Agrobacterium-mediated transformation of rice Xiangbing meng 2009.04.02
Outline • General transformation steps • Model of Agrobacterium-mediated genetic transformation • selectable marker and promoter • Factors affecting transformation frequency • Transgene integration and expression • Other transformation technology • Application • Marker-free transgenic plants
Summary of rice transformation • Chan et al. (1992, 1993) showed regeneration of Agrobacterium-transformed calli from root explants and immature embryos. • Already about 40 different genotypes of indica, japonica and javanica rice have been transformed using this approach (2006). • Several useful traits have been introduced in rice by using Agrobacterium, including genes for abiotic stress tolerance, biotic stress tolerance, herbicide tolerance, nutritional enhancement, enhanced photosynthesis and rice functional genomics.
These seedlings come from one resistant callus and are defined one line.
Transformantion elements • Explant • Agrobacterium tumefacien and Agrobacterium rhizogenes • DNA: plasmid; DNA fragment • Selection marker and promoter
Agrobacterium • Agrobacterium tumefaciens: The Gram-negative soil bacterium as pathogen results in crown gall tumors in plants. T-DNA, a part of the bacterial tumor-inducing (Ti) plasmid, is transferred from the bacterium to the plant cell with the aid of a number of Virulence (Vir) proteins encoded by the Ti-plasmid. 根癌农杆菌 • Agrobacterium rhizogenes: Root inducing (Ri) plasmid.发根农杆菌
A model for the Agrobacterium-mediated genetic transformation Recognition and attachment Vir genes expression by host signals T-strand produce T-complex export into host Transport through cytoplasm and nuclear T-DNA uncoating and integration.
Overexpression of key host proteins is useful for increasing the transformation efficiency of model plants. • histone H2A ,uncoating Vir proteins • VIP1 ,a plant protein specifically interacts with VirE2 and is essential for T-DNA nuclear import. • VirB2-interacting protein (VirB2 is the main T-pilus protein).
Selectable marker genes • The systems to select the transformed cells, tissues or organisms from the non-transformed ones are indispensable and selectable marker genes are vital to the plant transformation process. Marker genes enable the transformed cells to survive on medium containing the selective agent, while non-transformed cells and tissues die. • Antibiotics (kanamycin or hygromycin) and herbicide (phosphinothricin, PPT) are mostly used. • Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional in the presence of external substrates.
A conditional-positive selection system consists of a gene encoding for a protein, usually an enzyme that confers resistance to a specific substrate that may be toxic to the untransformed plant cells or that facilitates the growth as well as differentiation of the transformed cells alone. This system includes antibiotics, herbicides, toxic and non-toxic drugs or metabolite analog or a carbon source. • Non-conditional-positive selection systems do not require external substrates but promote the selective growth and differentiation of transformed cells.
Selectable marker genes commonly used in cereal transformation
Plant origin selectable markers • EPSP synthase (Petunia hybrida), aroA (Zea mays ) • Acetolactate synthase (Arabidopsis thaliana ) • 5-MT : ASA2 (Tobacco ), OASA1D (Rice) • 5MT/Cadmium cholride: Tryptophan synthase (Arabidopsis TSB1) • AtTPS1 (Glucose ) • kn1 (Hormone-free medium ,Maize ) • Arabidopsis thaliana ATP-binding cassette (ABC) transporter gene , Atwbc19, confers antibiotic (kanamycin) resistance to transgenic plants.
Promoters • Transgenic rice with multitraits need different promoters. • CaMV35S, rice ACT1 and maize UBI1 gene promoters have been used extensively to drive high and constitutive expression of transgene in rice. • Promoter of rice cytochrome c gene was highly active in almost every plant tissue. It was even more active than rice ACT1 gene promoter in leaves, root, embryo and calli (2002). • Activity of the OsAct2 promoter region is much higher than that of the OsAct1 promoter region(2009).
