SUPPLEMENTARY MATERIAL
Morpho-genetic and chemical stability of long term maintained Agrobacterium
mediated transgenic Catharanthus roseus plants
1Priyanka
Verma*, 1Abhishek Sharma, 1Shamshad Ahmad Khan, 1Ajay Kumar Mathur and
2Karuna Shanker
1
Department of Plant Biotechnology,
2
Department of Analytical Chemistry, Central Institute of
Medicinal and Aromatic Plants (CIMAP), Council of Scientific and Industrial Research, PO
CIMAP, Lucknow- 226015, India
*Corresponding author:
Dr. Priyanka Verma
Scientist
Department of Plant Biotechnology
Central Institute of Medicinal and Aromatic Plants
PO CIMAP, Kukrail Picnic Spot Road
Lucknow- 226015, India
Tel. ++91-522-2717557, Fax. ++91-522-2342666
Email: priyankaurbest@gmail.com
Abstract
Transgenic Catharanthus roseus plants (DT and NT) obtained from the Agrobacterium tumefaciens
and A. rhizognenes mediated transformations, respectively, have been maintained in vitro for five
years. Plants were studied at regular intervals under various parameters like plant height, leaf size,
multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually losses the
GUS gene expression and it was not detected in the fifth year while NT plant even in the fifth year
showed the presence of rol gene A, B and C indicating the more stable nature of Ri transgene.
Vindoline content in the DT was twice more than in control plants. Alkaloid and tryptophan profile
was almost constant during the five years. The cluster analysis revealed that the transgenic Dhawal
(DT) plant is more close to the control Nirmal plant followed by transgenic Nirmal plant (NT).
Key words: Catharanthus roseus; Transgenic plants; ISSR; Terpenoid indole alkaloids
Experimental
Plant material
Transgenics developed from Agrobacterium mediated genetic transformation (Verma and Mathur
2011b; Verma et al. 2012b) namely DT (var. Dhawal derived A. tumefacience transformed) and NT
(Nirmal derived A. rhizogenes transformed) were maintained on ½ strength and full strength MS
basal medium, respectively. The untransformed plants belonging to two varieties of C. roseus i.e.
Dhawal and Nirmal were maintained on modified full strength MS (Murashige and Skoog 1962)
media supplemented with 1 mg l-1 BAP and 0.1 mg l-1 NAA. Nodal segments of both the transgenic
plants were cut and placed on respective maintenance media and regularly sub-cultured after every
30 days. The plants were maintained for five years and observed under different parameters such as
plant height; root length; shoot multiplication etc. on regular intervals. Data shown in Table 1 has
been taken in fifth year of maintenance.
ISSR Profiling
Seventeen ISSR markers (Table S1) were utilized to amplify products from the isolated DNA of all
the four plants (transgencis with their respective control plants). Plant genomic DNA was isolated
using standard C-TAB method. The PCR mixture consisted of 2.5 µl of 10× taq buffer, 1.0 µl of 10
mM dNTPs mix, 1.25 unit of taq DNA polymerase, 6 pmol of primer i.e.1 µl of the primer. ISSR
reactions were performed with 25 ng of DNA. PCR was carried out by denaturation at 94˚C for 5
min followed by 45 cycles of 1 min denaturation at 94˚C, annealing at 45˚C for 1 min and extension
at 72˚C for 2 min with the final extension of 5 min at 72˚C. PCR reactions were performed in 90
well thermocycler (Eppendorf, Germany). Isolated DNA and the amplified products were separated
by electrophoresis on 0.8 % (DNA yield) and 1.2 % (PCR product) agarose gel, respectively, stained
with 0.5 mg/l ethidium bromide using 50 mA current for 3 h in 1x tris EDTA buffer and visualized
by fluorescence under UV light. To calculate the band sizes, double digested k DNA with EcoRI and
HindIII was loaded as standard molecular weight marker. The bands were visualized and
photographed using Gel Documentation System (XR-Quantity One, Bio-Rad Laboratories). A
similarity matrix was obtained after multivariate analysis using Nei and Li’s coefficient (Nei and Li
1979). These similarity coefficients were used to generate a tree for cluster analysis using the
UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method and the software NTSYS
2.1 (Numerical Taxonomy and Multivariate Analysis System).
Tryptophan estimation and alkaloid analysis
Tryptophan estimation in the free amino acid pool was done by a modified procedure of Bieleski and
Turner (1966) for sample preparation and Dalby and Tsai (1975) for colorimetric estimation. One
gram of oven dried tissue was grounded in mortar pestle and extracted twice with 10 ml MCW
mixture (methanol: chloroform: water 12:5:3) and thrice with 10 ml 80% ethanol during each
centrifugation at 12,000–15,000 rpm for 15 min. To the combined extracts of MCW mixture, 7 ml
distilled water and 5 ml chloroform was added and centrifuged to separate phases. Top layer of
centrifuged solution was taken and added to the pooled methanolic extracts. The volume was
reduced to 10 ml under vacuum in a flash evaporator. The concentrated sample was then loaded onto
a DOWEX 50X-2 columns (15 cm) and allowed to stand for 30 min before fractionating out at 1.0
ml/min for complete adsorption on the resin. To remove soluble carbohydrates, the column was then
washed with 25 ml distilled water. Tryptophan was eluted from the column by 4:9:25 ml washing
with 0.3 % ammonia solution. The pooled ammonia fractions were evaporated to dryness on a water
bath at a temperature 80˚C. The residue was then re-dissolved in 2.0 ml distilled water. For
colorimetric estimation, 0.5 ml of that sample was mixed with the 2.0 ml of a reagent made freshly
by mixing equal volume of reagent A and B [Reagent A- 270 mg of FeCl3.6H2O dissolved in 0.5 ml
water mixed with 1 liter of 2% acetic anhydride in glacial acetic acid; Reagent B- 30 N Sulphuric
acid]. The reaction mixture was kept on vortex and incubated at 60˚C on a water bath for 15 min.
