High throughput genetic transformation of sorghum (Sorghum bicolor L.)

Srinivas Belide

CSIRO AGRICULTURE AND FOODDO NOT C

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• High-yield, nutrient use efficient

• Drought tolerant (Waxy layer, small stomata, extensive root system)

• Can be cultivated on over 80% of the world’s agricultural land (including marginal lands)

• Used for food, feed, fodder and fuel (4F)

• Emerging as genetic model for C4 grass bioenergy crops.

Advantages of Sorghum

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* Vegetative oil (Leaf and stem)

* Modification of seed composition-oil, starch etc (Gene editing and GM)

* Insect resistance (RNAi)* Rust resistance (Lr67) and

* Yield increase

Sorghum traits engineering at CSIRO*

* Owns IP DO NOT C

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Challenges in sorghum transformation

• Immature embryos are most preferred explants (Liu and Godwin. 2012; Ping Che et al 2018)

• Continuous supply of stock plants required, influenced by environmental conditions (Quality of IE)

• Isolation of immature embryos : labour intensive and time consuming.

• Green tissue methods in other cereals (Rice-Cho et al 2004; Barley-Cho et al 1998)

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Differentiating embryonic callus (DEC tissue)-flexible explant

• Embryogenic callus with nodular structures induced from immature embryos (Tx430)

• DEC tissue can be maintained more than 18 months with minimal impact on it its regeneration potential.

• Subculture-biweekly and use

Srinivas et al 2016 (WO2017/201719 1A)DO NOT C

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DEC Tissue

Callus initiation from immature embryo Embryogenic callus with nodular structures

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Maintenance of DEC tissue on CIM

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• Lipoic acid (LA) is a sulfur-containing compound involved in several multienzyme complexes

• In animals, free LA and dihydrolipoic acid are metabolic antioxidants that are able to scavenge reactive oxygen species

• Recycles other antioxidants such as vitamin C, glutathione, and vitamin-B

• Increases the expression of genes involved in the regulation of normal growth and metabolism (Packer et al., 1995, Packer and Tritschler 1996, 1997)

• As a plant transformation enhancer-Dan et al., 2004, 2009, 2015; Dutt 2011 etc.

Lipoic acid-promoting differentiation and proliferation

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Comparison of DEC tissue induction frequency and quality with and without LA

Immature embryo size

Parameter Media

CIM CIM +1 mg/l LA

1.4-2 mm DEC tissue induction frequency

61.3 ± 3.2 79.0 ± 6.5*

DEC tissue quality Average quality High quality

>2 mm DEC tissue induction frequency

52.3 ± 3.1 59.0 ± 5.5**

DEC tissue quality Low quality Good quality

*Significant at P < 0.013** Not significant

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Effect of LA on callus yield from* immature embryos of Tx430

* Forty five immature embryos 1.4 - 2 mm in size (15/replica)

Culturing time (weeks)

DEC yield in number (~5 mm each and ready to use)

CIM + 1 mg/l LA CIM without LA4 36A 29a

6 90B 45b

8 120C 45c

10 218D 150d

12 825E 435e

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Effect of LA on regeneration frequency and number of shoots/calli

DEC tissue age in months (from

immature embryo isolation)

Regeneration frequency (%) Number of shoots/callus

No LA in SIM and SRM

LA in SIM and SRM

No LA in SIM and SRM

LA in SIM and SRM

2 92.3 ± 3.05 97.6 ± 2.5 22.0 ± 3.6a 40.0 ± 4.5b

4 86.0 ± 2.0* 97.0 ± 2.6* 16.0 ± 2.0c 40.0 ± 1.5d

6 81.0 ± 1.0** 94.3 ± 2.0** 14 .0± 2.0e 33.0+2.5f

* Significant at P=0.004; **Significant at P=<0.001

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Effect of LA on regeneration(2 months old calli)

No LA With LA Multiple shoots fromone single callus

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Detection of somaclonal variation (ploidy) by flow cytometery

Number of the callus line

Age of the callus line from induction

nDNA content (pg)

CL-09 18 months 1.72 +0.014

CL-10 12 months 1.71+ 0.028

CL-11 6 months 1.78+ 0.169

CL-12 6 months 1.76+0.007

CL-13 5 months 1.98+0.169WT Sorghum (3 weeks old plant) 1.74 pg

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WT CL-9(18 m)

CL-10(12 m)

CL-11(6 m)

CL-12(6 m)

CL-13(5 m)

Histograms of flow cytometeryof DEC tissue

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Plasmids used for bombardment of sorghum DEC tissue

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Post bombardment recovery of DEC tissues and GUS gene expression

Number of tissues

bombarded

Number of brown tissues* Number of GUS spots/tissue**

CIM no L-cysteine and ascorbic acid

CIM + L-cysteine and ascorbic acid

CIM no L-cysteine and ascorbic acid

CIM + cysteine and ascorbic

acidExp-A: 75 15 8 12 25Exp-B: 75 18 11 13 20Exp-C: 75 23 14 7 38Average 18.6 ± 4.04 11.0 ± 4.04 10.6 ± 3.2 27.6 ± 9.2

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Geneticin selection

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Improved recovery of transgenics

Yukoh and Toshihiko, Nat Protoc, 2008DO NOT C

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Transformation efficiency by PDS

Srinivas et al, 2017, Plant Methods

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PDS mediated transformation and regeneration

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Molecular characterization and copy number analysis by dPCR

Copy number of NPT-II gene in 31 independent T0 transgenic plants analysed by digital droplet PCRDO N

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Copy number analysis by dPCR

Percentage of plants Copy number

22.5% <2 copies

26% 2-3 copies

48.3% > 4 copies

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Fast-track trait development-leaf oilConstruct1 Construct 2 No. of events

generatedCo-transformation

efficiency

Core construct 1

Variable construct 1 40

65-70%

Variable construct 2 65

Variable construct 3 28

Variable construct 4 100

Variable construct 5 50

Variable construct 6 85

Variable construct 7 15

Variable construct 7 45

II nd Gen CC2-CC7

-- ~40 each NA

III rd Gen CC8-12

In progress

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Time line for sorghum to T1 seed

3-4 months

IE isolation,Transformation, Plant regeneration

4 months

3-4 months

T1 Seed~10-12 months

Transformation and regeneration

4 months

3-4 months

T1 Seed~7-8 months

CSIRO method “saves time and resources”

DECTissue

IEBased

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Oil (TAG) from biomass crops

3.3% oil 8.4% oil 12% oil 7% lipids

Potato Sorghum Sugarcane Ryegrass

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Oil (TAG) from biomass crops

8.4% oil R/A ? Progress

Ist gen 2nd gen 3rd gen

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Accumulation of lipid droplets in Sorghum bicolor leaf mesophyll cells

TV and SB et al., Submitted to PBJDO NOT C

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Networking opportunity

• CSIRO interested to explore R&D partnerships with academic/private sector to promote sorghum for the benefit of developing world.

• Please contact Dr. Surinder Singh, Group leader, CSIRO for further information

Surinder.singh@csiro.auOr

Srinivas.belide@csiro.au

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Thank youCSIRO Agriculture and FoodSrinivas Belide

Ph: +61 2 6246 49 49Email: Srinivas.belide@csiro.au

AcknowledgmentsSurinder SinghJames PetrieThomas VanherckeAnu MathewJasmine RajamonyFreddie LoymanIngrid Venables

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