Engineering topics

Krikorian, A. D.

Publications and source records attributed to Krikorian, A. D..

At least 19 records

NASA NTRS2001

Cell-wall architecture and lignin composition of wheat developed in a microgravity environment

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.

manned↗

NASA NTRS1998

Plants and somatic embryos in space: what have we learned?

Space provides a unique environment that can affect the interplay between cell cycle controls and environment and can thus modify the processes of cell division, development and growth. It is proposed that the chromosomal and nuclear abnormalities frequently encountered in cells of various plants exposed to space are due to a combination of factors including the biological status of the systems and the way in which they are grown, exposed to, and ultimately, the way in which they experience multiple stresses. The extent to which space-specific changes become manifest is dependent on the extent of pre-existing stresses in the system. This has become evident in a variety of plant species grown in space but has been particularly amenable to study using in vitro systems, especially in developing embryoids. The following observations allow us to harmonize disparate results from a variety of space experiments:- (a) the more completely developed a system, the less likely it is to show cell stress during growth; the less morphologically complex, the greater the vulnerability; (b) the size/"packaging" of the genome (karyotype) are significant experimental variables; plants with larger genomes (e.g. polyploids) seem to be more space-stress tolerant; (c) a single space-associated stress is inadequate to produce a significant adverse response unless the stress is severe, or a biological parameter necessary to 'amplify' it exists. On this view, an appropriate "stress match" with other non-equilibrium determinants, much like a 'tug of war', can result in genomic variations in space. All this emphasizes that fastidiously-controlled growing environments must be devised if one is to resolve the matter of direct versus indirect effects of space. Better understanding of the novel physico-chemical equilibrium phenomena associated with space will allow those interested in space cell and developmental biology to pick and choose procedures best suited to their exploitation for specific objectives.

Flight Experiment↗

NASA NTRS1996

Space stress and genome shock in developing plant cells

In the present paper I review symptoms of stress at the level of the nucleus in cells of plants grown in space under nonoptimized conditions. It remains to be disclosed to what extent gravity "unloading" in the space environment directly contributes to the low mitotic index and the chromosomal anomalies and damage that is frequently, but not invariably, demonstrable in space-grown plants. Evaluation of the available facts indicates that indirect effects play a major role and that there is a significant biological component to the susceptibility to stress damage equation as well. Much remains to be learned on how to provide strictly controlled, optimal environments for plant growth in space. Only after optimized controls become possible will one be able to attribute any observed space effects to lowered gravity or to other significant but more indirect effects of the space environment.

Review, Tutorial↗

NASA NTRS1996

Enhanced root production in Haplopappus gracilis grown under spaceflight conditions

The production and growth of roots in two aseptically maintained clonal populations of Haplopappus gracilis (family Compositae), each with a distinctive pattern of root production, were studied after they had been exposed to space for 5 days aboard a NASA Space Shuttle. Total root production of both populations was 67-95% greater when compared with their Earth-grown controls. Roots were generated: (1) laterally from pre-formed roots, the tips of which had been severed at the time of plantlet insertion into a "horticultural foam" substrate supplied with a nutrient solution; (2) adventitiously from the basal or cut-end portion of shoots; (3) de novo, i.e. from primordial which were non-existent at the outset of the experiment. Roots grew in all directions in space but were uniformly positively gravitropic in ground controls. In space and on Earth, both clonal populations maintained their clone-specific root formation and growth characteristics and produced an equivalent amount of tissue when compared to each other. As on Earth, and as expected, there were fewer and shorter roots on plantlets that formed floral buds. The significance of altered moisture distribution in the "horticultural foam" substrate in space for root growth and the significance of our findings for growing plants in altered gravity environments are discussed.

Flight Experiment↗

NASA NTRS1996

Growth and photosynthetic responses of wheat plants grown in space

Growth and photosynthesis of wheat (Triticum aestivum L. cv Super Dwarf) plants grown onboard the space shuttle Discovery for 10 d were examined. Compared to ground control plants, the shoot fresh weight of space-grown seedlings decreased by 25%. Postflight measurements of the O2 evolution/photosynthetic photon flux density response curves of leaf samples revealed that the CO2-saturated photosynthetic rate at saturating light intensities in space-grown plants declined 25% relative to the rate in ground control plants. The relative quantum yield of CO2-saturated photosynthetic O2 evolution measured at limiting light intensities was not significantly affected. In space-grown plants, the light compensation point of the leaves increased by 33%, which likely was due to an increase (27%) in leaf dark-respiration rates. Related experiments with thylakoids isolated from space-grown plants showed that the light-saturated photosynthetic electron transport rate from H2O through photosystems II and I was reduced by 28%. These results demonstrate that photosynthetic functions are affected by the microgravity environment.

NASA Discipline Plant Biology↗

NASA NTRS1996

Strategies for "minimal growth maintenance" of cell cultures: a perspective on management for extended duration experimentation in the microgravity environment of a Space station

