A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions

Abstract

The Braun Biostat BF2 bioreactor system employs a novel aeration and agitation system, designed to enhance gaseous exchange and reduce shear stresses on submerged cell suspension cultures. The Biostat BF2 bioreactor employs a central pivoting spindle, around which the aeration tubing is wound forming a large paddle-type structure suspended from the top-plate and swung in a circle by a solid-state magnetic stirrer.The aeration tubing is a polypropylene capillary membrane, which has a unique microporous structure and is ideal for aeration, permitting two-way, bubble-free, gaseous exchange of the medium. This tubing can be rendered porous and can be used in the perfusion of aqueous solutions, enabling cell-free media exchange to be conducted. Thin-walled silicone rubber tubing, although gas permeable to a degree, cannot be made porous to aqueous solutions.The bioreactor was inoculated with a suspension culture of Sitka spruce (Picea sitchensis [Bong.] Carr.) known to be embryogenic and capable of maturing to plantlets on solidified medium. The perfusion capability of the bioreactor was employed to replace the inital proliferation medium with maturation medium in order to induce the development of the somatic embryos in submerged cell culture. The size ratio of the somatic embryo heads was monitored over 7 weeks. This cell line was found to mirror just the initial elongation, previously observed in shake-flask culture.Abbreviations: BAP = 6benzylaminopurine; 2,4-D = 2,4-dichlorophenoxyacetic acid; SSPM = Selby Sitka proliferation medium; SSMM = Selby Sitka maturation medium

Application of bioreactor systems for large scale production of horticultural and medicinal plants

“…In addition, somatic embryos have the potential for long-term storage through cryopreservation or desiccation, which facilitates flexibility in scheduling production and transportation and therefore fits large-scale production. The production of somatic embryos in bioreactors has been reported for a number of species (for reviews see Denchev et al, 1992;Cervelli and Senaratna, 1995;Moorhouse et al, 1996;Timmis, 1998;Ibaraki and Kurata, 2001;Paek and Chakrabarty, 2003), but many improvements are needed for the practical automatic somatic embryo production systems that can cope with synchronization of the somatic embryo development, identifying the occasional embryo abnormality during culture, and overcoming the difficulties in embling acclimatization.…”

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

“…The negative effect from liquid culture conditions on the development of gymnosperm somatic embryos was previously noted for Sitka spruce (Picea sitchensis) using a Braun Biostat BF2 system which resulted in similar performance of the bioreactor culture to a suspension culture in Erlenmeyer flasks (Moorhouse et al, 1996). No mature embryos could be obtained, indicating that the somatic embryos were not reaching the developmental stage permissive to maturation stimuli.…”

“…However, before the technique can be effectively implemented on a large scale, it is necessary to eliminate the costly and time consuming manual handling of somatic embryos. To automate the laboratory procedure for handling somatic embryos, it is essential to establish an efficient system for propagation and plant formation in liquid culture medium allowing the use of bioreactors (Gupta and Timmis, 2005;Moorhouse et al, 1996;Tautorus et al, 1992).…”

Mass propagation of conifer trees in liquid cultures?progress towards commercialization

“…However, embryos from ESM grown in bioreactors tended to be of larger size and germinated better, although the smaller embryos accounted for the improved germination . Moorhouse et al (1996) used a Biostat BF2 (magnetic stirrer base) bioreactor for development of Sitka spruce (Picea sitchensis) somatic embryos. The maturation medium was perfused in the bioreactor to replace the initial proliferation medium in order to induce the development of somatic embryos in a submerged cell culture.…”