Toyoki Kozai

Automation and environmental control in plant tissue culture

Preface. Glossary. 1. Automation in plant tissue culture -- general introduction and overview J. Aitken-Christie, T. Kozai, S. Takayama. 2. Economic analysis of automated micropropagation I. Chu. 3. Economic aspects of somatic embryogenesis R. Cervelli, T. Senaratna. 4. Systems analysis and engineering P.N. Walker. 5. Engineering aspects of plant propagation in bioreactors P.H. Heyderdahl, O.A.S. Olsen, A.K. Hvoslef-Eide. 6. Mechanical engineering approaches to plant biotechnology Y. Miwa, Y. Kushihashi, T. Kozai. 7. Image analysis for plant cell culture and micropropagation M.A.L. Smith. 8. Image analysis for embryogenesis K. Kurata. 9. Automation of the bioreactor process for mass propagation and secondary metabolism R.R. Leathers, M.A.L. Smith, J. Aitken-Christie. 10. Delivery systems for tissue culture by encapsulation Y. Sakamoto, N. Onishi, T. Hirosawa. 11. A delivery system for naked somatic embryos of interior spruce D.R. Roberts, F.B. Webster, D.R. Cyr, T.K. Edmonds, S.M.A. Grimes, B.C.S. Sutton. 12. Automated systems for organogenesis K. Kurata. 13. Commercialisation of tissue culture and automated systems K.S. Wilson. 14. Environmental control in plant tissue culture -- General introduction and overview T. Kozai, M.A.L. Smith. 15. Physical microenvironment and its effects K. Fujiwara, T. Kozai. 16. Vessels, gels, liquid media, and support systems M.A.L. Smith, L.A. Spomer. 17. The chemical microenvironment R.R. Williams. 18. Carbon nutrition in vitro: Regulation and manipulation of carbon assimilation in micropropagated systems Y. Desjardins, C. Hdider, J. De Riek. 19. Ethylene D. Matthys, J. Gielis, P. Debergh. 20. In vitro acclimatization M. Ziv. 21. Low temperature storage of plant tissue cultures B.W.W. Grout. 22. Environmental measurement and control systems T. Kozai, Y. Kitaya, K. Fujiwara, M.A.L. Smith, J. Aitken-Christie. Index.

Micropropagation under photoautotrophic conditions

Micropropagation has many advantages over conventional vegetative propagation and its commercial use in horticulture, agriculture and forestry is currently expanding worldwide. However, its widespread commercial use for major crops is still restricted as a result of its relatively high production costs. The high production costs in conventional micropropagation are mainly due to high labor costs, limited rates of growth during multiplication, poor rooting, and low survival rates of the plantlets during acclimatization.

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

Abstract:  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 (280–315 nm) 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 3 months 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 ≫ blue ≥ white light. Interestingly, in a more mature plant (6 months) 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 3 days followed by low intensity UV‐B radiation for 15 days. 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.

Environmental control for the large-scale production of plants through in vitro techniques

Leafy or chlorophyllous explants of a number of plant species currently micropropagated have been found to have high photosynthetic ability. Their growth and development have been promoted on sugar-free medium rather than on sugar-containing medium, provided that the environmental factors, such as CO2 concentration, light intensity and relative humidity, are controlled for promoting photosynthesis and transpiration of explants/shoots/plantlets in vitro. Thus, environmental control is essential for promoting photosynthetic growth and development of in vitro plantlets.Several types of sugar-free (photoautotrophic) culture systems for large-scale micropropagation of plants have been developed. Advantages of sugar-free over conventional (heterotrophic or photomixotrophic) micropropagation systems are as follows: growth and development of plantlets in vitro are faster and more uniform, plantlets in vitro have less physiological and morphological disorders, biological contamination in vitro is less, plantlets have a higher percentage of survival during acclimatization ex vitro, and larger culture vessels could be used because of less biological contamination. Hence, production costs could be reduced and plant quality could be improved significantly with photoautotrophic micropropagation. Methods for the measurement and control of in vitro environments and the beneficial effects of environmental control on photosynthetic growth, development, and morphogenesis in large-scale production of micropropagated plantlets are presented.

The In Vitro Environment and its Control in Micropropagation

The reasons for high production costs of micropropagated plantlets are analyzed. Problems requiring solutions by proper control of the in vitro environment for improving shoot/plantlet quality and cost effectiveness are revealed. Features of the in vitro environment and responses of shoots/plantlets cultured in vitro to the environment in conventional micropropagation are discussed in detail. The features and responses are interpreted from an environmental control point of view. Similarity of growth patterns between plantlets and seedlings under different in vitro environmental conditions is shown. Strategies for environmental control in micropropagation are discussed. The environmental effect on growth and development of shoots/plantlets cultured in vitro under controlled environments is shown. Advantages and disadvantages of heterotrophic, photomixotrophic and photoautotrophic micropropagation are discussed. Some novel environmental control systems are introduced.

