Tripta Jhang

Genetic differentiation of Vigna species by RAPD, URP and SSR markers

Seventy genotypes belonging to 7 wild and cultivated Vigna species were genetically differentiated using randomly amplified polymorphic DNA (RAPD), universal rice primer (URP) and simple sequence repeat (SSR) markers. We identified RAPD marker, OPG13 which produced a species-specific fingerprint profile. This primer characterized all the Vigna species uniquely suggesting an insight for their co-evolution, domestication and interspecific relationship. The cluster analysis of combined data set of all the markers resulted in five major groups. Most of the genotypes belonging to cultivated species formed a specific group whereas all the wild species formed a separate cluster using unweighted paired group method with arithmetic averages and principle component analysis. The Mantel matrix correspondence test resulted in a high matrix correlation with best fit (r = 0.95) from combined marker data. Comparison of three-marker systems showed that SSR marker was more efficient in detecting genetic variability among all the Vigna species. The narrow genetic base of the V. radiata cultivars obtained in the present study emphasized that large germplasm collection should be used in Vigna improvement programme.

Evaluation of the genetic fidelity of in vitro-propagated gerbera ( Gerbera jamesonii Bolus) using DNA-based markers

The genetic fidelity of in vitro-raised gerbera clones was assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 35 RAPD and 32 ISSR primers screened, only 12 RAPD and 10 ISSR primers produced clear, reproducible and scorable bands. The 12 RAPD primers produced 54 distinct and scorable bands, with an average of 4.5 bands per primer. The number of scorable bands for ISSR primers varied from 3 (ISSR-14) to 9 (ISSR-07), with an average of 5.5 bands per primer. The number of bands generated per primer was greater in ISSR than RAPD. All banding profiles from micropropagated plants were monomorphic and similar to those of the mother plant. A similarity matrix based on Jaccard’s coefficient revealed that the pair-wise value between the mother and the in vitro-raised plantlets was 1, indicating 100% similarity. This confirmed the true-to-type nature of the in vitro-raised clones.

Pyramiding of two bacterial blight resistance and a semidwarfing gene in Type 3 Basmati using marker-assisted selection

A traditional Type 3 Basmati rice cultivar grown in India is tall and lodges even under low nitrogen fertilizer dose. In addition to lodging, it is highly susceptible to several diseases and pests including bacterial blight (BB). BB resistance genes (Xa21 and xa13) and a semidwarfing gene (sd-1) were pyramided in Type 3 Basmati from a rice cultivar PR106-P2 using marker-assisted selection (MAS). Foreground selection for BB resistance genes, Xa21 and xa13 and reduced plant height gene, sd-1 was carried on the basis of linked molecular markers pTA248, RG136 and ‘h’, respectively. The BC2F3 progenies with both the BB resistance genes were highly resistant with lower lesion length than either of the genes individually. Background profiling of the selected 16 BC2F3 progenies was done using 95 anchored SSR and 12 ISSR markers. Among the selected 16 BC2F3 progenies, 38-5-2 and 38-5-36 closely clustered along with the recipient parent Type 3 Basmati showing above 85% genetic similarity with the same. Further selection was continued till F5 generation for higher recovery for Type 3 Basmati characteristics. The desirable alleles of intermediate amylose content (wx) and aroma (fgr) loci of Type 3 Basmati were also tracked using the linked SSR markers. The BC2F5 pyramid lines T3-4, T3-5, T3-6 and T3-7 homozygous for the three target genes Xa21, xa13 and sd-1 from the donor parent with wx and fgr alleles of Type 3 Basmati had excellent cooking quality and strong aroma.

Evaluation of Somaclonal Variation for Genetic Improvement of Patchouli (Pogostemon patchouli), an Exclusively Vegetatively Propagated Aromatic Plant

