Báo cáo lâm nghiệp: " An for attempt at generating haploid lines of Poplar species genetic manipulation and breeding program"

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Nội dung Text: Báo cáo lâm nghiệp: " An for attempt at generating haploid lines of Poplar species genetic manipulation and breeding program"

Physiological aspects of somatic polyembryogenesis in suspension cultures of conifers D.J. Durzan Department of Environmental Horticulture, University of California, Davis, CA 95616, U.S.A. Introduction cell suspension cultures for the multipli- cation of tree crops has been reviewed by Durzan (1988a, b) and Durzan and Gupta (1988). Using cell suspension cultures of Somatic polyembryogenesis (SPE) is a embryonal-suspensor masses (ESMs) new cell culture technology that needs to from lobiolly pine and Douglas fir, morpho- be distinguished from somatic embryo- genic protoplasts have been prepared that genesis and other forms of regeneration enable the recovery of somatic embryos (cf. Durzan, 1988a). SPE, involving the and the transient expression of a foreign reconstitution of multiple embryos by gene (luc) (Gupta and Durzan, 1987a; cleavage or budding of a proembryo, is Gupta et aG, 1988). Somatic embryos one of 3 categories of regeneration re- have also been recovered from cryopre- cognized by Sinnott (1960). It is one of 4 served ESMs (Gupta et al., 1987). In this broad categories of polyembryogenesis review, several recently recognized phy- (see Table I). Process control of SPE in
aspects of fractal siological aspects of SPE in conifers Fluid dynamics: streak lines (Douglas fir, Pseudotsuga menziesii; Nor- way Spruce, Picea abies; loblolly pine, Pinus taeda; and sugar pine, P. lamber- ESMs, transplanted just after fertilization tiana) presented. are with plant growth regula- onto agar plates tors, proliferate in darkness with the pro- duction of a clear, mucilaginous fluid. The viscosity of this mucilage is indicated by a of polyem- Physicochemical aspects fluid bridge when forceps are used to bryogenesis remove some of the cells. The viscous fluid can be removed and studied with reference to known phenomena in fluid In the evolution of the seed habit, fertiliza- dynamics (Batchelor, 1967). Among phy- tion has become independent of water as sicochemical properties displayed by the a medium for the process (Heslop-Harri- fluid are streak lines, i.e., patterns created son, 1983). Furthermore, the development by inanimate particles settling in the fluid of the zygote of gymnosperms occurs in (cf. Fig. 1 A-E). In suspension cultures, darkness and in a viscous, mucilaginous where viscosity is increased by high levels fluid. This fluid has interesting dynamic of sucrose, myoinositol and casein hydro- properties, with physicochemical implica- lysate, streak lines reflect the characteris- tions for the polyembryonic processes. tically polyembryonic shapes of ESMs The fluid surrounds the zygotic proembryo (Fig. 1 F-J). as it grows and develops within the ero- The factor that estimates the relative sion duct of the nourishing female game- of non-viscous (inertial) and tophyte or on a semi-solid medium. Sus- importance pension cultures of the ESM also contain viscous forces created by settling particles or cells acting on the fluid is the Reynolds this mucilage, which contributes to the vis- number, R. R is based on the length of cosity of the liquid medium. flow, density and viscosity of the medium. mucilage and cells of the ESM have The Surface tension and the heterogeneity of affinity for water. This affinity was an cells in the inoculum (ESM) particles or exploited by earlier investigators who pre- also factors. are soaked seeds to facilitate the removal of According to Batchelor (1967), when R embryos from bracts of (cf. Dogra, cones equals one, the viscous, inertial and pres- 1967). which the stream-tube passes. The current also shows the peripheral formation of new caps. F. The emergence of somatic embryos produced by polyembryogenesis from an ESM transplanted onto the surface of an agar medium is not unlike the start of vertical density current formation in A and B (x 13). G. Fragmentation of a loblolly pine polyembryonic mass in cell suspension culture, as viewed with polarized light, compares with B above (x 24.7). H. A developing loblolly pine embryo (e) with a large suspensor, as viewed under polarized light. The suspensor morphologically resembles the flow patterns (stream lines) of a solid sphere falling in a viscous fluid (x 44.2). 1. Polyembryonic mass, excised from the erosion zone of sugar pine and placed on an agar surface, shows the heaviest embryo at the bottom and the lighter cleavage and budding products on top. Multiple embryos are pro- duced by cleavage and budding polyembryogenesis on a ’thread’ of cells in the ESM (x 6.5). J. Embryos trans- planted from sugar pine seeds continue to develop and can be rescued, i.e., regenerated, by the process of reconstitution (Sinnott, 1960) and multiplied by somatic polyembryogenesis (Durzan, 1988a) (x 7.8).