Inducible or tissue or stages specific promoter • Promoters of rice POXA and POXN were found to be root-specific and their expression was also enhanced in the leaves on UV and wounding treatment. • Wound inducibility has also been reported in promoter of MPI gene. Use of this promoter to drive the expression of cry1B in transgenic rice showed better performance. • Endosperm-specific, root/shoot-specific, seed-specific, anther-specific, and early development stage specific promoters. Seed storage glutelin pmoters, GluA,B,C, containing endosperm specificity-determining motifs(GCN4,AACA,and P-box). • The specific promoter activity is controlled by introns in the case of rice α-tubulin gene family.
Factors for transformation frequency • Ti plasmid type and binary vector • Selectable marker • Bacterial strains: A. rhizogenes (LBA9402 and Ar2626) and A. tumefaciens (LBA4404 and EHA101, EHA105 from EHA101, AGL0 and AGL1 from LBA4404). • Inoculation and co-cultivation medium, culture conditions prior to and during inoculation et al.. • Activation of T-DNA transfer process by exogenously added acetosyringone. • Genotype and explant are considered to be the major limiting factors.Strong influence on the transformation frequency is exerted by the plant genetic background in indica rice. The genotypic influence is often overcome by modifying the nutrient medium or transformation conditions.
Transgene integration and expression • The integrated DNA was stable and inherited in a Mendelian fashion in the majority of Agrobacterium-derived lines. • The stable inheritance and expression of foreign genes are of critical importance in the application of genetically engineered cereals to agriculture. • The factors contribute to variation in transgene expression: tissue culture-induced variation or chimerism in the primary integration site (position effects), transgene copy number (dosage effects), transgene mutation and epigenetic gene silencing. • A pre-determined genomic locus can be achieved by the useof site-specific recombinase systems, such as Cre/lox and FLP/frt. • MARs (matrix attachment regions ) are DNA elements that arethought to influence gene expression by anchoring activechromatin domains to the nuclear matrix. • Devoidof vector backbone sequences to produce so-called ‘clean’ transgenic plants: using minimal linear transgene constructs (promoter, coding region of the gene and terminator). Fu x.D. et al.,2000. • Transgene expression can be controlled temporally and spatially by the use of cell- and tissue-specific or chemically inducible promoters.
Particle bombardment • The microprojectile bombardment or biolistics genotype independent and less labor intensive. • But: arrangement of multiple copies of transgenes, particularly in the form of inverted repeats and problem of high copy number of the transgene. • The problem of rearrangement also has been overcome up to large extent using minimal linear cassette (including promoter, open reading frame and terminator only) to coat microcarriers. Such ‘clean gene’ technology would be of great importance in avoiding undesirable effects of vector backbone.
Other methods • Rice protoplasts can be transformed with naked DNA by treatment with PEG in the presence of divalent ions such as calcium. protoplasts are not easy to work with and regeneration of fertile plants is problematic. • pollen tube pathway • LASER , imbibitions of embryo or seeds in the presence of DNA and WHISKERSTM. • needle dipped in Agrobacterium culture to prick the seed’s embryonic portion. • Bioactive beads-mediated transformation (100 kb large DNA fragment).
Enhancement of stress tolerances • Insect resistance:proteinase inhibitor, lectin, Insecticidal protein( cry). • Disease resistance: Xa1 to Xa29, OSWRKY71,resistance (R) gene, Pathogenesis-related (PR) genes, CP orreplicase-mediated resistance to virus infection. • Abiotic stresses: various compatible solutes (glycine betaine, trehalose, proline, and polyamines), Late embryogenesis proteins, membrane transporters, regulators of signal transduction or transcription. • Herbicide Tolerance: bar;P450 monooxygenase and glutathione S-transferase
Improvement of grain quality • Nutritional Enhancement: Milled rice contains no β-carotene (provitamin A) daffodil phytoene synthase gene. Golden rice, phytoene synthase (PSY) and lycopene β-cyclase (LCY) genes from daffodil and phytoene desaturase (crt1) from bacterium, Erwinia uredovora • Alteration of Starch Content: WX genes.
