This document summarizes Samwel Muiruri Kariuki's research at IITA Kenya on inducing early flowering in cassava. The document discusses two methods: using LED light supplementation to induce flowering within 4 months, compared to 10 months without light; and developing a CMV-inducible CRISPR-Cas9 system to edit cassava genes in a virus-activated manner. Preliminary results show light supplementation significantly increased the number of flowering plants compared to the control. The researcher is working to assemble constructs using a CMV promoter to drive Cas9 expression and test them in Nicotiana benthamiana transformations. The goal is to create a virus-inducible gene editing system for cassava.

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Induction of early flowering in cassava
through light supplementation and CMV-
inducible CRISPR-Cas9 system
Samwel Muiruri Kariuki, IITA Kenya

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Presentation Summary
• Introduction (Light induced flowering)
ücassava flowering
üLight in flowering
• Materials and methods
• Preliminary results
• Introduction (CMV inducible CRISPR-Cas9)
ü CRISPR-Cas9 technology
ü CMV inducible promoter
• Progress
• Acknowledgements

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Introduction
• Cassava is critically important for food
• Cassava is a long day crop
• Flowering is troublesome:
üLack of flowering
üLack of female flowers
üAsynchronous
üHigh abortion rate
• No flowering = No Sexual breeding

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Role of light in plant flowering
Pineda et al., 2020
• LED of 625–635 nm induce early
flowering in cassava
• Red and blue light constitute
Photosynthetically Active Radiation
(PAR)
• Towards low-cost approaches for
accelerating plant growth/inducing
flowering?
• Speed breeding being used in cereal
crops

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Materials and methods
• Clone/cultivar selection
• Experimental set-up
• Data and sample collection

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Clone selection
Clone Nature of flowering (Thika) Months to flowering
DSC 258 Late flowering 10
DSC 120 Late flowering 10
DSC 272 Early flowering 6
DSC 269 Non-flowering Not flowered at 12 months
DSC 257 Medium 8
DSC 196 Non-flowering Not flowered at 12 months

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Experimental set-up
• Twelve replicates/clone, three plants per plot, 72 plots
• Plots are 0.5 X 1 M
• Six 100 W white LED lights, 3.5 M above ground

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Experimental set-up – ctd’ (control)
• No light in the control set-up

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Light distribution
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
380 430 480 530 580 630 680 730 780
umol
m-2
s-1
Wavelength(nm)
Light wavelength distribution
- Most light is within blue
to near-red (430 – 630
nm)
- PAR averaged 1 – 3
umol/m2/s
- Three hours night break

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Progress

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Light induces early flowering - 4 months AP
41.17647059
58.82352941
No light supplementation
Flowered Not flowered
89.71962617
10.28037383
Light supplementation
Flowered Not flowered

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Flowering over time
0
5
10
15
20
25
30
35
40
45
50
88 95 102 109 116 123 130 137 144 151 158 165 172
Number
of
flowering
plants
DPP
Number of flowering plants over time
Light
No light

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Branching per clone 5 Months post planting
0
5
10
15
20
25
30
35
40
DSC 120 DSC 196 DSC 257 DSC 258 DSC 269 DSC 272
Number
of
plants
Clones
Number of branched plants
Treatment Branched Treatment Not branched Control Branched Control Not branched
Clone Nature of flowering (Thika) Months to flowering
DSC 258 Late flowering 10
DSC 120 Late flowering 10
DSC 272 Early flowering 6
DSC 269 Non-flowering Not flowered at 12 months
DSC 257 Medium 8
DSC 196 Non-flowering Not flowered at 12 months

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Beautiful reward in the field

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Cassava Mosaic Virus (CMV) inducible CRISPR-
Cas9

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CRISPR-Cas9 technology
• Two-component technology:
üProgrammable guide RNA
(gRNA)
üNuclease (Cas9)
• gRNA directs Cas9
• Cas9 introduces DSB in target
DNA – repaired by:
üNon-homologous end joining
(NHEJ)
üHomologous recombination (HR) Doudna and Charpentier, 2014

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Cassava Mosaic Virus activatable promoter
Rey and Vanderschuren, 2017
Bidirectional promoter
Fact sheet
• AC1 represses its own
expression
• AC2 aka TraP activates
sense promoters
• A 104 fragment within the
TraP gene ensures universal
activation
• Different virus AC2s can
transactivate
• AC2 can activate some
eukaryotic cells

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Towards a fully
inducible
promoter
• A 104 bp TraP element is
required for inducibility in
vasculature
• If the 104 element is fused to
sense promoter, the promoter
could be fully inducible

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Overview
§ Initial goal was a
virus kill switch in
cassava
§ Now proof of
concept
§ Transformations in
Nicotiana
benthamiana

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Progress
Trap-104 CR-sense Cas9
AtU6 NbgRNA polyT NosT
2X35S Cas9
AtU6 NbgRNA polyT NosT
• Constructs assembled
• N. benthamiana
transformations
104-Trap-Cas9 2X35s-Cas9 (+ve control) -ve control

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Acknowledgements
• FLAIR Fellowship grant No. FLRR1201370
• FLAIR Collaboration grant No. FCGR1211038
Prof. Wendy Harwood - JIC
Prof. Cathy Martin - JIC
Dr. Leena Tripathi
Dr. Morag Ferguson
Trushar
Ivan Juma (Morag’ s PhD student)
IITA Nairobi Team
Team at KALRO Kandara
Angelyne Rose

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Thank you