Rajwant K. Kalia

Evaluation of genetic fidelity of in vitro raised plants of Dendrocalamus asper (Schult. & Schult. F.) Backer ex K. Heyne using DNA-based markers

Dendrocalamus asper, an edible bamboo is valued for its tender edible shoots in the food industry. However, overexploitation of natural stands of D. asper coupled with minimal conservation and reforestation efforts has led to its rapid depletion in nature. Therefore protocol for rapid multiplication of D. asper via direct regeneration using nodal segments from mature clumps was standardized and more than 25,000 plants were transferred to the field (Singh et al. 2012a). However, genetic fidelity of these in vitro raised plants needs to be authenticated for commercial scale application of the developed micropropagation protocol. PCR-based molecular markers have emerged as simple, fast, reliable and labor-effective tools for testing the genetic fidelity of in vitro raised plants. This study report the genetic fidelity analysis of in vitro raised plants of D. asper for the first time using arbitrary (Random Amplified Polymorphic DNA, RAPD), semi-arbitrary (Inter-Simple Sequence Repeat, ISSR; Amplified Fragment Length Polymorphism, AFLP), and sequence-based (Simple Sequence Repeat, SSR) markers. Bulked DNA samples of 20 in vitro raised shoots (collected after every three subculture cycles starting from 3rd to 30th passage) and field transferred plantlets were compared with the mother plant DNA using 90 primer combinations (25 each of RAPD, ISSR, SSR, and 15 AFLP) and scorable bands were produced by 78 (22 RAPD, 24 ISSR, 21 SSR, and 11 AFLP) primers. A total of 146 distinct and scorable bands were produced by 22 RAPD primers with an average of 6.6 bands per primer while the number of bands for ISSR primers varied from 3 (ISSR-4 and 9) to 13 (ISSR-17), with an average of 7.1 bands per primer. Similarly, SSR markers also showed wide variation in number of bands, ranging from 2 (RM 261) to 12 (RM 44, 140, and 224) with an average of 7.8 bands. AFLP primer combinations could generate 35–72 bands with an average of 48.7 bands per primer pair. Amplification of monomorphic bands with all primer combinations authenticated the true to type nature of the in vitro raised plants of D. asper which underwent up to 30 subculture passages over a period of approximately 2 years thereby supporting the commercial utilization of the developed micropropagation protocol.

Investigation of heavy metals in frequently utilized medicinal plants collected from environmentally diverse locations of north western India

The increasing prevalence of environmental pollution, especially soil contamination with heavy metals has led to their uptake in the human food chains through plant parts. Accumulation and magnification of heavy metals in human tissues through consumption of herbal remedies can cause hazardous impacts on health. Therefore, chemical profiling of nine heavy metals (Mn, Cr, Pb, Fe, Cd, Co, Zn, Ni and Hg) was undertaken in stem and leaf samples of ten medicinal plants (Acacia nilotica, Bacopa monnieri, Commiphora wightii, Ficus religiosa, Glycyrrhiza glabra, Hemidesmus indicus, Salvadora oleoides, Terminalia bellirica, Terminalia chebula and Withania somnifera) collected from environmentally diverse regions of Haryana and Rajasthan states in North-Western India. Concentration of all heavy metals, except Cr, was within permissible limits in the tested stem and leaf samples. Leaf samples had consistently more Cr compared to respective stem samples with highest concentration in leaf samples of Bacopa monnieri (13.19 ± 0.0480 ppm) and stem samples of Withania somnifera (4.93 ± 0.0185 ppm) both collected from Bahadurgarh (heavy industrial area), Haryana. This amount was beyond the permissible limit of 2.0 ppm defined by WHO for raw herbal material. Other two most perilous metals Pb (2.64 ± 0.0260) and Cd (0.04 ± 0.0274) were also recorded in Bahadurgarh region, although below permissible limits. Concentration of Hg remained below detectable levels in all the leaf and stem samples tested. These results suggested that cultivation of medicinal plants and other dietary herbs should be curtailed near environmentally polluted especially industrial areas for avoidance of health hazards.

Collection and Characterization of Citrus indica Tanaka and C. macroptera Montr.: Wild Endangered Species of Northeastern India

Citrus indica and C. macroptera are the wild endangered species of Citrus occurring in northeastern India. Surveys were undertaken in this region for ascertaining distribution, studying variability and for collection of germplasm of these two species. C. indica, an endemic species of this region, was collected from the Citrus Gene Sanctuary located in buffer zone of Nokrek Biosphere Reserve in the Garo hills of Meghalaya. In addition, a putative natural hybrid of C. indica and C. limon was collected for the first time from the south Garo hills. C. macroptera had much wider distribution and was collected from Mizoram and Meghalaya states. In Jantia hills of Meghalaya, natural populations of this species are in a highly threatened state. The two species were unevenly distributed all over the explored territory. Morphological characterization of leaves, fruits and seeds indicated the presence of sizable variability within collected accessions of these two Citrus species. Indigenous technical knowledge gathered on the use and socio-economic importance indicated commercial potential for these two species in northeastern India. However, lack of cultivation of these species and clearing of forest cover at an alarming rate has led to an urgent need to adopt complementary conservation strategies to safeguard these species and to ensure their availability for future utilization. A major emphasis on developing methods for their propagation, multiplication and regeneration in in situ and ex situ conditions is required.

