Post on 19-Apr-2022
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