D. P. Barik

In vitro regeneration of medicinal plant Centella asiatica

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm−3 N6-benzylaminopurine (BAP) and 0.05 mg dm−3 α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm−3 indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.

High-frequency plant regeneration by in vitro shoot proliferation in cotyledonary node explants of grasspea (Lathyrus sativus L.)

SummaryMultiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N6-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl−1 (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl−1 (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.

ED-XRF spectrometric analysis of comparative elemental composition of in vivo and in vitro roots of Andrographis paniculata (Burm.f.) Wall. ex Nees—a multi-medicinal herb

The multi-elemental composition of in vitro--proliferated root tissues of Andrographis paniculata (Burm.f.) Wall. ex Nees was compared with that of the naturally grown in vivo plants. Trace elements namely Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Rb, Sr and Pb in addition to two macro-elements K and Ca were identified and quantified in root tissues of both sources using the energy dispersive X-ray fluorescence (ED-XRF) technique. ED-XRF analysis was performed using Mo K X-rays generated from a secondary molybdenum target. The elemental content of in vitro roots was found to be at par with that of naturally grown plants of the same species. This opens up a possibility of exploiting in vitro root cultures as a viable, alternative and renewable source of phytochemicals of relevance, besides providing a means for conservation of the valuable natural resources.

Analysis of Genetic Diversity among Selected Grasspea (Lathyrus sativus L.) Genotypes Using RAPD Markers

Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variability among five selected genotypes of grasspea. Out of 30 random decamer primers tested for the present investigation 20 showed reproducible DNA amplification. A total of 257 loci were amplified of which 159 were polymorphic including 57 genotype-specific unique bands. Amplicons had molecular weights ranging from 3.0 kb to 0.1 kb. Majority amplicons were shared by most of the genotypes which indicated a very narrow genetic gap between them. The dendrogram constructed on the basis of RAPD data showed two clusters. The local genotype collected from Nayagarh was grouped along with IC-120451 and IC-120453, sharing a common node at an 82% similarity level. The other genotypes, IC-120478 and IC-120487, were located in the second clade having a common node at 84% similarity level. The investigation showed that though all the genotypes of grasspea were of apparently similar morphology there exists polymorphism at the molecular level, which can be exploited in breeding programmes aimed at crop improvement.

High frequency in vitro regeneration of Lathyrus sativus L.

A simple and efficient protocol for high frequency plant regeneration of a grain legume grasspea (Lathyrus sativus L.) is described. Of different explant types tested epicotyl segments were most responsive. Murashige and Skoog’s (1962) medium augmented with 17.76 µM 6-benzyladenine + 10.74 µM α-naphthaleneacetic acid showed the highest percentage of direct shoot regeneration. Among cultivars IC-120487 showed the highest regeneration frequency (80 %) with maximum shoot numbers (8.2 shoots per explant) and maximum average shoot length (4.1 cm). About 78 % of the regenerated shoots were rooted in half-strength MS medium containing 2.85 µM indole-3-acetic acid. After primary hardening the plantlets were established in soil with a survival rate of 75 %.

Direct shoot regeneration from epicotyl explants of grasspea (Lathyrus sativus)

A reproducible procedure is described for adventitious shoot organogenesis in epicotyl segments resulting in prolific plant regeneration of a grain legume grasspea (Lathyrus sativus L.). Among seedling explant types examined, epicotyl segments were most responsive. The highest percentage of direct shoot regeneration was elicited on Murashige–Skoog (MS) medium augmented with 4.0 mg L–1 6-benzyladenine (BA) + 2.0 mg L–1 α-naphthaleneacetic acid (NAA). Compared with four other genotypes examined, IC-120487 showed the highest shoot regeneration frequency (approximately 80%) with maximum shoot numbers (averaging eight shoots per explant) and longest average shoot length (approximately 4 cm). Rhizogenesis was induced in ~78% of the regenerated shoots in half-strength MS medium containing 0.5 mg L–1 indole-3-acetic acid (IAA). Plantlets were acclimated in vermi-compost and 75% of those transferred to soil survived and set viable seeds.

