Search

We present results on the analysis of 100 mL medium samples extracted from sterilized foam (Smithers-Oasis, Kent OH) used to support the growth of a representative dicotyledon (Haplopappus gracilis) and a representative monocotyledon (Hemerocallis cv Autumn Blaze) in NASA’s Plant Growth Unit (PGU) during a 5-day Space Shuttle flight and ground experiments. At recovery, the media remaining within replicate (n = 5) foam blocks (for both the spaceflight and ground experiments) were extracted under vacuum, filtered and subjected to elemental analyses. A unique aspect of this experiment was that all plants were either aseptically-generated tissue culture propagated plantlets or aseptic seedling clones. The design of the PGU facilitated the maintenance of asepsis throughout the mission (confirmed by post-flight microbial sampling) and thus any possible impact of microorganisms on medium composition was eliminated. Concentration levels of some elements remained the same, while some decreased and others increased. There was a significant two-fold difference between the final concentrations of potassium when the Earth-based and microgravity experiments were contrasted.

The microgravity environment encountered during space-flight has long been considered to affect plant growth and developmental processes, including cell wall biopolymer composition and content. As a prelude to studying how microgravity is perceived - and acted upon - by plants, it was first instructive to investigate what gross effects on plant growth and development occurred in microgravity. Thus, wheat seedlings were exposed to microgravity on board the space shuttle Discovery (STS-51) for a 10 day duration, and these specimens were compared with their counterparts grown on Earth under the same conditions (e.g. controls). First, the primary roots of the wheat that developed under both microgravity and 1 g on Earth were examined to assess the role of gravity on cellulose microfibril (CMF) organization and secondary wall thickening patterns. Using a quick freeze/deep etch technique, this revealed that the cell wall CMFs of the space-grown wheat maintained the same organization as their 1 g-grown counterparts. That is, in all instances, CMFs were randomly interwoven with each other in the outermost layers (farthest removed from the plasma membrane), and parallel to each other within the individual strata immediately adjacent to the plasma membranes. The CMF angle in the innermost stratum relative to the immediately adjacent stratum was ca 80 degrees in both the space and Earth-grown plants. Second, all plants grown in microgravity had roots that grew downwards into the agar; they did not display "wandering" and upward growth as previously reported by others. Third, the space-grown wheat also developed normal protoxylem and metaxylem vessel elements with secondary thickening patterns ranging from spiral to regular pit to reticulate thickenings. Fourthly, both the space- and Earth-grown plants were essentially of the same size and height, and their lignin analyses revealed no substantial differences in their amounts and composition regardless of the gravitational field experienced, i.e. for the purposes of this study, all plants were essentially identical. These results suggest that the microgravity environment itself at best only slightly affected either cell wall biopolymer synthesis or the deposition of CMFs, in contrast to previous assertions.

As part of the "Cellular Mechanisms of Spaceflight-Specific Stress to Plants" experiment, nine BRIC (Biological Research in Canisters) 100VC canisters, each containing four 100 mm dia polycarbonate petri dishes with embryogenic daylily (Hemerocallis sp.) cultures, were launched on 12 Jan 97 (STS-81), transferred to 'Mir' and returned on 24 May 97 (STS-84). Pre-flight, flight and ground control data for temperature, relative humidity, CO2 and ethylene in the BRIC canisters are presented.

Summary Protoplasts of soybean and an indole-alkaloid-free Madagascar periwinkle (vinca) were fused by electromanipulation. Callus cultures of these protoplasts were maintained in hormone-free semi-solid medium for nearly thirteen years. The stability of these cultures was tested by analysis of nuclear ribosomal DNA internal transcribed spacer (ITS) region. One fusion, out of the five finally tested, was regarded as exhibiting the characteristics of both the genomes from the original fused protoplasts while the others presented dominantly the ITS region of soybean.

