S.M.A. Zobayed

Melatonin in Glycyrrhiza uralensis : response of plant roots to spectral quality of light and UV-B radiation

Abstract:u2002 Melatonin (N‐acetyl‐5‐methoxytryptamine) is known to be synthesized and secreted by the pineal gland in vertebrates. Evidence for the occurrence of melatonin in the roots of Glycyrrhiza uralensis plants and the response of this plant to the spectral quality of light including red, blue and white light (control) and UV‐B radiation (280–315u2003nm) for the synthesis of melatonin were investigated. Melatonin was extracted and quantified in seed, root, leaf and stem tissues and results revealed that the root tissues contained the highest concentration of melatonin; melatonin concentrations also increased with plant development. After 3u2003months of growth under red, blue and white fluorescent lamps, the melatonin concentrations were highest in red light exposed plants and varied depending on the wavelength of light spectrum in the following order red ≫ blueu2003≥u2003white light. Interestingly, in a more mature plant (6u2003months) melatonin concentration was increased considerably; the increments in concentration were X4, X5 and X3 in 6‐month‐old red, blue and white light exposed (control) plants, respectively. The difference in melatonin concentrations between blue and white light exposed (control) plants was not significant. The concentration of melatonin quantified in the root tissues was highest in the plants exposed to high intensity UV‐B radiation for 3u2003days followed by low intensity UV‐B radiation for 15u2003days. The reduction of melatonin under longer periods of UV‐B exposure indicates that melatonin synthesis may be related to the integrated (intensity and duration) value of UV‐B irradiation. Melatonin in G. uralensis plant is presumably for protection against oxidative damage caused as a response to UV irradiation.

Photoautotrophic (sugar-free medium) Micropropagation as a New Micropropagation and Transplant Production System

Preface Contributors Acknowledgements 1. Introduction T. Kozai 2. Units and terminology use for the studies of photoautotrophic micropropagation T. Kozai, C. Kubota 3. Concepts, definitions, ventilation methods, advantages and disadvantages T. Kozai, C. Kubota 4. In vitro aerial environments and their effects on growth and development of plants T. Kozai, C. Kubota 5. In vitro root zone environments and their effects on growth and development of plants T. Kozai, C. Kubota 6. Physiological and anatomical characteristics of in vitro photoautotrophic plants F. Afreen 7. Photoautotrophic plant conversion in the process of somatic embryogenesis F. Afreen, S.M.A. Zobayed 8. Photoautotrophic micropropagation of woody species Q.T. Nguyen, T. Kozai 9. Ventilation in micropropagation S.M.A. Zobayed 10. A commercialized photoautotrophic micropropagation system using large vessels with forced ventilation Y. Xiao, T. Kozai 11. Low temperature storage of plants under dim light C. Kubota 12. Modelling and simulation for production planning in photoautotrophic micropropagation C. Kubota 13. Modelling and simulation in photoautotrophic micropropagation G. Niu 14. Frequently asked questions C. Kubota, T. Kozai 15. Plant species successfully micropropagated photoautotrophically C. Kubota, F. Afreen, S.M.A. Zobayed 16. Reconsideration of conventional micropropagation systems T. Kozai 17. Closed systems with lamps for high quality transplant production at low costs using minimum resources T. Kozai 18. Concluding remarks S.M.A. Zobayed, F. Afreen Subject Index

Leaf anatomy of in vitro tobacco and cauliflower plantlets as affected by different types of ventilation