Physical microenvironment and its effects

Growth and development of plant tissue cultures are intimately coupled to the microenvironment in the culture vessels. The microenvironment, in turn, is largely dependent on mass and energy exchange processes. Maximum potentials for growth and developmental rates of cultures are determined by their genes, however their actual rates are limited by their surrounding microenvironment. Systematic comprehension and control of the microenvironment is, therefore, required to realize the full genetic potential of cultures, and to establish a procedure which will make cultures exhibit their hereditary characteristics in a highly efficient and stable way to meet our purposes.

Automation in plant tissue culture — general introduction and overview —

Plant tissue culture is now a proven technology for the in vitro production of large numbers of genetically identical plants. Two distinctly different biological methods are traditionally employed involving: 1) organogenesis, and 2) embryogenesis, and the choice of either method depends on the species, the success rate of the method for producing plants at a realistic cost, and local laboratory conditions. Tissue culture is frequently more expensive than other forms of propagation using cuttings or seed, because it is more labour intensive and requires more specialised environmental control throughout the numerous stages of development. This has been a major constraint to the larger scale deployment of tissue culture. However, there are many commercial companies exploiting the technology and most of the operations are still being carried out manually.

Development and application of photoautotrophic micropropagation plant system

Research has revealed that most chlorophyllous explants/plants in vitro have the ability to grow photoautotrophically (without sugar in the culture medium), and that the low or negative net photosynthetic rate of plants in vitro is not due to poor photosynthetic ability, but to the low CO2 concentration in the air-tight culture vessel during the photoperiod. Moreover, numerous studies have been conducted on improving the in vitro environment and investigating its effects on growth and development of cultures/plantlets on nearly 50 species since the concept of photoautotrophic micropropagation was developed more than two decades ago. These studies indicate that the photoautotrophic growth in vitro of many plant species can be significantly promoted by increasing the CO2 concentration and light intensity in the vessel, by decreasing the relative humidity in the vessel, and by using a fibrous or porous supporting material with high air porosity instead of gelling agents such as agar. This paper reviews the development and characteristics of photoautotrophic micropropagation systems and the effects of environmental conditions on the growth and development of the plantlets. The commercial applications and the perspective of photoautotrophic micropropagation systems are discussed.

Photoautotrophic and photomixotrophic growth of strawberry plantlets in vitro and changes in nutrient composition of the medium

Explants excised from strawberry (Fragaria x ananassa Duch.) plantlets were cultured in vitro for 21 days on half-strength MS (Murashige & Skoog 1962) basal liquid medium with 20 g l-1 sucrose and without sugar in the vessels capped with gas permeable microporous polypropylene film. The experiments were conducted under CO2 nonenriched (350–450 μmol mol-1 in the culture room) and CO2 enriched (2,000 μmol mol-1 during the photoperiod in the culture room) conditions with a PPF (photosynthetic photon flux) of 200 μmol m-2 s-1. The CO2 concentration in the vessels decreased to approximately 200 μmol mol-1 during the photoperiod on day 21 under CO2 nonenriched conditions. The fresh and dry weight, net photosynthetic rate (NPR) per plantlet, NPR per g leaf fresh weight, NPR per g leaf dry weight, the number of unfolded leaves, and ion uptake of PO43-, NO3-, Ca2+, Mg2+ and K+ on day 21 were the greatest under photoautotrophic (no sugar in the medium) and CO2 enriched conditions. The residual percent of PO43- was 3% on day 21 under photoautotrophic and CO2 enriched conditions.

Effects of CO2 enrichment and supporting materialin vitro on photoautotrophic growth ofEucalyptus plantletsin vitro andex vitro

SummaryEucalyptus camaldulensis shoots were cultured photoautotrophicallyin vitro for 6 wk with four different types of supporting materials (agar matrix, Gelrite matrix, plastic net, or vermiculite) under CO2-nonenriched or CO2-enriched conditions. Plantlets from each treatmentin vitro were then grownex vitro in a greenhouse for 4 wk. The growth and net photosynthetic rate of plantletsin vitro, as well as subsequent growth, survival percentage, transpiration rate, and net photosynthetic rate of plantletsex vitro were evaluated. CO2 enrichment significantly increased growth (total dry weight and number of primary roots) and net photosynthetic rate of plantletsin vitro, as well as the growth and survival percentage of plantletsex vitro regardless of the type of supporting materials. The growthin vitro was greatest in the vermiculite, followed by the plastic net, Gelrite matrix, and agar matrix (in descending order) under either the CO2-nonenriched or CO2-enriched conditions. The growth and survival percentage of plantletsex vitro were highest in the vermicultie under the CO2-enriched condition. The extensive root system producedin vitro was necessary for growth and survival of plantletsex vitro.