Foliar Micromorphological Studies Research Articles

In vitro propagation of Farsetia macrantha Blatt. & Hallb.: an endemic and threatened plant of Indian Thar Desert

Farsetia macrantha Blatt. & Hallb. is an endemic and critically endangered plant species of Indian Thar desert. Being an endemic and threatened plant species, there is need to develop an efficient in vitro regeneration protocol for its conservation. Since, there is no published report on in vitro regeneration of F. macrantha, therefore, present study was designed to develop an efficient micropropagation method of F. macrantha along with foliar micromorphological studies. Cotyledonary node taken from 15 to 20 days old seedlings was used as an explant for in vitro regeneration. MS medium supplemented with additives ascorbic acid (50 mg l−1), citric acid (25 mg l−1), l-arginine (25 mg l−1) and adenine sulphate (25 mg l−1) and 0.5 mg l−1 BA was best for shoot bud induction. Different types, concentrations and combinations of cytokinins (BA, Kin and mT) and auxin (IAA) tested for shoot multiplication, maximum number of shoots (25.10 ± 0.69) regenerated on MS medium containing additives and combination of two cytokinins 0.5 mg l−1 BA + 0.25 mg l−1 Kin. Microshoots were rooted successfully with highest rooting response (75.59%) on ½ MS medium containing 2.0 mg l−1 IBA. In vitro rooted plants were hardened with ~ 75% survival rate. Comparative foliar micromorphological studies conducted among in vitro grown and hardened plants suggest the developmental changes of micropropagated plants during acclimatization. The present in vitro regeneration protocol could be used for large-scale propagation and conservation of F. macrantha. Farsetia macrantha is an endemic and critically endangered plant species of Indian Thar desert and presently it is restricted only to the Barmer district of Rajasthan, India. In the present study, an in vitro regeneration method has been developed first time for its conservation.

In vitro multiplication, micromorphological studies and ex vitro rooting of Hybanthus enneaspermus (L.) F. Muell. – a rare medicinal plant

AbstractHybanthus enneaspermusis a rare medicinal plant. We defined a protocol for micropropagation,ex vitrorooting of cloned shoots and their acclimatization. Surface-sterilized nodal segments were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kin). Medium supplemented with 1.5 mg L−1BAP was found optimum for shoot induction from the explants and 6.4±0.69 shoots were regenerated from each node with 97% response. Shoots were further proliferated maximally (228±10.3 shoots per culture bottle with 7.5±0.43 cm length) on MS medium augmented with 1.0 mg L−1each of BAP and Kin within 4–5 weeks. The shoots were rootedin vitroon half strength MS medium containing 2.0 mg L−1indole-3 butyric acid (IBA). The cloned shoots were pulse-treated with 300 mg L–1 of IBA and cultured on soilrite® in a greenhouse. About 96% of the IBA-pulsed shoots rootedex vitroin soilrite®, each shoot producing 12.5±0.54 roots with 5.1±0.62 cm length. Theex vitrorooted plantlets showed a better rate of survival (92%) in a field study thanin vitrorooted plantlets (86%). A comparative foliar micromorphological study ofH. enneaspermuswas conducted to understand the micromorphological changes during plant developmental processes fromin vitrotoin vivoconditions in terms of variations in stomata, vein structures and spacing, and trichomes. This is the first report onex vitrorooting inH. enneaspermusand the protocol can be exploited for conservation and large-scale propagation of this rare and medicinally important plant.

Optimization of factors affecting in vitro regeneration, flowering, ex vitro rooting and foliar micromorphological studies of Oldenlandia corymbosa L.: a multipotent herb

The conditions were optimized for efficient in vitro regeneration of shoots and roots of Oldenlandia corymbosa L. using nodal shoot explants. Murashige and Skoog’s (MS) medium augmented with additives and 2.0 mg L−1 BAP was recorded optimum for shoot bud induction from the nodal meristems. The shoots were proliferated by subsequent subcultures on half strength MS medium fortified with 1.0 mg L−1 BAP and 0.5 mg L−1 kinetin + additives. This media combination yielded maximum number of shoots (223 ± 4.12 shoots/culture bottle) with 13.4 cm average length. Flower buds were induced (4.2 ± 0.28 flowers) from the in vitro multiplied shoots on MS medium contained 1.0 mg L−1 BAP and 0.5 mg L−1 of kinetin and IAA under 50 µmol m−2 s−1 SFPD light intensity for 12 h/day photoperiod. In vitro regenerated shoots were rooted on half strength MS medium conjunct with IAA, IBA and NAA singly at different concentrations. The best rooting response was observed on half strength MS medium containing IBA at 2.0 mg L−1 with activated charcoal. Roots were also induced from the cut ends of the shoots using ex vitro rooting techniques in O. corymbosa by pulse treating the shoots with 300 mg L−1 IBA for 4 min. Better roots were achieved in this method than the in vitro roots in terms of numbers (14.7 ± 0.21) and firmness. The foliar micromorphological studies could help to understand the structural adaptations of micropropagated O. corymbosa plantlets towards field environments. The in vitro induced anomalies in stomatal apparatus were repaired during hardening of plantlets in the greenhouse and after field transfer. Decrease in stomatal density (from 70.78 ± 0.55 to 55.6 ± 0.10), and increase in veins, trichomes and crystals/raphides densities revealed the developments of structural changes in the leaves to withstand in the harsh field conditions. The acclimatized plantlets with well developed root systems were successfully shifted to the natural soils with 98% survival rate.