Abstract Tobacco and cauliflower plantlets were grown in vitro under forced ventilation (fast flow; 10 cm 3 xa0min −1 and slow flow, 5 cm 3 xa0min −1 ), diffusive ventilation and in airtight vessels. Culture vessel headspace atmosphere (CO 2 and ethylene concentrations, relative humidity), leaf-stomatal structure, density and function, internal leaf anatomy, epicuticular waxes of leaves have been studied and compared with those grown in vivo (growth room conditions). No ethylene accumulation was noticed in the headspace of the culture vessel under forced ventilation (slow or fast); however, ethylene accumulation was noticed in airtight vessels. During the photoperiod, CO 2 depletion occurred in the headspace of the airtight vessels, the CO 2 concentration increased with increasing the efficiency of ventilation. Relative humidity was nearly 100% in the airtight vessel at the end of the experiment; the value was 92% in the fast forced ventilation. The stomatal density in both the species was significantly higher in plantlets subjected to the airtight vessel and diffusive treatments, and densities decreased with increasing efficiency of ventilation. In general stomatal sizes appeared to be inversely related to efficiencies of ventilation; comparatively large in the leaves grown in airtight and diffusive treatments. Relatively larger areas were occupied by stomata in both the airtight condition and diffusive ventilation in both the species and was significantly lower in in vivo grown plantlets and in forced ventilation treatment. Permanently widely-open stomata which do not respond normally by closing in the dark or under conditions of low relative humidity, and which are in abnormally exposed positions on the leaf were noticed in the airtight and the diffusive ventilation; stomata were functional in the forced ventilation and in vivo treatments.

Cauliflower shoot-culture : Effects of different types of ventilation on growth and physiology

Abstract Hypocotyl cuttings of in vitro grown cauliflower ( Brassica oleracea var. botrytis L.) were cultured in 60 cm 3 glass vessels containing MS medium supplemented with BAP (1.0 mg l −1 ) and NAA (0.5 mg l −1 ) which led to callus induction and new shoot proliferation. The effects of various methods of ventilations (sealed, diffusive and humidity-induced convective throughflow ventilation) on growth and physiology of the regenerated shoots and callus were investigated and described. The humidity-induced convective throughflow ventilation (HICT-ventilation) system proved to be the most effective to procure best growth and photosynthesis of the regenerated shoots. On the contrary the sealed vessels showed very poor growth with little leaf and shoot number, fresh weight and callus volume. The diffusive treatment is being intermediate between the sealed and the HICT-ventilation treatment. Throughout the 30 days experiment, the ethylene, CO 2 and oxygen concentrations in the culture atmosphere were also monitored. High ethylene accumulation was observed in sealed (1.75 μl l −1 ) and diffusive system (0.55 μl l −1 ), which could not accumulate in the culture vessels grown under HICT-ventilation system. Thus the inhibition of growth observed in the sealed and diffusive system is thought to be associated with the accumulated ethylene. Very high CO 2 concentration (5%) was observed in sealed system despite the continuous illumination; maximum CO 2 output originated from the callus. In the HICT-ventilation system it was below atmospheric. Oxygen concentrations in the sealed and the diffusely vented vessels fell (7.1 and 15.1%, respectively) as CO 2 level rose, which remained fairly constant at levels a little below atmospheric in HICT-ventilation.

Evaluation of a closed system, diffusive and humidity-induced convective throughflow ventilation on the growth and physiology of cauliflower in vitro

The effects of ethylene inhibitors (silver nitrate – AgNO3 and silver thiosulphate – Ag2S2O3 as inhibitors of ethylene activity, cobalt chloride – CoCl2 as inhibitor of ethylene biosynthesis) and ethylene stimulator (aminocyclopropane-1-carboxylic acid – ACC) were studied on the growth of cauliflower (Brassica oleracea L.) seedlings cultured in closed vessels (60 cm3). The addition of ethylene inhibitors have significant stimulatory effects on the growth and development of seedlings and the effects were greatest with 10 μM AgNO3, the fresh weight of leaves was 2.6×, and the leaf area 2.8× those of the control (no additives). The effects of various methods of ventilation (humidity-induced convective through-flow ventilation, diffusive ventilation and sealed condition) on the growth and physiology of in vitrocauliflower seedlings were also investigated. The seedlings were cultured either in the presence or absence of AgNO3 (inhibitors of ethylene activity) and ACC (a precursor). Ethylene and CO2 levels in the head-space of the culture vessels were monitored. The humidity-induced through-flow ventilation system has shown to be effective for improving growth, leaf chlorophyll content and the rate of net photosynthesis and preventing symptoms of hyperhydricity, such as leaf epinasty, and franginess, reduction of leaf area etc. In contrast, the results also indicated that the sealing of culture vessels could have serious inhibitory effects on growth and development, induce hyperhydricity and reduce leaf chlorophyll content.In the light period, CO2 depletion occurred in the head-space of the sealed vessels (ca. 40 μl l-1), the CO2 concentration increased with increasing efficiency of the ventilation. No ethylene accumulation was noticed in the head-space of the culture vessels when humidity-induced throughflow ventilation was applied; however, high ethylene accumulation occurred in sealed vessels.

