Gene Transfer System for Plant Seed, Egg and Microbe SONIDEL Limited Resarch cooperation: National Institute of Agrobiological Sciences Plant Genetic Engineering Unit Improvement Research is being Made.

(2/24) 1) Development of Concept Agrobacterium Rice : Agrobacterium Particle Gun Wheat, Barley : Particle Gun Current Gene Transfer Method Microinjection Silk Worm : Microinjection Need Tissue Culture (time-consuming !) (time-consuming !) Need High Skill & A Lot of Labor (tedious !)

(3/24) 1) Development of Concept No Need Tissue Culture Plant : No Need Tissue Culture No Need Microinjection Animal (silk worm) : No Need Microinjection A New Gene Transfer Method Simple Efficient Low-cost National Institute of Agrobiological Sciences Japan – patents pending * *”Electroporation method including the use of depressurization / presurization” PCT/JP03/08937

2) Basic Technique and Feature ① Soak mature seeds in water overnight. ② Soak mature seeds in electroporation buffer ③ Strong Vacuum treatment→DNA permeate tissues ・・・ Vacuum Chamber （ Fig.1 ） ④ Electroporation→DNA is transferred into cells ・・・ Electroporator （ Fig.1 ）, Chamber Having Platinum Electrode （ Fig.2 ） Basic Procedure (e.g. Rice Plant ） This new technique uses Vacuum + Electrical pulse Conventional Electroporation uses Electrical pulse only (4/24)

Fig.1: Vacuum Chamber (left) and Electroporator, CUY21EDIT(right) Fig.2: Chamber Having Platinum Electrode, CUY495P10 2) Basic Technique and Feature (5/24)

Fig. 4: GUS gene expression reference (Wheat) Fig.3: The level of GUS gene expression by fluorescence assay (Wheat) Effectiveness VEE-- N: Negative control, --: Buffer only, V: Vacuum, E: Electroporation 2) Basic Technique and Feature (6/24)

Fig.1 Geneticine selection of rice seeds and/or seedlings Fig2. Fertilizable transformed plant Fig3. Southern analysis in T0(existent) generation Fig4. Next generation (T1) individual Fig5.. Southern analysis of T1(next) generation Transferred pWI vector integrated npt II gene M wt wt cp -10 cp M cp -2 cp ) Transformed Rice Plant Cultivation (not transformed) (npt II gene introduced ) (7/24)

No. of T0 T1 of T1 PCR+No. RT-PCR+No (ongoing) RT-PCR : T1 npt Ⅱ gene detection (Rice Plant) 3) Transformed Rice Plant Cultivation (8/24)

・ When we started our research In water Vacuum EP Growing ・ After improvement In water Ultrasonic Vacuum Polarity exchange EP Ultrasonic Growing (9/24) 4) Improvement of the method of gene transfer Fig1. Hexagonal chamber type electrode, CUY602 Fig2. Ultrasonic treatment Fig.3 Polarity exchange device, CUY901 Fig.4 Result of improvement (simple determination) Score Koshi hikari Kiat ake When we started our research After improvement

5)New experiments in 2007 or later Gene transfer to Silk Worm Eggs Gene transfer to Silk Worm Eggs Gene transfer to Arabidopsis seeds Gene transfer to Arabidopsis seeds Gene transfer to Yeast Gene transfer to Yeast Contact-free Ultrasonic Apparatus Contact-free Ultrasonic Apparatus (10/24)

Gene Transfer to Silk Worm Eggs ① Current common method After making a hole by a thin needle, inject DNA by glass capillary A lot of care ！ Inject Vector and Plasmid into an egg Breeding Mounting Mating Extraction Screen by fluorescence stereomicroscope (11/24) Feeding Artificial diet

A. 2 days after gene transfer B. 10 days after gene transfer C. Hatchling Larva The capacity is approx. 2,000 eggs at one time Make mass transfer possible Gene Transfer to Silk Worm Eggs ② Our method (Vacuum and Electroporation) (12/24)