Field evaluation of plants produced in vitro initiated from a phenotypically distinct, apparently healthy dwarf `French-type' plantain clone `Dwarf Superplatano' (Musa `AAB') was carried out at three sites in Puerto Rico. The use of the floral axis tip from a `mother plant' versus vegetative apices from lateral buds of the same plant as a source of the primary explant was compared and contrasted. Material from floral axis tip consistently showed high phenotypic uniformity whereas materials from vegetative apices of `sword' suckers were less so. `Virus-like symptoms' that became apparent in much of the material just before flowering (shooting stage) were determined to be due to the badnavirus banana streak virus (BSV), a dsDNA pararetrovirus. The `good news' is that a primary explant taken from the floral axis tip was quicker in its initial response to yield a multiplication system in vitro, and produced significantly fewer virus-infected plants, ca. 5%. By contrast, primary explants obtained from the vegetative sucker-derived apices were later in their production of initial buds, and produced many more virus-infected plants, an average of 32%. Comparison of vegetative-apex-derived plants and floral axis tip-derived plants disclosed no evidence that apices from vegetative suckers or floral stem tips gave rise to genetic off-types due to mutations were brought about by the in vitro process per se. Phenotypic differences were due to virus infestation. The `bad news' is that this dwarf plantain clone of considerable interest and potential in Puerto Rico can show very severe BSV symptoms. The occurrence of BSV infection in tissue culture-derived plants may be related to the presence of viral sequences integrated into the host genome, in which case there is no apparent strategy to rid the clone of this `virus'.

F. C. Steward and Jakob Reinert in the late 1950s, independently and with different degrees of scientific exactness, demonstrated that somatic cells of cultivated carrot can produce embryo-like structures in aseptic culture. Growth substances in the nutrient medium were viewed as central to the process. The now classic papers of Steward and Reinert have found a special and enduring place in the literature of plant development. But Harry Waris also deserves credit for his observation that vegetative cells sloughed off from aseptically germinated seedlings reared in liquid nutrient medium can produce ‘embryos’. In his studies, seedlings of Oenanthe aquatica (Umbelliferae) were maintained in culture for protracted periods under nutrient conditions designed to foster imbalance in protein metabolism, but without exogenous growth hormones. Seedlings placed in media with high concentrations of glycine grew normally for 3–4 months; after this a “period of morbidity” occurred, followed by production of new plants from the root tips. These new plants, later called “neomorphs”, in turn reproduced by colorless outgrowths of leaf epidermis. Such outgrowths, and “nodules” formed in a callus produced by the original seedlings, passed through stages described as “nodule”, “fusiform”, and a stage with two or more “lobes”. Transfer of the neomorphs to a medium lacking glycine resulted in the development of normal plants. We show that Waris was among the first, if not the first, to observe and recognize somatic embryo production in aseptic culture, and indeed to call them “embryos”. We also discuss his investigations in the context of understanding development at the cellular level, then and now.

A commonly held view is that the ideas and basis for the practice of orchid micropropagation arose de novo in 1960 from the work of Georges Morel in France. In this paper we argue that the crucial developments in micropropagation were made by Gavino Rotor in 1949 in the USA and Hans Thomale in 1957 in Germany, and that Morel's work needs to be seen in the context of a long line of research achievements in the in vitro culture of a wide range of explanted tissues and organs from plants of many species. A critical, historical, analysis of the events as they relate to clonal orchid multiplication is offered here. Two important technical innovations for orchid micropropagation—the use of activated charcoal to darken nutrient media and the adoption of liquid culture environments for part of the process—are examined in detail. In addition, an unusual US patent claiming invention of ‘a method for rapidly reproducing orchids’, especially cattleyas, is analysed. The origin of the micropropagation process claimed in this patent, said by the nominal inventor to go back as far as 1950, is discussed, but the claim remains unsubstantiated. Finally, consideration is given to the problems of adjudicating unequivocal priority for ‘discovery’ of a process as complicated and as broad as micropropagation.

