It is also used as a plant model for tissue culture and embryogenesis studies [5–8], as well as genetic and genomic studies looking for understanding the evolution of domesticated carrots [9–
11]. Other researches have been centered on studying gene function and regulation of carotenoid synthesis in the storage root
through over expressing or down regulating carotenogenic genes
[12–16]. Genetic engineering of carrots has been also successful for
producing astaxanthin [17] and human interferon alpha-2b protein
as medical treatment for several virus diseases [18]. On the other
hand, carrots expressing disease resistance genes have provided
defense against abiotic and biotic stresses [19, 20]. Recently, the
CRISPR/Cas technology was established in carrot targeting the
flavanone-3-hydroxylase (F3H) [21]. For most of these researches,
carrot genetic transformation is required. Among the diverse techniques, those based in Agrobacterium tumefaciens are the most
common methods used elsewhere since the first transformation
protocol was reported [22]. Thereafter, many optimized transformation procedures have been established using roots, cotyledons,
hypocotyls, and petioles as explants as well as different cocultivation
time and culture medium [19, 23–25]. In this work we present a
detailed method for carrot nuclear transformation through somatic
embryogenesis for basic and applied research.
2 Materials
All culture media should be prepared with deionized water and
analytical grade reagents, then, sterilized (150
C for 15 min in
autoclave) before use. Glass petri dishes, tweezers, and scalpels
should be sterilized in autoclave as well.
2.1 Carrot
Transformation
1. Liquid MS 1Â media: 1Â Murashige and Skoog medium with
vitamins (MS), 2% Sucrose, 0.05% myoinositol, pH 5.8. Weigh
2.215 g of MS, 10 g of sucrose, 50 mg of myoinositol and add
400 mL of deionized water. Stir the solution at room temperature until all components are dissolved, then adjust pH with
KOH (see Note 1). Make up to 500 mL with deionized water
and sterilized.
2. Solid MS 1Â media: 1Â MS, 2% sucrose, 0.05% myoinositol,
0.7% agar, pH 5.8. The procedure is the same as the previous
culture media, but after adjusting pH and making up the
volume, 7 g/L agar is added and then sterilized.
3. Coculture plates: Solid MS 1Â media supplemented with
1 mg/L 2.4-dichlorophenoxyacetic acid (2.4-D). Melt the
solid MS 1Â media and let it cool (see Note 2), then measure
30 mL of media inside a 50 mL, sterile plastic tube, and add
2.4-D to a final concentration of 1 mg/L (use a stock solution
314
Christian Gonzalez-Calquin and Claudia Stange
11]. Other researches have been centered on studying gene function and regulation of carotenoid synthesis in the storage root
through over expressing or down regulating carotenogenic genes
[12–16]. Genetic engineering of carrots has been also successful for
producing astaxanthin [17] and human interferon alpha-2b protein
as medical treatment for several virus diseases [18]. On the other
hand, carrots expressing disease resistance genes have provided
defense against abiotic and biotic stresses [19, 20]. Recently, the
CRISPR/Cas technology was established in carrot targeting the
flavanone-3-hydroxylase (F3H) [21]. For most of these researches,
carrot genetic transformation is required. Among the diverse techniques, those based in Agrobacterium tumefaciens are the most
common methods used elsewhere since the first transformation
protocol was reported [22]. Thereafter, many optimized transformation procedures have been established using roots, cotyledons,
hypocotyls, and petioles as explants as well as different cocultivation
time and culture medium [19, 23–25]. In this work we present a
detailed method for carrot nuclear transformation through somatic
embryogenesis for basic and applied research.
2 Materials
All culture media should be prepared with deionized water and
analytical grade reagents, then, sterilized (150
C for 15 min in
autoclave) before use. Glass petri dishes, tweezers, and scalpels
should be sterilized in autoclave as well.
2.1 Carrot
Transformation
1. Liquid MS 1Â media: 1Â Murashige and Skoog medium with
vitamins (MS), 2% Sucrose, 0.05% myoinositol, pH 5.8. Weigh
2.215 g of MS, 10 g of sucrose, 50 mg of myoinositol and add
400 mL of deionized water. Stir the solution at room temperature until all components are dissolved, then adjust pH with
KOH (see Note 1). Make up to 500 mL with deionized water
and sterilized.
2. Solid MS 1Â media: 1Â MS, 2% sucrose, 0.05% myoinositol,
0.7% agar, pH 5.8. The procedure is the same as the previous
culture media, but after adjusting pH and making up the
volume, 7 g/L agar is added and then sterilized.
3. Coculture plates: Solid MS 1Â media supplemented with
1 mg/L 2.4-dichlorophenoxyacetic acid (2.4-D). Melt the
solid MS 1Â media and let it cool (see Note 2), then measure
30 mL of media inside a 50 mL, sterile plastic tube, and add
2.4-D to a final concentration of 1 mg/L (use a stock solution
314
Christian Gonzalez-Calquin and Claudia Stange