Gene Transfer to Silk Worm Eggs ③ Gene Transfer Condition Fig.1 Electrical condition, GFP expression rate and Hatching rate Voltage （ V) ％ Optimized Voltage GFP Expression Rate Hatching rate Setup ： Pulse length 10ms, Pulse interval 50ms, No. of Pulses 10 (13/24)

5)New experiments in 2007 or later Gene transfer to Silk Worm Eggs Gene transfer to Silk Worm Eggs Gene transfer to Arabidopsis seeds Gene transfer to Arabidopsis seeds Gene transfer to Yeast Gene transfer to Yeast Contact-free Ultrasonic Apparatus Contact-free Ultrasonic Apparatus (14/24)

(15/24) Fig.1 Platinum Plate Electrode for 2ml Tube, CUY280 Fig.3 GUS gene expression in Arabidopsis Seeds Fig.2 GUS gene expression in tobacco BY-2 cells Gene Transfer to Arabidopsis Seeds ① Micro seeds, Electrode for Microbe and Tube

Gene Transfer to Arabidopsis Seeds ② Gene Transfer Condition Fig.1 GUS Gene Expression in Arabidopsis Seeds Transfered EL2Ω(35S+Ω+GUS)99 Pulses leads to a good result Setup Voltage ： 30 Ｖ Pulse length ： 50ms Pulse interval ： 75ms GFP Expression Rate GFP Expression Rate (%) Number of Pulses GFP and npt II gene are also transfered. (16/24)

5)New experiments in 2007 or later Gene transfer to Silk Worm Eggs Gene transfer to Silk Worm Eggs Gene transfer to Arabidopsis seeds Gene transfer to Arabidopsis seeds Gene transfer to Yeast Gene transfer to Yeast Contact-free Ultrasonic Apparatus Contact-free Ultrasonic Apparatus (17/24)

(18/24) Specific name Saccharomyces cerevisiae Strain BY4700 (Sequenced all its genome) Genotype MATa ura3Δ0 Ura3 / Orotidine-5‘-phosphate decarboxylase missing Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University Fig.1 Procedure for Gene Transfer to Yeast Gene Transfer to Yeast ① Gene Transfer Procedure Vector DNA Amylase gene Uracil synthesis gene Host Yeast Yeast that can not synthesize uracil Unable to culture in urasil missing medium (minimum medium) Gene Transfer Select transferred gene in minimum medium Minimum medium Amylase gene Uracil synthesis gene recombinant Recombinant secretes mold amylase Amylolysis part by amylase Starch iodine reaction Starch medium

(19/24) Transfectant Wild type Gene Transfer to Yeast ② Cultivation of Yeast

(20/24) HS : Heat Shock V: Vacuum EP : Electroporation V + EP : Vacuum + Electroporation Fig.1 Efficiency of Yeast Transformation V+EP is best A. Heat Shock Method （ conventionally ） B. Vacuum + Electroporation Gene Transfer to Yeast ③ Efficiency of Yeast Transformation No. of Colony

5)New experiments in 2007 or later Gene transfer to Silk Worm Eggs Gene transfer to Silk Worm Eggs (21/24) Gene transfer to Arabidopsis seeds Gene transfer to Arabidopsis seeds Gene transfer to Yeast Gene transfer to Yeast Noncontact type Ultrasonic Processor Noncontact type Ultrasonic Processor

A. AppearanceB. Inside of the tank (22/24) Gene Transfer by Noncontact type Ultrasonic Processor ① Apparatus

Supersonic probe Tube or vial in which biologic material (e.g. seeds) are contained Temprature- controlled water tank Supersonic from 3 ways (left, right, bottom) crossing in the material position gives maximum energy. Attachment for fixing (23/24) Gene Transfer by Noncontact type Supersonoc Processor ② Structure Supersonic probe

(24/24) C. Cultured tobacco cells : EL2Ω B. Cultured rice cells : pWI-GUS A. Rice mature seeds CNT 5min １０ min Gene Transfer by Noncontact type Ultrasonic Processor ③ Transfer Results