Arun Kumar Kukreja

Genetic transformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor

The study was undertaken to induce hairy roots in Glycyrrhiza glabra in leaf explants and to optimize the nutritional requirement for its growth kinetics at shake flask and bioreactor level. Pathogenecity of Agrobacterium depends upon transformation ability of strain and age, type, and physiological state of explants. Agrobacterium rhizogenes strain K599 was used to infect leaf explants of G. glabra . Explants of different age groups were obtained from 2 to 5 weeks old in vitro grown cultures. Bacterial strain K599 could induce hairy roots in 3 and 4 weeks old leaf explants cultured on B 5 , MS, NB and WP basal semi-solid medium. Leaf explants of 2 and 5 weeks old culture were not responsive to bacterial infection in terms of hairy root induction. Maximum transformation frequency (TF) of tested bacterial strain was 47% obtained in 3 weeks old explants after 25 days of incubation on MS basal semi solid medium. NB and B 5 both media composition showed 20% of transformation frequency after 28 and 38 days respectively. WP medium did not support induction of roots in cultured leaf explants infected with A. rhizogenes strain K599even after 50 days of incubation. Further, when all the four media combinations were tested for root growth it was found that though WP was not responsive for hairy root induction, yet all four basal media supported hairy root growth and a gradual increase in fresh weight biomass was observed with an increase in culture duration. However amongst all, the NB medium composition supported best growth of hairy roots followed by MS, B 5 and WP media. About 20 times increase in root biomass on fresh weight basis was recorded after 45days of culture in NB medium. Initial inoculum of roots (0.18 g. F.wt./ flask) containing 50 ml of liquid culture medium produced 3.59 g (F. wt.) biomass. A fast growing hairy root clone G6 was grown in a 5 l capacity mechanically agitated bioreactor provided with a nylon mesh septum. After 30 days of sterile run, 310 g of root biomass was harvested from the bioreactor culture vessel, recording about 20 times increase over initial inoculum (16.0 g).

Elicitor-induced cellular and molecular events are responsible for productivity enhancement in hairy root cultures: an insight study.

A wide range of external stress stimuli triggers a plant cell to undergo a complex network of reactions that ultimately lead to the synthesis and accumulation of secondary metabolites. These secondary metabolites help the plant to survive under stress challenge. The potential of biotic and abiotic elicitors for the induction and enhancement of secondary metabolite production in various culture systems including hairy root (HR) cultures is well-known. The elicitor-induced defense responses involves signal perception of elicitor by a cell surface receptor followed by its transduction involving some major cellular and molecular events including activation of major secondary message signaling pathways. This result in induction of gene expressions escorting to the synthesis of various proteins mainly associated with plant defense responses and secondary metabolite synthesis and accumulation. The review discusses the elicitor-induced various cellular and molecular events and correlates them with enhanced secondary metabolite synthesis in HR systems. Further, this review also concludes that combining elicitation with in-silico approaches enhances the usefulness of this practice in better understanding and identifying the rate-limiting steps of biosynthetic pathways existing in HRs which in turn can contribute towards better productivity by utilizing metabolic engineering aspects.

Screening and evaluation of agronomically useful somaclonal variations in Japanese mint (Mentha arvensis L.)

SummaryA procedure has been standardized for high frequency plant regeneration response from nodal explant cultures of Mentha arvensis Linn. var. piperascens Holmes. Murashige and Skoogs medium supplemented with IAA or NAA (0.5–2.0 mgl−1) alone, supported axillary shoot elongation while BAP (2.0–3.0 mgl−1) alongwith IAA (1.0 mgl−1) supported multiple shoot production. In vitro-derived shoots readily developed roots when cultured on NAA (1.0 mgl−1) fortified MS medium. Regenerated plantlets were successfully transferred to glasshouse (90–95% survival rate) and ultimately to the field. Among 280 plants transferred to the field a wide range of variation was observed for various agronomic traits i.e. plant height (32.0–92.0 cm), leaf-stem weight ratio (0.53–2.32), herb yield (105.0–870.0 g), oil content (0.32–1.10%) and oil yield (0.66–5.22 ml/plant). In addition, variations were also recorded for four major constituents of the essential oil i.e. menthol (65.2–94.77%), menthone (1.40–20.89%), isomenthone (0.96–5.14%) and menthyl acetate (0.75–8.52%). A positive correlation is found for oil yield with plant height and herb yield, whereas a negative correlation exists between herb yield and oil content. Based on the initial agronomic assessments on individual plant basis, 27 somaclones were selected and further evaluated in a replicated plant to row trial with parent plant CIMAP/Hy-77 as standard check. Somaclones Sc 59 and Sc 179, selected on the basis of higher herb yield in the initial screening, recorded 55.8% and 64.3% increase in oil yield over the control, respectively. Somaclones Sc 93, Sc 114, Sc 121 and Sc 124 that were selected for their better oil content exhibited 47.2%, 50.6%, 57.5% and 48.2% increase in oil yield over the parent variety, respectively. The performance of these clones in evaluation trials is discussed in relation to the possibility of genetic improvement of mints through somaclonal breeding.

An extensive case study of hairy-root cultures for enhanced secondary-metabolite production through metabolic-pathway engineering.