Patchouli (Pogostemon patchouli) is an important, exclusively vegetatively propagated aromatic plant, whose essential oil is widely used in perfumery and cosmetic products. Forty SC1 generation (first generation following in vitro phase) somaclones selected randomly from about 400 somaclones developed from the variety Johore, were multiplied through stem cuttings and evaluated in SC2 and SC3 generations to study the extent of somaclonal variation generated for plant height, herb yield, essential oil content, essential oil yield, and seven constituents of the essential oil. Significant or highly significant somaclonal variation was observed for plant height, herb yield, essential oil content, essential oil yield, and contents of patchouli alcohol, α-guaiene, α,δ-patchoulene, and α-bulnesene in the essential oil. The number of somaclones significantly superior to the parental variety for plant height, herb yield, essential oil content, and patchouli alcohol content in the essential oil ranged from 8–16 and the maximum superiority over the parental variety for these traits ranged from 21–79%. Broad-sense heritability estimates of plant height, herb yield, and essential oil content were 0.60–0.70 while those of essential oil yield and patchouli alcohol content were 0.44 and 0.47, respectively. Heritability estimates of other studied essential oil constituents were generally low (0.12–0.38). A high positive correlation was observed between essential oil yield and herb yield suggesting that selection for herb yield would be effective in improving essential oil yield. Patchouli alcohol content in the essential oil was negatively correlated with all the studied traits. Somaclonal variation, heritabilities of traits, and inter-trait correlations are reported for the first time in patchouli.

Inter Simple Sequence Repeat Analysis to Confirm Genetic Stability of Micropropagated Plantlets in Three Grape (Vitis spp) Rootstock Genotypes

Three grape rootstock genotypes — Dogridge (Vitis champini), SO4 (V. beriandieri × V. rupestris) and H-144 (V. vinifera × V. labrusca), and their 30 in vitro regenerated plantlets were subjected to Inter Simple Sequence Repeat (ISSR) analysis in order to ascertain the genetic stability of micropropagated plantlets. Out of 35 primers screened initially with three mother plants, 10 were finally selected based on sufficient polymorphism and appearance of clear and scorable banding patterns. Each primer generated a unique set of amplification products ranging in size from 100 to 1800 bp. These ten ISSR primers produced 81 distinct and scorable band classes with an average of 8.1 bands per primer. Based on similarity matrix and cluster analysis the rootstock genotypes and their tissue culture derivatives formed three distinct genetic groups indicating their genetic relationships. Furthermore, no variation was detected among in vitro regenerated grape plantlets and their field-grown mother plants corroborating the high level of clonal fidelity of the in vitro regenerated plantlets and supporting the multiplication protocol utilizing nodal segments as in vitro culture initiation material.

Breeding medicinal plant, periwinkle [Catharanthus roseus (L) G. Don]: a review

Periwinkle [ Catharanthus roseus (L) G. Don] has become one of the very extensively investigated medicinal plants after the discovery of two powerful anti-cancer alkaloids, vinblastine and vincristine, in its leaves more than 50 years ago. These alkaloidal drugs are still in clinical use. Also, periwinkle is still the only source of these alkaloids and their precursors, catharanthine and vindoline. Low concentrations of these alkaloids in the plant and, therefore, high costs of their extraction have led to tremendous efforts towards understanding their biosynthesis and exploration of alternate ways of their production such as, chemical synthesis, cell, tissue and hairy root cultures, and metabolic engineering of heterologous organisms. Literature on this plant is quite voluminous, with an average of about 80 publications per year during last three decades (1985–2015). Nearly 60% of these publications are on physiology, biochemistry, cell and tissue culture, phytochemistry, metabolic and genetic engineering aspects. In spite of these efforts, an economically viable alternative to field-grown periwinkle plants as a source of these alkaloids has not yet been found. Biosynthesis of C. roseus alkaloids is a complex process involving many genes, enzymes, regulators, inter- and intra-cellular transporters, cell types, organelles and tissues and its current understanding is still considered to be incomplete to produce C. roseus alkaloids through metabolic engineering/synthetic biology. Till such time, breeding periwinkle varieties with higher concentrations of anti-cancer alkaloids for cultivation can be an alternate approach to meet the demand for these alkaloids and reduce their costs. While literature on cell and tissue culture, phytochemistry, metabolic and genetic engineering aspects of periwinkle has been reviewed periodically, crop production and plant breeding aspects have received little attention. In this paper, an attempt has been made to bring together published information on genetics and breeding of periwinkle as a medicinal plant. Some probable constraints which may have hindered taking up periwinkle breeding are identified. Initially, quite a few attempts have been made at genetic improvement of periwinkle through induced polyploidy, and subsequently through induced mutagenesis. Mutations, both natural and induced, provide a valuable resource for use in breeding and in functional and reverse genomics research. It is only during last 6–7 years, genetic diversity has been assessed using molecular markers and very recently molecular markers have been identified for marker-assisted selection for alkaloid yield.

