Meira Ziv

Reconsideration of the term ‘vitrification’ as used in micropropagation

The term vitrification is currently used to describe two types of processes related to tissue-cultured plant material. The first is used to describe organs and tissues having an abnormal morphological appearance and physiological function. The second is used to describe the transition from liquid to solid state, i.e. the formation of ice during low temperature storage of in vitro cultured cells, tissues and organs. Use of the same term to define two greatly different processes in the same research area can only lead to confusion, especially for key words. Thus it is appropriate to reconsider the usage of vitrification in the first sense mentioned above. It is recommended that the term vitrification should no longer be used to indicate plant material with an abnormal morphological appearance and physiological function, and should be substituted by the term ‘hyperhydricity’.

Factors influencing the production of hardened glaucous carnation plantlets in vitro

Medium type, its water status and the relative humidity in the culture vessel modified carnation leaf development in vitro. Carnation shoot apices cultured on liquid or on 0.8% agar solidified media developed into plantlets having succulent and translucent leaves which are not transplantable to non-aseptic conditions. Increasing the agar and/or sucrose concentration in the medium as well as decreasing the relative humidity in the culture vessel by a desiccant promoted glaucous leaf production. Increased water status (ψH2O and relative humidity) increased shoot proliferation and translucency of leaves. Decreased water status reduced shoot proliferation but induced the formation of glaucous leaves. The culture of apices for 5–6 days on liquid medium prior to their sub-culture to 1.5% agar medium improved shoot proliferation and normal leaf development. An agar slant prevented the submergence of apices in water accumulating on the medium and thus reduced leaf translucency. Survival was further increased by the transfer of plantlets in uncapped culture vessels to a desiccator for 1–2 weeks prior to transplanting to soil.

Plant Biotechnology and In Vitro Biology in the 21st Century

During rooting of apple microcuttings, we applied 24h pulses with ACC (l-aminocyclopropane-I-carboxylic acid, an ethylene precursor) or STS (silverthiosulfate, an inhibitor of ethylene action). The timing of the pulse and the auxin concentration in the medium determined the effect (promotive, inhibitory, or no effect). Another major factor was submergence of the basal 4 mm of the stem in the medium: the results indicated that this led to entrapment and accumulation of ethylene in the basal part of the stem (the portion that produces the roots) at high auxin concentration. In stem-segments or slices that are both cultured on top of the medium, ethylene accumulation did not occur. The detrimental effect of auxin on shoot and roots was -at least partlydue to auxin-induced ethylene synthesis.

Malfunctioning stomata in vitreous leaves of carnation (Dianthus caryophyllus) plants propagated in vitro; Implications for hardening

Abstract Carnation ( Dianthus caryophyllus L. var. ceris royallete ) shoot apices cultured in liquid or semi-solid proliferation media often develop into vitreous plants with translucent or succulent leaves. These leaf types lack cuticular waxes and develop stomata with non-functioning guard cells. In the present work, the guard cells were highly variable in morphology and size. Stomata in epidermal peels from vitreous leaves did not close in response to darkness, abscisic acid (ABA) or Ca 2+ , signals which usually cause the closure of functional stomata. In functional stomata, reduction in the guard cells turgor and concomitant reduction in their volume and a change in shape causes closure of the stomatal pore. Stomatal guard cells of translucent and succulent leaves did not close even when the turgor was reduced to zero by plasmolysis. Guard cell osmotic potential increased in the normal fashion in response to 10 −4 M ABA indicating that the protoplasts of the non-functional stomata respond to the closing signal but the stomata fail to close. These results indicate that the cause for the failure of stomata from vitreous leaves to close lie mainly in the guard cell wall and not in the protoplast. Apices cultured in media with reduced minerals but with elevated Ca 2+ developed normal leaves with functioning guard cells. Reduced humidity in the culture tube and higher agar concentration in the medium, induced normal leaf and stomata development with improved carnation plantlet survival after transplanting.