To document and analyse objectively F.C. Steward's influence on the course of plant physiology is a task too great to be undertaken here. A detailed analysis would amount to a critical history of modem plant physiology. As Steward said of F.F. Blackman, F. R. S., in a memorial published in 1947 ‘To appraise the work and influence of F.F. Blackman one must look beyond the papers which bear his name, important as those were and are.’ Indeed, in a notice of a Festschrift published on the occasion of Steward’s retirement from Cornell University, a reviewer who knew him well aptly noted that ‘it is not so much the reviews themselves which form the real tribute to F.C. Steward as the fact that it was appropriate to invite experts in five different areas of plant physiology in order to cover the fields which Steward himself has covered and often led’. He later said that it would be difficult to imagine what contemporary plant physiology would be like had F.C. Steward not lived. It is easy to argue that Steward’s greatest contribution to plant physiology was that as a result of the scope and magnitude of his research, writings and lecturing activities, and his classroom teaching, he was able to create and sustain for half a century, a broad, highly personalized and extremely attractive context for the study of plant growth and development. Steward went far toward pulling the subject together in a comprehensible modem context, giving it historical continuity, while helping push it into the twentieth century through his unique research style and many contributions. He gave the field considerable glamour through his pioneering research, dynamic lecturing and literate, even graceful prose.

Recognition of a phenotypically distinct 'French-type' plantain (Musa AAB) designated 'Superplatano' (Superplantain) prompted evaluation of in vitro micropropagation as a means of generating sufficient numbers of plants for field evaluation in three locations in Puerto Rico. A multi-faceted study designed to evaluate relationships between different aseptic culture procedures and morphological origins of primary explants was carried out. Vegetative buds from various positions relative to the mother corm (defined by cardinal points on the compass) and explants from the floral axis of 'Maricongo' (the 'False-Horn', or florally determinate type 'progenitor' of 'Superplatano'), and 'Superplatano' (a 'French-type') were used as starting materials. Responses under field conditions were studied using a number of parameters including yield of commercially marketable fruits. We compared four populations of shoots, each of which derived from at least three different shoots from within one mat, shoots derived from vegetative and floral material from the same mat for both 'Maricongo' and 'Superplatano', and shoots derived from a number of floral buds of the same clone ('Maricongo') all of which were in culture for the same length of time. 'Superplatano' was stable whether from vegetative corm or floral bud apex. This shows conclusively that if the starting point in the micropropagation process is a stable Musa clone, our tissue culture procedure is reliable. Considerable variation in bunch phenotype was observed, however, in plants regenerated from ten of 12 shoot and floral meristems started from the 'False-Horn'-type 'Maricongo'. Change from 'False-Horn'-type (determinate) to 'French'-phenotype (indeterminate) was evident in each of the three locations. Frequency of bunch reversion varied from 0·4 to 100%, but was confined to individual originating stem tips rather than clones. The most dramatic bunch phenotypic change occurred in plants regenerated from clone 3. All plants regenerated from shoot 3-North bore 100% 'French-type' bunches. However, reversion in plants regenerated from shoot 3-West was only 1·8%, and no bunch phenotypic change was observed in plants from shoot 3-East. Plants regenerated from both shoot and male floral axis tips in 'Maricongo' clone 4 also bore 'French-type' bunches. Frequency of bunch reversion from shoot 4-East was 0·4% as compared to 2·6% from 4-floral. Bunch reversion occurred at the frequency of 2·0% when plants were regenerated from clone 6-floral. No bunch reversion was observed in plants regenerated from a single shoot tip in clones 1-West and 5-floral. No dwarfism was encountered in any of the tissue culture-derived plants. We conclude that tissue culture per se plays a very small role, if any, in the direct induction of off-types. Pre-existing characteristics of the primary explant determines whether products of a multiplication show fidelity or not. Our data suggest that 'Maricongo' is a chimera and that 'Superplatano' is revertant off-type that results when breakdown of the chimera occurs. Large numbers of stable 'Superplatano' were produced from unstable 'Maricongo' and this affirms the value of micropropagation for generation of clones with desirable bunch phenotype.

