Characterisation of Exacum bicolor Roxb.(KANNANTHALI) for aesthetic ,medicinal value and refinement of domestication practices

Abstract

India is blessed with a wide variety of endemic plant species due to the presence of four out of 34 biodiversity hotspots in the world. Endemic species with narrow geographical range of area are most likely to become extinct due to fragmentation of habitat, human interferences, deforestation, and expansion of agricultural land. Special attention must be given to such small natural populations before they get vanished. Many of such native plant species may also form an important source for sustainable landscaping and xeriscaping. Exacum bicolor which belongs to Gentianaceae family is an herbaceous perennial, endemic to peninsular India. The flowers which are a mix of colors imparted by the white petals with purple tips and bright, yellow-colored stamens, are the most beautiful part of the plant. Mature sturdy stem that ensures the non-lodging habit of Eaxcum bicolor even during heavy rainfall, makes the plant more suitable for gardening. The plant also holds enormous medicinal properties and was traditionally used by “vaidyas” for the treatment of various diseases. Though being frequently quoted by Malayalam literary writers, the plant is not that familiar to many common people due to its restricted occurrence. Exacum bicolor is an inhabitant of the dry grasslands of the lateritic hillocks and the shola grasslands in the high ranges of Kerala. The plant is enlisted as endangered due to its rapid shrinking population imposed by enmasse collection of the plants that happened decades back for herbal formulations, never ending, severe habitat destruction and also mass uprooting of the plant for its beauty. Discontinuous and isolated geographical situation of the habitats are other unique characteristics of the plant. The majority of the habitats in the lateritic hillocks are under private possession and are very likely to be exploited soon. Therefore, ex-situ conservation is of urgent need for conservation of Exacum bicolor. Evaluation of genetic variability among the available genotypes of the species and standardization of domestication practices are of utmost importance. In this context, the present study was undertaken with the objectives of morphological characterization of Exacum bicolor genotypes for aesthetic value, metabolite profiling for medicinal value, and standardization of propagation and management practices for popularization as a novel ornamental plant. The study was organized into three major experiments as morphological characterization, phytochemical analysis and standardization of domestication practices. In the first experiment, morphological characterization, eleven available genotypes of Exacum bicolor were collected and evaluated for various vegetative and floral parameters. A significant difference was noticed among the genotypes for both vegetative and floral characters. Plant characters such as stem girth (2.29 cm), number of primary branches (4.11), leaf length (8.76 cm), and floral characters such as number of flowers per plant (184.00) and duration of flowering (47.21 days) were found higher in the genotype KAU Ex 11. Qualitative characters like shape of the leaves, leaf tip as well as petal shape also differed among the genotypes. Most of the genotypes possessed linear-lanceolate leaves, whereas KAU Ex 7 and KAU Ex 14 (oblanceolate leaves), KAU Ex 9 (ovate-elliptic leaves) and KAU Ex 10 (ovate leaves) showed other leaf forms. Overall scoring of Exacum bicolor genotypes based on the number of flowers per plant and the duration of flowering resulted in the highest rank to KAU Ex 11, followed by KAU Ex 15. Wide variation in genetic parameters of various characters indicated occurrence of considerable diversity among the genotypes for these traits. The difference between phenotypic coefficient of variance (PCV) and genotypic coefficient of variance (GCV) was narrow for characters viz. number of leaves, leaf width, leaf length, number of flowers per plant and duration of flowering, indicating the impact of genetic makeup for these traits rather than influence of environment. High heritability (more than 90%) coupled with high GAM were recorded for number of leaves, leaf length, leaf width, days to bud initiation, duration of flowering which indicate the additive and dominance gene action for these characters and possibility of selection based on these