3.1 Materials
3.1.1 Plant materials
Representative samples of A. paniculata collected from various regions in India and other countries of Asia were used for the study. Seeds from foreign sources were either collected (Malaysia) or received as gift. Four-month-old plants raised through seed propagation and reared under identical conditions served as the source of young leaf tissues used in various experiments. The plants collected from natural habitats and then grown in the garden campus were treated as ex situ populations (? germplasm accessions) while those taken from the natural habitats and directly used in various experiments were considered as in situ populations (= natural populations). For mutation and possible selection of high yielding variants, based on the yield performance a sample collected from Tamil Nadu (AP36) was used.
3.1.2 Chemicals and glassware
Chemicals and glassware used for the experiments were procured from the following sources:
3.1.3 Instruments
Precision scientific instruments used for observations, extraction and analysis of various plant samples were:
3.1.4 Software
POPGENE Version 1.0 (Yeh & Boyle, 1997) and PCO3D Version 1.2 (R.P.Adams, Baylor University, USA) were used for genetic data analysis. ChemiImager 4000i Version 4.04 (Alpha Innotech Corporation, USA) was used to document and analyse isozyme, protein and RAPD gels. Statistical analysis was performed using Microsoft® Excel 97. Estimation of plant pigments and calculation of purity and amount of DNA required for PCR assay were carried out using PlantPack Version 1.1 (Sabu and Seeni, 2001).
3.2 Methods
3.2.1 Germplasm collection
Germplasm collection of A. paniculata was organized as an assemblage of seeds or plants collected through explorations,2 seed donations and exchanges from other collections. While collecting plants from their natural habitats, stratified random sampling method (Moss and Guarino, 1995) was followed. Representative samples from distinct populations (separated by at least 10 km distance) were collected and entered into logbook and used for all the experiments. Of the 56 populations of A. paniculata collected/ obtained, the present study was confined to 15 (Table 3.1), short-listing being based on the geographical location (e.g. all the foreign populations were included except the one from Sri Lanka which showed low rate of germination; if more than one population was collected from a habitat, any one having more number of plants/seeds were included) and morphological diversity (e.g. variation in leaf size, shape and colour; plant height; number of branches; number of seeds per fruits etc.). Seed samples collected from such populations were germinated, raised in pots and maintained under identical growing conditions in an experimental area within the institute campus (80 45’ N, 770 02’ E; 220 m above msl) and were used for analyzing intraspecific variations, if any. For analyzing genetic diversity, differentiation, theoretical gene flow and degree of selfing within as well as between populations and subpopulations, two metapopulations (= very large populations) were located in Sirumalai (AP54), Tamil Nadu and Nallamalai (AP56), Andhra Pradesh (Table 3.1). Each of the metapopulations consisted of many scattered local populations, which in turn composed of many continuous subpopulations.
3.2.2 Biology
Plant material: Seeds of A. paniculata collected from various localities (Table 3.1) were germinated in petri-plates. Three week-old seedlings were transplanted and grown in pots (20 cm wide and 30 cm long), the potting medium consisting of sand and farmyard manure in 3:1 ratio. Seeds were also sown in plots of 2 x 3 m2 within the institute campus and seedlings were raised under identical conditions. Plants were irrigated regularly on alternative days except during the rainy season.
Distribution map: Details for the construction of distribution map of A. paniculata were collected from published systematic works, floristic manuals and a personal communication from Krishna Murti, Royal Botanic Gardens, UK. Observations on flowering and fruiting were made on potted as well as field-grown plants. Details of the morphology of leaves and fruits were collected from plants of uniform age.
Ploidy check: To check the ploidy level of the collected plants, excised young flower buds were fixed in Carnoy’s fixative (Sharma & Sharma, 1980) as described by Ziaciddin et al. (1997) with appropriate modifications. Flower buds were kept in Carnoy’s I fixative (absolute alcohol and acetic acid, 3:1) for 30 min followed by the addition of saturated aqueous solution (10 µl/ml) of ferric chloride (4 gm/ml) to the fixative containing the buds and incubation for 7.5 h at room temperature. After this period, the fixative was replaced with fresh fixative and the buds were incubated for additional 16 h. After fixation, the buds were transferred to 70% ethanol and stored at 40C. Flower buds retrieved from storage were squashed under coverslip in 2% acetocarmine, sealed with transparent nail polish and the anther cells observed under the microscope.