Plantlets of daylily (Hemerocallis cv. Autumn Blaze) regenerated from cell suspensions, and 4 clonal populations of Haplopappus gracilis were aseptically cultivated aboard the Shuttle "Discovery" during a 5-day mission within NASA's Plant Growth Unit (PGU) apparatus. Daylily was selected as a representative herbaceous perennial monocotyledon and the haplopappus clones represented an annual dicotyledon. The latter included 4 strains with different physiological and morphological characteristics: two aseptic seedling clones (each generated from a single seedling) and two tissue culture-derived lines. Mean daily growth rates for the primary shoots of all plantlets averaged 4.13 mm day-1 (SD = 2.20) for the flight experiment and 4.68 mm day-1 (SD = 2.59) for the ground control. Comparable growth rates calculated by summing both the primary and secondary shoots for all plantlets were 5.94 mm day-1 (SD = 2.89) for the flight experiment and 6.38 mm day-1 (SD = 3.71) for the control. Statistically significant differences existed between: (1) flight vs control primary shoot growth (the controls growing more than plantlets subjected to spaceflight conditions), (2) the different populations (the daylily gaining more shoot material than any of the haplopappus populations and the haplopappus seedling clones outperforming the tissue culture-derived haplopappus lines), and (3) the individual Plant Growth Chambers contained within the PGU. The data suggest that some spaceflight-associated factor(s) increased the tendency for primary shoot apices to degrade or senesce, resulting in the release of apical dominance and permitting the emergence of axillary branches, which subsequently partially compensated for the reduced primary axis growth. In addition to spaceflight-associated factors, the physiologically diverse nature of the experimental material as well as environmental heterogeneities within the culture apparatus contributed to the variation in growth results. The findings could explain some discrepancies reported from various plant culture experiments conducted in space.

Embryogenic cultures of cultivated carrot (Daucus crota cv. Scarlet Nantes) were initiated from seedling hypocotyls on hormone-containing nutrient medium and from wounded zygotic embryos on hormone-free medium. Both of these cultures were maintained with continuous multiplication as unorganized, embryogenic cell masses on hormone-free medium at pH 4.0, containing NH+4 as the sole nitrogen source. When grown on hormone-free medium at pH 4.0, neither culture contained any elongated cells. Virtually all cells were densely cytoplasmic and nearly spherical. Some cells were enlarged, not densely cytoplasmic, but always spherical. When either culture was transferred to an auxin-containing medium at pH 5.8, numerous elongated cells were produced. Elongated cells were observed when either naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was used, and whether the nitrogen source was NH+4 alone or a combination of NH+4 and NO−3. Elongated cells were more abundant when a combined nitrogen source was used. When cultures containing elongated cells were transferred to and multiplied on hormone-free or hormone-containing medium buffered at pH 4.0, all elongated cells disappeared after 2 weeks. No elongated cells were observed in any of the lines tested at pH 4.0. These results clearly show that it was the pH of the culture medium and not the presence or absence of an auxin or the nitrogen source(s) that permitted or prevented cell elongation in the embryogenic cultures tested.

Racimos tiernos del 'Superplátano' se podaron a 4, 5 y 6 manos para determinar el efecto de la poda en el tamaño de las frutas y en el rendimiento. Como control se incluyeron racimos sin podar del plátano 'Maricongo' tipo Cuerno. Las frutas en los racimos podados a 4 y 5 manos excedieron el peso medio de 270 g., criterio utilizado localmente para cualificar como comercial los plátanos tipo Cuerno. Los racimos del 'Superplátano' podados a 4 y 5 manos produjeron 146,800 y 180,200 frutas vendibles/ha., respectivamente. Estos rendimientos representan un aumento de 26,000 y 59,400 frutas/ha. en comparación con el de 'Maricongo' sin podar. La reducción de manos en el 'Superplátano' aumentó substancialmente el largo, grosor y peso medio de las frutas en la mano proximal y en la distal del racimo.

