S. M. A. Zobayed

RECENT ADVANCEMENT IN RESEARCH ON PHOTOAUTOTROPHIC MICROPROPAGATION USING LARGE CULTURE VESSELS WITH FORCED VENTILATION

SummarySuccessful fundamental or basic research, while being stimulated by applied studies, provides the development of new technologies for the benefit of mankind. Photoautotrophic micropropagation or micropropagation using sugar-free medium is no exception from this generalization. The concept of photoautotrophic micropropagation is derived from research that revealed the relatively high photosynthetic abilities of chlorophyllous cultures such as leafy explants and plantlets in vitro. To meet the ever-increasing demand for quality transplants, the scaling-up of photoautotrophic micropropagation systems, for commercialization, has become necessary. This article reviews the recent advancement in the development and utilization of large culture vessels for photoautotrophic micropropagation with special emphasis on the feasibility of the system for the commercial-scale propagation. The review also includes choices for supporting material, ventilation type, planting density, vessel volume, and vessel sterilization procedure, and problems and solutions to achieve uniform growth in a large culture vessel. A case study of the commercial application of a photoautotrophic micropropagation system using large culture vessles, which recently has been established in Kunming, China, is also presented in this article.

Supporting material affects the growth and development of in vitro sweet potato plantlets cultured photoautotrophically

SummaryA comparative study was conducted to optimize the vegetative growth of sweet potato (Ipomoea batatas L. (Lam), cv. Beniazuma) plantlets cultured in vitro in five different types of supporting materials: agar matrix (a seaweed derivative; Kanto Chemical Co. Inc., Tokyo), gellan gum (a Pseudomonas derivative; Kanto Chemical Co. Inc., Tokyo), vermiculite (a kind of hydrous silicates), a mixture of vermiculite and cellulose fiber (Florialite; Nisshinbo Industries, Inc., Tokyo) and cellulose plug (Sorbarod; Baumgartner Rapiers SA., Switzerland). Single nodal cuttings were cultured photoautotrophically (without any sugar in the medium and with enriched CO2 and high photosynthetic photon flux) for 21 d on MS basal medium. Plantlets exhibited the greatest growth when Florialite was used as supporting material. The leaf and root fresh and dry mass were 2.4× and 2.9×, and 2.2× and 2.8× greater, respectively, than those of the plantlets grown in the agar matrix (control). Plantlets cultured in Sorbarod supporting material exhibited the second greatest fresh and dry mass of leaves and roots followed by vermiculite and gellan gum supporting material. The most interesting feature was the development of a large number of fine lateral roots from the main adventitious root in the Florialite treatment. Among the treatments, the highest net photosynthetic rate was evident in the Florialite grown plantlets. The percent porosity of the supporting materials was highest in Sorbarod followed by Florialite and vermiculite. Plantlets transplanted from the Florialite supporting material exhibited the highest acclimatization percentage followed by that of the Sorbarod treatment.

Stomatal characteristics and leaf anatomy of potato plantlets cultured in vitro under photoautotrophic and photomixotrophic conditions

SummaryPotato plantlets (Solanum tuberosum L. cv. Benimaru) were cultured under photoautotrophic (without any sucrose in the nutrient medium and with enriched CO2 and high photosynthetic photon flux) and photomixotrophic conditions (20 g 1−1 sucrose in the medium). Leaf anatomy and stomatal characteristics of the leaves were studied in relation to stomatal size and density. Leaf diffusive resistance, transpiration rate, and wax content of the leaves were also investigated. In the photoautotrophic treatment, stomata behaved normally by closing in the dark and opening in the light. The stomatal density increased twofold compared to that of the photomixotrophic treatment. Relatively thick leaves and an organized palisade layer were observed and the epicuticulal wax content was remarkably higher in this treatment, i.e., seven times greater than that of photomixotrophic treatment. In general, higher diffusive resistance of the leaves was observed than under photomixotrophic conditions; also the resistance increased in darkness and decreased in the light. All these characteristics led the plantlets to have a normal and controlled transpiration rate, which was exceptionally high in the photomixotrophic treatment throughout the light and the dark period.

DEVELOPMENT OF A FORCED VENTILATION MICROPROPAGATION SYSTEM FOR LARGE-SCALE PHOTOAUTOTROPHIC CULTURE AND ITS UTILIZATION IN SWEET POTATO

SummaryA forced ventilation system has been developed for large-scale photoautotrophic micropropagation of chlorophyllous plants. The major goal of the system is to provide a uniform supply of CO2 inside a large culture vessel (volume 3480 ml) to achieve uniform growth of the plantlets. The system has been designed such that sterile nutrient solution can be supplied throughout the culture period, which is essential for long-term culture. Sweet potato (Ipomoea batatas L. Lam., cv. ‘Beniazuma’) was used as a model plant for photoautotrophic culture with stagnant and nonstagnant nutrient solution in large vessels. Growth and net photosynthetic rates of the plantlets were compared with those of the plantlets grown in a small vessel under photoautotrophic conditions (with natural ventilation) and conventional photomixotrophic conditions. The results indicated that the large vessel with the forced ventilation system was effective for improving growth and uniformity of the plantlets and the rate of net photosynthesis. The stagnant nutrient solution condition under photoautotrophic forced ventilation treatment significantly increased the fresh mass of the plantlets; however, percent dry mass was highest in the treatment with nonstagnant nutrient solution condition. The results demonstrated that the conventional photomixotrophic culture system can cause seriously inhibited growth and development.