Comparative foliar micromorphological studies of in vitro and field transferred plants of Morinda citrifolia

Micromorphological studies revealed the developmental changes in micropropagated plants of Morinda citrifolia L. from in vitro conditions to field environment. The lower relative humidity, higher light intensity and septic stressful conditions in the field environment could make gradual changes in the micropropagated plants so as to adapt the external environment. Arrested stomatal development, single guard cells and indistinct subsidiaries were observed in vitro with highest stomatal density (52.0±0.11) obtained in the ambient in vitro environment. The development of tissues, epidermal ornamentation, efficient stomatal functionality and vein-islets numbers (12.0±0.0) during in vivo transfer may help in acclimation of micropropagated plants under field conditions. Raphides were observed in the in vitro propagated as well as in vivo transferred plantlets. The gradual acclimatisation and ex vitro rooting technique increased the survival rate of plantlets in the field. The micromorphological changes resulting from in vitro to field environments are important to understand the development of tissues and adaptation of micropropagated plants, which could help in improvement in survivability during field trials.

Comprehensive analysis of in vitro to field transition of micromorphology and leaf architecture in Passiflora edulis Sims. f. flavicarpa Deg.

This study elucidate the micromorphological changes and leaf architectural developments of in vitro (multiplication phase) and in vivo transferred (acclimatized plants in the field) micropropagated plantlets of Passiflora edulis Sims. f. flavicarpa Deg. The in vitro leaves were small, ovate and pale green in color; but glossy, bright green colored leaves with conspicuous midrib were observed in field transferred plants. Constantly opened stomata throughout the culture period were found under in vitro conditions, these were fully functional after field transfer of the plantlets. In vitro environment favoured increased stomatal density (80.5), stomatal index (28.8), under-developed trichomes, reduced vein islet density (7.0) and irregular venation pattern. Reduced stomatal density (63.4) and index (25.3), well developed trichomes, increased vein islet density (22.0) and regular venation pattern were observed in field acclimatized plantlets. Crystals were absent in the in vitro grown leaves but well developed rhombohedral and sphaerocrystals were observed throughout the major and minor veins in the cells of field plants. The foliar micromorphological study revealed that the in vitro environmental conditions favoured the development of abnormal features. These anomalies have been adjusted and plants developed structural and morphological changes needed for successful adaptation in the field conditions.

Optimization of in vitro and ex vitro regeneration and micromorphological studies in Basella alba L.

The optimum concentrations of the plant hormones for in vitro regeneration and subsequent effect of auxins on rooting (in vitro and ex vitro) of shoots of Basella alba L. have been investigated in present study. Nodal shoot segments were used as explants to initiate the cultures. The bud breaking from explants was observed within 1week of incubation on agar gelled Murashige and Skoog's (MS) medium. Multiple axillary shoots (7.30±0.56 shoots per explant) were induced on MS medium supplemented with 2.0mg/L 6-benzylaminopurine (BAP). The shoots were multiplied (maximum 17.10±0.44 shoots per explant) on the same medium fortified with 0.5mg/L each of BAP and Kin (Kinetin) +0.1mg/L IAA. These shoots were excised and rooted in vitro (10.73±0.92 roots per shoot) on half-strength MS medium augmented with 2.0mg/L indole-3 butyric acid (IBA). Hundred percentage success rates have been achieved by ex vitro rooting of the in vitro regenerated shoots with IBA at 300mg/L. The in vitro and ex vitro rooted shoots were acclimatized in greenhouse and subsequently transferred to the natural field conditions where 100% survival rate was reported. The ex vitro rooting method was found more advantageous than in vitro rooting in terms of time, energy and survival percentage of B. alba. A comparative foliar micromorphological study of B. alba was conducted to understand the micromorphological changes in plants while shifting from in vitro to the in vivo conditions in terms of variations in stomatal index, venation pattern and vein density, and the arrangement of crystals. The study could help in understanding the response of in vitro raised plants towards in vivo environment.