How cells manage without gravity and how they change in the absence of gravity are basic questions that only prolonged life on a Space station will enable us to answer. We know from investigations carried out on various kinds of Space vehicles and stations that profound physiological effects can and often to occur. We need to know more of the basic biochemistry and biophysics both of cells and of whole organisms in conditions of reduced gravity. The unique environment of Space affords plant scientists an unusual opportunity to carry out experiments in microgravity, but some major challenges must be faced before this can be done with confidence. Various laboratory activities that are routine on Earth take on special significance and offer problems that need imaginative resolution before even a relatively simple experiment can be reliably executed on a Space station. For example, scientists might wish to investigate whether adaptive or other changes that have occurred in the environment of Space are retained after return to Earth-normal conditions. Investigators seeking to carry out experiments in the low-gravity environment of Space using cultured cells will need to solve the problem of keeping cultures quiescent for protracted periods before an experiment is initiated, after periodic sampling is carried out, and after the experiment is completed. This review gives an evaluation of a range of strategies that can enable one to manipulate cell physiology and curtail growth dramatically toward this end. These strategies include cryopreservation, chilling, reduced oxygen, gel entrapment strategies, osmotic adjustment, nutrient starvation, pH manipulation, and the use of mitotic inhibitors and growth-retarding chemicals. Cells not only need to be rendered quiescent for protracted periods but they also must be recoverable and further grown if it is so desired. Elaboration of satisfactory procedures for management of cells and tissues at "near zero or minimal growth" will have great value and practical consequences for experimentation on Earth as well as in Space. All of the parameters and conditions and procedural details needed to meet all the specific objectives will be the basis of the design and fabrication of cell culture units for use in the Space environment. It is expected that this will be an evolutionary process.

STS-61A Shuttle Project↗

NASA NTRS1992

Shoot growth in aseptically cultivated daylily and haplopappus plantlets after a 5-day spaceflight

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.

short duration↗

NASA NTRS1991

Generation and multiplication of plantlets from callus derived from Haplopappus gracilus (Nutt.) Gray and their karyotype analysis

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.

NASA Discipline Plant Biology↗

NASA NTRS1991

Growth and maintenance of an embryogenic cell culture of daylily (Hemerocallis) on hormone-free medium

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.

Non-NASA Center↗

NASA NTRS1990

Somatic embryogenesis of carrot in hormone-free medium: external pH control over morphogenesis

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.

NASA Discipline Number 29-20↗

NASA NTRS1990

Biological research on a Space Station

A Space Station can provide reliable, long duration access to ug environments for basic and applied biological research. The uniqueness of access to near-weightless environments to probe fundamental questions of significance to gravitational and Space biologists can be exploited from many vantage points. Access to centrifuge facilities that can provide 1 g and hypo-g controls will permit identification of gravity-dependent or primary effects. Understanding secondary effects of the ug environment as well will allow a fuller exploitation of the Space environment.

Krikorian, A. D.↗

NASA NTRS1990

Low external pH replaces 2,4-D in maintaining and multiplying 2,4-D-initiated embryogenic cells of carrot

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.

Non-NASA Center↗

NASA NTRS1990

Somatic proembryo production from excised, wounded zygotic carrot embryos on hormone-free medium: evaluation of the effects of pH, ethylene and activated charcoal

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.

NASA Program Space Biology↗

NASA NTRS1989

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

Non-NASA Center↗

NASA NTRS1989

Ethylene-associated phase change from juvenile to mature phenotype of daylily (Hemerocallis) in vitro

Hemerocallis plantlets maintained in vitro for extended periods of time in tightly closed culture vessels frequently show a phenotype, albeit on a miniaturized scale, typical of more mature, field-grown plants. The positive relationship of elevated ethylene in the headspace of such vessels to the phase shift from juvenile to mature form is established. Rigorous restriction in air exchange with the external environment by means of silicone grease seals hastens the phase change and improves uniformity of response. Although some plantlets may take longer to accumulate enough ethylene in sealed jars to undergo change, added ethylene and ethylene-releasing agents promote it. Ethylene adsorbants (e.g. mercuric perchlorate) block the shift of juvenile to mature form. Critical ambient ethylene level for the shift is ca 1 microliter l-1. Levels up to 1000 microliters l-1 do not hasten the response but are not toxic. The phase change is fully reversible when air exchange permits ethylene to drop below 1 microliter l-1. At least 1 microliter l-1 ethylene is required to sustain the mature phenotype. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) prevents the phase change, while the ethylene biosynthesis intermediate 1-aminocyclopropanecarboxylic acid (ACC) improves it. KOH, as a CO2 absorbent, does not prevent the phase change. Histology sections demonstrate subtle changes in the form of shoot tips of plantlets undergoing phase change.

NASA Discipline Plant Biology↗

NASA NTRS1988

Chromosomes and plant cell division in space

The objectives were: examination of chromosomal aberrations; development of an experimental system; and engineering design units (EDUs) evaluation. Evaluation criteria are presented. Procedures were developed for shuttle-based investigations which result in the procurement of plant root tips for subsequent cytological examination.

Krikorian, A. D.↗

NASA NTRS1985

Tissue culture apparatus for flight experimentation

The development of an apparatus for in-flight treatment of cells, tissues, or small organisms for microscopic and chemical analyses is discussed. The hardware for the apparatus is to have: (1) automated functions, (2) the capability to interface with ground-based facilities, (3) independently controlled chambers, (4) variable chamber configurations and volumes, and (4) the capabilities for processing the materials. The components of the equipment used on Skylab 3 for the study of animal cells are described. The design of an apparatus which incorporates all the required capabilities is proposed.

Scheld, H. W.↗

NASA NTRS1985

Concepts, strategies and potentials using hypo-g and other features of the space environment for commercialization using higher plants

Opportunities for releasing, capturing, constructing and/or fixing the differential expressions or response potentials of the higher plant genome in the hypo-g environment for commercialization are explored. General strategies include improved plant-growing, crop and forestry production systems which conserve soil, water, labor and energy resources, and nutritional partitioning and mobilization of nutrients and synthates. Tissue and cell culture techniques of commercial potential include the growing and manipulation of cultured plant cells in vitro in a bioreactor to produce biologicals and secondary plants of economic value. The facilitation of plant breeding, the cloning of specific pathogen-free materials, the elimination of growing point or apex viruses, and the increase of plant yield are other O-g applications. The space environment may be advantageous in somatic embryogenesis, the culture of alkaloids, and the development of completely new crop plant germ plasm.

Krikorian, A. D.↗