A combination of vermiculite and paper pulp supporting material for the photoautotrophic micropropagation of sweet potato

A mixture of vermiculite (hydrous silicates) and paper pulp (waste product of paper industry) was used as a supporting material for the in vitro photoautotrophic micropropagation of plantlets. Sweet potato was used as a model plant to find out the appropriate proportion of vermiculite and paper pulp for the optimum growth of the plantlets. The plantlets grown in the conventional supporting material, agar, were used as the control. The study revealed that in all aspects, the plantlets grown in vermiculite mixed with 30% (w/w) paper pulp exhibited the highest growth performance. The shoot and root fresh mass were x2.7 greater than those in agar (control); the leaf, stem and root dry mass were also greater and at least two fold in this treatment compared with those in the control. The net photosynthetic rate per plantlet was highest in this treatment, and on day 20 it was 15.3 µmol CO(2) h(-1) as compared with 9.8 µmol CO(2) h(-1) in the control. The growth of both shoots and roots decreased gradually with the increase or decrease of percentage of paper pulp in the supporting material. In general, the growth was significantly poorer in the plantlets grown in 100% vermiculite than that in vermiculite mixed with 30% paper pulp but still greater than in the control. The porosity of the supporting materials increased with the increase in the percentage of paper pulp in the supporting material. After transplanting to the ex vitro condition the survival percentage did not vary significantly (90-100%) among the treatments, except in control where it was only 73%. The number of unfolded leaves and the stem height were similar among the treatments except those in the control.

Physiology of Eucalyptus plantlets grown photoautotrophically in a scaled-up vessel

SummaryNodal cuttings of Eucalyptus camaldulensis L. plantlets were cultured photoautotrophically (sugar-free nutrient medium and with enriched CO2 and high photosynthetic photon flux) in a scaled-up vessel (volume 4.0 liters) under forced ventilation (SV-treatment). After 28 d of culture, physiological aspects of the plantlets were compared with plantlets grown photomixotrophically (20 g l−1 sucrose in the medium) in a Magenta vessel (volume 0.4 liters) under natural ventilation (control). In the SV-treatment net photosynthetic rates were enhanced, normal stomatal closing and opening were observed, and the epicuticular leaf-wax content was significantly higher than the control. The anatomical study showed well-organized palisade and spongy mesophyll layers of SV leaves. The SV-treatment also allowed in vitro acclimatization, and after transplanting ex vitro, the transpiration rate and the percent water loss was lower than those of the control and thus the SV plantlets acclimatized easily ex vitro.

ENHANCED GROWTH AND QUALITY OF ST. JOHN'S WORT (HYPERICUM PERFORATUM L.) UNDER PHOTOAUTOTROPHIC IN VITRO CONDITIONS

SummaryPhotomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many rescarchers for transplant production of St. Johns wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21d of culture, Pa conditions enhanced the growth and quality of St. Johns wort plantlets in vitro, and these plantlets showed faster growth after transplantaing ex vitro compared with those cultured under Pm conditions.

Evolution of Culture Vessel for Micropropagation: From Test Tube To Culture Room

To improve the culture conditions for micropropagation, different types of culture vessels and capping systems have been designed. Some of these designs improve the aerial composition in the culture vessel and some for recycling the nutrient medium. This article describes the evolution of different culture vessel and culture systems, with special emphasis on forced ventilation to improve the culture atmosphere and thus to improve the growth and multiplication and also the quality of propagules. By altering the aerial environment of the culture vessel, plantlets can be grown photoautotrophically (sugar free medium) which has many advantages over the photomixotrophic or heterotrophic system. By using forced ventilation and a photoautotrophic culture system, the scaling-up of the culture vessel is possible with high growth rate and survival percentage and with minimum time and space. More recently, this scale-up system has been further extended making the aseptic culture room itself a large culture vessel containing many small sterile trays with plants on the culture shelves and with a common headspace. By using this enlarged system, the production of even more quality transplants was achieved relatively easily.

NaCl ENHANCES GROWTH AND MORPHOGENESIS POTENTIAL OF ALHAGI GRAECORUM

SummaryThe contamination of soils with excess salts is one of the greatest challenges to plant survival, but some unique species have evolved to thrive in highly saline environments. One such species, Alhagi graecorum Boiss., has been previously shown to accumulate high levels of sodium while growing in salt marshes. The aim of this research was to investigate the effects of saline conditions on the growth and the regeneration capacity of this species. Plantlets and explants of A. graecorum were cultured on a medium supplemented with various concentrations of NaCl, where A. graecorum tissues accumulated up to 8% Na+. The capacity for regeneration was enhanced by the excess sodium, indicating a requirement of salt for optimal growth and development in this species. Further study of this species may provide new concepts and understanding of the metabolism of sodium in higher plants.

Physiology of in Vitro Plantlets Grown Photoauto-Trophically

Earlier efforts to improve the growth and multiplication of in vitro grown plantlets have focused mainly on the composition of the nutrient medium and the use of growth regulators. The physiological characteristics of micropropagated plantlets cultured under conventional photomixotrophic/heterotrophic conditions in airtight vessels are often abnormal. Low rates of photosynthesis and transpiration, poor water and mineral uptake, non-functional stornata, lack of organisation of palisade and mesophyll tissues in the leaves and poor root quality are the major short-comings of plantlets grown under conventional micropropagation conditions. Recent research has revealed that the aerial environment of the culture vessel can significantly affects the growth and quality of the plantlets, the duration of culture and the cost of production. The use of a more suitable supporting material than conventional agar can also improve the root growth and quality as well as shoot growth. Most recently, the use of a photoautotrophic culture system (sugar free medium) with forced ventilation and with fibrous rooting substrates was proved to be the best for improving the growth and quality of the plantlets and for reducing production costs. This article discusses various ways of improving the physiological conditions and quality of micropropagated plantlets with special emphasis on the culture atmosphere and the rooting substrate.