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May 3, 2023 Department of Genetics and Plant Breeding 1
WELCOME
May 3, 2023 Department of Genetics and Plant Breeding 2
Poornima.R.N.PALB 4214
Senior M.Sc
May 3, 2023 3
Flow Of Seminar
Department of Genetics and Plant Breeding
Introduction. Need of Synthetic Chromosomes. Requirements for Synthetic
chromosomes. Methods to develop Synthetic
chromosomes Advantages and limitations. Application of Synthetic
chromosome technology in crops- Case studies.
Future Aspects. Conclusion.
May 3, 2023 Department of Genetics and Plant Breeding 4
Food In Fifty Years…??
May 3, 2023 Department of Genetics and Plant Breeding 5
Why make Synthetic chromosomes…???
May 3, 2023 Department of Genetics and Plant Breeding 6
Synthetic Chromosomes
Knowledge
Products
May 3, 2023 Department of Genetics and Plant Breeding
Need of Synthetic/Artificial chromosomes
Direct method
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May 3, 2023 Department of Genetics and Plant Breeding
• Gene stacking- difficult• Transgene position
effects
First Generation Genetic Engineering
Second Generation Genetic Engineering
Synthetic Chromosomes
Delivery of large DNA sequences
Complete metabolic pathways
Genetic changes 100-kb to megabases (Mb)
Need.....???
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May 3, 2023 Department of Genetics and Plant Breeding
Synthetic chromosomes are chromosome based non-integrating vector system that is transmissible and suitable for transfer of large genes, gene complexes and/or multiple gene together with regulatory element for safe, controlled and persistent gene expression
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03/05/2023 DEPARTMENT OF PLANT BIOTECHNOLOGY 10
Demonstrated the successful use of telomere truncation in maize plants to produce minichromosomes (2006)
created a synthetic chromosome from yeast paving the way for a new
field of study(2014)
Pillars of this new technology
James A Birchler
J. Craig Venter
May 3, 2023 Department of Genetics and Plant Breeding
“This has been sought for a long time in the plant world, and it should open a number of new avenues. If we can do this in plants, a number of advances could be done in agriculture that would not otherwise be possible, from improved crops to inexpensive pharmaceutical production to other applications in biotechnology”
James BirchlerMU College of Arts and Science
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May 3, 2023 Department of Genetics and Plant Breeding
Requirements for Synthetic Plant Chromosome
Basic requirements for Synthetic Plant Chromosome
1.Centromere
2. Telomere
3. Sufficient Chromatin
4. Selectable Marker transgene
5.Site specific recombination system
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May 3, 2023 Department of Genetics and Plant Breeding
Centromere Components In Plants
Centromere – Requisite component of SC
Contains tandem repeated sequences of various sizes, ~1 to 3 Mb.
CENH3,the Histone H3 varient of plants typical of active centromeres, associates with this repeat region. Retrotransposons (Maize-CRM elements)
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May 3, 2023 Department of Genetics and Plant Breeding
Telomere Components In Plants
Specialized structure which cap the ends of eukaryotic chromosome.
Consist of highly conserved long array of short tandemly repeated sequences. e.g. TTTAGGG in A. thaliana
TTAGGG in Homo sapiens TTAGG in insects
Average length: 3-40 kb (Burr et al., 1992) Functions: 1. Maintaining the structural integrity
2.Ensure complete replication of extreme ends of chromosome
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May 3, 2023 Department of Genetics and Plant Breeding
Methods To Develop Synthetic Chromosome
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May 3, 2023 Department of Genetics and Plant Breeding
Bottom up method
• de novo assembly of cloned chromosomal components, such as
centromeric and telomeric sequences, a selective marker gene and genomic DNA that contains a replication origin.
This method is well established inYeast (Murray and Szostak, 1983) Mammalian cells (Harrington et al.,1997)
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Reasons for failure
• Construction yeast artificial chromosome is used as a model for bottom-up method.
• Centromere specification in yeast is unusual among eukaryotes and Centromeres in higher eukaryotes have diffuse organization (Henicoff et al., 2001).
e.g. Centromeric repeats of barley (Hordeum vulgare) have been shown to be neither necessary nor sufficient to establish a functional centromeric activity (Nasuda et al., 2005) .
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Epigenetic Aspects Of Centromere Specification
• Mechanism by which centromeres are established, maintained,
and function remain a mystery.
• A chromosome was found that did not contain detectable
centromere repeats but till organized a kinetochore at a specific
site that was associated with centromere activity in barley
(Nasuda et al.,2005).
• Substitution of the histone H3 by CENH3(CENP-A) in
centromeric nucleosomes is crucial for kinetochore formation.
18Department of Genetics and Plant BreedingMay 3, 2023
So what is the solution…???