Problems, progress and future prospects of improvement of Commiphora wightii (Arn.) Bhandari, an endangered herbal magic, through modern biotechnological tools: a review

Commiphora wightii (Arn.) Bhandari syn. C. mukul Engl. (Burseraceae) is an economically and pharmacologically important slow growing, dioecious, balsamiferous woody, multipurpose shrub heading towards extinction. Commonly known as “Guggul” due to the presence of steroidal compound guggulsterone in the oleo-gum resin, it has been used in treating various ailments and disorders since ancient times (2000 B.C.). Evaluation and confirmation of hypolipidemic effects of guggul based on Ayurvedic text in 1960s provided a new insight into its pharmacological applications. Two bioactive isomers of guggulsterone, E and Z, are responsible for lipid- and cholesterol-lowering activities. Recently, it has been shown to have anti-cancerous activity also. It is found in the dry regions of Indian subcontinent, namely India, Pakistan and Bangladesh. Ruthless and unscientific harvesting of oleo-gum resin from the wild, by local populations, for economic benefits with negligible conservation efforts has made this species endangered and has led to its inclusion in Red Data Book of IUCN. Although this plant has many excellent traits, adequate attention has not been focused on its conservation and improvement. Conventional propagation methods i.e., seeds, cuttings and air layering are in place but have many limitations. Therefore, application of modern biotechnological tools needs to be standardized for harnessing maximum benefits from this pharmaceutically important plant. An efficient regeneration system needs to be in place for improvement of this genus through genetic transformation and production of useful metabolites in cell cultures. Studies are in progress for micropropagation through shoot multiplication and somatic embryogenesis, as well as for secondary metabolite (guggulsterone) production in callus cultures and bioreactors. No selected germplasm is available for C. wightii since it is a wild plant. Breeding programs have not yet been started due to lack of systematic cultivation and conservation programs. Moreover, little information has been gathered regarding the genetic variability in this species using RAPD and ISSR markers. No details are available about genetic makeup and QTL linkage maps. Investigations are in progress to search sex linked markers in this dioecious species. Research is also in progress to decipher the molecular mechanisms underlying various pharmacological actions of guggul. Since the approval of use of guggul as a food supplement by United States Food and Drug Administration in 1994, an exponential increase in research publications on various aspects of research on guggul have been published. Present communication summarizes the problems, progress made and suggests some future directions of research for this important endangered medicinal plant.

Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers

Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.

Understanding Tecomella undulata: an endangered pharmaceutically important timber species of hot arid regions

Tecomella undulata (Sm.) Seem. (family Bignoniaceae) is an economically and pharmaceutically important small tree of arid regions of India, Pakistan and Arabia. It is well known as ‘Desert teak’ or ‘Marwar teak’ being the main source of timber amongst the tree species of desert region of Rajasthan, India. T. undulata has occupied a reputed position of having valuable medicinal properties in both folk and classical streams of indigenous medicinal systems. Plant is well-known for its wide range of therapeutic activities like hepatoprotective, antibacterial, antimicrobial, antifungal and anti-termite, immunomodulatory, anticancer, cytotoxic, analgesic, anti-inflammatory, anti-obesity, etc. Rohitakarishta, an ayurvedic drug obtained from T. undulata, is the classical compound which is being prescribed in liver and spleen diseases, oedema and anaemia. This well accepted agroforestry tree of the arid regions is heading towards extinction due to its increasing demand in timber and pharmacological industries coupled with negligible conservation efforts. This species has now been designated as “threatened” in Rajasthan, India. Susceptibility to many pathogens and pests is one of the major problems and detrimental factor in successful establishment and growth of this tree. Conventionally, it is propagated through seeds; however, natural regeneration of plants is poor due to air dispersal of seeds to remote areas, extreme environmental conditions during seed dispersal, improper harvest and storage of seeds, and short seed viability. The tree is very slow growing and suitable vegetative propagation methods are not available for its rapid multiplication. Alternatively, attempts have been made to propagate this tree through in vitro approaches; however, it also has many limitations including difficulties in culture establishment, slow growth, low rooting rates as well as field establishment rates. Moreover, little information is available regarding genetic diversity in this species using morphological and molecular markers. Efforts to undertake breeding programs for improvement of T. undulata have not been initiated yet. Some literature is available regarding cytology, role in agroforestry and silviculture, association with vesicular arbuscular mycorrhiza and application of biotechnological tools in T. undulata, however, an exponential increase in research publications on identification of bioactive constituents and verification of pharmacological effects has been recorded in the recent past. In this communication, we emphasize the research progress made in T. undulata on various aspects and suggest some future directions of research.