Determination of moisture content of seeds: To study the effect of moisture content on seed germination, lots of 25 seeds were sown on filter paper discs moistened with 7.5 ml deionised water in disposable petri-plates (10 cm wide; Laxbro, Mumbai, India). The seeds were germinated in the dark at a temperature regime of 330C / 230C (day / night) under laboratory conditions. The seed moisture content of various samples was determined (Anonymous, 1985) by incubating seeds at 103 ± 20C for 17 h in a thermostat controlled Universal electric oven (Narang Scientific Works, India).
Breeding system: For investigating the breeding behaviour of the species, individual groups of plants (6-8 plants in each of the groups) were given various treatments. To check the extent of self pollination, one group of plants bearing flower buds was bagged to prevent entry of foreign pollen grains and another group manually selfed and then covered with a bag. Yet another group without any treatment (bagging / selfing) was kept as control for various experiments. Pod set and seed development in these plants were monitored periodically up to 3 months. Many bees of the genus Apis were found visiting flowers during daytime. In order to study their influence on pollination, one group of plants was covered with fine nylon net (9 strands per cm) while the other set was not covered. To find out likelihood of cross-pollination, flower buds of one group of plants were emasculated and dusted with pollen from flowers of another distinct group of plants. In the control plants flower buds were emasculated but not treated with pollen grains.
In order to study the effect of wind on pollination, 7-8 plants were transferred to the laboratory and maintained in a closed room devoid of free air circulation while another set of 2 plants was kept outside the laboratory as control. The mature fruits from various treatments were collected and fertilization success was determined by scoring large, developing ovules as fertilized and shrunken or small as infertile ones.
To assess percentage of pollen viability, undehised anthers were collected before anthesis from 4 different plants from the study site. Pollen viability was estimated by staining the samples in 2% glycero-acetocarmine (1:1). Grains not staining or shrunken were recorded as non-viable and the fully stained ones as viable.
Seed germination: Various treatments were tried to induce germination of freshly harvested mature seeds of A. paniculata. Organic infusion of growth regulators with acetone was performed as reported by Persson (1988). Lots of 25 seeds were treated for two hours by immersion at room temperature in 50 ml conical flasks containing growth regulator solutions. The seeds were then taken out, air-dried and stored at room temperature until tested for germination. Acetone (Merck, India) was used for making solutions of giberellic acid (GA3) (Sigma, USA) at 1 mM concentration and kinetin (Sigma, USA) at 0.5 mM.
Effect of surfactants on germination was studied by treating the seeds with 0.1% solution of CTAB (Sigma) and Tween-80 (Hi Media), for 3 h. Acid scarification was also tried to break dormancy due to seed coat factors if any, by immersion in concentrated sulphuric acid for 2, 10, 30 and 60 min. After the treatment, the seeds were separately rinsed 4-5 times in distilled water and then tested for germination.
3.2.3 Analysis of intraspecific variations morphological variation
In order to analyse the morphological variations between the selected 15 populations, ten quantitative morphologic markers, viz. plant height, number of primary branches, number of leaves, leaf length, leaf breadth, leaf area, number of fruits per plant, fruit length, number of seeds per fruit and weight of 25 seeds were selected (Table 3.2). Seeds collected from selected plants were germinated and the characters analysed through three generations. The mean values then obtained for each character were used for plotting polygraphs, an ideal representation of phenotypic variations using Microsoft® Excel.
phytochemical variation
Seed propagated plants of identical age (6 months) grown under uniform conditions were screened for the major active principle, andrographolide (C20H30O5; MW 350.44), a bicyclic diterpenoid lactone. The methanolic extract of the shade-dried leaves was analysed spectrophotometrically at 223 nm to estimate its concentration following the standard protocol of Gained et al. (1963). A detailed method of extraction and estimation is depicted in Fig 3.1. Further analyses of selected samples were carried out through HPLC (Handa and Sharma, 1990). Authentic sample of andrographolide served as the standard.
physiological variation
Young (2-month-old) and mature (6-month-old) plants were selected for analyzing age-related changes in chlorophyll, carotenoid and anthocyanin content among different populations. Chlorophyll and carotenoids were extracted using 80% acetone (Sadasivam and Manikkam, 1996). 5 gm fresh leaves were homogenized in 40 ml of 80% acetone and then the extract was centrifuged at 1000 rpm for 2 min in Sorvall RC5C centrifuge. The supernatant was collected and used for measuring optical density at 490, 645 and 663nm using Shimadzu UV2000 spectrophotometer. Chlorophyll a, chlorophyll b, total chlorophyll and carotenoids from young as well as old plants of 15 populations of A. paniculata were estimated following Arnon (1949) using PlantPack computer package.
