The wealth of India. A dictionary of Indian raw materials and industrial products. Vol. 7; N-Pe.

Magnolia sirindhorniae Noot. & Chalermglin is an endangered species with high ornamental and commercial value that needs to be urgently protected and judiciously commercialized. In this study, a protocol for efficient regeneration of this species is standardized. The lateral buds of the M. sirindhorniae plant were used as an explant. Half-strength Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 6-benzyladenine (BA), 0.1 mg/L α-naphthaleneacetic acid (NAA), and 2.0 mg/L gibberellic acid (GA3) was found to be the optimal medium for shoot induction. The maximum shoot multiplication rate (310%) was obtained on Douglas-fir cotyledon revised medium (DCR) fortified with 0.2 mg/L BA, 0.01 mg/L NAA, and additives. The half-strength DCR medium supplemented with 0.5 mg/L NAA and 0.5 mg/L indole-3-butyric acid (IBA) supported the maximum rate (85.0%) of in vitro root induction. After a simple acclimatization process, the survival rate of plantlets in a substrate mixture of sterile perlite and peat soil (1:3; v/v) was 90.2%. DNA markers were used for assessment of genetic uniformity, confirming the genetic uniformity and stability of regenerated plants of M. sirindhorniae. Thus, the described protocol can safely be applied for large scale propagation of this imperative plant.

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An efficient micropropagation protocol for an endangered ornamental tree species ( Magnolia sirindhorniae Noot. & Chalermglin) and assessment of genetic uniformity through DNA markers

The primary issues regarding the lack of protocol reproducibility among laboratories are environmental factors. Light (quantity and particularly quality), is one of those main factors, and studies seldom present the spectral quality of the light sources used. With the advent of light-emitting diode (LED) technology, impressive progress has been made in environmental controls and morphogenetic responses, as directed by the light used in the culture shelves. A wide array of LED lights with different spectra are currently available and light is important in large-scale propagation, especially liquid bioreactor systems. LED technology continues to evolve rapidly and has created additional possibilities. This laboratory has dedicated extensive efforts to implement photoautotrophic propagation, and light is a key component of the system. This review presents relevant topics on the influence of light in various plant tissue culture-based techniques.

Light quality in plant tissue culture: does it matter?

The present review illustrates the implementation of synthetic seed technology for mass propagation and short-term storage of several medicinal plants, popularly grown throughout the world. Biotechnology-based research with special reference to in vitro plant cell and tissue culture intervention created a new outlook in terms of mass propagation, germplasm storage and cryoconservation, production of secondary metabolites as well as genetic transformation. Synthetic seed technology involving alginate encapsulation of in vitro or in vivo generated explants proved to be a competent system to deal with multiplication, storage and exchange of seedless medicinal plants having traits of choice that are intricate to propagate via conventional approach. Nevertheless, optimization of production, storage and exchange of synthetic seeds are influenced by several factors. Manipulation of those factors such as explant selection, encapsulating agent and matrix determined the success of synthetic seed technology in medicinal plants. The present review elucidates an outline of past progress, present status and future prospects of synthetic seed technology intervention in medicinal plants with special emphasis on the factors which determine the success of this technology.

Synthetic seed production of medicinal plants: a review on influence of explants, encapsulation agent and matrix

Plant tissue culture has been used for conservation, micropropagation, and in planta overproduction of some pharma molecules of medicinal plants. New biotechnology-based breeding methods such as targeted genome editing methods are able to create custom-designed medicinal plants with different secondary metabolite profiles. For a long time, humans have used medicinal plants for therapeutic purposes and in food and other industries. Classical biotechnology techniques have been exploited in breeding medicinal plants. Now, it is time to apply faster biotechnology-based breeding methods (BBBMs) to these valuable plants. Assessment of the genetic diversity, conservation, proliferation, and overproduction are the main ways by which genetics and biotechnology can help to improve medicinal plants faster. Plant tissue culture (PTC) plays an important role as a platform to apply other BBBMs in medicinal plants. Agrobacterium-mediated gene transformation and artificial polyploidy induction are the main BBBMs that are directly dependent on PTC. Manageable regulation of endogens and/or transferred genes via engineered zinc-finger proteins or transcription activator-like effectors can help targeted manipulation of secondary metabolite pathways in medicinal plants. The next-generation sequencing techniques have great potential to study the genetic diversity of medicinal plants through restriction-site-associated DNA sequencing (RAD-seq) technique and also to identify the genes and enzymes that are involved in the biosynthetic pathway of secondary metabolites through precise transcriptome profiling (RNA-seq). The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce user-designed medicinal plants. These current targeted genome editing methods are able to manage plant synthetic biology and open new gates to medicinal plants to be introduced into appropriate industries.

Application of genetics and biotechnology for improving medicinal plants.

