Agrobacterium-mediated transformation of Camelina sativa for production of transgenic plants

Camelina sativa (C. sativa), an oilseed species rich in poly-unsaturated fatty acids, has gained great importance as an industrial oil platform crop in recent years. Despite the potential benefits of C. sativa for bioenergy applications, limited research has been conducted to improve its agronomic qualities. Hence, a simple and efficient technique for production of transgenic C. sativa plants is warranted. In the present study, shoot apical meristems of two C. sativa cultivars (Pl650159 and Pl650161) were transformed with Agrobacterium strain ‘EHA 105’ harboring the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase II (nptII) genes. After two days of co-cultivation in the dark, explants were transferred to selection medium. Transgenic shoots were identified on the basis of green fluorescence and kanamycin resistance. Shoots were then rooted and transferred to potting mix soil for acclimatization. This protocol describes an efficient method to generate transgenic C. sativa plants in as little as 4 weeks.


BACKGROUND
About 85% of our energy needs are met by petroleum-based fossil fuels such as oil, coal and natural gas. Continuous use of these nonrenewable fuels is unsustainable due to depleting supplies, rising prices, and the contribution of these fuels to atmospheric pollution [1]. Such environmental and economic concerns have driven interest in renewable bioenergy, which could serve as a substitute for fossil fuels and alleviate greenhouse gas emissions. Of the various sources of biofuel, oilseeds have recently emerged as promising sources for efficient biodiesel production. One of these crops, Camelina sativa (L.) (C. sativa) Crantz, is a member of the Brassicaceae family whose unique characteristics have fueled its rise as an important biofuel feedstock [2]. Seeds produce an oil rich in poly-unsaturated fatty acids [3,4] thereby making it a valuable renewable energy crop for the emerging biofuel industry [5,6].
While C. sativa accessions have been investigated for their agronomic traits and oil profiles, very little research has been conducted to improve value-added qualities due to limited breeding studies [7]. Genetic transformation enables the stable incorporation of these traits resulting in varietal improvement and requires efficient in vitro propagation to generate a large number of transgenic plants. In addition to enhancing single traits, this process serves as an important tool for studying gene function and expression of characteristics such as disease resistance and stress tolerance. Somatic hybridization studies [7,8], plant regeneration from leaf explants [9], and floral dip transformation [10,11] have been reported; however, plant regeneration and Agrobacterium-mediated genetic transformation from apical and axillary meristems has not been optimized. In addition, use of shoot meristems would be more advantageous than the floral dip method, which is plagued by very low transformation efficiency [12]. In this study, we describe a transformation system for C. sativa using shoot apical meristem explants from rapidly growing micropropagation cultures. Transgenic plants were selected based on expression of enhanced green fluorescent protein (EGFP) and kanamycin resistance (Fig. 1). HINT: To simplify media preparation, stock solutions of 1 mg/L can be prepared separately for BAP and NAA. These chemicals can be dissolved in 1 N NaOH and diluted to final volume with distilled water.

NOTE:
The cultivars used in the study exhibited a high capacity for shoot regeneration.

Antibiotic stock solutions
9 Rifampicin: 20 mg of antibiotic dissolved in 500 µl of methanol.

CAUTION:
Rifampicin is light sensitive and should be stored and used away from light. 9 Kanamycin sulfate: 100 mg of antibiotic dissolved in 1 ml of water and sterilized using a 0.2 µm filter membrane. 9 Cefotaxime: 100 mg of each antibiotic dissolved in 1 ml of water and filter sterilized.

NOTE:
Antibiotic stock solutions are filter-sterilized and stored at −20°C. Antibiotics are added after autoclaving and cooling the culture medium to 55°C.
Agrobacterium culture 9 Binary vector containing an egfp/nptII fusion gene under the control of a cauliflower mosaic virus 35S (CaMV 35S) promoter. 9 Agrobacterium culture stock containing the binary vector (stored in glycerol at −80°C).

1.2.
Transfer seeds to 25% commercial bleach solution containing one drop 100% Tween 20 (added using 1 ml micropipette) and surface-sterilize for 15 min with constant agitation.

1.3.
Rinse twice for 5 min with sterile distilled water.

1.4.
Blot dry seeds on filter paper and transfer to CR medium.

1.5.
Cover Petri dishes in aluminum foil and incubate in darkness at 25°C for 2 d.

1.6.
Transfer Petri dishes containing the germinated seedlings to cool white fluorescent light (75 mm m -2 s -1 and 16 h photoperiod) at 25°C for 5 d.

1.7.
Excise meristems from germinated seedlings and transfer to fresh CR plates under conditions described in the previous step 1.6.
CRITICAL STEP: Avoid any contact of the scalpel blade with the shoot apical meristem. This could result in significant decrease or lack of proliferation in the subsequent steps.

1.8.
Proliferate cultures by subculture to fresh CR medium at two week intervals.

NOTE:
It is critical to transfer shoot cultures to fresh CR or selection (Sel) medium at 1-2 week intervals. Failure to do so will lead to a decrease in regeneration potential of explants and subsequent reduction in transformation efficiency and plant regeneration.