during the protoplasmic streaming of forces in the system contribute to the sure embryonal tube cells in the budding pro- motion of the fluid around descending par- ticles. When R is less than one, inertial cess. forces are negligible. We can postulate As cells settle in viscous culture a that similar physicochemical forces are medium, the fall is compensated by a imposed on embryogenic cells of trans- rising current from the bottom. In static planted embryonal-suspensor masses in suspension cultures of an ESM, a critical suspension culture under the influence of viscosity is required to create vertical den- gravity. These forces are imposed on the sity currents by proembryos falling under histogenic algorithm and translated into the influence of gravity. By contrast, the ontogeny that is characteristic for each proembryos in the erosion duct of a seed conifer type (Durzan, 1988a). The end maintain contact with cells of the female result is that proembryonal developmental are being digested for gametophyte that patterns mimic, with their dense proem- nutrients. Under static conditions and in bryonal cells and their bouyant elongated highly viscous culture media, the fall of capillary-like suspensors, the fractal dy- particles, cells and isolated proembryos is namic forces found in viscous fluids in slowed to the amount of energy required suspension cultures, as revealed by streak to overcome the viscous resistance to flow lines and vertical density currents. We can and shearing stress. The resultant drag now study these forces in artificial systems establishes the direction of the falling outside the seed. The expectation is that material. new principles will arise for the improved As organelles, protoplasts and cells fall design of ’artificial seeds’, especially in a vertical density current, a flow pattern in relation to suspended nutrients and amino arises, whereby the heavier particles form acid chelates in the mucilage. a bulbous cap at the lower end, not unlike the dominant p!roembryo in an embryonal- suspensor mass. The shape of the cap contributes to the flaring, involution and Verticai density currents surface discontinuities that emerge. These forces create a sheath or boundary layer Vertical density currents in viscous fluids for the stream tube. are created by particles and/or cells set- Stream lines may appear and irregular tling under the influence of gravity. With fractal, reflecting the constrictions and vertical density currents, some cells may nodes of the settling particles enlarging fall much more rapidly than settling ac- and cells (Batchelor, 1967). Rings on cording to Stoke’s law (Bradley, 1963). eitheir side of the stream arise from the This phenomenon occurs in limnology and peripheral breakdown (instability) of cell is evident in other natural events (Bradley, masses. 1963; Thompson, 1942). in closed Vertical Shearing stresses are associated with density currents occur systems where the fluid is incompressible, the property of viscosity (Kay and Nedder- i.e., its density is not affected by changes man, 1985). For somatic polyembryogene- in pressure. Such currents are found in sis, the analogy is proposed that, at points suspension cultures and possibly on a of instability on the ESM created by shear micro scale in the erosion duct of seeds. stresses, new embryos could form by Inside cells, currents may also be dis- cleavage or budding. The analogy remains played by nuclei and ergastic materials to be tested experimentally. Where
stresses are great, as in shake cultures cell, at least under certain conditions (see with Erlenmeyer flasks, cells of the ESM Batchelor, 1967). However,I am not yet tend to lignify (Gupta and Durzan, 1987b). how far to compare the behavior of sure inanimate particles with polyembryonic Settling particles in a viscous fluid form systems in explaining the physicochemical a torus at the front (Batchelor, 1967). A processes underlying the histogenic algo- torus of particles may divide into 2 compo- rithms. nents, not unlike a cleaved proembryonal
growth factors contribute to the age. The in Plant mucilage growth regulators production of a callus and possibly can induce embryogenesis in cells of the Nurse cultures, or contact of mucilaginous explant. Moreover, when cells of the ESM ESMs with explants from the same or dif- are cultured in liquid medium, the mucil- ferent species, will promote morpho- polymerize under the influence of genetic activity in the ESM and/or in the can age plant growth regulators. Products of the explant (Fig. 2). The cause of this con- polymerization can mimic shapes of the tinued response is uncertain, but suggests that growth factors are found in the mucil- embryo (Fig. 3;.
