A. D. Krikorian

Hormones in Tissue Culture and Micro-Propagation

Aseptic culture techniques have figured prominently in the study of plant growth and development. The identity of hormones and role of growth regulators came about in large measure as a result of these studies. The first successful aseptic cultures were those of excised root tips. Somewhat later it became possible to grow callus cultures derived from storage organ explants or the cambial region of woody species. These cultures grew slowly and the stimuli to cell division usually entailed the addition of auxins such as indole-3-acetic acid (IAA) or naphthaleneacetic acid (NAA) to otherwise simple media comprised of mineral salts, sucrose and a few vitamins (16).

Kat and its use: An historical perspective

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 used 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.

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.

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

SummaryWounded 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 proembryos 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 (≈ 4) is detrimental to proembryo production, but is essential to maintaining PGSPs on hormone-free nutrient medium, whereas a sustained pH >5.7 allows continued development of PGSPs into later embryo stages.

Plant regeneration via somatic embryogenesis in the seeded diploid banana Musa ornata Roxb.

Somatic embryos of a seeded diploid ornamental banana (Musa ornata Roxb.) were obtained from zygotic embryos cultured on semi-solid Murashige and Skoog (MS) (1962) medium with the auxin 2,4-D (0.5, 1, 2 mg/l) and 5% CW. Removal of 2,4-D and transferral to Schenk and Hildebrandt (SH) (1972) salts with CW followed by basal MS led to embryo germination and growth. Plantlet production was obtained using filter paper bridges in liquid half-strength SH medium with 1% sucrose. The remarkable phenotypic fidelity of somatic embryos to that of zygotic embryos and the presence of a haustorium-like outgrowth on the somatic embryos is described.

Somatic embryos from cultured tissues of triploid plantains (Musa ‘ABB’)

Somatic embryos of two triploid plantain clones ABB ‘Saba’ and ‘Pelipita’ were produced in liquid from cells derived from multiplying shoot cultures. The medium consisted of a modified Murashige and Skoog (1962) medium with the auxins 2,4-D (1 mg/l) and 2,4, 5-T (1 mg/l) and the cytokinin BAP (0.1, 1 mg/l). Whereas proembryonic masses formed in each instance, green somatic embryos formed only in those treatments containing 2,4,5-T.

Effects of Spaceflight on Growth and Cell Division in Higher Plants

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.

Morphogenetic responses of cultured totipotent cells of carrot /Daucus carota var. carota/ at zero gravity

An experiment designed to test whether embryos capable of developing from isolated somatic carrot cells could do so under conditions of weightlessness in space was performed aboard the unmanned Soviet biosatellite Kosmos 782 under the auspices of the joint United States-Soviet Biological Satellite Mission. Space flight and weightlessness seem to have had no adverse effects on the induction of embryoids or on the development of their organs. A portion of the crop of carrot plantlets originated in space and grown to maturity were not morphologically different from controls.

Aseptic culture techniques for banana and plantain improvement

There is an urgent need to identify or to produce ‘Black Sigatoka’ diseasetolerant or -resistant cooking and dessert bananas. Since bananas are perhaps the most conspicuously sterile of all cultivated fruits, breeding of resistant stock is fraught with great difficulties. An overview is provided of the potential value that may be derived from the use of aseptic culture techniques for generating and/or multiplying specific pathogen-tolerant clones. Special emphasis is given to the principles underlying various strategies and to the several levels of sophisticated methods presently available or that still need to be further developed before substantive practical benefits accrue. While the stance adopted in this paper is conservative, so-called tissue culture approaches to banana breeding and improvement may well serve as a model not only forMusa but for other recalcitrant crop plants.

pH Control of Carrot Somatic Embryogenesis

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.