Larry C. Fowke

Embryogeny of gymnosperms: advances in synthetic seed technology of conifers

Synthetic seed technology requires the inexpensive production of large numbers of high-quality somatic embryos. Proliferating embryogenic cultures from conifers consist of immature embryos, which undergo synchronous maturation in the presence of abscisic acid and elevated osmoticum. Improvements in conifer somatic embryo quality have been achieved by identifying the conditions in vitro that resemble the conditions during in ovulo development of zygotic embryos. One normal aspect of zygotic embryo development for conifers is maturation drying, which allows seeds to be stored and promotes normal germination. Conditions of culture are described that yield mature conifer somatic embryos that possess normal storage proteins and fatty acids and which survive either partial drying, or full drying to moisture contents similar to those achieved by mature dehydrated zygotic embryos. Large numbers of quiescent somatic embryos can be produced throughout the year and stored for germination in the spring, which simplifies production and provides plants of uniform size. This review focuses on recent advances in conifer somatic embryogenesis and synthetic seed technology, particularly in areas of embryo development, maturation drying, encapsulation and germination. Comparisons of conifer embryogeny are made with other gymnosperms and angiosperms.

The development of somatic embryos in tissue-cultures initiated from immature embryos of Picea abies (Norway spruce)

Embryos of Picea abies at various developmental stages were cultured on defined media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (10−5 M) and N6-benzyladenine (BA) (5×10−6 M). The immature embryos gave rise to a highly friable and embryogenic callus which could be maintained by subculture and contained polarized and organized structures (somatic embryos) consisting of long highly vacuolated cells at one end (suspensor) and a group of small meristematic cells at the other (embryonal end). These structures closely resembled the early stages of normal zygotic embryogeny. Upon further culture these structures formed a bipolar shoot-root axis with an independent and closed vascular system. In many instances either the shoot or the root meristems failed to differentiate. Embryogenic tissues obtained on agar media could be transferred to liquid media and maintained by subculture for at least 6 months. The development of somatic embryos was observed in the liquid cultures also.

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa.

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 μm) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.

Endocytosis of cationized ferritin by coated vesicles of soybean protoplasts

Soybean (Glycine max (L.) Merr.) protoplasts have been surface-labelled with cationized ferritin, and the fate of the label has been followed ultrastructurally. Endocytosis of the label occurs via the coated-membrane system. The pathway followed by the label, once it has been taken into the interior of the protoplast, appears to be similar to that found during receptor-mediated endocytosis in animal cells. Cationized ferritin is first seen in coated vesicles but rapidly appears in smooth vesicles. Labelled, partially coated vesicles are occasionally observed, indicating that the smooth vesicles may have arisen by the uncoating of coated vesicles. Structures which eventually become labelled with cationized ferritin include multivesicular bodies, dictyosomes, large smooth vesicles, and a system of partially coated reticula.

Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance.

In order to enhance post-germinative vigour, somatic embryos of Picea glauca (Moench) Voss. were matured under in-vitro conditions that stimulated triacylglycerol (TAG) biosynthesis. In P. glauca seeds over 90% of the TAG was stored within the megagametophyte, and isolated zygotic embryos contained twice the amount of TAG of somatic embryos cultured for four weeks on basal medium containing 16 μM abscisic acid (ABA). Polyethylene glycol-4000 (PEG) as a non-permeating osmoticum with ABA promoted TAG biosynthesis by somatic embryos and sustained maturation throughout an eight-week culture period. Treatments that promoted TAG biosynthesis also prevented precocious germination and promoted desiccation tolerance. Thus, the optimal culture conditions for maturation, desiccation survival, and plantlet regeneration were 16–24 μM ABA and 7.5% PEG for eight weeks, followed by desiccation. Under these conditions the levels of TAG per somatic embryo were raised ninefold to about five times the zygotic-embryo level, and the TAG fatty-acid composition became similar to that of zygotic embryos. A study of sectioned material, using light and transmission electron microscopy, showed that the structure and distribution of lipid bodies within these somatic embryos and the degree of embryo development were similar to mature zygotic embryos. Up to 81% of the desiccated somatic embryos regenerated to plantlets during which time the TAG was utilised in a manner similar to zygotic seedlings.

Induction of Somatic Embryos and Plantlets from Cryopreserved Cell Cultures of White Spruce (Picea glauca)

Summary A cell suspension culture of Picea glauca with potential for sustained somatic embryo production has been successfully cryopreserved retaining very high levels of viability even after 1 year storage in liquid nitrogen. The following cryobiological components were identified as optimal for the recovery of viable cells postcryopreservation: 1) preculturing the cells for 24 h prior to freezing, in liquid nutrient medium enriched with 0.4 M sorbitol, 2) treating the cells after preculture with a cryoprotectant combination of 0.4 M sorbitol + 5 % DMSO, and 3) controlled freezing at a cooling rate of 0.3 °C min −1 to −35 °C followed by storage in liquid nitrogen. Dimethylsulfoxide alone at concentrations ranging from 5 to 20 %, in addition to being cytotoxic, was ineffective as a cryoprotectant. The callus re-established from cryopreserved cells maintained embryogenic potential identical to the unfrozen controls. Light and scanning electron microscopic examination of somatic embryos revealed structural and organizational conformity with those induced from unfrozen controls. Calli regrown from cryopreserved cells were induced to differentiate into plantlets through embryogenesis, and into shoots by organogenesis.