Enhanced shoot and cormlet proliferation in liquid cultured gladiolus buds by growth retardants

Gladiolus bud explants propagated in agitated liquid medium, in the presence of the growth retardants daminozide, ancymidol, paclobutrazol or Majic, proliferated into massive bud aggregates without leaves. The buds developed into protocorms and after subculture to agar-solidified medium formed cormlets 8–10 mm in diameter. The corms were not dormant and, after transplanting to pots in the greenhouse, developed into plantlets, 5–6 months after explant isolation.

Simple bioreactors for mass propagation of plants

Bioreactors provide a rapid and efficient plant propagation system for many agricultural and forestry species, utilizing liquid media to avoid intensive manual handling. Large-scale liquid cultures have been used for micropropagation through organogenesis or somatic embryogenesis pathways. Various types of bioreactors with gas-sparged mixing are suitable for the production of clusters of buds, meristems or protocorms. A simple glass bubble-column bioreactor for the proliferation of ornamental and vegetable crop species resulted in biomass increase of 3 to 6-fold in 3–4 weeks. An internal loop bioreactor was used for asparagus, celery and cucumber embryogenic cultures. However, as the biomass increased, the mixing and circulation were not optimal and growth was reduced. A disposable pre-sterilized plastic bioreactor (2–5-l volume) was used for the proliferation of meristematic clusters of several ornamental, vegetable and woody plant species. The plastic bioreactor induced minimal shearing and foaming, resulting in an increase in biomass as compared to the glass bubble-column bioreactor. A major issue related to the use of liquid media in bioreactors is hyperhydricity, that is, morphogenic malformation. Liquid cultures impose stress signals that are expressed in developmental aberrations. Submerged tissues exhibit oxidative stress, with elevated concentrations of reactive oxygen species associated with a change in antioxidant enzyme activity. These changes affect the anatomy and physiology of the plants and their survival. Malformation was controlled by adding growth retardants to decrease rapid proliferation. Growth retardants ancymidol or paclobutrazol reduced water uptake during cell proliferation, decreased vacuolation and intercellular spaces, shortened the stems and inhibited leaf expansion, inducing the formation of clusters. Using a two-stage bioreactor process, the medium was changed in the second stage to a medium lacking growth retardants to induce development of the meristematic clusters into buds or somatic embryos. Cluster biomass increased 10–15-fold during a period of 25–30 days depending on the species. Potato bud clusters cultured in 1.5 1 of medium in a 2-l capacity bioreactor, increased during 10–30 days. Poplar in vitro roots regenerated buds in the presence of thidiazuron (TDZ); the biomass increased 12-fold in 30 days. Bioreactor-regenerated clusters were separated with a manual cutter, producing small propagule units that formed shoots and initiated roots. Clusters of buds or meristematic nodules with reduced shoots, as well as arrested leaf growth, had less distortion and were optimal for automated cutting and dispensing. In tuber-, bulb- and corm-producing plants, growth retardants and elevated sucrose concentrations in the media were found to enhance storage organ formation, providing a better propagule for transplanting or storage. Bioreactor-cultures have several advantages compared with agar-based cultures, with a better control of the contact of the plant tissue with the culture medium, and optimal nutrient and growth regulator supply, as well as aeration and medium circulation, the filtration of the medium and the scaling-up of the cultures. Micropropagation in bioreactors for optimal plant production will depend on a better understanding of plant responses to signals from the microenvironment and on specific culture manipulation to control the morphogenesis of plants in liquid cultures.