May 3, 2023 Department of Genetics and Plant Breeding 19
May 3, 2023 Department of Genetics and Plant Breeding
Top down methodBased on chromosome fragmentation or truncation.
This method utilizes the insertion of telomere sequences into existing chromosomes.
This sequence signals for new telomeres which causes the truncation of the chromosome.
insertion of new genes for desired traits.
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May 3, 2023 Department of Genetics and Plant Breeding
Telomere-mediated Truncation
Aim- To whittle away the chromosome arms using transformation of telomere repeats.
It bypasses the complications of the epigenetic aspects of centromere specification.
It works robustly in plants & can be used to produce engineered minichromosomes with endogenous centromeres.
Construct- Genes of interest, Site-specific recombination cassettes, Telomere repeats.
(Yu et al., 2006)
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May 3, 2023 Department of Genetics and Plant Breeding 22
Adding of genes to a engineered minichromosome
This approach of telomere mediated chromosome truncation was first shown by Farr et al.,1991 i.e. introduction of cloned telomeric repeats into cultivated cells may truncate the distal portions of chromosomes by the formation of new telomeres at integration sites in mammalian cell lines.
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Type of truncation
Description Pros Cons
Truncation of Bchromosomes
In plant species that harbour Bs, truncation of B chromosome arms can be selected.
Loss of chromosome segments from Bs does not affect the viability of the plant.
Bs are not present in all plant species. This approach is applicable only to those speciesthat have or will accept Bs.
Truncation of Achromosomes in
tetraploid background
Truncation of Achromosome can be rescuedin a tetraploid background.
Tetraploid plants can tolerate truncation of chromosome arms. This approach will work readily in natural polyploids.
Tetraploids would need to be generated in diploid species.
(Gaeta et al.,2012)24Department of Genetics and Plant BreedingMay 3, 2023
25Department of Genetics and Plant BreedingMay 3, 2023
A chromosome truncation B chromosome truncation
Particle bombardment
Truncating plasmid
Telomere truncation of different types of chromosomes
(a) A chromosome
(b) B chromosome
(c) Telocentric chromosome
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27Department of Genetics and Plant BreedingMay 3, 2023
Minichromosomes
• extremely small version of a chromosome.• By minimizing the amount of unnecessary genetic information
on the chromosome and including the basic components necessary for replication,we can construct this chromosomal plotform,so that we can insert new genes.
28Department of Genetics and Plant BreedingMay 3, 2023
B Chromosome Based MinichromosomesB chromosome based minichromosome is interesting-Reasons:- 1. Naturally occurring supernumerary chromosome
2. Basically inert, small size – no phenotype (Jones et al., 2003) 3. No developmental & transmission problem
4. Easy detection of B chromosome derivatives (Kato et al., 2005) 5. No report of recombination with A chromosome set
Minimal detrimental effect on host genome
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May 3, 2023 Department of Genetics and Plant Breeding 30
Generalized scheme for the production of engineered minichromosomes in maize
J. A. Birchler et al.,2010
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Maintenance Of mini B chromosomes in the population
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Advantages and Limitations
• Fecilitates in understanding fundamental questions about chromosomal structure and function.
• Synthetic chromosomes in plants are likely to have more applications than in other taxa.
• This approach for construction of engineered chromosomes can be easily extended to other plant species
• allows for the stacking of genes side-by-side on the same chromosome thus reducing likelihood of segregation of novel traits.
Advantages
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• Functional genomic studies could use minichromosomes as a platform for adding specific genes
• Next generation vectors for human gene therapy & plant genetic engineering
• stable expression & maintenance of multiple transgenes in one genome.
• Mass production of foreign proteins, pharmaceuticals, or useful metabolites.
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Limitations
• Small chromosomes do not always pair homologously in meiotic prophase.
• low meiotic transmission rate.
• pollen abortion.
• Regeneration of plants is difficult.
May 3, 2023 Department of Genetics and Plant Breeding 35
Case Studies
May 3, 2023 Department of Genetics and Plant Breeding 36
May 3, 2023 Department of Genetics and Plant Breeding 37
• In this report, they demonstrated that 2.6 kb of Arabidopsis telomeric repeats were efficient for telomere-mediated chromosomal truncation in maize.
• They also showed that internally integrated telomeric sequences are stable in the genome.
May 3, 2023 Department of Genetics and Plant Breeding 38
May 3, 2023 Department of Genetics and Plant Breeding 39
Constructs Transgenic plants
Transgenic loci
pWY76 93 123(57 at distal loci)
pWY86 83 108 (61 at distal loci)
pWY96 44 58 (11 at distal loci)
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Cytological detection of chromosomal truncations
pWY86 pWY76
pWY86 pWY86
B77
T87
B37B44
May 3, 2023 Department of Genetics and Plant Breeding 41
Restriction mapping of the positions of transgenes by a Southern blot.
