Genetic transformation using engineered plant chromosomes

Poornima K N 9869

Introduction
Food security through better crops - achieved by continuous up gradation of genomic technologies and fine tune existing ones.

Bioengineering- plant resistance to herbicides, insects , viruses as well as entire biosynthetic pathways.

Milestone
Plant transformation methods Agrobacterium mediated and biolistic methods revolutionized crop improvement. Technical challenges:
Transfer of only single or few genes Insertion at random sites Gene silencing Linkage of transformed genes to selectable markers Difficulty in transfer of several genes togetherbiosynthetic pathways.

Solution
Engineering nonintegrating minichromosomes
especially for transfer of several genes

Minichromosomes: A broad range of small chromosomes with all elements essential for their replication and autonomous existence within a cell.
In E.Coli Small circular plasmid with oriC. In eukaryotes often linear and require functional centromere, telomere in addition to ori and exist separately from the normal karyotype of a cell.

Engineering minichromosomes in plants

Two approaches
Top-down: Engineering of existing chromosomes Bottom up: De novo assembling of chromosome

Basis for minichromosome formation

Ways to generate minichromosomes:
Gamma irradiation - broken chromosomes heal during DNA repair. (Brock and Pryor., 1996) Breakage-fusion-bridge (BFB) cycle- de novo repair of broken chromosome ends result in formation of minichromosomes. (Zheng et al., 1999)

Current Opinion in Biotechnology 2007, 18:425431

Principle of top-down approach

Minichromosomes are generated by inducing truncation of endogenous chromosome followed by de novo telomere synthesis. These minichromosomes require subsequent targeting with transgenes to be used as vectors or platforms for biosynthetic pathways. Telomere-mediated truncation using engineered construct offers distinct advantage that minichromosome formation is coincident with transgene delivery.

The Plant Cell, Vol. 19: 23232327, August 2007

Objectives
Development of minichromosomes from truncated maize A chromosomes Development of minichromosomes from truncated maize B chromosomes Test gene expression from Normal B and MiniB chromosomes Test transmission of minichromosomes Test for site-specific recombination of minichromosome.

Minichromosomes from truncated Maize A and B chromosomes

2.6kb

Chromosomal truncation constructs

YU et al., 2006, PNAS , November 14, vol. 103 , no. 46 , 1733117336

Maize A minichromosome
Chromosomal truncation by Agrobacterium-mediated transformation using truncation constructs Minichromosome (MC) obtained from maize chromosome 7 long arm truncation MC recovered in spontaneous tetraploid production named as R2

R2 crossed to diploid gave triploids with MC (no gametophyte abortion)

Triploids crossed to diploids gave aneuploids that yielded diploids with MC when crossed to normal diploids R2 never paired with chr 7 or any other chr due to lack of pairing . Therefore minimal chances of recombination with normal set depicting its use for chromosome engineering. R2 showed stability during mitosis and meiosis.

Minichromosome R2 produced by telomere truncation of an A chromosome

Pachynema

Diakinesis R2 in meiotic cells

Metaphase I

Homozygote of R2

PNAS , May 22, 2007 ,vol. 104 no. 21 , 8925

Minichromosomes from maize B chromosome

B chromosome based MC preferred for chromosome engineering than A MC : No developmental or transmission problems as they are inert. Recombination of B chr with the A set never been observed The insert size may not be crucial as there will be no residual endogenous genes that might interfere with plant development and transgene expression. Amenable for dosage manipulation

Experiment
Biolistic transformation of immature embryos with avrg 3.3 B chromosomes using truncation constructs + pHAC25 plasmid 281 transgenic events bialophos-resistant calli

7 fragments from A chromosome 7 A-B translocations

45 events with transgenes on 55 normal B or miniB chr 10 truncated B chr without transgene signal

86-14

86B93

86B23

MC at pachynema and anaphase I

Normal B and miniB chromosomes

Progeny of MC

Gene expression from normal B and miniB chromosome

FISH screening of root tips of regenerated seedlings using pWY96 and pAHC probes separately showed trangenes integration at same location in 84% of transformants. 17 transgenic events had only transgenes on B chromosome 9 of the 17 transgene expression confirmed by GUS expression. GUS expression - leaves, roots, shoots, and mature kernel embryos and endosperm. GUS expressed from introduced genes on B and miniB chromosomes in both embryo and endosperm tissues has significant agricultural implications demonstrating foreign gene expression in the kernel, the major harvested product.

Transmission of MC
Meiotic transmission varied from 12% to 39% through male parent enables pollen selection system. The non disjunction property of B type MC provides a mechanism to create a dosage series of the engineered construct for increased expression of the resident genes.

Recombination of MC

Cre/lox-mediated site-specic recombination Red fluorescence protein expression from recombination

Sequence alignment of two recombination products amplified by nested PCR

Plant Cell Rep (2011) 30:799806

Bottom-up approach
Two reports
Carlson et al. introduced a circular DNA with maize centromeric repeat sequences, selection marker gene, a phenotypic marker gene and filler DNA. Functioned in vivo as maize minichromosome (MMC) with no clear autonomous nature. But high frequency of meiotic transmission (50% as hemizygote and ~93% as homozygote ) suggesting it may have integrated into the maize genome. Anaiev et al. In maize- introduced circular constructs without telomere repeats only resulted in stable integrations into the chromosomes.
Further studies are needed to confirm feasibility of this approach in plant systems
PLoS Genetics, October 2007 | Volume 3 | Issue 10 | e179

Applications
Minichromosomes can serve as a platform for multiple, large, complex, regulatory transgene transformation. An extra chromosome would allow plants to be used as a factory for the production of multiple foreign proteins and also as efficient tool for gene stacking. Minichromosomes could also be used to add/modify whole biochemical pathways to plants. Engineered chromosomes can be transferred to other crop plants by conventional hybridization without problems of cis linkage drag or position effects. Excellent platform for studying gene expression , organization of centromeres , genome dosage and other basic phenomena like recombination. MC allow for copy number control, site specific recombination and rapid removal of genes when they are no longer needed in a particular genetic background.

Questioned to be answered in future

Optimal conditions for formation, transmissibility of engineered plant MC. Size restrictions for MC stability and

Maximum copy number that could be maintained in the cell and its effect on transgene expression.
Can extensive and elaborate biosynthetic pathways be built.

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