traits for further crop improvement programmes. K-mean cluster analysis of eleven genotypes yielded three clusters. Four genotypes each were included in cluster 1 and cluster 2, and three genotypes in cluster 3. The clustering pattern indicated that, geographical origin had no influence on grouping of genotypes. Association among various vegetative and floral characters analyzed via genotypic and phenotypic correlation revealed that Genotypic correlation coefficients of all the attributes were greater than or equal to the phenotypic correlation coefficients which indicated that influence of environment is negligible on phenotypic variation. The second experiment was phytochemical analysis. With respect to phytochemical analysis, extractive yield in methanol, contents of total polyphenols, alkaloids, flavonoids and saponins were estimated. The extractive yield in methanol (18.98 g/100 g) and alkaloids (0.77%) were found higher in the genotype KAU Ex 7, total polyphenols were higher in KAU Ex 10 (397.03 mg GAE/100g), flavonoids in KAU Ex 16 (1541.50 mg QE/100 g) and saponins in KAU Ex 12 (3.19 %). Gas chromatography-mass spectrometry analysis of sequential extracts of two genotypes of Exacum bicolor yielded a number of possible compounds which possess various therapeutic properties. The presence of secoiridoid namely, gentiolacotne [1H,6HPyrano[ 3,4-c]pyran-1,6-dione, 5-ethyl-3,4,5,8-tetrahydro-5-hydroxy-, (.+-.)] was first reported in Exacum bicolor. The third experiment, refinement of domestication practices, was attempted on enhancement of seed germination, seedling growth, standardization of seed germination media, potting media, vegetative propagation through cuttings, spacing & pinching, elucidation of floral biology and assessment of seed dormancy. The seeds were subjected to treatments viz. priming with certain chemical/bio agents, heat scarification, and light stratification by exposing to fluorescent and ultraviolet (UV) light. Priming with GA3 at 750 mg/L for 24 h recorded higher seed germination (77.50%), seedling survival (69.25%), shoot length (23.03 mm), root length (25.63 mm), dry weight of seedlings (1.60 mg), vigour index I (3771.14) and II (124.04). Among heat scarification treatments tested, significantly highest seed germination (73.93%) and seedling survival (67.86%) was exhibited when the seeds were preheated at 120ͦC for 15 minutes. Preheating at 80ͦC for 15 minutes duration recorded significantly longest shoots (41.40 mm) and roots (21.24 mm). Exposure of seeds at 80 ͦC for 30 minutes recorded highest vigour index I (3149.54), and 100 ͦC for 15 (108.07) minutes and 120 ͦC for 15 minutes (99.90) duration of exposure yielded higher vigour index II. Light stratification using fluorescent light was found to be more beneficial for seed germination and seedling survival. Fluorescent light exposure at 12/12 h light/dark photoperiod for 6 days recorded higher seed germination (92.38%), seedling survival (92.14%), and vigour index I (7876.43) and II (296.72), whereas shoot length (41.09 mm), root length (67.01 mm), and seedling dry weight (4.80 mg) were found higher when seeds were exposed to UV light for 45 minutes. Analysing the germination media for seed germination in Exacum bicolor, seeds were sown in germination media comprised of various combinations of cocopeat, sand, FYM, perlite and vermiculite alone and along with arbuscular mycorrhizal fungus (AMF) and Phosphobacterium. Highest seed germination was observed in media composed of cocopeat and sand (1:1) (76.21%). Seedling survival, shoot length, root length and seedling dry weight were recorded higher in media containing cocopeat and sand (1:1) along with AMF (10g/pot), and cocopeat, perlite, vermiculite (3:1:1) along with AMF (10g/pot). Analysing the nutrient management for growth enhancement, seedlings were applied with nutrients viz. N: P: K as 30: 10: 10 and 19: 19: 19 and ‘Amino rich’ alone and in combination with different concentrations of plant growth regulators viz. GA3 and IAA. Foliar application of N: P: K as 30: 10: 10 alone in progressive doses (0.5 g/L in week 1 of germination; 1 g/L in week 2; 2 g/L from week 3 onwards) recorded highest seedling survival (70.12%), fresh weight (63.70 mg), and dry weight of seedlings (3.62 mg). Various combinations of growing media were tested to analyse the effect of different potting media on the growth and flowering of Exacum bicolor. Plant height was found highest in media composed of