The reaction was stopped using an ice bath and the absorption was read at 575 nm. The free
tryptophan was calculated using a standard curve of L-tryptophan (SIGMA-USA) prepared in the
range of 0–50 mg 0.5-1 ml.
For determining the total alkaloids content, 1.0 gm oven-dried (50–60˚C) tissue was extracted thrice
with HPLC grade methanol (3:9:30 ml; 12 h each) at room temperature. The methanolic extracts
were pooled and dried in vacuum to 10 ml, mixed with 10 ml distill water, acidified with10 ml of 3
% HCl and washed thrice with hexane (3:9:30 ml). The aqueous portion was basified with ammonia
(till pH 8.0), extracted with chloroform (3:9:30) dried over anhydrous sodium sulphate, concentrated
in vacuum and weighed. For HPLC resolution and quantification, a modular HPLC apparatus
(Waters Corp., Milford, MA, USA) equipped with a 600E multi-solvent delivery system and a 2,996
photodiode array (PDA) detector was used. Data obtained were processed using Empower Pro
(Waters Corp.) chromatographic software. The injector, solvent delivery controller, and Empower
Pro chromatographic manager were integrated to provide precise and reproducible results. On-line
degassing of solvents was done with helium. An RP-18e reversed-phase chromolith Performance
HPLC column (100-x4.6 mm i.d.) was used for all the analysis. A constant flow rate of 1.2 ml/min
was used during analysis. An optimum mobile phase composition was achieved by using 21:79 (v/v)
acetonitrile-0.1 M phosphate buffer containing 0.5% glacial acetic acid. The other conditions were
pH 3.5, flow rate of 1.2 ml.min, and detector wavelength at 254 nm. Entire run took 40 min (Gupta
et al. 2005).
GUS assay
The leaves of DT plant were immersed overnight at 37ºC in an assay mixture containing 1 mM Xgluc, 50 mM sodium phosphate buffer (pH 7.0) and 0.1% Triton X- 100. Stained tissues were
transferred to 70% (v/v) ethanol and incubated at room temperature to remove residual chlorophyll
(Verma and Mathur, 2011b).
PCR based rol/ GUS gene detection
PCR based detection of rol A, B and C in NT plant and GUS gene in DT plant was performed
according to the method followed by Verma et al.
(2012b) and Verma and Mathur (2011b),
respectively.
Reference
Bieleski RL, Turner NA. 1966. Separation and estimation of amino acids in crude plant extracts by
thin layer electrophoresis and chromatography. Ann Biochem. 17: 278-293.
Dalby A, Tsai CY. 1975. Acetic anhydride requirement in the colorimetric determination of
tryptophan. Ann Biochem. 63: 283-285.
Gupta MM, Singh DV, Tripathi AK, Pandey R, Verma RK, Singh S, Shasany AK, Khanuja SPS.
2005. Simultaneous determination of vincristine, vinblastine, catharanthine and vindoline in leaves of
Catharanthus roseus by high performance liquid chromatography. J Chromato Sci. 43: 450-453.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue
cultures. Physiol Plant.15: 473-492.
Nei M, Li WH. 1979. Mathematical model for studying genetic variation in terms of restriction
endonucleases. PNAS. (USA) 76: 5269–5273.
Figure S1
A
B
C
D
E
F
G
K
H
L
I
M
J
N
Figure S2
D
DT
N
NT
D
DT
UBC 855
D
DT
DT
N
NT
M
D
M
D
DT
D
DT
N
NT
M
DT
D
N
DT
N
UBC 807
NT
NT
NT
M
D
DT
N
NT
N
NT
UBC 848
N
T-2
N
G-3
UBC 843
UBC 823
M
NT
UBC 844
A-4
D
N
NT
M
D
DT
UBC 834
Legend to Figures
Fig. S1. Morphological and histological changes occurred in the Agrobacterium mediated transgenic
plants of C. roseus over a period of five years. Control Nirmal Plant (A);Transgenic NT plant during
first (B), second (C), third (D), fourth (E) and fifth (F) year of growth; Control Dhawal Plant (G);
Transgenic DT plant at first (H) third (I) and fourth (J) year of growth; Gradual loss of GUS
expression in transgenic DT plant during the course of five years (K)-(N)
Fig. S2. ISSR profile generated by primers UBC 855, UBC 844, UBC 843, UBC 848, UBC 823,
UBC 834, UBC 807, T-2, G-3and A-4 and Nei and Li’s similarity matrix based UPGMA
dendrogram. Lanes D and N represents the ISSR profile of parent varieties Dhawal and Nirmal with
respective transgenic plants DT and NT.