In the growth condition(s) of plants, numerous secondary metabolites (SMs) are produced by them to serve variety of cellular functions essential for physiological processes, and recent increasing evidences have implicated stress and defense response signaling in their production. The type and concentration(s) of secondary molecule(s) produced by a plant are determined by the species, genotype, physiology, developmental stage and environmental factors during growth. This suggests the physiological adaptive responses employed by various plant taxonomic groups in coping with the stress and defensive stimuli. The past recent decades had witnessed renewed interest to study abiotic factors that influence secondary metabolism during in vitro and in vivo growth of plants. Application of molecular biology tools and techniques are facilitating understanding the signaling processes and pathways involved in the SMs production at subcellular, cellular, organ and whole plant systems during in vivo and in vitro growth, with application in metabolic engineering of biosynthetic pathways intermediates.

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Stress and defense responses in plant secondary metabolites production

Plant tissue culture as an important tool for the continuous production of active compounds including secondary metabolites and engineered molecules. Novel methods (gene editing, abiotic stress) can improve the technique. Humans have a long history of reliance on plants for a supply of food, shelter and, most importantly, medicine. Current-day pharmaceuticals are typically based on plant-derived metabolites, with new products being discovered constantly. Nevertheless, the consistent and uniform supply of plant pharmaceuticals has often been compromised. One alternative for the production of important plant active compounds is in vitro plant tissue culture, as it assures independence from geographical conditions by eliminating the need to rely on wild plants. Plant transformation also allows the further use of plants for the production of engineered compounds, such as vaccines and multiple pharmaceuticals. This review summarizes the important bioactive compounds currently produced by plant tissue culture and the fundamental methods and plants employed for their production.

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In vitro plant tissue culture: means for production of biological active compounds

Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid

High frequency regeneration protocol for Dendrobium nobile: A model tissue culture approach for propagation of medicinally important orchid species

A newly developed and novel DNA marker technique, i.e. start codon targeted (SCoT) polymorphic markers that target plant gene regions were used to assess genetic stability of in vitro raised plants of Cleome gynandra multiplied by enhanced bud proliferation from nodal segments. Seven randomly selected micropropagated plants, following at least 2 months of growth in the greenhouse along with mother plant were subjected to molecular analysis. Of 24 primers screened, 15 primers produced unambiguous and reproducible bands. All 15 primers generated a total of 65 fragments, with a mean of 4.3 ranging 2–7 per primer. No polymorphism was detected in regenerated plants and the mother plant, revealing the genetic fidelity of the in vitro raised plantlets. To verify the results of SCoT analysis, random amplified polymorphic DNA (RAPD) markers were also used for the assessment of genetic fidelity of tissue culture raised plants. The monomorphic banding pattern in micropropagated plants and the mother plant obtained from SCoT and RAPD analysis confirms the genetic stability of the in vitro raised plants and demonstrates the reliability of our micropropagation system for C. gynandra, an important C4 plant.

Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis

The proponents of anthropocene (the era of human-induced climatic changes) suggest that unless humanity changes its behavior according to earth system rules there will be no more holocene-type relative stability. Even the sustainability of the Earth’s life support systems is threatened. Humans and their use of land have transformed more than three quarters of the terrestrial biosphere into anthropogenic biomes. According to Rockstrom and Karlberg, humanity has entered a new phase of sustainability challenges, the Anthropocene, in which human development has reached a scale where it affects vital planetary processes. The earth system is under the pressure from a quadruple squeeze. The human-induced carbon and nitrogen (N) fertilizers are generating a strong imbalance with phosphorous (P). Global fresh water resources are, and will increasingly be, a fundamental limiting factor in feeding the world. By 2025, two third of the world’s population could be living under water-critical conditions. Arid regions (drylands) cover about 45 % of earth’s land surface; they constitute the largest biome on the planet. The dryland forests takes up carbon at rates similar to those of pine forests. In this millennium, global drylands face a myriad of problems that present research, management and policy challenges. Climate change/global warming is great threats to life supporting systems of the desert: drought and heat-induced die-back/drying/deaths and climate-driven vegetation/tree mortality in fragile ecosystems are great concerns. Introduction of alien/invasive species and cultivation of plants with unfavorable water footprint are threat to fragile ecosystems. The study of plant biology in the 21st century is, and will continue to be, vastly different from that in the 20th century. One driver for this has been the use of genomics methods to reveal the genetic blueprints for not one but dozens of plant species. Plant genomics researchers have readily embraced new algorithms, technologies and approaches to generate genome, transcriptome and epigenome datasets for model and crop species that have permitted deep inferences into plant biology. At the Department of Botany, Jai Narain Vyas University, Jodhpur; with the help of Department of Biotechnology, Department of Science and Technology, Government of India and the University Grants Commission of India, we have developed biotechnological methods for characterization, conservation, propagation and improvement of plants of Rajasthan. These are described and discussed in this paper. A few recommendations, useful for the desert ecosystem and bio-resources are submitted.

Bioresearches of Fragile Ecosystem/Desert

We developed a micropropagation protocol for Cleome gynandra, a C4 model plant with medicinal importance. Surface-sterilized nodal segments obtained from 1 to 2-month-old field grown plant were used as explants for culture establishment and plant regeneration. Multiple shoots differentiated through bud breaking on Murashige and Skoog (MS) medium with different concentrations of benzyladenine (BA) and kinetin (Kin). The optimum shoot differentiation occurred on medium with 1.5 mg l−1 BA. Out of various concentrations and combinations of cytokinins and auxins, MS medium containing 0.5 mg l−1 BA and 0.1 mg l−1 IAA (indole-3-acetic acid) was found best for shoot multiplication. However, the differentiated shoots exhibited hyperhydration, leaf curling and early leaf fall during subculturing. To overcome these problems, regenerated shoots were transferred to the modified MS medium with reduced nitrates (825 mg l−1 NH4NO3 and 950 mg l−1 KNO3) and 100 mg l−1 (NH4)2SO4. The micropropagated shoots were rooted (i) in vitro on one-fourth strength of MS salts with 0.25 mg l−1 each of IBA (indole-3 butyric acid) and NOA (2-naphthoxyacetic acid) + 100 mg l−1 activated charcoal, and (ii) ex vitro, by treating the shoot base(s) with 200 mg l−1 of IBA for 3 min and transferred to soilrite moistened with one-fourth strength of MS macro salts in culture bottles. The plants were hardened in the greenhouse with 85 % survival rate. Micromorphological studies of the plants were conducted during hardening with reference to development and changes in vein spacing, glandular trichome and stomata. In comparison to leaves under in vitro condition, higher density of veins and glandular trichomes was observed in the leaves of hardened plants. In addition, stomata became functional during hardening which were non-functional under in vitro condition.

In vitro propagation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l−1 of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l−1 ammonium sulphate {(NH4) 2SO4} (MMS3) + BA (0.25 mg l−1), Kinetin (Kin; 0.25 mg l−1), Indole-3-acetic acid (IAA; 0.1 mg l−1) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l−1 of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.

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Nisha Rathore

Papers

Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis

Bioresearches of Fragile Ecosystem/Desert

In vitro propagation and micromorphological studies of Cleome gynandra: a C4 model plant closely related to Arabidopsis thaliana

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

In vitro flowering and seed production in regenerated shoots of Cleome viscosa