An intrinsic improvement is taking place in the methodologies for the development of culture systems with first‐rate production of plant‐based molecules. The blending of HR (hairy root) cultures with ME (metabolic engineering) approaches offers new insights into, and possibilities for, improving the system productivity for known and/or novel high‐value plant‐derived active compounds. The introduction and expression of foreign genes in plants results in improvement of cellular activities by manipulating enzymatic, regulatory and transport function of the cell. The rational amendments in the rate‐limiting steps of a biosynthetic pathway as well as inactivating the inefficient pathway(s) for by‐product formation can be accomplished either through single‐step engineering or through the multi‐step engineering. The hierarchical control of any metabolic process can lead the engineer to apply the ME ideas and principles to any of the strata, including transcriptional, moving on to translational and enzymatic activity. The HR culture systems offer a remarkable potential for commercial production of a number of low‐volume, but high‐value, secondary metabolites. Taking HR as a model system, in the present review, we discuss engineering principles and perceptions to exploit secondary‐metabolite pathways for the production of important bioactive compounds. We also talk about requisites and possible challenges that occur during ME, with emphasis on examples of various HR systems. Furthermore, it also highlights the utilization of global information obtained from ‘‐omic’ platforms in order to explore pathway architecture, structural and functional aspects of important enzymes and genes that can support the design of sets of engineering, resulting in the generation of wide‐ranging views of DNA sequence‐to‐metabolite passageway networking and their control to obtain desired results.

Genetic Improvement of Mints: On the Qualitative Traits of Essential Oil of In-Vitro Derived Clones of Japanese Mint (Mentha arvensis var. piperascens Holmes)

ABSTRACT A wide range of variations was recorded in the somaclones of Mentha arvensis L. var. piperascens Holmes for menthol (65.2–94.8%), menthone (1.4–20.9%), isomenthone (1.0–5.2%) and menthyl acetate (0.8–8.5%). Correlation analysis of these four constituents revealed a negative significant correlation of menthol with menthone (r= −0.856) and isomenthone (r=−0.837), while menthone had a positive significant correlation with isomenthone (r=0.853). Eight somaclones possessed a higher menthol content than the parent, while five of these had also been ranked promising based on the agronomic characters. On the basis of quantitative and qualitative traits, 27 somaclones were further evaluated in a replicated plant to row trial along with the parent clone. A decline in menthol content along with 2–4 fold increase in menthone and isomenthone was observed in a randomized block design trial with somaclones. Two somaclones (Sc 93 and Sc 179) showed stability in terms of percent composition of the volatile oil. T...

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or α-naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or α-napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with α-naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).

Establishment and multiplication of colchi-autotetraploids of Rauvolfia serpentina L. Benth. ex Kurz. through tissue culture

A tissue culture procedure was developed for the establishment and propagation of a colchi-autotetraploid of Rauvolfia serpentina for possible commercial exploitation. Multiplication of autotetraploid shoots was obtained either through axillary bud elongation on Murashige and Skoog [1] medium (MS) containing 2.65 μM (0.5 mgl−1) α-naphthaleneacetic acid and 0.33 μM (0.05 mgl−1) kinetin, or via multiple shoot formation on MS medium supplemented with 4.44 μM (1.0 mgl−1) 6-benzylaminopurine and 0.53 μM (0.1 mgl−1) α-naphthaleneacetic acid. Rooting could be induced by transferring the shoots to MS medium containing 7.95 μM (1.5 mgl−1) α-naphthaleneacetic acid alone. The plantlets, thus formed, were tetraploid in nature by cytological observations of the root tips. They exhibited 80–90% success in establishment under glass house and field conditions.

Biomass yield, essential oil yield and resource use efficiency in geranium (Pelargonium graveolens L. Her. ex. Ait), intercropped with fodder crops

The objective of this study was to determine the effect of intercropping of fodder crops on growth and yield attributes of the essential oil-yielding multi-harvest aromatic plant geranium (Pelargonium graveolens L. Her. ex. Ait) under field conditions during 2005–2007. In addition aggressivity, land equivalent ratio (LER), area time equivalent ratio (ATER) and land use efficiency % (LUE%) as an index of intercropping advantage were determined to assess the resource use efficiency of intercropping systems. The main crop geranium was intercropped with oat (Avena sativa L.) and berseem or Egyptian clover (Trifolium alexandrum L.) in different combinations. All crops were also grown in pure stands. The study indicated that the growth and yield of geranium was affected at first harvest compared to sole. But the second harvest in different intercropping systems compared to sole did not affect biomass and oil yield of geranium. Intercrops produced bonus yields over and above that of geranium. The resource use efficiency values were higher in intercropping systems over sole geranium. The study clearly showed that geranium-based intercrop treatments might provide the highest total yield as well as resource use efficiency.

Essential Oil Composition of the Inflorescence of Artemisia capillaris Thunb. Collected at Different Stages of Flowering from Kumaon Region of Western Himalaya

Abstract The essential oils obtained from Artemisia capillaris at different stages of flowering were analyzed by GC and GC/MS. The main components of this oil were capillene, γ-terpinene, eugenol, limonene, p-cymene, myrcene and α-pinene. Capillene (40.1%), α-pinene (2.4%) and p-cymene (2.5%) were higher in the oil obtained from plants collected during full bloom stage, while γ-terpinene (24.6%) and eugenol (15.0%) were the main components of the oil from the seed setting stage. Characteristic of the oil was the presence of the rarely occurring compounds like capillene and norcapillene.