Cassia sophera Linn. is an important medicinal plant belonging to family Fabaceae. It is extensively used in Homeopathy and is well known for its medicinal properties. The present study describes a simple, efficient and reproducible regeneration system for in vitro propagation of C. sophera through cotyledonary node (CN) explant excised from 21 d old axenic seedlings. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). Multiple shoots were produced on all the concentrations of TDZ; however 2.5 μM concentration proved to be optimal for the production of maximum number of shoots. To avoid adverse effects of prolonged exposure to TDZ in long term establishment, the cultures were transferred to TDZ free MS medium fortified with various concentrations of 6- benzyl aminopurine (BA) for multiplication, proliferation and elongation of induced shoots. Emergence of new shoot buds and multiplication continued up to second subculture passage and maximum number (14.9 ± 1.4) of shoots obtained on MS + BA (1.0 μM). Best rooting response was observed on half strength MS containing Indole-3-butyric acid (IBA) (1.0 μM). Regenerated plantlets were successfully acclimatized and hardened off inside the culture room and then transferred to green house with 100 % survival rate.

TDZ-induced high frequency shoot regeneration in Cassia sophera Linn. via cotyledonary node explants

The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS + TDZ (1.0 μM) were transferred to shoot regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS + BA (2.5 μM) + NAA (0.6 μM) wherein a maximum of 42.76 ± 1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63 ± 0.75 shoots per explant and average shoot length of 5.43 ± 0.20 cm. Elongated microshoots were successfully rooted under ex vitro conditions by pulse treatment in 200 μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation of shoot buds in large number and thereafter conversion into healthy shoots.

A micropropagation protocol for Cassia angustifolia Vahl. from root explants

A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, excised from 14-day old aseptic seedlings, has been established. Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) singly or in combination with auxins was used for regeneration studies. Among the single treatment of three cytokinins BA at 1.0 µM was found to be optimum for direct shoot regeneration as it induced an average of 8.20 ± 0.66 shoots per explant. The regeneration frequency further enhanced with the application of auxin along with optimal BA concentration. The highest frequency for shoot regeneration (90%), the maximum number of shoots per explant (12.20 ± 0.73) and the maximum shoot length (6.40 ± 0.07) cm were obtained on the medium consisted of MS + 1.0 µM BA + 0.5 µM NAA. Successful in vitro rooting was induced from cut end of the microshoots when placed on half-strength MS + IBA (2.5 µM). The regenerated shoots with well developed root system were successfully acclimatized and established in pots containing sterilized garden soil and garden manure (1:1) and grown under greenhouse conditions with 85% survival rate.

In vitro plant regeneration system for Cassia siamea Lam., a leguminous tree of economic importance

The present study described the encapsulation of nodal segments of Cassia angustifolia Vahl. excised from 1-month-old in vitro raised cultures for short-term conservation and propagation. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) were tested to prepare uniform beads. The ideal beads were obtained through a combination of 3 % sodium alginate and 100 mM calcium chloride. The maximum conversion response (94 %) of encapsulated beads was obtained in Murashige and Skoog’s medium (MS medium) supplemented with 2.5 μM benzyladenine (BA) and 0.4 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. The encapsulated and non-encapsulated nodal segments were also stored at 4 °C for different time periods (0, 1, 2, 4, 6 and 8 weeks). The regenerated microshoots were best rooted in optimized rooting medium that comprised half-strength MS + 1.0 μM indole-3-butyric acid (IBA) + 5.0 μM phloroglucinol (PG) for the production of complete plantlets. The regenerated plantlets were successfully hardened and acclimatized in natural conditions with 70 % survival rate.

Encapsulation of nodal segments of Cassia angustifolia Vahl. for short-term storage and germplasm exchange

In 1902 Gottlieb Haberlandt proposed the idea to culture individual plant cells on artificial nutrient medium. Although he failed to culture them due to poor choice of experimental materials and inadequate nutrient supply, he made several valuable predictions about the nutrients’ requirement for in vitro culture conditions, which could possibly induce cell division, proliferation and embryo induction. Tissue culture has now become a well-established technique for culturing and studying the physiological behaviour of isolated plant organs, tissues, cells, protoplasts and even cell organelles under precisely controlled physical and chemical conditions. Micropropagation is one of the most important applications of plant tissue culture. It provides numerous advantages over conventional propagation like mass production of true-to-type and disease-free plants of elite species in highly speedy manner irrespective of the season requiring smaller space and tissue source. Therefore, it provides a reliable technique for in vitro conservation of various rare, endangered and threatened germplasm. Micropropagation protocols have been standardized for commercial production of many important medicinal and horticultural crops. Somatic embryogenesis is an extremely important aspect of plant tissue culture, occurring in vitro either indirectly from callus, suspension or protoplast culture or directly from the cell(s) of an organized structure. Advantages of somatic embryogenesis over organogenesis include several practical means of micropropagation. It reduces the necessity of timely and costly manipulations of individual explants as compared to organogenesis.

Shahina Parveen

Papers

TDZ-induced high frequency shoot regeneration in Cassia sophera Linn. via cotyledonary node explants

A micropropagation protocol for Cassia angustifolia Vahl. from root explants

In vitro plant regeneration system for Cassia siamea Lam., a leguminous tree of economic importance

Encapsulation of nodal segments of Cassia angustifolia Vahl. for short-term storage and germplasm exchange

Historical Perspective and Basic Principles of Plant Tissue Culture