeks. After excised, it can grow independently on hormone-free MS basic medium, and TLC can be used to detect that the tumor tissue contains octopine induced by Agrobacterium. The formation of tumors involved the transfer of plasmids, and it was shown that genes that control certain metabolites (such as opines and plant hormone synthesis) carried by plasmids were expressed in tumor cells. This is the earliest report o...

AI Technical Summary

Problems solved by technology

From 1994 to 1998, Arokiaraj and others successfully transferred the genes of β-glucuronidase (GUS) and neomycin phosphotransferase into rubber by gene gun method and Agrobacterium-mediated method. It is still expressed normally in the line and shows a high degree of stability, which is an important feature required for transgenic animals and plants. The genetic transformation system of rubber has been initially established. The basic technical route of rubber conversion, but the conversion efficiency is extremely low

In 2000, Montoro et al. of the French Tropical Agricultural Research Center used the subcultured fragile inner integument dedifferentiated callus as the recipient for transformation, studied the role of calcium in the transformation of Agrobacterium rubber, but did not obtain transgenic plants

In 2000, Li Weiguo of the China Rubber Research Institute used the anther dedifferentiated callus as a receptor to introduce the cassava SOD gene into rubber, and obtained transgenic embryoids and bottle seedlings, but the transgenic bottle seedlings could not proliferate.

In 2005, Montoro et al. successfully obtained transgenic plants using the Agrobacterium-mediated method on the basis of dedifferentiated callus subculture in the rubber integument and related suspension culture techniques. However, a large number of vitrified seedlings were produced during the transformation process, and related tissue culture technology is not perfect

In 2006, Chen Xiongting from the Institute of Biotechnology of the Chinese Academy of Tropical Agricultural Sciences used the GUS gene as a reporter gene and the NPTII gene as a screening marker gene to bombard the dedifferentiated callus of rubber anthers with a gene gun for transformation experiments, and introduced the GAI dwarf gene into Brasilia nigra. The method of gene gun transformation of rubber callus was explored, and it was preliminarily determined that the GAI gene had been integrated into the rubber genome, but after the rubber callus was bombarded by the gene gun, the degree of browning was serious, and the embryoid body regeneration rate and emergence rate is relatively low

[0003] At the end of the 1970s, Chinese scientific and technological workers used isolated anthers to successfully cultivate pollen haploid and anther diploid plants for the first time. Now, regenerated plants can be obtained from anthers, inner integuments, and cotyledons through somatic embryogenesis. However, it is difficult to establish embryogenic cell suspension lines and transformation systems

[0004] For a long time, gene gun or Agrobacterium transformation using dedifferentiated callus as the receptor has been the basic method of the world rubber tree genetic transformation system, but the transformation efficiency of this method is extremely low, which has become a bottleneck restricting the research of rubber tree genetic engineering. The main factors that seriously affect the transformation efficiency are: the frequency of dedifferentiated callus induction to mature embryos is low; In the transformation operation, the callus is easily broken during the operation, and the toxic effect of Agrobacterium can easily lead to tissue browning and necrosis; the dedifferentiated callus is very sensitive to bacteriostatic agents and screening agents, and the bacteriostatic and screening processes are likely to affect the callus growth and differentiation

[0005] In addition, the practice of rubber tissue culture for many years has shown that it is difficult to induce normal embryoid bodies from callus cultured from other parts such as stems and leaves, and is generally not used for genetic transformation.