This document summarizes a student project to transform tomato plants using CRISPR-Cas9 to enhance shelf life. The objectives are to standardize tomato hypocotyl regeneration and optimize genetic transformation using a CRISPR construct targeting two tomato pectate lyase genes. The methodology involves germinating tomato seeds, taking hypocotyl explants, co-cultivating with Agrobacterium containing the CRISPR construct, regenerating plants on selection media, and rooting regenerants to obtain transgenic plants with increased shelf life. Preliminary results showed regeneration of putative transgenic shoots from several batches of explants.

1. ICAR – INDIAN INSTITUTE OF HORTICULTURAL
RESEARCH, HESSARAGHATTA ,BENGALURU.
N
UNIVERSITY OF AGRICULTURAL SCIENCES ,GKVK, Bengaluru
COLLEGE OF AGRICULTURE ,Hassan
Submitted from: Guided by:
Dr. T R Usharani
SHREYAS H K Senior scientist
IV B Tech (Biotechnology) Division of Basic sciences
College of agriculture ,Hassan ICAR - IIHR
Hessaraghatta, Bengaluru

2. PROJECT;
“TRANSFORMATION OF TOMATO WITH
CRISPER-CAS9 CONSTRUCT TO ENHANCE
SHELF LIFE”

3. INTRODUCTION
●Tomato(Lycopersicon esculentum) belongs to family Solanaceae
●Chromosome number - 2n =24
●Rich in vitamin A and C
●Seeds are kidney shaped and 3-5mm long and 2-4mm wide
●The tomato Pectate lyase (PL) gene is crucial for fruit softening in tomato
●The silencing of this PL alters texture without affecting other aspects of
ripening

4. OBJECTIVES
●To standardize the protocol for regeneration of hypocotyl explants in
tomato cv Arka Vikas
●To optimize the genetic transformation protocol in tomato cv Arka Vikas
using CRISPER- Cas9 construct

5. Agrobacterium tumefaciens is a gram-negative soil bacterium which
causes crown gall tumors
Tumor formation is the result of the transfer, integration and expression
of a gene of the A . tumefaciens called T-DNA
CRISPER- Clustered Regularly Interspersed Short Palindromic Repeats
Cas9- Protein can target and cleave invading DNA in a sequence-
specific manner based on the guide RNA
In our work A. tumefaciens strain LBA 4404 having guide RNA targeting
PL1 and PL2 along with Cas9 gene were used

6. Tomato seed Inoculation
Materials required
●Seed sample
●Autoclaved water
●1% Bavistin solution
●Tween-20
●70% alcohol
●4% Sodium hyphochlorite solution
●MS media (½ MS)

7. Sterilization of seeds
●Seeds of cv Arka vikas were washed with water followed by 1% Bavistin
solution for an hour
●Seeds were washed with autoclaved water 3 times
●Then seeds were treated with a drop of Tween-20 for a minute with shaking
●Wash the seeds for 3 times with Autoclaved water
●Treat the seeds with 70% alcohol for 1 minute

8. ●Wash the seeds with Autoclaved water for 3 times
●Treat the seeds with 4% sodium hypochlorite for 10 minutes
●Seeds were then rinsed 3-5 times with Autoclaved water to remove all the
traces of chlorine
●Seeds were then blot dried

9. Inoculation
●50mL of ½ MS media was prepared in each tissue culture bottles
●Seeds were inoculated in tissue culture bottles(15-25 seeds per bottle is
optimum)
●Seeds were then allowed to germinate in a growth chamber (keep the
inoculated bottle in dark for 4 days and keep in light condition from 5th day)

10. Selection of explants
●From the 10-12 days old germinated seedlings, hypocotyls of 2-3 cm length
were taken as explants

11. Agrobacterium mediated transformation
Strain
●Agrobacterium tumefaciens strain LBA 4404 harboring guide
RNA targeting PL1 and PL2 along with Cas9 gene were used.