Unopened flower heads of Haplopappus gracilis (2n = 4) provided primary explants for callus production and subsequent induction of organized growth. Callus was initiated from small (3-5 mm in length) floral buds with benzylaminopurine (BAP) (44.4 micromoles; 10 mg/l) and naphthalene acetic acid (NAA) (0.54 micromole; 0.1 mg/l). Lowering the BAP level to 4.44 micromoles (1 mg/l) but maintaining the NAA level, gave rise to organized but highly compressed shoot growing points from an otherwise undifferentiated callus mass. Shoots selected from such cultures were maintainable and could be proliferated by growing 1-1.5-cm stem tip cuttings on Murashige and Skoog basal medium (solidified with agar) containing 0.444 micromole (0.1 mg/l) BAP and 0.054 micromole (0.01 mg/l) NAA. The stem tip multiplication rates obtainable by these means permit reliable strategies for shoot multiplication or production of rooted plantlets. Prolonged subculture and maintenance of shoots on growth regulator-free medium leads to in vitro flowering and greatly reduces rooting capacity. Karyotype analysis of chromosomes from root tip cells at metaphase and chromosome measurements show that karyologically uniform plantlets (based on chromosome number and morphology) can be obtained.

Cultures of preglobular stage proembryos (PGSPs) were initiated from mechanically wounded mature zygotic embryos of carrot, Daucus carota, on a hormone-free, semisolid medium. These PGSPs have been maintained and multiplied for extended periods without their progression into later embryo stages on the same hormone-free medium containing 1 mM NH4+ as the sole nitrogen source. Sustained maintenance of cultures comprised exclusively of PGSPs was dependent on medium pH throughout the culture period. Best growth and multiplication of PGSP cultures occurred when the pH of unbuffered, hormone-free medium fell from 4.5 to 4 over a 2-week period or when buffered medium was titrated to pH 4. If the hormone-free medium was buffered to sustain a pH at or above 4.5, PGSPs developed into later embryo stages. Maintenance with continuous multiplication of PGSPs occurred equally well on medium containing NH4+ or NH4+ and NO3-, but growth was poor with NO3- alone. Additional observations on the effects of medium components such as various nitrogen sources and levels, sucrose concentration, semisolid supports, type of buffer, borate concentration, activated charcoal, and initial pH that permit optimum maintenance of the PGSPs or foster their continued developmental progression into mature embryos and plantlets are reported. The influence of the pH of the hormone-free medium as a determinant in maintaining cultures as PGSPs or allowing their continued embryonic development are unequivocally demonstrated by gross morphology, scanning electron microscopy, and histological preparations.

A mixed culture comprised of both embryonic globules and nonembryogenic callus was derived from seedling hypocotyls of Daucus carota cv. Scarlet Nantes on 2,4-D- containing medium using well-established methods. Then the mixed cultures were transferred to, and serially subcultured on, a hormone-free medium near pH 4. The medium contained 1 mM NH4+ as the sole nitrogen source. When cultured in this way, embryonic globules were able to multiply without development into later embryo stages. Nonembryogenic callus did not survive. Continuous culture of embryonic globules on this low pH hormone-free medium yielded cultures consisting entirely of preglobular stage proembryos (PGSPs). PGSP cultures have been maintained as such with continuous multiplication for nearly 2 years without loss of embryogenic potential. These hormone-free-maintained PGSPs continue their development to later embryo stages when cultured on the same hormone-free medium buffered at pH 5.8. We show that hormone-free medium near pH 4 can replace 2,4-D in its ability to sustain multiplication of 2,4-D-initiated embryogenic cells of carrot at an acceptable growth rate without their development into later embryo stages. This procedure provides selective conditions that do not permit the growth of non-embryogenic cells while providing an adequate environment for embryogenic cell proliferation and should prove invaluable in studying habituation.

