1. Transformed explants grown on selective medium Transformation strategy
“Transformed sweet potato plant tissues potentially able to produce anthocyanins as nutraceutic compounds”
­Ninel Peralta1, Arletys Verdecia2, Néstor Mora2, Andrys Martínez1, Laritza Domínguez2 and Rolando Morán2.
1. Camagüey University Ignacio Agramonte Loynaz, 2. Centre for Genetic Engineering and Biotechnology, Camagüey.
Corresponding author:
Abstract
The consumption of natural colorants with antioxidant capacity is showing a growing interest due to its benefits for human health. The main plant metabolic pathway for these substances is related with the production of flavonols and anthocyanins. The use of biotechnological tools allows their production in cells usually unable to naturally produce them. The present work describes the development of a sweet potato, variety CEMSA , regeneration and genetic transformation methodology with the aims of improving its nutritional and medicinal quality as well as tissues potentially useful for antioxidant compound production. It was demonstrated that the best in vitro regeneration efficiency was achieved when stem segments are used as explants. They were cultivated first in MS solid medium supplemented with 0.4 mg.L-1 of Naphtalene Acetic Acid and then transferred to a similar medium containing 0.2 mg.L-1 of the same hormone. 25mg.L-1 of Kanamycin was established as the minimum concentration able to inhibit the in vitro sweet potato cell regeneration. Through an Agrobacterium tumefaciens mediated transformation methodology, a DNA fragment was incorporated to cells of this crop. Foreign DNA contained the coding genes of two Transcription Factors of the anthocyanin biosynthesis pathway from the plant Antirrhinus majus. The growth in antibiotic selection medium of tissues resulting from transformation reflects the obtainment of sweet potato cells able to produce anthocyanins as a consequence of interest gene insertion. This result confirms the validity of the proposed methodology as a way to obtain natural food colorants in plants by means of the biotechnology.
Results
Regeneration
Rooting percentage and shoot formation in stem segments (S) and petioles (P) under different hormonal combinations
Treatment
Rooting (%)
Shooting (%)
Control S
33 c
25 b IS
95 a
35 a
IIS
10 e
0 d
Control P
20.8 d IP
83 b
4.2 c
IIP
8.3 e
CS and CP control explants after 4 weeks in the dark , CS .1 and CP .1 control explants after 8 week in the light; IS and IP explants cultivated in 0.4 mg.L-1 NAA after 4 weeks in the dark , IIS ans IIP explants in 0.1 mg.L D after 4 weeks in the dark, IS.1, IIS.1, IP.1 and IIP.1 explants cultivated in 0.2 mg.L-1 NAA after 8 weeks in the light
In vitro stem segments regeneration under Kanamycin c
A and B, control (0 mg.L-1),
C (12.5 mg.L-1) y D (25 mg.L-1)
Materials and Methods
Molecular confirmation of transformed Agrobacterium tumefaciens LBA 4404 strain carrying the binary plasmid pJAM 2025
General strategy
Artificials
Naturals
Anthocyaninss
Sweet potato
(Ipomoea batatas L.)
Genetic transformation by
Agrobacterium tumefaciens
Food colorants bp
PCR Amplification of the 1153 bp fragment coding for the Sporamin promoter. 1- Molecular Weigth Marker Bench Top Ladder (Promega). 2- PCR product from recombinant A.t. plasmid DNA. 3- PCR product from native A.t. plasmid (negative control).
Transformed explants grown on selective medium
Transformation strategy
Rosea 1
MYB
Delilah
bHLH
Promoters
Map of the T-DNA region of the construct pJAM2025 for plant transformation
in vitro CEMSA sweet potato plants of four weeks under culture
27 mL liquid MS medium, 15 – 20 explantes
Infection
3 mL of bacterial culture recombinant strain LBA4404 containing pJAM (OD620nm 0.8 – 1.0)
Co- culture
24 h resting in the dark, 280C
Incubation
30 mL liquid MS, 500 mg.L-1 of Cefotaxime for 30′
Washing
Sterile water 3-5 washing
Regeneration
MS mg.L-1 NAA mg.L-1 Cefotaxime
Wiping
Sterile filter paper
Slow stirring 1′
stem segments
Untransformed control
Conclusion
Sweet potato plant tissues from the variety CEMSA 78354, resistant to in vitro selection conditions and potentially able to produce and accumulate anthocyanins, were obtained