Quality of micropropagated plants : vitrification

SummaryVitrification-Hyperhydrous shoot development, effects the survival and quality of several micropropagated plants ex-vitro. The leaves which are the immediate organ to be affected, exhibit abnormal morphology and physiology. Leaf malfunction is apparently a stress response to very rich media and high relative humidity. The understanding of the underlying mechanism of vitrification and its control in vitro can contribute to a more efficient micropropagation. Vitrification was found to be associated with elevated ethylene production which was related to hypolignification and poor cell wall development. Liquid and low agar media induced callose formation along with reduced and disoriented cellulose biosynthesis, manifested also in non-functioning guard cells. Malfunctioning stomata, in addition to defective cuticle contributed to increased transpiration and desiccation of in vitro formed leaves. The activity of various enzymes, associated with cell wall synthesis, was low and total proteins in normal leaves was higher than in vitreous ones. Various measures were found to reduce vitrification; lowered matrix and water potential in the medium, reduction in RH, low NH4+, changes in Ca++ levels and the removal of ethylene. These measures improved leaf morphogenesis, survival and the quality of several micropropagated plant species.

Rescue of interspecific lens hybrids by means of embryo culture

A two-stage in vitro technique was established for the development of interspecific hybrid embryos in the genus Lens. The culture of 14-day-old fertilized ovules on MS medium supplemented with zeatin, followed by the release of the embryos from the ovular integuments, allowed the development of viable and vigorous plants.

Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media.

Abstract The regeneration potential of excised aspen (Populus tremula L.) roots cultivated in liquid medium, as affected by plant growth regulators and by the position of the isolated root explant on the main root, was investigated. The effect of various levels of benzyladenine (BA) and thidiazuron (TDZ) on bud regeneration in root explants was studied. TDZ in the medium had a marked effect on bud development as compared with BA, inducing a tenfold increase in the number of buds regenerated from various root explants. TDZ enhanced both root and root-borne shoot biomass production but reduced further shoot development and elongation. The position of the isolated root sections on the main root affected regeneration, the proximal sections further away from the root tip producing the highest number of buds per explant in both BA and TDZ treatments. Buds regenerated in close proximity to the site of lateral roots in BA-treated roots, while in TDZ-treated root sections, the buds formed all over the root regardless of the presence of lateral roots. The buds developed from inner cortical and sub-epidermal cell layers, disrupting the epidermis and the inner layers. Root biomass production and growth was greatly enhanced in well-aerated bioreactor culture in the presence of 4.5×10–2 μM TDZ. A high number of the root-borne shoots could be rooted and converted to plantlets. However, while shoots regenerated in a medium with BA rooted well in a growth regulator-free medium, shoots formed in a medium with TDZ required auxin for rooting. Roots cultured in the presence of ancymidol, a gibberellin biosynthesis inhibitor, regenerated non-hyperhydric bud clusters and hyperhydric shoots. These were separated mechanically, subcultured to growth and rooting medium and transplanted ex vitro resulting in phenotypically true-to-type plantlets. The potential of liquid cultures for aspen shoot biomass production from roots is discussed.

Regulation of somatic embryogenesis in celery cell suspensions

Embryogenic suspension cultures of celery (Apium graveolens L.) were established from petiole and leaf callus. Suspensions were routinely subcultured in a ‘maintenance medium’ (with 2.3 μM 2,4-D and 0.88 μM BA). Somatic embryogenesis occurred in this medium, but was considerably improved in a ‘regeneration medium’ (2.3 μM kinetin, without 2,4-D). Cultures thus maintained, contained embryogenic clumps, aggregated somatic embryos, and few free-floating singular somatic embryos. Addition of mannitol (3–4% w/v) prevented cell lysis, greatly increased the number of singular somatic embryos, improved their normal differentiation, and accelerated torpedo embryo development. Experiments to reveal the nature of the mannitol effect demonstrated that the decreased osmotic potential was an important factor, but not the only one: iso-molar solutions of sucrose alone were not as effective. The mannitol effect could be manifested after a short (2–3 days) exposure period, suggesting a ‘trigger’ (induction) mechanism. Several pathways of somatic embryogenesis in celery and its regulation by subculturing, with the addition of mannitol, are outlined. Cultures thus maintained resulted in a high rate of normal somatic embryogenesis and production of normal transplantable celery plants.