High frequency shoot proliferation from cotyledonary node of Lawsonia inermis L. and validation of their molecular finger printing

An efficient and reproducible protocol for in vitro plant regeneration was developed for Lawsonia inermis L. using cotyledonary node explant derived from axenic seedlings. Highest shoot proliferation frequency (ca 96.6%) was achieved on Murashige and Skoog’s, 1962 (MS) basal medium supplemented with 8.88 μM 6-Benzyladenine (BA) + 2.68 μM Napthalene acetic acid (NAA). Up-scaling of shoots was carried out using in vitro nodes on MS medium supplemented with 4.44 μM BA. So overall, an average of 238 shoots was produced at 75 days. Of the four different forms of cotyledonary node explants evaluated, highest shoot multiplication was observed in cotyledonary node explant with two whole cotyledons. In vitro regenerated shoots were best rooted (ca 34.3 roots / shoot) on ½ MS medium devoid of any growth regulator. The plantlets were successfully acclimated in sand:soil:: 1:1and established in the garden soil. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis revealed a homogeneous amplification profile for all micropropagated plants validating the genetic fidelity of the in vitro-regenerated plants and supporting the regeneration protocol for economic commercial exploitation.

Comparative thin-layer chromatographic studies and development of a high-performance thin-layer chromatography method for the quantification of lawsone in natural and micropropagated plant parts of Lawsonia inermis L.

A simple, sensitive, and precised high-performance thin-layer chromatography (HPTLC) method has been developed for the analysis of lawsone in natural vis-à-vis micropropagated plant parts of Lawsonia inermis L. Separation of the components was perfectly achieved on high-performance thin-layer chromatography (TLC) plates using optimized tertiary mobile phase of benzene–ethyl acetate– acetic acid (7.5:2.5:0.1, v/v). Densitometric scanning was performed before derivatization of the plate in absorption/reflection mode, and lawsone was quantified at its maximum absorbance of wavelength of 275 nm. Linearity of the method was obtained in the concentration range of 50 to 350 ng spot−1 with a correlation coefficient (r2) of 0.9999, indicating good relationship between concentrations in opposition to the peak area. The limit of detection and limit of quantification were found to be 16 and 50 ng spot−1, respectively. The obtained recovery ranges from 95.09% to 96.90% with an average value of 96.02% proved the excellent accuracy of the method. The developed method was found to be highly sensitive, and the mobile phase enables outstanding separation of lawsone from other components present in the mixture. The International Conference on Harmonization (ICH) guidelines were followed for validation of the HPTLC method in terms of precision, repeatability, and accuracy. The maximum content of lawsone was reported in the leaves of the micropropagated plant.

Genetic diversity analysis of 60 ginger germplasm core accessions using ISSR and SSR markers

Abstract Molecular techniques play a critical role in studies of phylogeny and, thus, have been applied to understand the distribution and extent of genetic variation within and between species. In the present study, a genetic analysis was undertaken using molecular markers (9 ISSR and 13 SSR) on 60 ginger cultivars from different regions of the eastern coast of India (Odisha). The data obtained with 22 polymorphic markers revealed moderate to high diversity in the collection. Both ISSR and SSR markers were efficient in distinguishing all the 60 ginger cultivars. A total of 42 and 160 polymorphic bands were observed with ISSR and SSR markers, respectively. However, SSR markers were observed to be better at displaying average polymorphism (63.29%) than ISSR markers (55%). Analysis of molecular variance results showed that 52 and 66% of the variation occurred among different ginger populations, whereas 48 and 34% of the variation was found within populations, respectively, using ISSR and SSR markers, indicating that ginger cultivars display significant genetic diversity at the population level. Principal coordinates analysis and the dendrogram constructed out of combined data of both markers showed grouping of ginger accessions to their respective area of collection, indicating geographical closeness due to genetic similarity irrespective of the relationship that exists at the morphological level.

Adventitious Shoot Organogenesis and Plant Regeneration from Internode Explants of Paederia Foetida L.: A Valuable Medicinal Plant

A plant regeneration protocol via adventitious shoot organogenesis from internode explants of Paederia foetida (Skunk vine) is reported here for the first time. Three explants (leaf, mature internode and internode derived from axenic shoot cultures) were tested for shoot organogenesis. Leaf explants failed to induce adventitious shoots whereas axenic internode explant was found to be superior to mature internode explants for the induction of adventitious shoots. Axenic internode explants cultured on MS medium supplemented with 3.0 mg/l BAP showed maximum (86.7 %; 10.4 shoots per explant) adventitious shoot organogenesis. The regenerated shoots were best rooted (90 %; 14 roots per shoot) on half-strength MS medium. Eighty percent of the rooted shoots were successfully acclimatized in soil: sand (1:1) mixture. All these acclimatized plants were successfully transferred to larger pots containing garden soil and subsequently established in the field.