Gupta P.K. (1988) Somatic Durzan D.J. & Progress in understanding morpho- and polyembryogenesis in coni- embryogenesis genesis will depend upon the simplifi- fers. Adv. Biotech. Processes 9, 53-81 cation of such physiological systems, Gupta P.K. & Durzan D.J. (1987a) Somatic better conditions for physicochemical embryos from protoplasts of loblolly pine measurements, improved methods for 2 proembryonal cells. BiolTechnology5, 710-712 image analysis (e.g., Serra, 1982) and a Gupta P.K. & Durzan D.J. (1987b) Biotechnolo- better understanding of how abscisic acid gy of conifer-type somatic polyembryogenesis and other plant growth regulators contri- and plantlet regeneration in loblolly pine. bute to this process (Boulay et al., 1988; 1 BiolTechnology 5, 147-151 Durzan, 1987). Gupta P.K. & Durzan D.J. (1987c) In vitro esta- blishment and multiplication of juvenile and mature Douglas fir and sugar pine. Acta Hortic. 212, 483-487 References Durzan D.J. Gupta P.K., Dandekar A.M. & (1988) Somatic proembryo formation and tran- sient expression of a luciferase gene in Douglas Batchelor G.K. (1967) In: An Introduction to fir and loblolly pine protoplasts. Plant Sci. 58, Fluid Dynamics. Cambridge University Press, 85-92 Cambridge Gupta P.K., Durzan D.J. & Finkle B.J. (1987) Boulay M.H., Gupta P.K., Krogstrup P. & Durzan Somatic polyembryogenesis in embryogenic D.J. (1988) Conversion of somatic embryos cell masses of Picea abies (Norway spruce) from cell suspension cultures of Norway spruce and Pinus taeda (loblolly pine) after freezing in (Picea abies Karst.). Plant Cell Rep. 7, 134-137 liquid nitrogen. Can. J. For. Res. 17, 1130-1134 Bradley W.H. (1963) Vertical density currents. Heslop-Harrison J. (1983) The reproductive ver- Science 150, 1423-1428 satility of flowering plants: an overview. In: Dogra P.D. (1967) Seed sterility and distur- Strategies of Plant Reproduction, (Meudt W.J., bances in embryogeny in conifers with particu- ed.), Bare Symp. No. 6, Allanheld, Osmun, lar reference to seed testing and breeding in Totowa. pp. 3-18 8 Pinaceae. Stud. For. Suec. 45, 1-97 J.M. & Nedderman R.M. (1985) In: Fluid Kay Durzan D.J. (1987) Plant growth regulators in Mechanics and Transfer Processes. Cambridge cell and tissue culture of woody perennials. Univ. Press, Cambridge Plant Growth Regul. 6, 95-112 2 Serra J. (1982): In: Image Analysis and Durzan D.J. (1988a) Somatic polyembryogene- Mathematical Morphology. Academic Press, sis for the multiplication of tree crops. Biotech. New York Genet. Eng. Rev. 6, 339-376 (1960) In: Plant Morphogenesis. Sinnott Durzan D.J. (1988b) Process control in somatic McGraw Hill, New York polyembryogenesis. In: Molecular Genetics of Thompson D.W. (1942) In: On Growth and Forest Trees, (Hallgren J.E., ed.), Frans Kempe Form, Vol. 1. Cambridge Univ. Press, Cam- Symp. 1988, Swedish Agric. Univ., Umea. bridge pp. 147-186