NOTES Electrostatic Encapsulation and Growth of Plant Cell Cultures in Alginate Hamad A. Al-Hajry, † Salha A. Al-Maskry, † Latifa M. Al-Kharousi, ‡ Osman El-Mardi, ‡ Walid H. Shayya, † and Mattheus F. A. Goosen* ,† Department of Bioresource & Agricultural Engineering, Department of Agronomy, Horticulture, Entomology, and Plant Pathology, College of Agriculture, Sultan Qaboos University, P.O. Box 34, Al-Khod 123, Muscat, Sultanate of Oman The growth of callus tissue from African Violets, encapsulated in alginate using electrostatics, was investigated as well as the mechanism of alginate droplet formation. Alginate microbeads as small as 500 ((50) microns in diameter could be produced by electrostatic extrusion directly from a plastic syringe (1900 micron extrusion orifice), in the absence of a needle. Video analysis of the mechanism of electrostatic alginate droplet formation from the syringe showed the development of a Taylor cone-like droplet which extended to form a thin strand that then broke up into droplets. Autoclaving of the alginate/medium solution significantly reduced its viscosity, giving smaller beads. Calculated microbead diameters agreed well with experimental values. Callus tissue from leaf explants was successfully immobilized and cultured using electrostatic extrusion. Tissue immobilized using 4% alginate in medium and cultured on agar grew best, producing a complete plantlet within four months. The long-term aim is to develop an effective method for large production of artificial seeds. Introduction Encapsulation technology has been used in the bio- pharmaceutical industry for immobilized cell culture (Bickerstaff, 1997; Posillico, 1986) and in medicine for cell transplantation therapy (Christenson et al., 1993). Agricultural applications of microencapsulated cells, though, have only recently been reported. Encapsulation, for example, has been employed for the cryopreservation of potato shoot tips (Bouafia et al., 1996), for enhanced regrowth of plants (Piccioni and Standardi, 1995; Pic- cioni, 1997), for improved food production through chi- tosan seed-coating (Freepons, 1997), and for making artificial seeds using somatic embryos and tissue (Reden- baugh et al., 1993; Goosen et al., 1997). The technique of somatic embryogenesis in liquid culture, which is believed to be an economical way for future production of artificial seeds, may benefit from cell immobilization technology. Encapsulation may aid in the germination and growth of somatic tissue by allowing for higher cell densities, protecting cells from shear damage in suspension culture (Takeda et al., 1998), allowing for surface attachment in the case of anchorage-dependent cells, and being very suitable for scale-up in bioreactors (King et al., 1989). In a recent study, Shigeta (1995) found that shoot growth and root development of plantlets germinated from alginate-encapsulated somatic embryos of carrots were promoted by transferring the embryos- from a culture medium containing sucrose to a culture vessel with a similar medium without sucrose. A new procedure was also developed for cryopreservation of plant tissue in which prefreezing dehydration was re- placed by encapsulation-dehydration at room tempera- ture (Bouafia et al., 1996). Shoot tips were trapped in alginate beads, precultured in sucrose-enriched medium, air dehydrated, and directly cooled in liquid nitrogen. The encapsulation-dehydration procedure improved the sur- vival rate of plant tissue compared to the conventional nonimmobilized cryopreservation process. This could have been due to the effect of seed moisture on seed quality (Bai et al., 1997). A variety of techniques may be used to produce microbeads. These include air jet extrusion, emulsion technology, and, more recently, electrostatic methods (Bugarski et al., 1994a; 1994b). With the electrostatic method, the droplet emission process depends on such factors as the needle diameter, the distance from the collecting solution, and the applied voltage (i.e., the strength of electrostatic field) (Newab and Mason, 1958; Goosen et al., 1997). Electrostatic atomization and elec- trostatically assisted atomization have been employed in a variety of areas, including paint spraying (Balachan- dran and Bailey, 1982), electrostatic printing (Fillimore and Van Lokeren (1982), pesticide application in citrus * Current address of corresponding author: Mattheus (Theo) F. A. Goosen, College of Engineering, Sultan Qaboos University, P.O. Box 33, Al-Khod 123, Sultanate of Oman. Email: theog@ squ.edu.om. Fax: (968) 513-416. † Department of Bioresource & Agricultural Engineering. ‡ Department of Agronomy, Horticulture, Entomology, and Plant Pathology. 768 Biotechnol. Prog. 1999, 15, 768-774 10.1021/bp990069e CCC: $18.00 © 1999 American Chemical Society and American Institute of Chemical Engineers Published on Web 06/19/1999