12. Preparation of culture
•Single colony of Agrobacterium tumefaciens strains PL1 and PL2 was
inoculated in 5ml LB containing 100mg/L of Kanamycin and 10mg/L of
Rifamycin and 5mg/L of Tetracycline under aseptic condition
•Incubation at 28º C at 200rpm overnight.

13. Preparation of culture
●Culture (LB) of Agrobacterium tumefaciens strains PL1 and PL2 were
inoculated in autoclaved 100ml YEMA Broth containing 100mg/L of
Kanamycin and 10mg/L of Rifamycin and 5mg/L of Tetracycline under aseptic
condition
●Incubation at 28º C at 200rpm overnight.

14. Co-cultivation of explants
●The grown Agrobacterium culture was taken and allowed them to centrifuge
at 10000rpm for 10 minutes
● Take the pellet and adjust to an O.D600nm of 0.5 by diluting with
autoclaved MS solution(1/10 concentration)
●Then take the 10-12 days old germinated seedlings, hypocotyls of 2-3cm
were cutoff and incubated in the culture for 10 minutes and blot
●Completely dried hypocotyls were then plated in MS media containing
2mg/L BAP and 0.1 mg/L of IAA and plates were incubated for 48 hours

15. Suppression/ Elimination of Agrobacterium
●After transformation hypocotyls were transferred to
regenerating MS media supplemented with Tementin (400
mg/L) and incubated for 48 hours for suppression of over
growth of Agrobacterium

16. Selection of transformants
●Hypocotyls were transferred to MS media containing
Tementin (400 mg/L) and kanamycin (100mg/L) for selection
of putative transformants by regeneration of hypocotyls
●Hypocotyls were subcultured for every 15-20 days
periodically in a fresh media to obtain healthy transformants

17. Rooting of Regenerants
●The regenerated putative transformed shoots were excised and
transferred to rooting media containing ½ MS supplement with
0.5mg/L of IBA
●Tementin (400mg/L) and kanamycin (100mg/L) was also added
to the media for efficient selection

19. OBSERVATION AND RESULT
SEED
INOCULATION
SEEDS
GERMINATED
CO-
CULTIVATION
REGENARATION SELECTION ROOTING
BATCH 1 105 84 0 0 0 0
BATCH 2 90 71 0 0 0 0
BATCH 3 90 72 0 0 0 0
BATCH 4 75 58 53 51 43 14
BATCH 5 75 56 53 0 0 0
BATCH 6 60 54 51 51 47 11
BATCH 7 60 55 51 50 45 8

20. APPENDIX
Seed
germination
Co-cultivation Regeneration
media
Selection
media
Rooting
MS Salts 0.5X 1X 1X 1X 0.5X
Sucrose (g/L) 15g 30g 30g 30g 15g
Gelrite (g/L) 3g 3g 3g 3g 3g
BAP/ Zeatin (mg/L) - 2mg 2mg 2mg -
IAA (mg/L) - 0.1mg 0.1mg 0.1mg -
Timentin(mg/L) - 400mg 400mg 400mg
Kanamycin (mg/L) - - - 100mg 100mg
Ascorbic acid
(mg/L)
- 40mg 40mg 40mg -
L-cystiene (mg/L) - 40mg 40mg 40mg -
Thiamine (mg/L) - 1mg 1mg 1mg -
IBA (mg/L) - - - - 0.5mg

21. STOCK REQUIRED TO BE TAKEN
RIFAMYCIN 5Omg 10mg 20mg
KANAMYCIN 100mg 100mg 100mg
TETRACYCLINE 12.5mg 5mg 40mg
AGROBACTERIUM
STRAIN
- - 1ml
For YEMA Inoculation
FOR 100ml of YEMA

22. Refrences
Cortina et al (2004)- tomato transformation and transgenic plant production
Frary et al (1983) – Factors affecting efficiency of Agrobacterium-mediated
transformation of tomato
Oktem et al (1999)- regeneration and Agrobacterium mediated
transformation in tomato
Uluisik et al (2016)- CRISPER-Cas9 mediated transformation in tomato
Yu et al (2017)- regulation of ALC gene in tomato using CRISPER-Cas9 system