Morphological, agronomic, and yield characterization of cluster bean (cyamopsis tetragonoloba L.) germplasm accessions

Cluster bean is an important legume crop of the arid and semi-arid regions of the world. In the present study, 140 germplasm lines of cluster bean were evaluated for agronomic and yield traits. Wide variation was observed among agronomic and yield-related traits among the accessions, making selection possible for different characters. High heritability (< 85%) coupled with high genetic advance (< 30%) was observed for yield per plant (TY), pods per cluster (PDSCL), and clusters on main branch (CLMN). Pod length (PDLT) and total pods per plant (TDP) explained 30% and 72% of variability in seed number per pod (SDN) and TY, respectively. Principal component analysis revealed significant variation among the characters with the first four principal components explaining about 70.8% of the total variation. Projecting the germplasm accession onto the first two principal components revealed two groups: (i) accessions showing high PDSCL, TPD, and TY and (ii) accessions having high SDN. Among the 140 germplasm lines evaluated, IC-421815 (TY - 31.5 g plant-1; TPD - 139.8; and PDSCL - 6.6) and IC-370563 (31.2 g plant-1, 134.0 and 7.4) were the best performing lines compared to the checks. The information presented in this manuscript would be used for cluster bean breeding.

Evaluation of Clonal Variability in Shoot Coppicing Ability and in vitro Responses of Dalbergia sissoo Roxb.

Summary Clonal variations were observed amongst 12 clones of Dalbergia sissoo belonging to four states (U.P, Uttaranchal, Haryana and Rajasthan) of India, representing four different geographical zones in respect of ex vitro shoot coppicing ability and in vitro responses. Coppicing ability of shoot hedges of clones exhibited significant variation which ranged from average of 13.81 coppiced shoots (Clone 40, Uttar Pradesh) to 9.29 (Clone 64, Haryana). Comparative analysis of clones from different regions in respect to their coppicing ability revealed that clones from U.P had higher coppicing ability whereas those from Haryana proved to be least coppicers. Regional variations were also exhibited in the in vitro multiple bud induction ability on nodal explants excised from shoot hedges of clones (mean number of buds induced and percentage of cultures forming multiple buds). Regional as well as inter clonal variations were recorded in the shoot proliferation efficiency as well as rootability of microshoots of these clones as well as their optimal plant growth regulator requirements. BAP alone (2.5 μM) was sufficient for inducing multiple buds on cultured nodal explants of Uttaranchal and Uttar Pradesh region clones. On the contrary, clones from Rajasthan and Haryana had higher optimal requirement of BAP and in addition, they required media to be supplemented with auxin (NAA) for induction of multiple buds on explants. Correlation analysis between shoot coppicing ability of clones and in vitro performances of explants of these clones cultured on 2.5 μM BAP indicates a positive correlation. Observation lays credence to our view that these characters are genetically controlled and shoot coppicing can be used as a marker character in optimizing in vitro performance of clones. Using the information generated by this paper in vitro production of elite planting material can be maximized by ameliorating plant growth regulator requirement in the medium.

Genetic diversity of Indian jujube cultivars using SCoT, ISSR, and rDNA markers

Genetic variation and relationships among 37 cultivars of Ziziphus mauritiana (Lamk.) native of India were analyzed using start codon targeted (SCoT), inter-simple sequence repeats (ISSR), and ribosomal DNA (rDNA) markers. High level of polymorphism among SCoT (61.6%) and ISSR (61%) primers with higher PIC values ranging from 63.1 to 90.4% of SCoT and 47.3 to 88.8% of ISSR primers was recorded. SCoT and ISSR dendrograms revealed similarity coefficients ranging from 0.80 to 0.92 and 0.79 to 0.96, respectively, and clearly delineated all the cultivars of Z. mauritiana into well-supported distinct clusters. Greater Gst signifies higher amount of differentiation observed over multiple loci among seven Z. mauritiana populations. On the other hand, higher gene flow demonstrating a very high migration rate between Z. mauritiana populations indicated higher rates of transfer of alleles or genes from one population to another. The genetic diversity of population 1 (Rajasthan) was the richest among all the seven populations. The largest genetic distance was measured between Maharashtra and West Bengal and the least between Rajasthan and Punjab cultivars. Most of the genetic diversity exists within population rather than among populations. Substantial variation in the ITS-1 region signifies its phylogenetic utility specifically in assessing genetic diversity in Z. mauritiana. The clustering patterns using three molecular marker systems vis-à-vis place of origin exhibited no consistency in grouping of Z. mauritiana cultivars as cultivars from the same place of origin were genetically cataloged into different SCoT, ISSR, and ITS phylogram clusters indicating wide genetic diversity and distribution across agro-climatic zones validating the robustness of marker systems tested.