Indian J. Genet., 74(3): 362-370 (2014) DOI: 10.5958/0975-6906.2014.00854.2
Variability in morphological characters and alkaloid profile of EMS induced mutants of Catharanthus roseus (L.) G. Don 1
Ashutosh Kumar Verma*, R. R. Singh and S. Singh
1
Plant Genetics Unit, Department of Botany, University of Lucknow, Lucknow 226 007; National Research Centre on Plant Biotechnology, Pusa, New Delhi 110 012
(Received: January 2014; Revised: July 2014; Accepted: August 2014)
Abstract Seeds of Catharanthus roseus were mutagenised with ethyl methane sulphonate. On screening induced variability ten morphological variants (NV1-NV10) were isolated and their morphological variability was assessed. The hierarchical clustering analysis revealed four major groups based on morphological attributes. Genotypes with higher total alkaloid content in leaf were placed together in cluster-I and higher leaf yield plant–1, root yield plant–1 and total alkaloid content in root were placed in cluster-II. Genotypes with lowest mean values of all mentioned traits except leaf lamina width and petiole length were placed in cluster III. The consistency in performance of NV-1 and NV-10 from M1 to M3 without any significant variance in their progeny traits, showed that these were true breeding mutants. Alkaloid profiling of these ten variants was carried out by using recently developed DART MS technique. Marked variations in the alkaloid profile were also noted among them which signify the use of induced mutation in developing desirable ‘chemocultivars’.
Key words:
C. roseus, induced mutagenesis, EMS, variant, DART MS technique
Introduction Catharanthus roseus (L.) G. Don (periwinkle), a perennial tropical plant of family apocynaceae, produces more than 130 monoterpenoid indole alkaloids out of which 25 are dimeric in nature [1]. Two of the dimeric alkaloids vinblastine and vincristine mainly present in the aerial parts, have found extensive application in the treatment of human neoplasma. Among the monomeric alkaloids ajmalicine found in the roots has been confirmed to have a broad application in the treatment of circulatory diseases, especially in the relief of obstruction of normal cerebral blood flow. The annual world demand for vincristine, vinblastine
and ajamalcine is estimated 1kg, 12kg, 5000 kg and market value is about $3,500,000/kg, $1,000,000/kg, and $5000/kg respectively [2]. Vinblastine sulphate (sold as Velban) is used particularly to treat Hodgkin’s disease besides lymphocarcoma, choriocarcinoma, neuroblastoma, carcinoma of breast, lungs and other organs in acute and chronic leukemia. Vincristine sulphate (sold as Oncovin) arrest mitosis in metaphase and is very effective for treating acute leukaemia in children and lymphocytic leukemia. It is also used against Hodgkin’s disease, Wilkins’s tumor, neuroblastoma and reticulum cell sarcoma. Today India is the third largest manufacture of Vinblastine and Vincristine in the world and is exporting these alkaloids to European countries [3]. High demand and low yield of these alkaloids in the plant has led to research for alternative means for their production. The sustainable demand of these alkaloids, in present scenario and their high market price, promotes for intensive efforts for C. roseus improvement. Although considerable work has been done to improve alkaloid production through biotechnological means, very little work has been done for high alkaloid content in C. roseus through conventional approaches [1, 4]. Mutation breeding appears one of the promising approaches for development of improved ideochemovars [5]. Induced mutagenesis in C. roseus var. Nirmal was undertaken to produce improved genotypes. Ten variants were isolated from different generations. In order to make more comprehensive study of variants the growth and yield parameters were observed and their alkaloids profile analysis from leaf and root samples was carried out.
