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Single node cuttings (1 cm in length) of were cultured on gelled and liquid media to compare shoot multiplication efficiency. Liquid culture resulted in greater fresh weight, dry weight, shoot length and leaf area compared to gelled culture. Shoots from liquid culture grew vigorously without hyperhydricity, showing 100% survival. To determine optimal inoculation density of single nodes in a bioreactor, different numbers of single nodes (20 or 40 or 60 or 80) were placed into a 10-litre column-type bioreactor. Shoot length was greatest at the 80-node inoculation, with the least number of branches, indicating the best inoculation density tested for shoot multiplication in bioreactors. In the final experiment, single-node cuttings in bioreactors were treated with three different culture systems: ebb and flood, deep flow technique (DFT) culture and immersion. Results indicated that the DFT culture led to the greatest fresh weight, shoot length and leaf area, followed by the ebb and flood culture, while the immersion culture suppressed shoot multiplication due to the lack of oxygen and the high water potential. Our results suggested the possibility of large-scale production of shoots in bioreactors.

Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the context of propagating aggregates of plant tissue in liquid suspension bioreactors. Although micropropagated plants have a relatively low biological oxygen demand (BOD), the relatively large tissue size and localization of BOD in meristematic regions will typically result in oxygen mass transfer limitations in liquid culture. In contrast to the typical focus of bioreactor design on gas-liquid mass transfer, it is shown that media-solid mass transfer limitations limit oxygen available for aerobic plant tissue respiration. Approaches to improve oxygen availability through gas supplementation and bioreactor pressurization are discussed. The influence of media components on oxygen availability are also quantified for plant culture media. Experimental studies of polystyrene beads in suspension in a 30-litre air-lift and stirred bioreactors are used to illustrate design principles for circulation and mixing. Potential limitations to the use of liquid suspension culture due to plant physiological requirements are acknowledged.

Six identical bioreactors were constructed and built at the Agricultural University of Norway to provide optimal conditions for plant cell regeneration from cells into somatic embryos (“clonal or somatic seeds”). This was made possible through cooperation in COST87 by a European network in a working group on regeneration from plant cell cultures. The bioreactor design provides gentle stirring through a slow-speed stirrer that regularly changes direction of rotation to prevent “quiet” zones in the suspension in which cells can ssettle and grow. In addition, the oxygen is provided, bubble-free, through thin silicone tubing loops that are hanging loose, moving with the liquid to prevent cell growth on these tubes. We used off-the-shelf components whenever possible, to reduce the costs to a minimum, which was another aim of the construction. The result was a suite of relatively inexpensive computer-controlled bioreactors that could control temperature, oxygen, pH, stirrer speed and stirrer direction. In addition, we have provided different light spectral qualities by simple means of filtering the light. Using the present software, the parameters can be set up to alter every hour during the 24 h day/night cycle. All our cultures have improved growth in the bioreactors compared to identical cultures in Erlenmeyer flasks. The cultures used are: embryogenic cultures of carrot (), Norway spruce (, birch (), cyclamen and shoot cultures of Christmas begonia (. The paper also discusses recommendations for improvements of the current system for future revisions.

Bioreactors are an efficient tool for the production of plant propagules but, at present, their application is commercialized in only a few tissue culture companies. The present article reviews practical aspects of the use of bioreactors in the mass propagation of plants in relation to the responses of plant propagules in liquid medium, the characteristics of bioreactor culture techniques in plant propagation and discusses case studies of the use of bioreactors for several plant species including species, and . The establishment of plantlets from bioreactors and future prospects are also described.

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 to 5-litre 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 anti-oxidant 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 to 15-fold during a period of 25–30 days depending on the species. Potato bud clusters cultured in 1.5 litres of medium in a 2-litre capacity bioreactor, increased during 10–30 days. Poplar 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.

Automation of micropropagation organogenesis or somatic embryogenesis in a bioreactor has been advanced as a possible way of reducing costs. Micropropagation by conventional techniques is typically a labour-intensive means of clonal propagation. The paper describes lower cost and less labour-intensive clonal propagation through the use of modified air-lift, bubble column, bioreactors (a balloon-type bubble bioreactor), together with temporary immersion systems for the propagation of shoots, bud-clusters and somatic embryos. Propagation of , apple, , garlic, ginseng, grape, , and potato is described. In this chapter, features of bioreactors and bioreactor process design specifically for automated mass propagation of several plant crops are described, and recent research aimed at maximizing automation of the bioreactor production process is highlighted.

Membranes less attractive to embryos were tested as a replacement for nylon screens to prevent adherence of somatic embryos, cell clusters and cells to different sites of the bioreactor, a feature considered undesirable in plant cell suspension cultures. The results showed that the loss of embryogenic cell-mass could be halved by using silicone or track membranes. For aeration purposes, these membranes are as satisfactory as nylon screens conventionally used in cell lift impellers.

A low-cost Growtek bioreactor has been designed, patented and commercialised. It has unique features such as a floating and rotating explant-holder with perforated explant support and a side tube for medium changing, culture feeding and for content monitoring. The bioreactor can be operated both in static and agitated modes. Extensive performance studies have been conducted using representatives of trees (), commercial ornamentals (), monocotyledonous horticultural species (), tuber crops () and a medicinal plant (). In comparison to propagation in agar-gelled media as well as in liquid media using other culture vessels, this bioreactor exhibited 1.2 – 23.3 times shoot production, minimised root injuries by 32 – 48 %, reduced contamination by 12 – 18 % and reduced incubation time by 16– 42 %. Thousands of plantlets raised in this bioreactor have been field tested. Additionally, it was found to be effective for hairy root culture of .

Single node cuttings (1 cm in length) of were cultured on gelled and liquid media to compare shoot multiplication efficiency. Liquid culture resulted in greater fresh weight, dry weight, shoot length and leaf area compared to gelled culture. Shoots from liquid culture grew vigorously without hyperhydricity, showing 100% survival. To determine optimal inoculation density of single nodes in a bioreactor, different numbers of single nodes (20 or 40 or 60 or 80) were placed into a 10-litre column-type bioreactor. Shoot length was greatest at the 80-node inoculation, with the least number of branches, indicating the best inoculation density tested for shoot multiplication in bioreactors. In the final experiment, single-node cuttings in bioreactors were treated with three different culture systems: ebb and flood, deep flow technique (DFT) culture and immersion. Results indicated that the DFT culture led to the greatest fresh weight, shoot length and leaf area, followed by the ebb and flood culture, while the immersion culture suppressed shoot multiplication due to the lack of oxygen and the high water potential. Our results suggested the possibility of large-scale production of shoots in bioreactors.

The effect of physical and chemical environmental parameters on growth and differentiation of suspension cultures of the moss in bioreactors was investigated. By supplementation of the aeration gas with 2 % (v/v) CO as well as by continuous illumination, growth of this photoautotrophic growing batch culture was markedly enhanced, resulting in a doubling time of 1.2 d. The growth rate of semi continuously-growing bioreactor cultures was not affected by controlling the pH of the culture medium with set points at 4.5 or 7.0. However, growth of the culture at pH 7.0 resulted in increased caulonema development, thus showing a distinct effect on moss differentiation. The impact on research and plant biotechnological applications of the potential to control moss growth and differentiation by environmental parameters is discussed.