Camelina Sativa, common name "false flax” or “gold of pleasure," is

a spring planted annual oilseed crop that can be grown in cold and

marginal lands. First cultivated in Northern Europe during the

Bronze Age, Camelina seed oil was commonly used for food,

medicinal use and lamp oil.(2) Camelina's high percentage of linoleic

acid makes it a renewable resource of industrial raw materials.

Currently Camelina has been used in lubricants, soaps, surfactants

and most importantly, as a source of bio-diesel due to the high

erucic acid content.(4) This member of the Brassicaceae family has

great potential of becoming an important contributor to biofuels due

to its 30-40% oil yield and low input for production.(3) Though

Camelina needs little water and grows in fairly cold months, it does

not do as well in summer months experiencing the effects of abiotic

stress more in depth of water deficiency from drought often

accompanied by heat. Therefore the gene yeast S-

adenosylmethionine decarboxylase (ySAMdC) was introduced into

Camelina in hopes to increase Camelina's tolerance to abiotic

stress.(1)

Abstract

Introduction

Tissue culture of Camelina

Camelina seeds were washed with Tween 20® for 10 min and thoroughly rinsed

with tap water to remove the detergent. The seeds were rinsed with 70% ethanol

for one minute followed by four times with sterile water. The seeds were further

treated with 10% bleach for five minutes and rinsed with sterile water five times

before inoculating 20 seeds per jar containing germination medium 1. The

seeds were incubated in the dark at 25°C for two days to germinate. Later the

cultures were moved to light (12/12 – Light and dark). After two weeks,

cotyledons and young leaves were used for tissue culture and transformation

studies.

Agrobacterium-mediated transformation

Agrobacterium cultures containing GUS, eGFP, or ySAMdC were grown to an

optical density of 0.2. Explants (cotyledons or young leaves) were excised and

dipped in liquid Agrobacterium culture containing 10 µg/mL Acetosyringone.

Explants were blotted dry with sterile filter paper to remove excess culture and

plated on Medium 2 containing 100µM Acetosyringone. Plates were sealed with

parafilm and placed in the dark for 2 days. Explants were washed with liquid

Medium 2 and 10 µg/mL Cefotaxime (CEF) and sub-cultured on Medium 2

containing 100 µM CEF.

Preliminary results with marker genes GUS and GFP

were successful. Cotyledon explants are easier to

work with, not being as sensitive as young leaves, but

they did not give much shoot regeneration. Young

leaves on the other hand showed faster shoot

initiation and more regenerated shoots than

cotyledons. However due to their fragility, young

leaves are difficult to handle.

Furthermore, the first three transformations

performed with cotyledons showed callus

regeneration but little to no shoot regeneration. The

last transformations done with a mixture of cotyledons

and young leaves show more than double the amount

of shoot regeneration (all from young leaves).

Future work will include Molecular and Physiological

studies to confirm the presence of gene of interest

and conduct abiotic stress tolerance on transgenic

plants carrying the gene.

Conclusion

Results

Acknowledgements *National Science Foundation Research Experience Summer Undergraduate Program

*Penn State Harrisburg

*Dr. Shobha Devi Potlakayala *Dr. R. V. Sairam *Dr. Puthiyaparambil Josekutty

*Diego Morales *Imran Hussain *Matt Reitzel *Mike Chennavasin *Nasie Constantino

*Joe Meisenbach *Ben Tabatabi *Deepkamal Karelia *Aneel Maine

*Julianne Dauber *Alison Shuleri

*NSF-REU Students

Fig. 2 Fig.3 Fig.4 Fig. 5 Fig.6 Fig.7

Callus formation Developed callus Shoot Initiation Leaf formation Shoot Development Shoot Elongation

Camelina sativa is a spring planted annual oilseed crop that can be grown in cold and marginal lands.(4) Containing 30-40% oil yield, having a low input for production and rapid growth season of

85-100 days (from germination to harvest), Camelina has become a potentially important source of biofuel, though it can be grown in colder seasons and has little tolerance to drought in summer

months making it susceptible to abiotic stress.(3) Providing that Camelina's stress tolerance can be improved, research has been done to successfully incorporate S-Adenosylmethionine

decarboxylase (SAMdC), a drought and cold tolerant gene, to improve Camelina's stress tolerance. To do this, 6 agrobacterium-mediated transformations were performed with three liquid bacteria

cultures containing three separate genes: beta-Glucuronidase (GUS), a scorable maker that stains blue if the gene is present; green florescent protein (GFP), fluoresces green under UV light; and

SAMdC, gene of interest to enhance cold and drought tolerance. Explants (cotyledons/ young leaves) from the transformation were co-cultivated, washed, and subcultured for expected results of

callus, shoot, and root regeneration of a enhanced stress tolerant transgenic plant.