Multiple shoot induction and leaf and flower bud abscission of Annonacultures as affected by types of ventilation

Nodal explants of Annona squamosa L. and Annona muricata L. were cultured in vitro under various types of ventilation: airtight vessel (sealed condition; number of air exchange 0.1 h−1), natural ventilation (via a polypropylene membrane; number of air exchange 1.5 h−1), and forced ventilation (5.0 cm3 min−1 in a 60 cm3 vessel; number of air exchange 5.0 h−1). In both species, numbers of leaves, leaf areas and numbers of nodes per shoot increased with improving standards of ventilation, while leaf abscissions were substantially reduced; all the leaves had abscised in the airtight vessels after 12–15 days, but none had done so with forced ventilation. Flower-bud abscission in A. muricatashowed a similar trend after 21 days. These effects were associated with reductions in the accumulation of ethylene within the culture vessels, produced by increasing the efficiency of ventilation; ethylene was not detected in those fitted with a forced ventilation system. CO2 concentrations in culture headspaces and the net photosynthetic rates of the plantlets were also evaluated. CO2 concentrations decreased well below the ambient in the natural and airtight vessels; however, under forced ventilation, CO2 concentrations were significantly higher during the photoperiod, compared to those of the natural ventilation and airtight vessel treatments. In general, net photosynthetic rates per unit leaf area increased with increasing photosynthetic photon flux (PPF) and rates were highest in plantlets grown under forced ventilation, intermediate under natural ventilation and lowest in the airtight vessels.Eighteen different media were investigated for their effects on multiple shoot induction in both species. The best medium for multiple shoot induction and growth in A. squamosa was Murashige and Skoog medium (MS) + 6-benzylaminopurine (BA; 1.5 mg l−1) + casein hydrolysate (1.0 g l−1) and for A. muricata MS + BA (1.0 mg l−1) + naphthaleneacetic acid (NAA; 0.1 mg l−1).

Mass-Propagation of Coffee from Photoautotrophic Somatic Embryos

ABSTRACT The somatic embryos of Coffea arabusta were cultured and different stage embryos (torpedo, precotyledonary, cotyledonary and germinated) were selected. When these embryos were grown photoautotrophically (in sugar-free medium with CO 2 enrichment in the culture headspace and high photosynthetic photon flux) for 60 days the dry mass of each of the cotyledonary and germinated embryos increased by up to 10% and 50%, respectively. In contrast, the torpedo and precotyledonary stage embryos lost at least 20-25% of their initial dry mass. Thus we have suggested that cotyledonary stage embryos can be considered as the earliest stage, which can be cultured photoautotrophically in order to develop plantlets. In this article we have also discussed about a specially designed large culture vessel with forced ventilation system for the mass-propagation of coffee plantlets. The cotyledonary stage embryos were selected and grown in this specially designed large vessel having temporarily root zone immersing system with the view of large-scale embryo-to-plantlet development under photoautotrophic condition. The growth and survival of the plantlets after 45 days of culture were studied and compared with those grown photoautotrophically in a RITA vessel (Vitr Pic, CIRAD) and in a Magenta-type vessel. Results revealed that fresh mass and dry mass of leaves and roots of plantlets developed in the specially designed large vessel were significantly enhanced compared with those of plantlets developed in a RITA vessel. The poorest growth of the plantlets was observed when grown in a Magenta-type vessel. The survival of the plantlets after transplanting ex vitro followed a similar pattern and was highest in the large vessel followed by that in RITA vessel and in Magenta-type vessel.