In vitro regeneration frequency, micro-morphological studies and ex vitro rooting of Hemidesmus indicus (L.) R. Br.: a multi-potent endangered climber

Hemidesmus indicus is an endangered medicinal plant of Peninsular India. A rapid micropropagation method using nodal segments of a 4 year old plant has been achieved in the present study. The nodal explants (2.0–3.0 cm long) were harvested from actively growing shoots of conventionally raised plants and cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg l−1 6-benzylaminopurine (BAP). Hundred percent explants responded with 5.2 ± 0.25 cm long multiple shoots (9.0 ± 0.53) from the nodal meristems. The shoots were further multiplied enormously (272 ± 4.12 shoots per explant) by repeated subculture of mother explants and freshly induced shoot clumps on MS medium with the reduced concentrations of BAP (1.0 mg l−1), kinetin (Kin; 0.5 mg l−1) and indole-3 acetic acid (IAA; 0.1 mg l−1) within 4 weeks. Highest root induction (62.0 ± 0.54 roots per shoot) was observed on 1/4th strength MS medium supplemented with 3.0 mg l−1 indole-3 butyric acid (IBA). About 96 % shoots were rooted (45.0 ± 0.48 roots per shoot) by ex vitro methods when the cut ends of the shoots pulse treated with IBA (400 mg l−1) for 5 min. The in vitro as well as ex vitro rooted plantlets were acclimatized successfully in the greenhouse. Ex vitro rooted plantlets exhibited higher percentage (98 %) of survival as compared to the in vitro rooted plantlets (91 %) in the field conditions. There were not any observable differences between in vitro propagated and field transplanted plantlets. A comparative foliar micro-morphological study of H. indicus was conducted to understand the micro-morphological changes in the plants while shifting from in vitro to the in vivo conditions in terms of variations in stomata, venation pattern and vein density, and trichomes. The aforementioned protocol could be successfully used for the large-scale multiplication and conservation of germplasm of this endangered plant species.

Taxonomic studies of the genus Tephrosia Pers. (Papilionaceae) in Nigeria

The relationship between eleven Tephrosia species occurring in Nigeria was examined using a number of taxonomic tools. Fresh and herbarium specimens were used for this purpose and methods followed conventional taxonomic practice. Although the species occur in savanna ecosystems, herbarium collections revealed an abundant distribution in the southern part of Nigeria. Morphometric studies revealed that four quantitative characters viz: leaflet length, lamina length, fruit length and pedicel length can be used to delimit members of this genus. Based on the cluster analysis using average linkage within group, the closest species are T. leptostachya and T. purpurea with the shortest distance measure (0.713). T. linearis has the smallest leaflet, in length and width. Further morphological studies also showed that T. vogelii has the largest pod while T. barbigera has the highest number of seeds per pod. Generally, the species have very short petiolule ranging between 0.2cm and 0.3cm in length, on the average. Foliar micro-morphological studies also showed that the species generally possess polygonal cells with straight to curved anticlinal walls and anisocytic stomata types while pollen studies revealed tricolporate pollen grains to be predominant within the taxa. Although, the present study has added to the existing information regarding Tephrosia species, it also suggests further research to ascertain its taxonomic position within the Papilionaceae.

Micropropagation, Micromorphological Studies, and In Vitro Flowering in Rungia pectinata L.

A tissue culture protocol was developed for an important medicinal plant Rungia pectinata L. in the present study. Nodal shoots were used as explants and surface-sterilized with 0.1% HgCl2 solution. Murashige and Skoog (MS) medium was used to establish the cultures of R. pectinata. The bud break was reported on MS medium supplemented with 1.0 mg L−1 6-benzylaminopurine (BAP). About 98% response was observed with this media combination and maximum 3.2 shoots per explant with 4.3 cm length were recorded. The shoots were further multiplied using MS medium augmented with 0.5 mg L−1 each of BAP and kinetin (Kin) + 0.1 mg L−1 indole-3 acetic acid (IAA). Maximum 13.2 shoots per explant with 5.2 cm length were observed. All the shoots were rooted (4.9 roots per shoot with 3.5 cm length) on half strength MS medium fortified with 2.0 mg L−1 indole-3 butyric acid (IBA). In vitro flowering was induced from the shoots on half strength MS medium supplemented with same concentrations and combinations of growth regulators used for shoot multiplication under 12/12 hr light/dark photoperiod. The plantlets were hardened in the greenhouse for two months and finally transferred to the field. The foliar micromorphological studies revealed the developmental changes in stomata, vein density, and trichomes during the culture of shoots under in vitro conditions.

Taxonomic Evaluation of Fifteen Species of Ipomoea L. (Convolvulaceae) from South-Western Nigeria using Foliar Micromorphological Characters

Foliar micromorphological study was carried out on I. triloba, I. eriocarpa, I. alba, I. hederifolia, I. muricata, I. intrapilosa, I. asarifolia, I. batatas, I. aquatica, I. mauritiana, I. carnea, I.involucrata, I. nil, I. heterotricha and I. obscura to determine the patterns of variation in their epidermal characteristics and search for useful and stable anatomical characters for the identification of the species. Leaf epidermal features that provided useful specific distinctions are cell shape, anticlinal wall pattern, stomata shape, stomata type, trichome, cuticular striations, crystal, granular periclinal wall, stomata size, stomata index, scale and tannin. The characters revealed interrelationships among the Ipomoea species and also suggest their monophyly.