Volume 5 Number 1 2011

CONTENTS AND ABSTRACTS

Sunil D. Purohit (India), Jaime A. Teixeira da Silva (Japan) Nazima Habibi (India) Current Approaches for Cheaper and Better Micropropagation Technologies (pp 1-36)

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Review: There has been widespread use of plant tissue culture in micropropagation of plants of forestry, horticultural and medicinal importance. The success of mass propagation and its economic viability however, depends upon several factors which influence the scaling-up production. Poor rates of shoot multiplication, increasing cost of media ingredients, loss of cultures due to contamination and difficulties experienced during hardening and acclimatization are the major bottlenecks adversely affecting scaling-up of micropropagation technologies. A large number of in vitro technologies developed for a range of plant species have thus remained confined to laboratories because of such constraints. In order to extend tissue culture technology to large-scale propagation, it is necessary to develop methods that are relatively simple, have a high multiplication rate with high degree of reproducibility and give a high survival rate of microshoots or plantlets upon transfer to ex vitro conditions. Innovative approaches such as the use of liquid culture systems, replacing agar with other gelling agents viz. guar-gum, growing cultures under a CO₂-enriched environment, in vitro hardening, priming of tissue culture-raised plants using anti-transpirants or biopriming agents during the weaning period and improvement in the culture vessel environment by ventilation and other means have proved highly beneficial. Such approaches will not only allow production of micro-clones which are comparatively cheaper and better in their post-vitro performance in the soil environment but will also to help generate viable micropropagation technology suitable for mass production of desirable plant species.

Saswati Sengupta (India), Shamik Das (India/USA), Suparna Ghosh, Amita Pal (India) Phenolic Signals and Their Perception Leads to in Vitro Shoot Induction in Vigna radiata Cotyledonary Explants (pp 37-41)

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Original Research Paper: The differential regeneration potential of two cotyledons (Cot and Cot E) of Vigna radiata seed during in vitro shoot differentiation is well established in this study. However, the two explant types influenced both the efficiency and mode of regeneration of each other when 8 Cot explants were placed at the periphery and 4 Cot E explants at the centre of Petri dishes containing shoot induction medium. Such a cross-talk was assumed to be mediated by exudates released from these explants in the agar medium. This led us to investigate the nature of the diffusing compound(s). Biochemical analyses revealed the phenolic nature of these diffusing metabolites. Experimentally it was demonstrated that these phenol-exudates had the potential to alter the mode of regeneration from callus-mediated to direct shoot formation in Cot explants and to enhance the shoot-promoting activity of Cot E explants. In contrast to the well defined role of phenolic compounds in plant defense, this study further showed the involvement of a HPLC purified phenolic compound, present in the exudates, in triggering in vitro shoot differentiation. Such a compound may be used to induce in vitro organogenesis, particularly in recalcitrant grain legumes.

Monika Mahajan, Vinay Kumar, Sudesh Kumar Yadav (India) Effect of Flavonoid-mediated Free IAA Regulation on Growth and Development of in Vitro-Grown Tobacco Seedlings (pp 42-48)

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Original Research Paper: The role of exogenously applied two flavonoids, quercetin and epicatechin, was assessed on plant growth and development. For this, tobacco (Nicotiana tabacum) seedlings were supplemented with 50 and 100 µM epicatechin and quercetin individually and their shoot and root parts were analyzed for total flavonoids content, transcriptional regulation of flavonoid biosynthesis pathway, morphological and anatomical responses. Exogenous application of these two flavonoids increased the level of total flavonoids in the shoot and decreased it in the root. Similarly the transcript expression of genes encoding key regulatory enzymes of flavonoid biosynthesis pathway was increased in the shoot and decreased or not affected in the root. Interestingly, chalcone synthase (CHS) expression decreased in the shoot and increased in the root. Seedlings exposed to flavonoids were observed for the following changes: inhibition of lateral and adventitious roots, reduced plant growth and leaf expansion. Further, flavonoids increased the number of parallel vascular strands in the central and petiolar region of the leaf and decreased the cell size of various regions in the leaf. These observations were similar to the responses produced by synthetic auxin transport inhibitors. Therefore, free indole-3-acetic acid (IAA) content was estimated in tobacco seedlings. Exposure to flavonoids enhanced the endogenous free IAA content in tobacco shoots. In tobacco roots, exposure to only 50 µM epicatechin and quercetin enhanced the free IAA content, while 100 µM exposure decreased the free IAA content. Results suggest that recorded changes could be the consequence of decreased basipetal free IAA transport due to higher flavonoid content in tobacco seedlings.

