Improving cold storage and processing traits in potato

through targeted gene knockout(Clasen et al., 2015)

Plant Biotechnology Journal Published on 7 April 2015

Journal Club Meeting Presented by:

Sarbesh D. DangolJanuary 7, 2016.Nigde, Turkey.

Introduction

• Solanum tuberosum: Third most important food crop.

• Used by processors for potato chips, french fries and other processed products.

• Harvested once a year, it’s necessary to cold store tubers.

Cons of Cold storage of potatoes• Cold storage causes cold-induced sweetening

(CIS).• Processing at high temperatures form

unacceptable dark-pigmented products.• Formation of carcinogenic acrylamide during

processing.• Methods that reduce CIS and acrylamide?? How?? Reduce reducing sugar content. Target Invertase enzymes??

Formation of carcinogenic Acrylamide

Invertase enzymes accumulate reducing sugars

• CWIN (Cell-wall Invertase)• VIN (Vacuolar invertase) • CIN (Cytoplasmic invertase)

But why target VInv?

• VInv produce reducing sugars in cold-stored tubers (Kumar et al., 2004; Matsuura-Endo et al., 2006; Sowokinos, 2001).

• VInv knockdown lowers reducing sugars and dark-pigmented non-enzymatic browning (Bhaskar et al., 2010; Wu et al., 2011; Ye et al., 2010).

TALEN generated gene knockout is the approach of this research

• Knockout mutations in all alleles of the VInv gene.

• Use of transcription activator-like effector nucleases (TALENs).

TALEN vs ZFN and Gene Knockout

Method of synthesizing gene sequence specific TALEN pairs

Stepwise ligation to generate gene sequence specific TALEN pairs

Expression vector for TALEN

Why Solanum tuberosum cv Ranger Russet?

• Widely used for frying.

• Processing is hindered due to cold-induced sweetening.

Targeted mutagenesis in Exon I

454 deep sequencing of Exon 1 of VInv from Solanum tuberosum cv Ranger Russet.

Testing of TALEN activity

• Three TALEN pairs were designed. • TALEN encoding Plasmids (VInv_T1, VInv_T2 and

VInv_T3) were individually introduced into protoplasts.• Genomic DNA isolation, PCR amplification (272 bp).• 454 pyrosequencing.• Evaluation for NHEJ-induced mutations. • Mutations in all alleles.• Also tested in Russet Burbank, Atlantic and Shepody.

Transforming plasmid encoding TALEN pairs to protoplasts

Percentage mutagenesis in Ranger Russet

VInv_T2 induced consistently well mutagenesis in four tested varieties

Atlantic Ranger Russet Russet Burbank Shepody

Vinv_T2 for further experiments• Contained fewest SNPs in 3 different alleles.

• Consistently well mutagenesis in four tested varieties.

• Protoplasts were transformed with a plasmid VInv_T2 using PEG.

• Calli generation, shoot excision and rooting.

• DNA isolation from leaf sample for genotyping of VInv locus.

Vinv_T2 transformation into the protoplasts

Chromatograms with two or more peaks suggested mutation in alleles

Wild Type

Mutant

Initially screened for mutations by directly sequencing PCR amplicons.

Characterization of vacuolar invertase mutant plants

From appx. 600 regenerated shoots, 18 likely harbored mutations in at least one VInv allele.

For further characterization, PCR amplicons were cloned and sequenced.

Mutations in various transformation groups

St116_1 had frame shifts; Vinv was predicted to be consummately knocked out.

St116_1 was advanced to phenotypic characterization.

Did the plasmid integrate into the plant chromosomal DNA?

Analysis of Frc, Glc and Suc levels

St116_1 and St116_8 were cold-stored at 4 °C for 14 days.

Glucose and fructose were quantified using HPLC.

Analysis of Frc, Glc and Suc levels

Less brown potato chips after heat processing •Chips were prepared from tubers stored at 4 °C for 14 days.•Acrylamide levels were quantified using HPLC.

Discussion• Complexity of the autotetraploid genome. • High degree of heterozygosity.• Classic improvement require long breeding

cycles and screening large populations. • Transgenesis/cisgenesis, RNAi and TILLING, labor

intensive and integrate transgenes in a random fashion.

• Desired expression of the transgene required.• Targeted gene modification needs to be highly

efficient: Such as TALEN gene editing.

Discussion• TALENs cleaved multiple alleles with delivery to

potato protoplasts with over 80% efficiency.• About 3% of the plants were found to contain

mutations in one to four VInv alleles. • TALENs were highly efficient mutagens in

protoplasts from other commercial varieties. • Targeted mutations into a gene-of-interest in a

single generation in elite potato varieties.

Discussion• Off-target mutagenesis can occur when using TALENs

for genome modification (Frock et al., 2015; Mali et al., 2013).

• TALEN pairs could tolerate up to 3–4 mismatches (Juillerat et al., 2014).

• Searched publicly available potato genome sequences for potential TALEN-binding sites.

• No potential target sequences were found with four or less mismatches.

• To completely rule out the possibility, perform whole-genome sequencing.

Discussion• Previous studies with RNAi with partial suppression

did not control CIS effectively (Bhaskar et al., 2010; Wu et al., 2011).

• When all four VInv alleles were mutated, reducing sugars were undetectable.

• Low level of acrylamide generated within complete knockout lines.

• Low acrylamide suggests presence of reducing sugars, albeit at undetectable levels.

• Reducing sugars are most likely due to CWIN and CIN (Bhaskar et al., 2010; Roitsch and Gonzalez, 2004).

Future prospects

• Further reduce acrylamide content by targeting additional, nonessential invertases.

• Whole genome sequencing to elucidate whether there are mutations elsewhere.

• No foreign DNA and thus are not different from naturally occurring varieties.

• Non-GMO? Non-transgenic? Acceptance by regulators? Acceptance by anti-GMO activists/ consumers?

Critical Analysis1. It’s global VInv silencing, and not tuber-specific.2. Phenotypic disturbances? Wild type phenotype?

Stunted growth? Tuber sizes? Tubers per plant?3. There is stable integration in plant genome of

St116-1 and St116-8???4. Incorporation of promoter gene in plant

chromosome can also be called transgenic, isn’t it?

5. Cold storage experiment should have been done for 0th hour.

Critical Analysis6. If Ranger Russet is used as potato chips by

industries, and if it gives that dark chips as shown in wild type, how is that possible for consumers to consume such chips, or industry to process such a type? I think the wild type dark chips are merely exaggerated.

7. What if tubers are kept for months for cold storage?

8. Is it agronomically useful?

Critical Analysis

9. Is acrylamide really a carcinogen in humans? In rodents, yes. In humans? Possibly! Nevertheless, neurotoxic in humans.

10. The choice of the adjective “nonessential invertases” is a bit too extreme to use. CWIN and CIN aren’t non-essential invertases in tubers.

11. Efficiency of this experiment is too low to be reliable.

Research Article Details

Improving cold storage and processing traits in potato through targeted gene knockout

Benjamin M. Clasen, Thomas J. Stoddard, Song Luo, Zachary L. Demorest, Jin Li, Frederic Cedrone, Redeat Tibebu, Shawn Davison, Erin E. Ray, Aurelie Daulhac, Andrew Coffman, Ann Yabandith, Adam Retterath, William Haun, Nicholas J. Baltes, Luc Mathis, Daniel F. Voytas and Feng Zhang.

Cellectis plant sciences Inc., New Brighton, MN, USA Cellectis SA, Paris, France

Plant Biotechnology Journal (2016) 14, pp. 169–176 doi: 10.1111/pbi.12370

Thank You.