For estimating variation in anthocyanin content of leaves, 0.5 gm leaf tissue was homogenized in 4 ml of methanol containing 1.0 M HCl and was kept at 40C for 4 h. The homogenate was filtered and centrifuged at 10,000 rpm for 30 min and the absorbance of the supernatant was measured at 530 nm (Janda et al., 1996), after suitable dilution if necessary. The experiment was conducted for the young and mature plants of selected populations of A. paniculata.
Cytological variation
In order to study possible variations in chromosome number within A. paniculata, 4 populations (AP06, AP24, AP36 and AP48) that were morphologically and/or phytochemically different were chosen and used for preparing anther squashes. The procedure for fixing and squashing of flower buds was the same as that described under 3.2.2. Observations were made under a Leitz Orthoplan microscope with photomicrographic attachment. isozyme variation
Preparation of enzyme extract. Fully opened leaves of mature plants (3-4 months old) were used to prepare crude enzyme extracts for analysing variation between populations using eight enzyme systems (Table 3.3). Because all the ex situ populations were maintained as progeny arrays through controlled selfing or as clones, no within-population variation was expected and hence only four individuals per population were used at a time for preparing crude enzyme extracts.
The young leaves were washed once in deionised water and mashed in a pre-chilled mortar in 200 ?l of 0.2 M Tris-HCl buffer (pH 8.4) containing 0.056 M ?-mercaptoethanol. The resultant slurry was centrifuged at 12,000 rpm for 10 min at 40C in a Sorvall RC5C centrifuge and the supernatant was stored at ?700C before use.
For within population analysis, young 2-3 month old plants from the natural habitat were collected in wet paper and brought to the laboratory. After wash in tap water, the leaves were dissected out and the crude enzyme extracts were prepared by grinding in Tris-HCl buffer using a tissue-buffer ratio: 1 mg / 4 µl. The resultant slurry was centrifuged as described above. The supernatant was stored at -700C before use.
Electrophoresis. Vertical discontinuous polyacrylamide gel electrophoresis (PAGE) was carried out for separation of isozyme(s) (using crude extracts) with appropriate modifications (Anonymous, 1997). Composition of the gel and electrode buffers used for isozyme electrophoresis is presented in Table 3.4. Gels of 16 x 18 cm2 size and 1 mm thickness were cast using the gel mould of Hoefer electrophoresis system. For this the glass plates were assembled and fixed in a gel-casting stand. First, the resolving (separating) gel of the composition shown in the table was prepared and poured into the gap between the glass plates. The acrylamide solution was overlaid with thin film of distilled water in order to prevent oxygen from diffusing into the gel and inhibiting polymerisation. After polymerisation of the gel is complete, the overlay was removed and stacking gel components were mixed and poured over the already polymerised resolving gel. Immediately a clean Teflon comb was inserted into the stacking gel solution. After polymerisation, Teflon comb was removed and the wells were washed with distilled water. The gel was then mounted onto the electrophoretic apparatus.
Eight enzyme systems resolved in the 15 populations using Tris-Glycine electrophoresis buffer system were: alcohol dehydrogenase (Adh), aryl esterase (Est), glutamate dehydrogenase (Gdh), glutamate oxaloacetate transaminase (Got), malate dehydrogenase (Mdh), peroxidase (Per), succinate dehydrogenase (Sdh) and superoxide dismutase (Sod). Staining of the gels was done by following standard procedures (some with minor modifications) as listed in Table 3.5.
For the 2 natural populations (AP54 and AP56), only 3 enzymes (Est, Got, Sod) that revealed comparatively high polymorphism during previous experiments were analysed.
Genetic interpretation of enzyme phenotypes was based on observed differences in zymogram profile after activity staining of PAGE. Nomenclature of isozymes and allozymes was based on Wendel and Percy (1990). Locus encoding the most anodally migrating isozyme for each enzyme system was assigned the numerical designation 1, with additional loci numbered sequentially (2, 3…) in order of decreasing electrophoretic mobility. Similarly, the fastest moving allozyme at each locus was given character designation A, with following alleles (B, C…) named in order of decreasing electrophoretic mobility.