Ramesh Joshi, Bhanwar Lal Jat, Anshul Sharma (India), Dilip Nandwani (USA) In Vitro Propagation of Murraya koenigii L. Spreng (Curry Leaf Plant) through Adventitious Shoot Proliferation from Internode Explants (pp 49-52)

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Original Research Paper: Murraya koenigii (L.)Spreng, commonly known locally as “kurry patta” or “mitha neem” in India, is a valuable medicinal plant known for its biochemical and aromatic properties. Adventitious regeneration, which is a pre-requisite in most genetic transformation studies using Agrobacterium and ballistics, needs to be developed as a protocol for micropropagation of M. koenigii.This paper presents a procedure for the rapid, high frequency regeneration of M. koenigii plantlets from internode explants via adventitious shoot formation. The concentration of plant growth regulators (PGRs) in liquid MS medium exhibited a discrete role in the efficacy of adventitious shoot induction. N6-benzyle adenine (BA), kinetin, adenine sulphate and indole-3-acetic acid (IAA) in combination were the most effective PGRs for adventitious shoot induction. Murashige and Skoog (MS) liquid medium with 9.29 µM kinetin, 13.317 µM BA, 2.854 µM IAA and 70 mg/l adenine sulphate yielded the maximum number (18) of shoot buds from internode explants. The number of shoots was further increased (27.30) after sub-culturing them into semi-solid (containing 8 g/l agar-agar) MS medium fortified with similar concentrations and combinations of PGRs. Most in vitro shoots (2.5-3.0 cm long), rooted (90%) on semi-solid MS medium containing 19.68 µM indole-3-butyric acid within 28-30 days. The rooted plantlets were transplanted into pots containing a mixture of soilrite (mixture of peat moss + vermiculite + perlite in a 1: 1: 1 ratio that was mixed with natural soil in the ratio of 1: 1) at 70-80% relative humidity and 28 ± 2°C for hardening. 85% of in vitro-raised plantlets survived under field conditions.

Shesan John Owonubi, Omena Bernard Ojuederie, Mary Nkem Olayode (Nigeria) Organogenesis of Bambara Groundnut (Vigna subterranea (L.) Verdc.) through in Vitro Culture from Nodal Segments (pp 53-57)

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Original Research Paper: This study was designed to evaluate the effects of plant growth regulators – auxin (NAA) and cytokinin (BA) – on the regeneration potential of two accessions of bambara groundnut ‘TVSu1834’ (Nigeria) and ‘TVSu255’ (Niger) through in vitro culture. Nodal vine segments from 28-days-old plantlets derived from the embryonic axis were used as explants for nodal culture. The explants were cultured on half-strength MS basal salts supplemented with different concentrations and combinations of cytokinin and auxins for primary shoot proliferation. BA at 0.45 mg/l in combination with three different concentrations of NAA, namely 0 mg/l (M4), 0.15 mg/l (M2) and 0.25 mg/l (M3) were used for nodal culture. The control medium M1, had no plant growth regulators added. The shoots, leaves, and whole plant regeneration were evaluated at 4 weeks by means of percentage of explants producing shoot, callus and rooting per explant. The best shoot proliferation of nodal cuttings of ‘TVSu1834’ was observed in M3 medium containing half-strength MS, 0.45 mg/l BA, 0.25 mg/l NAA and 100 mg/l myo-inositol which produced multiple shoots with the longest shoots and most leaves compared to ‘TVSu255’. In M1 medium, the control, taller plantlets formed than at other concentrations and was the only medium that induced rooting. There was a genotype-dependent effect (P < 0.05) on shoot proliferation, shoot length and number of leaves in the two accessions.