Poor growth and nuclear abnormalities observable in some space-grown plants have been hypothesized as due to a combination of factors such as degree of development, the specific way the plants are grown and the way they experience multiple stresses, some of which are space-specific. Data from a 132-day experiment on 'Mir' using embryogenic cell cultures of daylily (Hemerocallis) allow seemingly contradictory evidence from earlier Shuttle missions to be harmonized: a) the more developed an embryo the less likely it is to suffer catastrophic cell stress during growth, whereas the less developed it is, the greater its vulnerability; (b) the extent to which the stress becomes manifest is also dependent on the extent of pre-existing stresses imposed by suboptimal growing conditions; (c) an appropriate, albeit undesirable, 'stress match' with other non-equilibrium determinants, much like a 'tug of war', can result in genomic variations in space. It is not understood what is/are the feature(s) of the space environment that cause the various cell division perturbations but they have not yet been mimicked on earth. The stress symptoms were found only in space materials and, as predicted, they were most frequently encountered in smaller, less-developed materials grown under non-optimized conditions. It is concluded that, while any substantial deviation from 'optimum' can be a 'stress', spaceflight subjects vulnerable materials to cell division or DNA-repair stress(es) that appear distinctive, but remain elusive so far. Fastidiously-controlled growing environments must be devised to resolve the matter of direct versus indirect effects of space. On a practical level, it is predicted that adapting plant biotechnologies to space conditions will not be a casual matter. Grant Numbers: NAG21026.

Structures now commonly referred to by tissue culture workers as somatic embryos were first discovered in cultures of cultivated carrot and related umbellifers in the late 1950s (Waris, 1957; Steward et al., 1958; Reinert 1959a and b). Many have since investigated the nature of somatic embryogenesis and aimed to develop its potential for applied uses like clonal micropropagation (see Krikorian, 1982; Halperin 1995; Minocha and Minocha, 1995; Neumann, 1995; Krikorian and Simola, 1999 and refs. there cited for some early history). Even so, the establishment and subsequent management of embryogenic systems remains empiric although there are some general underlying principles (cf. e.g. Bajaj, 1995; Thorpe, 1995 and all the volumes in this series).

Daylilies (Hemerocallis species and cultivars) are herbaceous perennials much sought after for their showy albeit very short-lived blooms. The mature plant is comprised of fans (ramets) consisting of an underground stem (rhizome), an expansive fibrous or fibrous-tuberous root system, and strap-shaped leaves. Axillary buds can be forced to yield vegetative separations. The deciduous or evergreen foliage consists of sheathed-at-the-base, heavily ribbed leaves which when mature (Fitter and Krikorian 1985; Smith et al. 1989) are long and two- ranked. Flowers are borne on a scape, often branched and largely leafless but with bracts, and shoot propagules (“proliferations”). Flower buds per scape may vary from around 5 to 20 (Wilkins 1985).

‘Root’ crops traditionally include plants with swollen hypocotyls (e.g. radish, beets), rhizomes, corms, cormels (e.g. Aroids) and stem tubers (e.g. true yams) as well as true roots (e.g. carrot) and root tubers (e.g. cassava, sweet potato). Because of its importance, potato is covered separately in Chapter 15, but some of the other major ‘root’ and tuber crops will be covered here. A few practical reasons as to why one might wish to carry out aseptic culture studies on these plants are provided under the specific entries. Two extremely valuable references with considerable insight for both broad and specific research needs that readers should be aware of, but might easily be overlooked, are Kay (1973) and Brucher (1989). For ideas on priorities of plant biotechnology research for developing countries reference may be made to National Research Council (1990) and Sasson and Costarini (1990).

A ‘plantation’ has traditionally been thought of as a large estate in a tropical or subtropical region that is generally cultivated or tended by semi-skilled or unskilled labor under the general direction of a ‘manager’. Anyone who has seen large plantation operations immediately recognizes that the field workers are far from ‘unskilled’. In some places, the term ‘estate’ crop is used synonymously with the term ‘plantation’ crop. Since both terms are steeped in history, and are often flavored with connotations of colonialism or unsavory exploitation (Brockway, 1979), it is perhaps better and more accurate nowadays to think of plantation crops as those wherein a very large or relatively large group of plants, usually but not always perennials, are maintained under cultivation using highly standardized or strictly controlled practices.