*Corresponding author’s e-mail:
[email protected] Published by the Indian Society of Genetics & Plant Breeding, F2, First Floor, NASC Complex, PB#11312, IARI, New Delhi 110 012 Online management by indianjournals.com
August, 2014]
Morphological diversity and alkaloid profile in induced variants of C. roseus
Materials and methods The seeds of C. roseus var. Nirmal (CIMAP 0865) procured from Central Institute of Medicinal and Aromatic Plants, Lucknow were treated with 0.50, 0.75 and 1.00% ethyl methane sulphonate (EMS). Seeds were shown in experimental field to raise M 1 and subsequent generations. On screening for induced variability from M1 to M2 generation for morphological traits ten morphologically distinct variants were isolated. Seeds from these variants were harvested separately and 100 randomly selected seeds were shown in six replications to raise their M1 generation (generation of selected variants). Five normal looking plants were selected (with respect of their parent i.e. selected variants) and harvested separately and shown in similar pattern as mentioned above to raise subsequent generations. Data were recorded on agronomic parameters namely, germination, plant height, stem perimeter, number of primary branches, –1 –1 root yield plant , leaf yield plant and biochemical parameters viz., total alkaloid content in leaf and root in M3 generation. Data were subjected to statistical analysis of components of variation i.e. genotypic coefficient of variation (GCV), phenotypic coefficient of variation (PCV) and environmental coefficient of variation (ECV). The groups were compared by one way analysis of variance (ANOVA) and the significance of mean difference between control group and other group was done by Dunnet’s post hoc test. Cluster analysis (Hierarchical clustering; Single linkage and Euclidean distances, standardized) was done to assess similarity among the variants. A two-tailed ( =2) probability P>0.05 was considered to be statistically significant. All analysis was performed on STASTICA (version 6.0). Total alkaloid content (µgm/g) estimation in leaf and root tissues was done following standard procedure [6]. At first 10 gm coarsely powdered plant material was extracted with 25 ml 2% aqueous acetic acid at room temperature for 10 minutes. The procedure was repeated three times. The extracts were mixed and diluted to 100 ml with 2% aqueous acetic acid. Then after, 5 ml amount of the extract/solution was taken and the pH was maintained at 2-2.5 with dilute HCl. Two ml of Dragendroff’s reagent (DR) was added to it, and the precipitate formed was centrifuged. The centrifugate was checked for complete precipitation by adding DR. After centrifugation, the centrifugate was decanted completely. The precipitate was further washed with alcohol. The filtrate was discarded and
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residue was then treated with 2ml of disodium sulfide solution (1%). The brownish black precipitate formed was then centrifuged. Completion of precipitation was checked by adding two drops of disodium sulfide. The residue of above mentioned step was dissolved in 2ml of concentrated nitric acid at (if required than warmed). The solution was diluted to 10ml in standard flask with distilled water; 1ml was then pipetted out, and 5 ml thiourea (3%) solution was added to it. The absorbance was measured at 435 nm against the blank containing nitric acid and thiourea. The amount of bismuth present in the solution was calculated by multiplying the absorbance values with the factor, taking suitable dilution factor in to consideration. The factor is obtained from the standard curve by using following formulaeFactor = Concentration/absorbance Leaf and root samples of these variants were collected and used for direct analysis in real time mass spectroscopy (DART-MS) analysis. DART MS is a new ambient ionization technique [7]. Using a helium plasma DART ionizes atmospheric water and generates water clusters which in turn ionize the sample held in the gas stream. The resulting spectra are relatively clean and simple. As DART ion source can ionize molecules directly from the surface, plant products can be analyzed directly without sample preparation [8-10]. DART-MS analysis had been carried out at SAIF division of Central Drug Research Institute, Lucknow and mass chromatograms were obtained. For which first leaves and root samples were washed and wiped dry. The mass spectrometer used was a JMS-100 TLC (AccuTof) atmospheric pressure ionization timeof-flight mass spectrometer (Jeol, Tokyo, Japan) fitted with a DART ion source. The mass spectrometer was operated in positive-ion mode with a resolving power of 6000 (full-width at half-maximum).The orifice 1 potential was set to 28 V, resulting in minimal fragmentation. The ring lens potentials were set to 13V. Orifice 1 was set to a temperature of 100°C. The RF ion guide potential was 300 V. The DART ion source was operated with helium gas flowing at approximately 4.0 l/min. The gas heater was set to 300°C. The potential on the discharge needle electrode of the DART source was set to 3000 V; electrode 1was 100 V and the grid was at 250 V. Freshly cut pieces of samples were positioned in the gap between the DART source and mass spectrometer for measurements. Data acquisition was from m/z 10 to 1050. Exact mass calibration was accomplished by including a mass
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spectrum of neat polyethylene (PEG) glycol (1:1 mixture PEG 200 and PEG 600) in the data file. mNitrobenzyl alcohol was also used for calibration. The mass calibration was accurate to within_0.002 u. Using theMass Center software, the elemental composition could be determined on selected peaks.
NV-6: Early flowering, and six petaled flower containing variant was isolated from 1% EMS (M 1) treatment. It showed (P< 0.05) reduction in pod length and highly significant (P< 0.01) reduction in leaf yield –1 plant , total alkaloid content in leaves and total alkaloid content in roots over control (Figs. 1G-G1).
Results and discussion
NV-7: This variant was found at 0.75% EMS treatment in M2. It is an early flowering, broad leaf and high leaf alkaloid accumulating variant. The pod length and pollen fertility were significantly higher (P< 0.05) –1 and leaf yield plant also showed highly significant (P< 0.01) reduction as compared to control. However, total alkaloid content in leaf showed highly significant increase over control (Fig. 1-H).
