Toshio Murashige

Plant tissue culture media

Plant tissue culture techniques have become vitally important for pursuing a wide range of fundamental and applied problems in research and development. The techniques encompass a variety of procedures used for specific purposes. The growing of masses of unorganized cells (callus) on agar or in liquid suspension is widely employed in biochemical and growth studies (1-5). The culture of segments of stems, roots, leaves or of callus provides systems to study differentiation, morphogenesis and plant regeneration (6, 7). Shoot apex culture methods leading to plant regeneration have been adopted for plant propagation and production of virusfree stock (8). The culture of anthers and pollen provides new approaches to haploid plant formation (9). Recently the technology has been extended to include the isolation and culture of plant protoplasts which are employed in fusion and somatic cell hybridization (10-13). The development of the various types of tissue culture has been based on empirical approaches, and some of the observations recorded in the literature may not be typical for plant cells. Differences in medium, environment, age, cell origin, and growth rates may explain the behavior of a particular line and need not represent a general characteristic of plant cells in culture. More uniformity in conditions of culture would assist in making data and observations more comparable.

Starch Accumulation in Shoot-Forming Tobacco Callus Cultures

Microscopic histochemical examinations of cultured tobacco callus disclosed a strong correlation between starch accumulation and shoot initiation. The accumulation started before any observable organized development and was heaviest in cells of loci which ultimately gave rise to organ primordia. Treatment of tissue cultures with gibberellin prevented starch accunmulation and organ formation.

Tissue culture propagation of tropical foliage plants

SummaryProcedures were established for clonal multiplication in vitro ofCordyline terminalis Kunth,Dracaena godseffiana Hort.,Scindapsus aureus Engler, andSyngonium podophyllum Schott. Shoot tips of actively growing terminals were selected as explants forCordyline andDracaena, and lateral buds were employed forScindapsus andSyngonium. The basal nutrient medium contained Murashige and Skoog salts, 3% sucrose, 100 mg per li-inositol, and 0.4 mg per l thiamine · HCl. The optima with respect to auxin, cytokinin, adenine sulfate · 2H2O, and NaH2PO4 · H2O addenda were determined. Also assessed were the influences of certain physical qualities of the nutrient medium and of the light intensity of the culture environment. The multiplication of each of the four plants was achieved by repeatedly subculturing the shoots that arose in vitro. Rates of plant increase per year per explant were calculated conservatively to be as follows:Syngonium, 5,000;Scindapsus, 100,000;Dracena, 300,000; andCordyline, 500,000.

Volatile emissions of plant tissue cultures

SummaryThe low-molecular-weight volatiles released by a variety of plant tissue cultures were examined by gas chromatography. Callus cultures invariably produced carbon dioxide, ethylene, acetaldehyde and ethanol. In cultures with developed shoots, ethanol was absent and acetaldehyde was detected only rarely.

Somatic embryogenesis in avocado (Persea americana Mill.) in vitro

A tissue culture procedure was developed for the regeneration of somatic embryos from callus cultures of the avocado,Persea americana. Immature zygotic embryos, 0.6–0.8 mm long, were used as original explants. Addition of 0.1 mg/l picloram (4-amino-3,5,6-trichloropicolinic acid) to culture medium appeared critical for callus initiation. Development of somatic embryos was accomplished in picloram concentrations of 0.01 to 0.1 ml/l. A few well developed embryos produced green shoots. Attempts to induce a higher incidence of germination were unsuccessful.

REPRESSION OF ASEXUAL EMBRYOGENESIS IN VITRO BY SOME PLANT GROWTH REGULATORS

SummaryA factor that represses asexual embryogenesis has been observed in the Rutaceae, with particularly high concentrations in the naturally monoembryonic cultivars. This investigation was an initial step towards identifying the factor.Citrus reticulata Blanco Ponkan mandarin nucellus explants andDaucus carota L. ‘Queen Annes Lace’ callus were employed to examine effects of known plant growth regulators and to determine possible identity of one or more of them with the repressive factor. The chalazal halves of ovules ofC. media L. ‘Citron of Commerce’ were used as control repressor source. Embryo initiation and growth of both test tissues were depressed markedly by 2,4-D, abscisic acid and ethephon. Slight inhibitions were obtained with IAA, kinetin and gibberellic acid. Recovery from the repressor did not occur readily inCitrus nucellus following recultures in citron-ovule-free medium; carrot callus resumed normal embryogenesis immediately upon transfer to suppressor-free medium. The repression by natural sources apparently involved the combined action of some or all natural hormones that are generically related to the above.

Suppression of Shoot Formation in Cultured Tobacco Cells by Gibberellic Acid

When tobacco pith was cultured on media containing gibberellic acid, shoot formation was observed. The formation of stem structures was strikingly suppressed by concentrations of 0.5 mg/lit. and above.

RESTORATION OF VIGOR AND ROOTING COMPETENCE IN STEM TISSUES OF MATURE CITRUS BY REPEATED GRAFTING OF THEIR SHOOT APICES ONTO FRESHLY GERMINATED SEEDLINGS IN VITRO

SummaryRepeated grafting of 0.2-cm shoot tips from fruiting-age trees ofCitrus reticulata Blanco ‘Ponkan’ mandarin andC. sinensis Osbeck ‘Liu Tseng’ sweet orange onto freshly germinated ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. X.C. sinensis Osbeck] seedlings in vitro resulted in progressive restoration of rooting competence and vigor of regenerated roots and shoots. The restored traits were retained through the course of the investigation and suggested a phase reversal phenomenon.

Volatile emissions of plant tissue cultures: II. Effects of the auxin 2,4-D on production of volatiles in callus cultures

SummaryThe levels of ethanol present in the gas phase of callus cultures were elevated by 2,4-D. Whereas carbon dioxide, ethylene and acetaldehyde also were affected, their changes in relation to auxin were inconsistent and usually smaller in magnitude than that of ethanol. Under conditions independent of auxin concentration, embryogenesis inDaucus carota andPhoenix dactylifera showed an inverse relationship with the concentration of ethanol in the cultures.

Morphogenic substances released by plant tissue cultures

The release of endogenous substances is a general phenomenon of plant tissue cultures, with some substances having significant developmental effects on the releasing tissues. Their systematic study was initiated with Nandina tissue cultures, and a yellow compound that accumulated in the culture medium was identified as the alkaloid, berberine. The rate of its release was related to the supplies of auxin, cytokinin, and nutrient salts. Addition of berberine \ HCl to nutrient media did not inhibit Nandina tissues, but suppressed shoot formation in Nicotiana stem segments. Growth of Nandina and Nicotiana callus, as well as rooting of Nicotiana stem segments, was promoted by alkaloid addenda.