Publisher Summary This chapter reviews the effects of spaceflight on growth and cell division in higher plants. The chapter focuses on the expression and modulation of the genetic information which is present in developing and growing systems. At the cellular level, the genetic information involves an understanding of the external and internal growth regulatory controls which operate in resting or quiescent cells as they are activated to grow again at rapid rates. Using an embryogenic system of an organism similar to carrot showed an increased biomass of embryonic structures generated in space in liquid cultures. Chromosomes and plant cell division is also discussed in the chapter. The CHROMEX experiment employed, for the first time in a space-based experiment, tissue culture-derived plantlets. For H. gracilis, comparably sized seed-derived plants, were included in the experimental protocol. In all cases, the experimental plants began as fully differentiated individuals, complete with leaves, shoots, and roots, which had been maintained under aseptic conditions from their initiation.

The culture of carrot cells in liquid suspension dates from 1953 and the recognition of their totipotency from 1956 [10]. By 1962 it was feasible to maintain in the laboratory, routinely, liquid cultures, heterogeneous as to their unit size, but in which large numbers of embryos readily developed from suspended cell clusters and single cells [8]. By this time, the role of synergistic combinations of the growth-promoting complex as it occurs in coconut water with auxins such as naphthaleneacetic acid (NAA) and 2,4-dichloro-phenoxyacetic acid (2,4-D) had become well-known [11, 23]. Moreover, the advantages to be gained in some otherwise morphogenetically recalcitrant cell cultures, of sequential treatments with different growth-promoting complexes and systems, became appreciated [23]. By these general means it was shown that a number of umbelliferous plants (family Apiaceae) and species or cultivars from other families, could yield cells and somatic embryos which in turn could give rise to whole plants. But when the main sequence of embryogenic development of carrot cells became known [24], it was found that its outcome could be greatly altered by the environmental conditions and the identity and mode of application of the growth and morphogenetic stimuli [11, 23].

Details of the plant cultivation system developed for the CHROMEX experiment flown aboard the Shuttle Discovery (March, 1989) in NASA's Plant Growth Unit (PGU) are presented. The physical regime as measured during Spaceflight, both within the orbiter cabin environment and within the PGU itself, is discussed. These data function as a guide to what may be representative of the environmental regime in which Space-based plant cultivation systems will be operating, at least for the near-term. Attention is also given to practical considerations involved in conducting a plant experiment in Space. Of particular importance are the differences expected to occur in moisture distribution patterns within substrates used to cultivate plants in Space vs on Earth.

Callus cultures of the diploid daylily (Hemerocallis) clone Autumn Blaze' were initiated and maintained in hormone-containing nutrient medium. At various times (from 6 weeks to 1 year) after being initiated, hormone-derived cultures were evaluated for their ability to be maintained and to multiply on hormone-free medium at low pH (between pH 4 and 4.5). Cultures had to be exposed to hormone-containing medium for at least 12 weeks before they could be maintained on hormone-free medium at low pH. The transition to maintainability on low pH hormone-free medium included the production of many aberrant embryonal forms ( neomorphs'). However, all hormone-derived cultures tested consisted entirely of preglobular stage proembryos (PGSPs) after 12-24 weeks on low pH hormone-free medium. PGSP cultures have been maintained and multiplied as such for over 1 year on low pH hormone-free medium. PGSPs continue their development into various somatic embryo stages when cultured on hormone-free medium buffered at pH 5.8. The production of well-formed somatic embryos was greatly enhanced when PGSPs were plated on activated charcoal impregnated filter papers that were placed on top of the agar surface. The gross morphology and histology of the PGSPs and stages of somatic embryo development are presented. The work shows that the ability of hormone-free medium at low pH to permit PGSP multiplication without development into later stages of embryo development is not restricted to carrot.