May 3, 2023 Department of Genetics and Plant Breeding
Summary• 2.6-kb direct repeat of Arabidopsis telomeric sequence
was used in two constructs, pWY76 and pWY86, to test the ability of telomeric sequences to cause chromosomal truncations.
• Direct evidence for chromosomal truncation came from the results of FISH karyotyping, which revealed broken chromosomes with transgene signals at the ends.
• This technology will be useful for chromosomal engineering in maize as well as other plant species.
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May 3, 2023 Department of Genetics and Plant Breeding 43
EXPERIMENTAL PROCEDURES
Plant material: Embryogenic calli derived from Immature embryos of rice telotrisomic line with Telo-12L derived from indica rice cultivar Zhongxian 3037.
Callus induction: • Immature embryos were cultured on N6D2 medium at 28 ͦC in
the dark for callus induction.• Embryogenic calli were subcultured every 2–3 weeks and used
for transformation by bombardment.
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Plasmid construction
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These three plasmids are mixed together in the ratio of 5:1:1
Gene transformation:• 1.5 mg 0.6 μM gold particles were coated with 2 μg plasmid mixture
and bombarded to rice calli with a PDS 1000/He particle delivery system.
• After 16 hours,the calli were transferred onto N6D2 medium supplemented with 25 mg/L hygromycin and cultured at 28 ͦC in the dark for 2 weeks.
• and then selected by subculture on N6D2 supplemented with 50 mg/L hygromycin every 3 weeks until the resistant calli emerged.
• The resistant clones were maintained on N6D2 medium supplemented with 25 mg/L hygromycin at 28 ͦC by subculture every 3 weeks.
Chromosome preparation from callus• Fast growing calli on selection medium after 7 days subculture were
selected to check chromosomal truncation and mini-chromosome formation in transgenic rice using FISH.
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(b)Transgenic clone with one distal signal
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(a)Transgenic clone with one internal signal
Fluorescence in situ hybridization(FISH) detection of transgenes
Rice transformation and transgene distributions
No. ofbombardedcalli
No. of resistance clones
No. ofTransgenic eventschecked
No. ofTransgene signals
No ofinternaltransgenes
No. ofdistaltransgenes
Ratio (%)of distaltransgenes
Control (a) 519 44 19 32 31 1 3.1
4–6-month-old calli (b)
4512 536 122 178 121 57 32.0
18-month-old calli (c)
854 111 94 138 84 54 39.1
Total no. (b + c)
5366 647 216 316 205 111 35.1
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The control was bombarded with only pCAMBIA1301
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(a–c) Mini-chromosomes with one transgene signal: (a) Clone 1008-100(b) Clone 1004-111(c) Clone 1004-015.(d) Clone 1004-011, mini-chromosome with transgene signals at both ends of the chromosome.
Rice mini-chromosomes from telomere truncation.
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Clone 1004-111
• One chromosome 12 and one Telo-
12L are shown in this clone.
• The mini-chromosome
(arrowhead) is probably originated
from chromosome 12 truncation
Origin of mini-chromosomes
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Clone 1008-100
• Three chromosomes 12L are identified
• Original Telo-12L was not found.
• The missing 12L is probably truncated to
produce the mini-chromosome
(arrowhead).
• Chromosome 12 is probably duplicated
to produce an extra chromosomes 12.
03/05/2023 DEPARTMENT OF PLANT BIOTECHNOLOGY 52
Distal transgene in control without
telomere sequence
Distal transgene with telomere
sequence Maize
chromosomal truncation
19.0 %
46.3-56.5 %
Rice chromosomal truncation
3.1 %
35.1 %
These results were compared with previous report of maize chromosomal truncations by Yu et al.,2006
Summary
• Telotrisomic rice line was used for mini-chromosome construction.
• minichromosomes were recovered from transgenic clones by monitoring hygromycin-resistant calli with FISH.
• All these mini-chromosomes were maintained stably in cell cultures for over 2 years.
• The construction of mini-chromosomes in rice will provide a platform for future artificial chromosome-based genetic engineering of this plant for multiple gene expressions.
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May 3, 2023 Department of Genetics and Plant Breeding
Future Direction
• develop a mini B chromosome-based genomic cloning system.
• combination with haploid breeding.
• Genome editing techniques. • Deliver genes that benefit the
agricultural, nutritional, energy and pharmaceuticals sectors.
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May 3, 2023 Department of Genetics and Plant Breeding 55
Conclusion
May 3, 2023 Department of Genetics and Plant Breeding 56
Thank You
May 3, 2023 Department of Genetics and Plant Breeding 57
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