On screening induced variability ten new (NV) variants were isolated. These variants were characterized by number of contrasting features and the results are presented here. NV-1: It is a dwarf, ovate leaf, late flowering, sepaloid flower, low leaf and root alkaloid accumulating variant found at 0.50% EMS treatment (M 1 ). Performance of all considered traits was significantly deviating from that of control except for stem –1 perimeter, number of primary branches plant , number –1 of secondary branches plant , leaf lamina width, petiole length, petal width, total alkaloid content in leaf. Mean values for leaf lamina length, corolla tube length, –1 petal length, pod length, seed pod , pollen fertility, –1 –1 leaf yield plant , root yield plant showed significant reduction over control (Figs. 1B-B1). NV-2: This variant was isolated at 0.50% EMS (M1) treatment. It was base branching and high root alkaloid accumulating variant. Performance of all considered traits was almost equal to that of control -1 except for pod length, leaf yield plant total alkaloid content in leaf and total alkaloid content in root. Total alkaloid content in root was found significantly higher (P< 0.01) than that of control (Fig. 1C). NV-3: Early flowering variant was isolated from 0.50% EMS (M2) treatment which showed significant reduction in petiole length (P< 0.05) and highly significant reduction in pod length and pollen fertility with respect of control (P< 0.01) (Fig. 1D). NV-4: It is a dwarf and low root alkaloid accumulating variant isolated from M2 generation at 0.75% EMS treatment and showed significant (P< 0.01) reduction in plant height, corolla tube length, pod –1 length, pollen fertility, leaf yield plant , total alkaloid content in root over control (Figs. 1E-E1). NV-5: Base branching containing variant found at 0.75% EMS (M2) treatment. The variant showed similarity in most of the traits with that of control except for pod length and pollen fertility which was significantly (P< 0.05) lower than that of control (Fig. 1F).
NV-8: Seven petals containing flowers, low root alkaloid accumulating variant was found at 0.75% (M1) –1 EMS treatment. Pod length, leaf yield plant and total alkaloid content in root showed highly significant (P< –1 0.01) reduction while seeds pod and pollen fertility showed significant reduction (P< 0.05) over control (Fig. 1I). NV-9: It is an early flowering, broad leaf with high leaf biomass yielding variant selected at 1.0% EMS (M2) treatment. Performance of all the studied traits –1 was equal to control except for leaf yield plant which was significantly (P< 0.05) higher than that of control (Fig. 1J). NV-10: Semi-dwarf variant was found at 1.0% EMS treatment in M2. Plant height, pod length, leaf –1 –1 yield plant , root yield plant and total root alkaloid content showed highly significant (P< 0.01) reduction, –1 while seeds pod , pollen fertility and total alkaloid content in leaf showed significant reduction (P< 0.05) over control (Fig. 1K). These variants and mutants NV-1 and NV-10 showed consistence performance from M 1 to M 3 generation without any significant variance in their progeny traits and therefore, they were true breeding and hence mutants. All the variants isolated from variety Nirmal showed marked variation in the morphological traits in contrast to the control. The newly created variation in present study is in accordance of previous reports in which mutants, with modified contents of alkaloid [11], monocotyledon [12] heterostyly with harkogamons flowers [13] and self tolerance [14] in C. roseus were induced through mutagenesis. Analysis of variance (ANOVA) The analysis of variance (one way ANOVA) revealed
66.25±1.55 19.35±0.55
Total alk. in leaf Total alk. in root
17.37±1.16 62.00±1.91 11.35±0.64**
21.10±1.94 59.33±4.61 22.95±1.26
25.75±1.57
65.95±4.52 41.33±3.90** 15.35±0.96* 30.52±1.33**
10.98±1.23** 52.70±3.45 18.58±0.33
25.67±2.05
88.13±1.06** 88.63±0.97** 89.58±0.80*
1.32±0.03
17.17±0.65
2.51±0.06*
1.39±0.10
2.25±0.05
2.39±0.11
5.63±0.07
*
2.00±0.05
12.33±1.54
16.33±0.56* 1.20±0.04
12.50±0.81 1.83±0.17
2.44±0.06* 17.58±0.58
10.83±1.38 12.00±1.63 2.19±0.11 6.48±0.25 2.74±0.05 1.51±0.07 2.71±0.09 2.25±0.06 1.69±0.04 2.31±0.08**
2.18±0.07 6.70±0.16 2.65±0.07 1.46±0.09 2.81±0.05 2.40±0.10 1.33±0.07 2.46±0.05*
17.17±0.48 16.17±0.79*
9.50±1.31 1.69±0.06 6.04±0.26 2.60±0.10 1.23±0.03 2.66±0.02 2.37±0.05 1.35±0.07 2.38±0.14* 19.67±0.67
26.29±2.39 64.65±1.82 19.67±1.27
25.44±2.21 24.82±1.72
44.03±2.89** 86.88±1.67** 66.47±3.30 13.83±1.31** 19.85±1.15 11.55±0.90**
22.46±2.46
1.07±0.14 90.90±0.45
1.42±0.04
1.40±0.10
90.04±0.82* 90.22±0.50*
1.08±0.09 92.74±0.48
1.40±0.07
2.31±0.13
2.52±0.10
1.45±0.11
2.60±0.16
6.32±0.14
7.33±0.61
7.83±0.79
8.17±1.40
7.67±1.33
51.18±4.24* 13.61±0.49**
30.97±2.03**
89.31±1.05*
2.23±0.07**
1.47±0.11
2.18±0.12
2.63±0.11
1.37±0.09
2.55±0.15
5.71±0.33
1.86±0.16
7.00±0.77
5.33±0.80
1.10±0.06
1.48±0.05
1.24±0.10
1.32±0.13
1.19±0.12
Characteristics marked in superscript are significant (*p0.05, * = p
