Immobilization of Flax Protoplasts in Agarose and Alginate Beads (Correlation between Ionically Bound Cell-Wall Proteins and Morphogenetic Response)

Abstract

Linum usifafissimum protoplast-derived colonies that are cultured in auxin-supplemented medium and immobilized in Ca2+-alginate matrix form round colonies that develop into polarized, embryo-like structures. On the other hand, protoplast-derived colonies that are immobilized i n agarose do not show an organized morphogenetic response, and unique, ionically bound cell-wall protein patterns match this response. Although only slight differences in neosynthesized or total constitutive polypeptides are observed, dramatic changes in ionically bound cell-wall proteins are seen. I n protoplasts grown on Ca'+-alginate-solidified, auxin-containing medium, several basic polypeptides were strongly induced and were found tightly bound t o the cell wall. In contrast, these basic proteins were found only weakly bound to the walls of protoplasts grown on agarose-solidified, auxin-containing medium or on Ca2+-alginatesolidified, auxin-free medium, i n which they were released into the medium. Our results suggest that plant cells can perceive and respond to the adjacent extracellular matrix, since we show that the growth of flax cells on Ca*+-alginate i n the presence of auxincontaining medium may promote the binding of specific proteins to the walls. This establishes a direct correlation of an embryo-like morphogenesis with ionically bound cell-wall basic proteins in flax protoplasts grown on Ca'+-alginate-solidified, auxin-containing medium.Virtually any cell can be immobilized (Nussinovitch et al., 1994). However, the gentlest methods that allow the best cell viability must be used, and this is achieved by using agar-type polysaccharides, including agarose and alginate, a family of co-polymers, that are most frequently used for plant cell entrapment.Flax protoplasts immobilized in alginate behave differently than those immobilized in agarose. We investigated the effects of agarose and alginate matrices on the morphogenetic response of phytohormone-controlled flax protoplast-derived cells that were obtained at optimized concentrations of growth regulators (5.5 p~ 2,4-D, 2.45 p~ NAA, 3.45 p~ BA, and 1.25 PM zeatin). We observed that cell colonies in agarose lacked defined outlines, since the clumps of actively dividing cells were surrounded by cells in an advanced state of lysis. In contrast, in alginate it was possible to observe the presence of compact and spherical colonies with actively dividing cells, as well as a periplasmic space with a very sinuous cell wall .Because of the presence of negative charges, Ca2+-alginate could affect the secretion of wall polysaccharides, thus modifying the composition and architecture of the newly formed walls, which has been reported for the increase of alkaloid synthesis by Coffea arabica cells (Haldimann and Brodelius, 1987). Indeed, it was demonstrated that alginate-induced colony differentiation is concomitant with a specific spatial and temporal distribution of acidic, methylesterified, and probably acetylated pectins (David et al., 1995a) by using the monoclonal antibody 2F4 (Liners ...

A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability

“…Meanwhile, Ca-alginate can be conveniently de-polymerized by potassium citrate, releasing encapsulated cells without damage. Although alginate has been widely used for the entrapment of protoplast in the form of beads [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] and film [2] , [3] , [4] , [12] , it is not firm enough to provide a good protection for entrapped cells [10] , especially when the experimental procedure needs repeated washing and cell collecting. Because silica is chemically inert, optically transparent, biologically resistant to microbial attack, mechanically strong, thermally stable and versatile in shape and size, silica-based matrices have been successfully used to directly encapsulate plant cells [21] , [22] , [23] , [24] or with a multiple step procedure [25] , [26] , [27] in recent years.…”

Plant cell and biotechnology studies in Linum usitatissimum – a review

“…PME and PMT enzyme activity have subsequently been Table 2. Key applications of flax protoplasts in biochemical studies Pectins in walls of protoplast-derived cells imbedded in agarose and alginate beads David et al (1995) Immobilization of flax protoplasts in agarose and alginate beads -correlation between ionically bound cell-wall proteins and morphogenetic response Roger et al (1996) Correlation of basic chitinases with the morphogenic response of flax cells. Roger et al (1998) Water deficit during root development: effects on the growth of roots and osmotic water permeability of isolated root protoplasts.…”

NAP enzyme recruitment in simultaneous bioremediation and nanoparticles synthesis

“…The lower activity of immobilized NAP could be attributed to the change in the three dimensional conformation of the protein, particularly in the catalytic active sites when linked with the support matrix which led to change in the enzyme properties. Further [ 60 ], suggested that the physical, chemical characteristics and polyanionic nature of the matrices are responsible for a particular physiology and performance of immobilized material (cells or proteins) with the space available for metabolic activity and ion exchange between the immobilized material and environment [ 61 ] also suggested that Ca-induced supramolecular conformation of alginate which influence on proteins behavior. In the light of such suggestion, the entrapment in alginate polymer seemed inadequate matrix or method for crude NAP-enzyme immobilization.…”