Yield improvement • ADP-glucose pyrophosphorylase in transgenic rice had been shown to increase seed weight per plant. • Transferring of C4 plant genes in C3 plants • Arabidopsis phytochrome A gene could increase the seed yield by 6–21% in transgenic rice (Garg et al., 2006). • A link between phytohormones (GA, cytokinin, brassinosteroids) metabolism and grain yield has been established in rice (Sakamoto, 2006).
Control of Plant Development • Flower development: OSMADS • Overexpression of floral control gene LEAFY in Arabidopsis is sufficient for the transformation of lateral shoots into flowers and causes early flowering • Plant architecture:OSTB1, teosinte branched 1 homologue in rice, has been found to be a negative regulator for lateral branching in rice • MONOCULM 1 • Histone acetylase • Expansins
Production of Novel Compounds • Antibody (ScFvT84.66) • Industrial valuable enzyme transglutaminase • Rice cell suspension culture systems also provide an alternative to animal/bacterial cell lines for the production of recombinant compounds for human use as risk of contamination is very low.
Environmental and biosafety aspects • Environmental release and biosafety have been subjects of debate regarding transgenic plants, especially for food crop like rice. • Demand for marker-free transgenic plants is high.
Marker-free transgenic plants • Excise or segregate marker genes from the host genome after regeneration of transgenic plants • Based on the strategy called marker-free transformation.
cre/lox recombination system • FLP/FRT recombination system • AC/DS transposon system • Twin T-DNA binary vector • Co-transformation • multi-autonomous transformation (MAT) vector, making use of Agrobacterium oncogenes as a positive selection marker.
Cre/loxP recombination system M: marker gene; GOI: gene of interest
Ac transposon-based expelling of nuclear marker genes M: marker gene; GOI: gene of interest
Homologous recombination based removal chloroplast marker M: marker gene; GOI: gene of interest
a dual binary vector system, pGreen/pSoup. pGreen is a small Ti binary vector that can only replicate in the presence of pSoup in the same bacterial strain. Co-transformation with both the vectors, one carrying the transgene and other carrying the selectable marker, followed by segregation in subsequent generation will produce marker-free plants. • The ‘clean gene’ approach, which involves only minimum linear cassette (promoter gene and terminator) • Recent studies have shown that plants have T-DNA border-like sequences in rice and Arabidopsis plant DNA (P-DNA) lacks any open reading frames and contains a high A/T content, it is likely the footprint of ancient Agrobacterium-mediated natural transformation events via horizontal gene transfer.
Co-transformation • Separate transformation of marker and transgene introduction of two T-DNAs, in separate Agrobacterium strains or biolistics introduction of two plasmids in the same tissue; • Introduction of two T-DNAs carried by different replicons within the same Agrobacterium strain; • Introduction of two T-DNAs located on the same replicon within an Agrobacterium • sexual reproduction • time consuming
negative selection • Removal of chloroplast marker genes: transgenes in plastids would not be disseminated via pollen. Mitochondria and chloroplasts have independent genomes. • positive selection systems:
Existing problems and future prospects • Tissue browning and necrosis after Agrobacterium infection are still major obstacles in the genetic transformation of cereals. • On pathogen infection, one of the earliest defence mechanisms activated is the production of reactive oxygen species, referred to as an oxidative burst, which activates programmed cell death (HR). A correlation between the reduction in cell death and the improved transformation frequency has been demonstrated. Necrosis inhibiting agents, such as silver nitrate, increased efficiency of transformation. • Suppression of the host defence response is a prerequisite to successful plant transformation.
Conceptions • Transformation frequency: Independent transgenic plants/inoculated immature embryo or callus. • Line: The seedlings from one transgenic cell, namely, one independent successfully transformation event, belong to one line, also are called clones. In fact, the seedlings from a piece of transgenic callus are called one line, even though several transformation events were happened. For this reason is that usually only one transgenic cell can successfully pass the selection, differentiation and finally become seedlings. • Generation: These transgenic seedlings are T0 generation seedlings, T0 generation mature seeds are T1 generation…Meiosis or pollination is the boundary of different generations.
Reference for highly efficient protocols for Agrobacterium tumefaciens-mediated transformation. • Yukoh Hiei & Toshihiko Komari. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nature Protocols 3, 824 – 834 (2008).