Large-Scale Photoautotrophic Micropropagation in a Scaled-Up Vessel

ABSTRACT A scaled-up culture vessel for the large-scale micropropagation has been designed and the method for the large-scale micropropagation of Eucalyptus under photoautotrophic condition (sugar-free medium) using this scaled-up culture vessel with forced ventilation (SV-treatment) is described. Growth, multiplication, epicuticular wax content, stomatal function of plantlets and transpiration rate and ex vitro survival of the plants grown under SV-treatment were studied and compared with those of the plantlets grown in a Magenta-type vessel with natural ventilation and under photomixotrophic condition (30xa0g l −1 sucrose in the medium). Results revealed that fresh and dry mass in plantlets grown under SV-treatment were significantly higher than those of plantlets from Magenta type vessel. Multiplication was enhanced and normal stomatal closing and opening were observed in the leaves of plantlets grown in scaled-up vessel. Epicuticular leaf-wax content was significantly higher compared with that of the leaves grown in Magenta type vessel. The anatomical study showed well-organized palisade and spongy mesophyll layers for leaves grown in SV-treatment. Importantly after transplanting ex vitro, plantlets survived well without any specialized ex vitro acclimatization treatment. This was because they were better able to control transpiration and thus lost less water and showed no signs of wilting. In contrast, plantlets cultured in Magenta type vessel had open stomata which were not functional, showed a higher transpiration rate and uncontrolled and rapid water loss immediately after transferring ex vitro. Minimization of large-scale plantlet production cost by using scaled-up vessel is also discussed in this article.

Microbial Contamination under Photoautotrophic Culture System

Microbial contamination is a serious and an unavoidable problem in the field of plant tissue culture. The use of antibiotic, commercial fungicide, thermotherapy, warm-water treatment and ultrasonic treatment are some of the commonly used method to control in vitro contamination. Almost all of these methods are expensive, with a low success rate and thus increased labor cost. We have found that the use of sugar free nutrient medium (photoautotrophic culture system) has significantly reduced the contamination in the multiplication and rooting stages of banana. In the conventional system (medium containing sugar and vitamins), atleast 34% of the vessels were contaminated (which is common in tissue culture of banana in our laboratory); 10% with fungal and 24% with bacterial contamination. However, by using the photoautotrophic culture system only 12% vessels were contaminated; 9% with fungal and only 3% with bacterial contamination. In this paper, we have discussed some common types of contamination in the sugar-containing medium and the possible use of photoautotrophic culture system for reducing the contamination rate in plant micropropagation of banana.

Mass propagation of coffee transplants under scaled-up photoautotrophic micropropagation system

Somatic embryos were obtained from leaf discs of coffee plants cultured in vitro. The main objective of our study was to develop a scaled-up micropropagation system under photoautotrophic conditions (PA) using Coffea arabusta somatic embryos as a model plant. At different developmental stages of somatic embryos (torpedo, precotyledonary, cotyledonary and germinated), the physiological variables in relation to the photosynthetic ability were investigated. Results revealed that cotyledonary and germinated stage embryos were physiologically capable to grow photoautotrophically. However, high photosynthetic photon flux (PPF: 100 μmol m 2 s -1 ) treatment for 14 days increased the photosynthetic efficiency of the somatic embryos and possibly make them more suitable to grow under PA conditions. To confirm these findings the somatic embryos were cultured under PA conditions (sugar-free medium with CO 2 enrichment in the culture vessel environment and high PPF). From the results it was concluded that cotyledonary stage is the earliest stage somatic embryo, which can be successfully grown photoautotrophically for conversion into plantlets. To scale up the plantlet conversion process of coffee somatic embryos under PA conditions a bioreactor was especially designed. The growth and conversion percentage of cotyledonary stage embryos cultured in the newly developed bioreactor were compared with those of commercially available systems such as RITA temporary immersion bioreactor and Magenta box. Results revealed that the growth, percent conversion and ex vitro survival of plants were highest in the newly developed bioreactor.