Regeneration Growth development

Seed Steilzatin

and Explant Preparn

Seed Surface Sterilization

and Explant

Preparation

Methods

Genetic Transformation

Seed Surface

Sterilization and

Germination

Preparation of

explants

(cotyledons and

young leaves)

Agrobacterium-mediated

transformation

Co-cultivation

Washing of explants

and subculture

Shoot

regeneration

Improving Stress Tolerance in Camelina sativa Krysta J. Haggins1, Shobha Potlakayala2,, Imran Hussain2, PC Josekutty2, Sairam Rudrabhatla2

Marygrove College1 8425 W. McNichols Detroit, MI 48221, Penn State Harrisburg2 777 West Harrisburg Pike Middletown, PA 17057

Website: http://harrisburg.psu.edu/reu/sustainable-bioenergy

Tran

sfo

rm

ati

on

#

Explant used (#)

Gene

# of calli

# of

regenerating shoots

Additional info

KC

T 1

10 None 7 0 Explants did not survive antibiotic

10 GUS 6 0

50 ySAMdC 10 0

KC

T2

10 None (negative control) 0 0 Explants did not survive antibiotic

10 GUS 3 0

50 ySAMdC 0 3

KC

T3

10 None (negative control) 6 0 Most explants did not survive antibiotic

10 GFP 6 0

50 ySAMdC 14 0

KC

T4

10 None (negative control) 7 2 1 explant has roots

15 GUS 2 2 1 shoot transferred to a jar

15 GFP 8 2 1 shoot transferred to a jar

50 ySAMdC 5 3 1 shoot transferred to a jar

KC

T5

10 None (negative control) 3 0

15 GUS 2 2 1 shoot transferred to a jar

15 GFP 2 3 1 shoot transferred to a jar

50 ySAMdC 4 3

KC

T6

10 None (negative control) 1 0

15 GUS 0 0

15 GFP 1 2 1 shoot transferred to a jar

50 ySAMdC 6 4 4 shoots transferred to jars

References

Fig. 8 A and B blue staining GUS expression in explants

Fig. 1 Protocol for seed surface sterilization

Fig. 10 Process for genetic transformation of Camelina

(1) Cheng L, Zou Y, Ding S, Zhang J, Yu X, Cao J, Lu G (2009). Polyamine accumulation in transgenic tomato

enhances the tolerance to high temperature stress. J. Integr. Plant Biol. 10.1111/j.1744-

7909.2009.00816.x.

(2) Ehrensing, D, & Guy, S. (2008). Camelina. Unpublished manuscript, Extension Service, Oregon State

University, Retrieved from http://extension.oregonstate.edu/catalog/pdf/em/em8953-e.pdf.

(3)Guy, S and Lauver, M. (2010). Camelina is a potential oilseed crop for washington. Unpublished

manuscript, Dept. of Crop and Soil Science, Washigton State University, Pullman, Washington.

Retrieved from

http://www.pacificbiomass.org/documents/Guy%20Bioenergy%20Research%20Symposium%20pos

ter.pdf.

(4) Putnam, D.H., J.T. Budin, L.A. Field, and W.M. Breene. 1993. Camelina: A promising low-input oilseed. p.

314-322. In: J. Janick and J.E. Simon (eds.), New crops. Wiley, New York.

Medium 1 Germination Media (per liter)

• 4.43g of MS + Vitamins

• 2% sucrose

• 0.7% of agar

• pH 5.8

Medium 2 Cultivation Media (per liter)

• 4.44g of MSB5 + vitamins

• 2% sucrose

• 0.7% agar

• pH 5.8

Hormones

• 0.4ml of Zeatin

• 0.4ml of AdSO4

• 0.1ml of IAA

Fig. 9 A, C, and E White light of explants(extracted callus, young leaf, callus on leaf

respectively). B, D, and F e GFP expression under UV light

A

F

E

D

C

B

Table 1 Explant regeneration analysis