Medicinal plants in the Mediterranean area: Synthesis of the results of the project Rubia

Almost half a century has passed sincein vitro tubers (microtubers) were first described in potato, but their adoption as a seed propagule has been uneven globally. Consensus is lacking regarding optimal production practices for microtubers and their relative productivity in relation to other propagules for minituber production. There is significant uncertainty regarding the utility of microtubers for evaluation of agronomic characters. However, the application of microtubers in germplasm conservation is widely accepted. Microtubers are producedin vitro in a plethora of different growing systems with varying environment, media constituents, and storage intervals. Many of the interactions between growth parametersin vitro and subsequent productivity appear to be genotype-specific. Accordingly, microtubers come in different sizes, have different dormancy requirements, and differ widely in relative growth potential and productivity. Despite these differences, there is evidence for strong analogies in growth responses between fieldgrown tubers and microtubers. The use of microtuber technology in seed tuber production, breeding programs, germplasm conservation, and research appears to have enormous potential. This review discusses microtuber production, yield and performance,in vitro screening, and germplasm storage and exchange.

Potato microtuber production and performance: A review

Bioreactors provide a rapid and efficient plant propagation system for many agricultural and forestry species, utilizing liquid media to avoid intensive manual handling. Large-scale liquid cultures have been used for micropropagation through organogenesis or somatic embryogenesis pathways. Various types of bioreactors with gas-sparged mixing are suitable for the production of clusters of buds, meristems or protocorms. A simple glass bubble-column bioreactor for the proliferation of ornamental and vegetable crop species resulted in biomass increase of 3 to 6-fold in 3–4 weeks. An internal loop bioreactor was used for asparagus, celery and cucumber embryogenic cultures. However, as the biomass increased, the mixing and circulation were not optimal and growth was reduced. A disposable pre-sterilized plastic bioreactor (2–5-l volume) was used for the proliferation of meristematic clusters of several ornamental, vegetable and woody plant species. The plastic bioreactor induced minimal shearing and foaming, resulting in an increase in biomass as compared to the glass bubble-column bioreactor. A major issue related to the use of liquid media in bioreactors is hyperhydricity, that is, morphogenic malformation. Liquid cultures impose stress signals that are expressed in developmental aberrations. Submerged tissues exhibit oxidative stress, with elevated concentrations of reactive oxygen species associated with a change in antioxidant enzyme activity. These changes affect the anatomy and physiology of the plants and their survival. Malformation was controlled by adding growth retardants to decrease rapid proliferation. Growth retardants ancymidol or paclobutrazol reduced water uptake during cell proliferation, decreased vacuolation and intercellular spaces, shortened the stems and inhibited leaf expansion, inducing the formation of clusters. Using a two-stage bioreactor process, the medium was changed in the second stage to a medium lacking growth retardants to induce development of the meristematic clusters into buds or somatic embryos. Cluster biomass increased 10–15-fold during a period of 25–30 days depending on the species. Potato bud clusters cultured in 1.5 1 of medium in a 2-l capacity bioreactor, increased during 10–30 days. Poplar in vitro roots regenerated buds in the presence of thidiazuron (TDZ); the biomass increased 12-fold in 30 days. Bioreactor-regenerated clusters were separated with a manual cutter, producing small propagule units that formed shoots and initiated roots. Clusters of buds or meristematic nodules with reduced shoots, as well as arrested leaf growth, had less distortion and were optimal for automated cutting and dispensing. In tuber-, bulb- and corm-producing plants, growth retardants and elevated sucrose concentrations in the media were found to enhance storage organ formation, providing a better propagule for transplanting or storage. Bioreactor-cultures have several advantages compared with agar-based cultures, with a better control of the contact of the plant tissue with the culture medium, and optimal nutrient and growth regulator supply, as well as aeration and medium circulation, the filtration of the medium and the scaling-up of the cultures. Micropropagation in bioreactors for optimal plant production will depend on a better understanding of plant responses to signals from the microenvironment and on specific culture manipulation to control the morphogenesis of plants in liquid cultures.

Simple bioreactors for mass propagation of plants

Part 1: Machine Vision Evaluation of photosynthetic capacity in micropropagated plants by image analysis/Y. Ibaraki Monitoring gene expression in plant tissues/J.J. Finer, S.L. Beck, M.T. Buenrostro-Nava, Y. Chi, P.P. Ling Applications and potentials of artificial neural networks in plant tissue culture/V.S.S. Prasad, S. Dutta Gupta Evaluation of plant suspension cultures by texture analysis/Y. Ibaraki Part 2: Bioreactor Technology Bioengineering aspects of bioreactor application in plant propagation/S. Takayama, M. Akita Agitated, thin-films of liquid media for efficient micropropagation/ J. Adelberg Design, development and applications of mist bioreactors for micropropagation and hairy root culture/M.J. Towler, Y. Kim, B.E. Wyslouzil, M.J. Correll, P.J. Weathers Bioreactor engineering for recombinant protein production using plant cell suspension culture/W.W. Su Types and designs of bioreactors for hairy root culture/Y-E Choi, Y-S Kim, K-Y Paek Oxygen transport in plant tissue culture systems/W.R. Curtis, A.L. Tuerk Temporary immersion bioreactor/F. Afteen Design and use of the wave bioreactor for plant cell culture/R. Eibl, D. Eibl Part 3: Mechanized Micropropagation Integrating automation technologies with commercial micropropagation/C.J. Sluis Machine vision and robotics for the separation and regeneration of plant tissue cultures/P.H. Heinemann, P.N. Walker Part 4: Engineering Cultural Environment Closed systems for high quality transplants using minimum resources/T. Kozai Aeration in plant tissue culture/S.M.A. Zobayed Tissue culture gel firmness: measurement and effects on growth/S.I. Cameron Effects of dissolved oxygen concentration on somatic embryogenesis/K. Kurata, T. Shimazu A commercialized photoautotrophic micropropagation system/T. Kozai, Y. Xiao Intelligent inverse analysis for temperature distribution in a plant culture vessel/H. Murase, T. Okayama, Suroso Part 5: Physical Aspects of Plant Tissue Engineering Electrical control of plant morphogenesis/C.G. Carmen The uses of ultrasound in plant tissue culture/V. Gaba, K. Kathiravan, S. Amutha, S. Singer, X. Xiaodi, G. Ananthakrishnan Acoustic characteristics of plant leaves using ultrasonic transmission waves/M. Fukuhara, S. Dutta Gupta, L. Okushima Physical and engineering perspectives of in vitro plant cryopreservation/E.E. Benson, J. Johnston, J. Muthusamy, K. Harding

Plant Tissue Culture Engineering

Bioreactor technology for plant micropropagation.

Single node stem segments fromin vitro potato shoots cultured in liquid medium in the presence of ancymidol (23.4 μM) developed into bud clusters in either shaken flasks or bioreactor cultures. Buds on the clusters developed tubers after subculture to a tuber induction medium with 23.2 μM kinetin, 19.5 μM ancymidol, and 6-8% sucrose. The number of tubers per cluster and their size were higher in agar induction medium on top of which a second layer of liquid medium was added, than in liquid shake or bioreactor cultures. The highest increase in tuber size (i.e., 720 mg fresh weight after 7 weeks), was obtained in agar cultures flushed twice with liquid tuber induction medium. The potential of bioreactor cultures for potato bud proliferation and enhanced tuber development in double layer agar-liquid cultures is discussed.

D. Shemesh

Papers

Propagation and tuberization of potato bud clusters from bioreactor culture