Variation at dna level
Extraction of genomic DNA. Total genomic DNA from the young leaves of the plants from 15 populations was isolated following modified Murray and Thompson (1980) method using CTAB (Table 3.6; Fig 3.2). Extraction buffer contained 1.2% PVP-40T (MW 40,000, Sigma, USA) to remove high phenolic contaminants and double CHCl3 extraction at 10,000 rpm helped to remove polysaccharides. After ethanol precipitation, DNA was resuspended in 100 ml of 1x TE buffer (pH 8.0).
RAPD assay. RAPD assay was carried out in 25 ml reaction mixture containing 2.5 ml 10x amplification buffer (500 mM KCl, 100 mM Tris HCl, 1.0% Triton X-100 and 15 mM MgCl2), 100 ml each of dATP, dGTP, dCTP and dTTP, 1.0 U of Taq DNA polymerase (Promega, USA), 15 pM of 10-mer primer (Operon Technologies Inc, USA) and 50 ng of genomic DNA. Amplification was performed in MJ Research Thermal Cycler (PTC 100). The sequential steps were: 1 cycle of 2 min at 93°C, 2 min at 35°C and 2 min at 72°C followed by 38 cycles of 1 min at 93°C, 1 min at 36°C and 2 min at 72°C. The last cycle was followed by 10 min extension at 72°C.
Agarose gel electrophoresis. The amplified products were resolved in 1.2% agarose gel (1x TBE) followed by EtBr staining and the bands detected were documented using Alpha ChemiImager. Amplified products that were reproducible and consistent in performance were chosen for data analysis.
3.2.4 Estimation of genetic diversity
POPGENE and PCO3D computer programs were used to estimate standard genetic variability measures from data obtained from isozyme and RAPD assays. The bands were represented by characters (A-E) for isozyme and numbers (0-1) for RAPD data analysis. The percentage of polymorphic loci (P), the mean observed number of alleles per locus (A or na) or the mean expected number of alleles per locus (ne), the mean gene diversity (Shannon’s information index) (I), the mean observed (Ho) and expected (He) heterozygocities and the average heterozygocity (h) were estimated. Nei’s original measures of genetic identity (i) and distance (d) (Nei, 1972) were also calculated. The distance values were calculated by the formula:
d = -loge[Jxy/(JxJy)0.5]
in which Jxy = ?xiyi, Jx = ?xi2, Jy = ?yi2, and x1 and y1 denote the frequency of the ith allele in populations X and Y, respectively.
The degree of genetic differentiation between populations was estimated following the F-statistics of Wright (1965) modified by Hartl and Clark (1989). Measures included the fixation indices - inbreeding in each subpopulation (FIS), and in the entire population (FIT), and degree of genetic differentiation among populations (FST). It is given by:
FST = 1 - (1-FIT) / (1-FIS).
The gene flow (Nm) among the subpopulations of AP54 and AP56 was calculated following Slatkin and Barton (1989) using the formula Nm = 0.25 x (1- FST) / FST. Cluster analysis using the UPGMA phenogram (Sneath and Sokal, 1973) was applied to estimate genetic distance values from Nei’s distance estimates.
3.2.5 Induction of mutation and selection of variants
One of the objectives of the study was to identify populations of enhanced productivity and further to improve the genotypes by inducing variations within an otherwise desirable population. Therefore, seeds collected from AP368 rich in andrographolide were subjected to chemical mutagenesis using EMS. The seeds were soaked in distilled water for 24 h kept over filter paper discs in petri dishes. Then they were immersed in 0.05 – 1.00% EMS (v/v) for varied duration of time. For each treatment (of the 16 combinations), 50 seeds were used with 50 ml of the mutagen. After the treatment, seeds were washed in running tap water for 2 h and sown immediately. The plants thus raised were provisionally designated as chemical mutants (CM).
The first-generation of chemical mutants (CM1) were not subjected to any analysis, but were selfed and seeds collected to raise second generation (CM2). The second-generation plants were analysed for resolving morphological, chemical (andrographolide) and biochemical (isozyme: esterase) variations if any. Parameters for all analysis were the same as that described under section 3.2.3. The herbage yield and andrographolide content were estimated in the second and third generation plants.