Prathap Reddy V., Krishna M. S. R. (India), Jaime A. Teixeira da Silva (Japan), Bheem Reddy I. N., Ravicharan A., Sokkareddy S., Sivaramakrishnan S. (India) Efficient in Vitro Plant Regeneration of Cotton Cultivar Narashima (Gossypium hirsutum L.) (pp 58-62)

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Original Research Paper: Development of a plant regeneration protocol of an elite Indian cotton (Gossypium hirsutum L.) cultivar ‘Narashima’ could help in genetic transformation for biotic stress tolerance and improve quality characteristics. In the present study, successful callus, shoot and root induction were achieved from cotyledonary nodes, hypocotyls, cotyledons and leaf explants cultured on MS medium supplemented with B5 vitamins, as well as combinations of auxins and cytokinins. Callus proliferation was best on Murashige and Skoog (MS) medium with 0.5 mg/l 2,4 dichlorophenoxyacetic acid, 0.4 mg/l thidiazuron (TDZ), 0.5 mg/l 6-benzyl amino purine (BAP) and 1.0 mg/l kinetin (Kn). Shoots were produced from organogenic callus, cotyledonary nodes, hypocotyls cultured on MS medium with 1.0 mg/l BAP, 1.0 mg/l TDZ, 2.0 mg/l KN and 0.4 mg/l α-naphthalene acetic acid (NAA) and 1 g/l activated charcoal. Profuse rooting was achieved on MS medium with 1.0 mg/l NAA and 0.4 mg/l indole-3-acetic acid. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization (68-70%) and hardened plantlets were obtained.

Shabir H. Wani (India), Jaime A. Teixeira da Silva (Japan), G. S. Sanghera, A. Haribhushan, N. B. Singh, Satbir S. Gosal (India) Regeneration Protocol for Whole Plants from Embryogenic Callus of Commercial Rice (Oryza sativa L.) Variety PR 116 (pp 63-66)

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Original Research Paper: The selection of highly regenerable rice varieties is a pre-requisite for success in rice biotechnology. In this report we developed a reproducible and effective plant regeneration system through somatic embryogenesis for Indica rice var. PR 116. Embryogenic calli derived from mature seeds served as explants. Callus was induced from mature seeds cultured on MS medium containing 30 g/l sucrose supplemented with 560 mg/l proline, 1.5-3.5 mg/l 2,4- dichlorophenoxy acetic acid (2,4-D) and 0.5-1.5 mg/l kinetin (Kin). The regeneration efficiency was tested on Murashige and Skoog (MS) medium containing 30 g/l sucrose fortified with 1.0-3.0 mg/l 6-benzyl aminopurine (BAP), 0.5-1.5 mg/l Kin and 0.5 to 1.5 mg/l α-naphthalene acetic acid (NAA). Highest callus induction (44.4%) was on MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest percentage shoot regeneration (42.5%) was on MS medium supplemented 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible.

Leyla Eshghi, Majid Pouryousef, Behnam Kamkar (Iran) Quantification of Stem Elongation Rate in Response to Temperature and Photoperiod by 24 Multiplicative Models (pp 67-72)

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Original Research Paper: The first step to quantify crop phenology is to precisely estimate the parameters which affect it. These main parameters are temperature and photoperiod. Therefore we aimed to formulate and validate 24 mathematical functions that can be used to determine cardinal temperatures, critical photoperiod (below which development rate decreases due to short photoperiods) and the effect of temperature and photoperiod on biological days required from emergence to stem elongation for wheat (cv. ‘Koohdasht’). For this purpose, 24 multiplicative non-linear regression models (including flat, logistic, quadratic, cubic, dent-like, segmented, curvilinear and beta) for response to temperature, and quadratic, dent-like and negative exponential, to assess the response to photoperiod, were used. Also, the phenological data obtained from an independent experiment were used for independent model evaluation. A multiplicative model that included a quadratic function for response to both temperature and photoperiod was the most adequate to describe the response of stem elongation rate to temperature and photoperiod. Using this function, a base temperature of 7.62°C, a ceiling temperature of 37.60°C, a critical photoperiod of 14.006 h and a photoperiod sensitivity coefficient of 0.11 h^-1 were obtained. This function and its parameters can be used in wheat simulation models to predict the duration of emergence to stem elongation based on a thermal time concept. Also, the required number of biological days from seedling to stem elongation using this model was 26.90.