Effects of abscisic acid and analogues on the maturation of white spruce (Picea glauca) somatic embryos

Abstract Somatic embryo-competent cultures of white spruce, Picea glauca (Moench) Voss, were grown on (±)-abscisic acid (ABA) and three analogues known to be biotransformed by intact plants into ABA: (±)-methyl abscisate (MeABA); (±)-ethyl-(2E,4E) and -(2E,4Z)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methylpentadienoate (epoxyester, EE); and (±)-(2E,4E) and -(2E,4Z)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methylpentadienocic acid (epoxyacid, EA). ABA between 8 and 12 μM most efficiently promoted embryo maturation and led to the maximum recovery (4%) of stage 4b embryos when used in the absence of auxin or cytokinin. MeABA in the absence of auxin and cytokinin also promoted embryo maturation, EE promoted only the early stages in maturation, and EA was unable to promote any maturation of somatic embryos. Phytohormone-free medium produced very similar responses to EA, though in rare cases stage 3 embryos were found. Neither ABA nor the analogues promoted embryo maturation when used in combination with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM N6-benzyladenine (BA).

Production of vigorous, desiccation tolerant white spruce (Picea glauca [Moench.] Voss.) synthetic seeds in a bioreactor

SummaryThis report describes a low-cost method for generating large numbers of high quality mature white spruce (Picea glauca [Moench.] Voss) somatic embryos which survived desiccation and grew to plantlets more vigorously than excised zygotic embryos cultured in vitro. Somatic embryos from suspension culture were supported within a culture chamber on a flat absorbent pad above the surface of a liquid culture medium containing 20–50 μM abscisic acid and 7.5 % polyethylene glycol. Throughout a 7 week culture period 3 L of fresh medium was pumped into one end of the chamber, while the spent medium exited by gravity from the opposite end. Over 6,300 cotyledonary stage white spruce somatic embryos were recovered after this time from a single culture chamber without manual manipulation. The somatic embryos were of excellent appearance with well developed cotyledons, and possessed high levels of storage lipids. They survived drying to about 8 % moisture content following treatment for 4 weeks at 63 % relative humidity, and following imbibition converted to normal plantlets at a frequency of 92 %, compared to 80 % for embryos grown in Petri dishes. Somatic embryos cultured within the bioreactor developed to plantlets that were 20 % longer than zygotic embryos excised from mature seed and grown in vitro, and were 38 % longer than somatic embryos cultured upon agar medium in Petri dishes.

Effects of co-expressing the plant CDK inhibitor ICK1 and D-type cyclin genes on plant growth, cell size and ploidy in Arabidopsis thaliana

Abstract.The cyclin-dependent kinase (CDK) plays a crucial role in regulating the cell cycle of eukaryotic organisms including plants. From previous studies, it is known that ICK1, the first plant CDK inhibitor identified in Arabidopsis plants, interacts with Arath;CycD3;1 (CycD3) and Arath;CDKA;1 (Cdc2a). Overexpression of ICK1 has major effects on cell division, plant growth, and morphology. In this study, approaches were taken to determine the effects on transgenic 35S::ICK1Arabidopsis plants of introducing another gene that could potentially modulate the activity of ICK1. F1 plants were obtained by crossing 35S::ICK1 plants with wild type (Wt) and transgenic plants expressing 35::GUS, 35S::CycD3, 35S::CycD2, or 35S::antiICK1 (antiICK1 refers to antisense-ICK1). The major effects on plant growth and morphology observed in the 35S::ICK1 plants were partially reversed in the F1 plants from the crosses [35S::ICK1 × 35S::CycD2] and [35S::ICK1 × 35S::CycD3], and completely restored in the F1 plants from the cross [35S::ICK1 × 35S::antiICK1]. This observation was further supported by the results of ploidy analysis and structural characterization. Overexpression of CycD2 and CycD3 had the opposite effect on leaf cell size to the overexpression of ICK1. In addition, in ICK1-overexpressing plants, the CycD2 and CycD3 transcript levels increased, indicating a possible feedback regulation. The present results demonstrate that the interactions between ICK1 and D-type cyclins previously observed by the yeast two-hybrid and in vitro techniques are biologically relevant. These results illustrate the possibility of modifying plant growth and architecture dynamically by adjusting the levels of positive and negative cell-cycle regulators.

The morphology of multivesicular bodies in soybean protoplasts and their role in endocytosis

SummaryMultivesicular bodies (MVBs) in soybean protoplasts are distinct organelles (generally 250–500 nm in diameter) consisting of a limiting membrane and a number of smaller internal vesicles (generally 40–100 nm in diameter). MVBs of soybean protoplasts are morphologically similar to MVBs of animal cell systems. They can have tubular protuberances which extend from the main body of the organelle and a lamellar plaque on the cytoplasmic surface of their limiting membrane. In addition, the internal vesicles can be labeled by a zinc iodide-osmium tetroxide postfixation and may form via invagination of the limiting membrane.The MVBs of soybean protoplasts are a major compartment in the endocytotic pathway. They accumulate, over time, exogenously applied cationized ferritin and may deliver it to the major lysosomal or lytic compartment of the plant cell, namely, the vacuoles.