In Situ and Ex Situ Conservation of Plant Genetic Resources and Traditional Knowledge

The efficient conservation and judicious use of plant genetic resources and related traditional knowledge is vital for food, health, and nutritional security. Plant genetic diversity available in gene banks, in the wild, and in farmer’s fields is crucial resource for food, health, and nutritional security, augmenting livelihoods and environmental services. These resources could support acclimatization of primary food crops to climate change impacts. In pursuit of improved cereal, pulse, horticultural, medicinal, aromatic, and other cash crop varieties suitable for particular agroecosystem, availability of a diversity of alleles/genes in terms of yield, quality, resistance against disease and pest, and tolerance for drought, heat, salt, and water in the wild relatives, indigenous, local land races, and farmer cultivars is the prime prerequisite, which limits the options for sustainable resilient agriculture management. To prevent their genetic erosion, a well-devised strong linkage between in situ resource conservation, collection and ex situ conservation in gene banks and their utilization for genetic improvement through research and breeding is required for their subsequent availability to farmer, pharmaceutical and seed community. Conserving plant genetic resources in situ as well as ex situ ensures efficient explorations in order to implement the International Treaty and Global Plan of Action on Plant Genetic Resources for Food and Agriculture. These collections are valuable genetic resource of resistance to diseases and pests, tolerance to climatic stresses, and improved targeted “trait” through crop improvement and assurance against genetic erosion. It is important to support in situ/on-farm conservation of agroecosystems because ex situ conservation cannot replicate the evolutionary processes and cultural practices but can act as crucial reservoir to safeguard them from calamities.

Medicinal and aromatic plants: A case example of evolving secondary metabolome and biochemical pathway diversity

The unique capability of plants, in spite of being immobile in strict sense, to defend and respond precisely to environmental stresses whether biotic or abiotic is relatable to their ability to synthesize an array of phytochemicals as metabolites. We find these plants compounds useful for health care as nutraceuticals, drugs and medicines and even most attractive fragrances and flavors. This huge diversity of low molecular weight compounds is represented by secondary metabolites that confer the power of responding to stimuli in plants. Hence, the network of metabolic pathways in plant species represents the pool of functions and chemical diversity leading to biomolecules such as alkaloids, flavanoids, terpenoids, glycosides, etc. Although the structures of approximately 50,000 such metabolites have already been elucidated, there are probably hundreds of thousands of such compounds which we are not able to detect or decipher within the existing limitation of detection. Only a few of these are part of ‘primary’ metabolic pathways (those common to all organisms). The rest are termed ‘secondary’ metabolites; this term is historical and was initially associated with inessentiality but we know today their necessity in defence to signals and stimuli.

RNA-Seq analysis of two elite lines of Withania somnifera to evaluate potential of heterosis

Metabolite targeted breeding is one of the prime need in Ashwagandha (Withania somnifera) to meet its fast growing demand at domestic and international level owing to its vast spectral pharmaceutical potential. Till date only scanty breeding efforts exists majority being introductions or selections from the wilds/landraces through which active bio constituent increase is limited. Hybridization based heterotic positive QTL alleles can be introgressed in a fine quality good agronomic genetic base using marker assisted selection. Investigations under this study generated a large resource of Withania specific genomic microsatellite markers and SNP through RNA seq. Using an RNA-seq approach, the expression of 101245 unigenes were quantified in parents. The expression levels of 30% of transcripts differed between parents, a majority of which had more than 1.5 fold-changes. RNA seq will help to decipher the Withanolide biosynthesis pathway and SNP responsible for functional polymorphism in pathway genes and hence predicting the potential of heterosis.

Random amplified marker technique for plants rich in polyphenols.

More than 10,000 publications using the random amplified polymorphic DNA (RAPD) or related arbitrary marker techniques have been published in two decades of its inception in 1990. Despite extensive use, RAPD technique has also attracted some criticisms, mainly for lack of reproducibility. In the light of its widespread applications, the objective of this chapter is to (1) provide a protocol for RAPD assay, (2) identify the potential factors affecting the optimization of the RAPD assays, and (3) provide proper statistical analysis to avoid false positives. It is suggested that after proper optimization, the RAPD is a reliable, sensitive, and reproducible assay having the potential to detect a wide range of DNA variations. Analyses of the relevant fragments generated in RAPD profile allow not only to identify some of the molecular events implicated in the genomic instability but also to discover genes playing key roles in genetic evolution and gene mapping. RAPD markers will continue to be boon for genetic studies of those organisms where yet no sequence information or scanty information is available.