Jaime A. Teixeira da Silva (Japan), Judit Dobránszki (Hungary) The Plant Growth Correction Factor. I. The Hypothetical and Philosophical Basis (pp 73-74)

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Hypothesis Paper: There are two possible reasons why regeneration ability in plant tissue culture (PTC) differs from study to study. In life, not all beings are born equal. In PTC, too, not all explants have the same regeneration capacity. A plethora of factors influence the organogenic outcome of an explant in PTC, but differences in production, yield and organogenic output are all measured by one factor, and one factor alone: the size of the explant. In this ground-breaking paper, we put forward a radical notion that would attempt to allow for the direct comparison of organogenic potential of PTCs of the same cultivar or species conducted in different studies or laboratories. The prototype concept, the growth correction factor or GCF, has been tested on a model species, apple (Malus sp.).

Subhasish Mondal (India), Jaime A. Teixeira da Silva (Japan), Partha Deb Ghosh (India) In Vitro Flowering in Rauvolfia serpentina (L.) Benth. ex. Kurz. (pp 75-77)

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Short Communication: Rauvolfia serpentina Benth. is an economically important medicinal plant renowned for curing cardiovascular diseases and hypertension, its pharmacological activity being due to the presence of different alkaloids. Multiple shoot regeneration and flower induction in vitro have been achieved in this study using combinations of cytokinin and auxin. Flowering was induced for the first time ever in Murashige and Skoog medium supplemented with 2.22 µM benzyl adenine (BA) + 2.32 µM kinetin (Kin) + 0.54 µM α-naphthalene acetic acid and 2.22 µM BA + 4.65 µM Kin under a 12-hr photoperiod.

Ahmed M. Eed, Hameedunnisa Begum, Subramoniam Sivaramakrishnan (India), Jaime A. Teixeira da Silva (Japan), Solipuram Amrender-Reddy, Ali Q. Al-gabal (India) Rapid Protocol for in Vitro Multiplication of Citrus limonia Osbeck Rootstock (pp 78-82)

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Techniques Paper: Rangpur lime (Citrus limonia Osbeck) is the most used rootstock in the world and in India, particularly in the state of Andhra Pradesh on account of its superior performance over other rootstocks. However, plants produced from seedlings are not used in orchards due to variations caused by polyembryony. To overcome such variation, in vitro propagation of Rangpur lime was performed using nodal segments and shoot tips as explants. After surface sterilization, the explants were inoculated on Murashige and Skoog (MS) medium supplemented with 3% sucrose, 100 mg/L benomyl and 250 mg/L streptomycin. The most effective concentrations of auxin (α-naphthalene acetic acid; NAA), cytokinin (6-benzyl adenine; BA) and gibberellic acid (GA₃) for in vitro shoot induction and multiplication in Rangpur lime rootstock were determined. Almost all NAA and BA treatments resulted in almost 100% shoot induction except for at 0.0 and 0.1 mg/L NAA and at 1.5 and 2 mg/L BA. Nodal segments induced a higher percentage of explant response with longer shoots in a shorter period of time than shoot tips, which produced more shoots and leaves than nodal segments. The effect of different BA and NAA concentrations on various parameters of proliferation were studied. Full-strength MS medium produced more regenerated shoots and leaves per shoot than half-strength MS medium. In addition, longer shoots formed with 0.1 mg/L GA₃ than culture medium without this plant growth regulator.