Wounded zygotic embryos of cultivated carrot produce somatic proembryos on hormone-free nutrient medium containing 1 mM NH4+ as the sole nitrogen source. Continued maintenance of proembryos on this medium leads to a "pure" culture of preglobular stage proembryos (PGSPs). Ethylene had no effect on this process. Also, somatic embryo production was not affected by growing cultures on activated charcoal-impregnated filter papers. However, somatic proembyros initiated on activated charcoal papers were not maintainable as PGSPs and developed into later embryo stages. Normally, medium pH dropped from 5.7 to 4 during each subculture period, but when using activated charcoal papers the pH endpoint was around 6 - 7 due to a leachable substance(s) within the filter papers. When powdered, activated charcoal was used in the medium as an adsorbent of products potentially released after wounding, pH dropped at the normal rate and to the expected levels; proembryos did not mature into later embryo stages and were maintainable exclusively as PGSPs. Low pH (approximately 4) is detrimental to proembyro production, but is essential to maintaining PGSPs on hormone-free nutrient medium, whereas a sustained pH > or = 5.7 allows continued development of PGSPs into later embryo stages.

Use of synthetic auxin to initiate and manage embryogenic cultures of carrot and other species is well established (1) even though a detailed understanding of what substances like 2,4-D do in these systems is obscure. Realization of a basic understanding of what is done procedurally and why it is done, i.e. knowing what physiological or developmental controls may be in place that need to be taken into account under a given set of circumstances, has been limited both because of the empiricism involved (2) and the unavailability for in depth study of cultures that comprise a given stage or conform to a narrow range of developmental parameters. Workers generally agree that it is not easy to achieve a highly responsive, morphologically uniform embryogenic culture of any plant, including the carrot. Efforts to synchronize cultures have been even less successful (3). Clearly a better understanding of induction, requirements for maintenance in a given growth mode, and control over somatic embryogenesis is required.

A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

Hemerocallis and Musa clones grown under aseptic conditions provide valuable test systems wherein both morphological and biochemical prerequisites for expression of morphological competence from protoplasts can be investigated. While there is considerable opportunity to study protoplast isolation and regeneration per se, the greatest challenge will be to induce, controllably, useful change(s) by co-use of molecular biology and protoplast culture-related methodologies. But the very way in which daylilies and bananas/plantains grow as plants imposes at present certain limitations on achieving cultures which can express their presumed, innate totipotency. Nevertheless, progress has been made and will continue, provided realistic strategies are adopted. When this is fully achieved, the true genetic engineering and use of protoplasts for Hemerocallis and Musa improvement can be realized.

Of the many Old World plants or plant products which have potential for abuse, kat has probably been the least publicized in so-called more economically developed countries. Because kat has rather localized use and because, unlike opium and hashish it has no documented history or well-developed tradition dating from remote antiquity, few outside the confined areas of use even know about it. While kat has been, and still is, used in traditional medicine, it cannot boast of any great potential for development as a drug for use in Western societies. In short, the incentives for detailed, serious investigation until recently have been lacking. Even so, there is a considerable, albeit scattered, body of literature which might help provide renewed insights in approaching what is seen by some as an increasingly serious kat problem. A multidisciplinary historical overview of the use, abuse and sociology of this important plant is presented. Special emphasis is given to European early knowledge of, and evolution of attitudes towards kat use. Alleged past use and development of contemporary use patterns, effects, legal aspects, and chemical composition is covered from an historical perspective as well.

This paper deals with problems of somatic embryogenesis (totipotency) of carrot cells in liquid culture and when dispersed in thin films of nutrient agar. The critical events that intervene between somatic cells and embryo plantlets during morphogenesis are delineated. Factors that affect the course of these events are described with special reference to the stimuli which permit the very smallest units to develop into organized structures. Special attention is directed to the importance of a protracted period of darkness and the use of an ‘embryo-conditioned medium’ as interacting stimuli that facilitate the early stages of development. Means of arresting and releasing the development of pro-embryonic units are discussed. Aspects of growth and form of the developing cultures are presented photographically at different levels. The behaviour of free protoplasts from pro-embryonic units is described and contrasted with that of intact totipotent cells. The significance of all these observations is examined in the light of practical applications and in relation to problems of development.