soil and AMF (10 g/ Kg medium) (39.36 cm), stem girth in soil + sand + vermicompost (1:1:1) + AMF (10g/kg medium), and number of leaves were found highest in soil + AMF (10g/kg medium) (25.44). Number of flowers per plant was found greatest in potting media comprised of soil + sand + vermicompost (1:1:1) (44.50). The potting media with soil alone (37.06 days) recorded longer duration of flowering. Larger flowers with longer petals were observed in potting media composed of cocopeat + perlite + vermiculite (3:1:1) + AMF (10g/kg medium). Based on the scoring and ranking of the twelve potting media treatments for vegetative and floral parameters, the media comprised of soil + sand + vermicompost (1:1:1) + AMF (10g/kg medium) recorded the highest rank (rank 1) followed by soil + sand + vermicompost (1:1:1) and coco peat + sand + vermicompost (3:1:1). Vegetative propagation of Exacum bicolor was performed through terminal stem cuttings treated with various concentrations of indole-3-butyric acid (IBA), charcoal powder, commercial hormone powder (Rootex). A significant difference was observed among the treatments, with 96.67 percent cuttings rooted when cuttings were treated with IBA at 5.0 mg/L. cuttings treated with IBA at 20.0 mg/L resulted in greatest number of roots (19.45) and IBA at 15.0 mg/L produced longer roots (42.86 cm). Influence of pinching and spacing on vegetative and floral parameters of Exacum bicolor was analyzed by adopting three levels of spacing viz. 20 × 20 cm, 30 × 20 cm and 30 × 30 cm and giving seven levels of pinching viz. no pinching, single pinching at 15, 30 and 45 days after transplanting (DAT), double pinching at 15 & 30, 15 & 45, and 30 & 45 DAT. Analysis of pinching and spacing on vegetative and floral parameters of Exacum bicolor showed significantly greater number of branches in all the double pinching treatments. However, plants spaced at 30 x 20 cm and double pinched on 15 & 45 days after transplanting (DAT) showed compact growth habit with stouter stem and a higher number of branches which are suitable for bedding plants. The treatment also recorded a greater number of flowers/plant (86.50) with longer duration of flowering (47.0 days). Floral biology and pollination behaviour of Exacum bicolor was studied analysing the anthesis time, pollen viability, stigma receptivity and mode of pollination. It was found that anthesis in Exacum bicolour was occurring between 4.00 am and 6.00 am with peak time of anthesis at 5.00 am (56.25%). Pollen viability gradually decreased with flower aging. Significantly higher viability of pollen grains was observed for flowers on the previous day of anthesis (D0), and flowers on the day of anthesis (D1) from 8.00 am to 1.00 pm, flowers on second day of anthesis (D2 flowers) up to 11.00 am, for third day of anthesis (D3) and fourth day of anthesis (D4 flowers) up to 9.00 am. The results of the stigma receptivity test revealed that pollinating flowers on the third day of anthesis at 9.00 am ensures successful seed set, capsule weight and seed weight per capsule. Pollination behaviour of Exacum bicolor was studied by assessing five methods of pollination such as controlled selfing, controlled out-crossing, forced open pollination, self-pollination (bagging) and open pollination. Cent percent fruit set was obtained in controlled selfing, controlled outcrossing and in open pollination indicating that the plant is both self and cross compatible. Lack of fruit set in self-pollination (bagging) is an indication of lack of the phenomenon of apomixis to promote natural self-pollination in this species. Seed dormancy was assessed by performing seed germination test immediately after seed extraction and on alternate days subsequent to seed extraction up to 35 days (D1, D3, D5,… D35) and thereafter at weekly intervals, until germination percentage reached greater than 50 percent. The germination test was performed without any seed treatment (control) and with seed priming using 750 mg/L GA3 for 24 h. Lack of seed germination in control even up to the 84th day of seed extraction indicated the occurrence of seed dormancy in this species. However, seeds treated with GA3 germinated from the first day seed extraction onwards. From the study, it could be concluded that Exacum bicolor can be effectively utilised as an ornamental potted plant, and standardisation of domestication practices ensures measures for successful cultivation of the plant for the plains in Kerala