NOTE:
Use fresh shoot tips and meristems for Agrobacterium-mediated transformation.

2.
Initiation of Agrobacterium culture 2.1. Thaw Agrobacterium culture containing the binary plasmid at room temperature.

2.2.
Spread approximately 20 µl of bacterial culture on a Petri dish containing solid YEP medium with 20 mg/L rifampicin and 100 mg/L kanamycin.

2.3.
Incubate dishes in the dark at 26°C for 3 d.

2.4.
Isolate a single colony growing on YEP medium and transfer it to a 125 ml conical flask containing 30 ml MG/L medium with 20 mg/L rifampicin and 100 mg/L kanamycin.

2.5.
Incubate on a rotary shaker at 180 rpm at 26°C for 16-20 h. The bacterial culture should appear cloudy at the end of the culture period.

2.6.
Transfer the culture to a 50 ml centrifuge tube and spin at 6000 rpm for 8 min at room temperature. Discard the supernatant and resuspend the pellet in 30 ml liquid MS medium. Adjust optical density at 600 nm (OD 600 ) value to 0.2 using liquid MS medium.
CRITICAL STEP: Low OD 600 can decrease transformation efficiency, while high OD 600 could lead to excess growth of Agrobacterium and decrease virulence.

2.7.
Transfer the contents of the tube to a 125 ml conical flask and incubate for additional 4 h in a rotary shaker under the same conditions as above. Use this culture for co-cultivation.

NOTE:
C. sativa shoots proliferate by micropropagation with new shoots emerging from the primary shoot. Thus, shoot tips containing apical meristems are the best target tissues for Agrobacterium-mediated transformation.

3.2.
Add 5.0 ml Agrobacterium culture to explants and mix thoroughly by swirling. Incubate for 7 min. Blot explants dry on filter paper to remove the excess bacteria.

NOTE:
Blot-drying explants on filter paper is beneficial as it reduces excessive growth of bacterial cells resulting in a dramatic decrease in cell necrosis and higher transformation efficiency.

3.3.
Transfer blot-dried explants to solid CR medium.

3.4.
Seal the Petri dish with Parafilm ® and co-cultivate in darkness at 26°C for 2 d.

3.5.
After 2 d observe explants for transient GFP expression using microscope equipped with fluorescence illumination system.

3.6.
Transfer co-cultivated explants to petri dishes containing solid Sel medium.

3.7.
Transgenic cultures can be identified on the basis of GFP fluorescence and kanamycin resistance and can be used to separate transgenic lines from non-transformed cultures. Designate each line as an independent event and transfer to Sel medium.

3.8.
Transfer shoots to Magenta GA7 vessels containing 30 ml Camelina rooting medium. Place vessels under conditions previously mentioned in step 1.6.

3.9.
Transfer plants to 7 cm plastic pots containing Pro Mix BX potting mix and acclimatize in a growth room for two weeks before transfer to a greenhouse.

4.1.
Total genomic DNA can be isolated using the QIAGEN DNeasy Plant Mini Kit.

4.3.
PCR products can be visualized by agarose gel electrophoresis.

ANTICIPATED RESULTS
Improvement of C. sativa germplasm for breeding programs requires the development and establishment of in vitro regeneration and transformation methods. Although the possibility of apical shoot meristems for plant regeneration and genetic transformation has long been suggested in other crops [13], there is no previous report on an egfp/nptII reporter-marker fusion transformation in C. sativa.
The procedure described herewith offers an optimized method of

Protocol
Agrobacterium-mediated transformation of apical shoot cultures as monitored by EGFP expression. Shoot tips that expressed EGFP produced a bright green fluorescence when observed under a microscope equipped with epi-fluorescence illumination ( Fig. 2A). Additionally, transgenic cells carrying these marker genes selectively grew on culture medium containing kanamycin while inhibiting the growth of non-transformed cells (Fig. 2B). Selectable marker genes such as npt II and hygromycin phosphotransferase are routinely used along with reporter genes in genetic transformation [14].   Transformation with an initial Agrobacterium optical density (OD 600 ) of 0.6 was excessive, while a reduction to 0.2 resulted in increased explant survival following cocultivation (Fig. 3A). In addition, reduction of co-cultivation interval from 3 to 2 d and a cefotaxime concentration to 200 to 50 mg/L (Fig. 3B) enhanced the survival of explants in both cultivars. A co-cultivation period of 3 d resulted in the death of all explants due to excess Agrobacterium growth. This report details a novel and efficient protocol for Agrobacterium-mediated transformation of C. sativa shoot meristems to generate transgenic plants in as little as 28 d (Fig. 4). Optimization of parameters for genetic transformation is essential for explant proliferation and improvement of value-added traits in plants [15].

TROUBLESHOOTING
Potential problems and troubleshooting suggestions are listed in Table 1.