Interactions of culture vessels, media volume, culture density, and carbon dioxide levels on lettuce and spearmint shoot growth in vitro.

The influence of culture chamber capacity, medium volume and culture density on the growth yields of lettuce (Lactuca sativa L.) and spearmint (Mentha spicata L.) shoots were determined in an environment containing either 350 or 10,000 µmol mol-1 CO2 after 8 weeks of incubation. High positive correlations occurred between the culture vessel capacity and spearmint fresh weight, leaf number, root number, and shoot number. Similarly, high positive correlations occurred between culture vessel capacity and lettuce fresh weight, leaf number, and root number. Higher fresh weights, leaf numbers, and root numbers were obtained from lettuce and spearmint shoots when cultured in 1-quart Mason jars containing 100- or 150-ml aliquots of medium compared to jars containing 25- or 50-ml aliquots of medium within an environment containing either 350 or 10,000 µmol mol-1 CO2. High culture density decreased growth yields, and this phenomenon could only be slightly off-set by the employment of an elevated CO2 environment or larger culture vessels.

Production of cucumber fruits from the culture of 'Marketmore-76' plantlets.

A procedure to produce fruits from cultured shoot tips of Cucumis sativus L. cultivar 'Marketmore-76' in vitro is described. Four-week-old shoot tips, derived from sterile germinated seedlings on a MS medium, were cultured in a 3.8-1 Mason jar using an automated plant culture system. Tips readily generated roots, leaves and flowers after another 4 to 8 weeks in culture. Administration of compressed air at a 300 ml/min flow rate for 30 min 10 or 15 times a day induced the development of parthenocarpic fruits from flowers. Fruits, up to 170 mm long by 35 mm diameter, were obtained within 30 to 45 d after flower opening.

Fruit organ cultures.

The culture of fruit tissues as whole organs or isolated tissue sections has been conducted with various species (1). Whole, isolated ovaries have been successfully cultured to give rise to mature fruits (e.g., strawberry). Typically, however, when an isolated portion of the fruit tissue is introduced into a sterile environment, it immediately loses structural integrity and degenerates into a rapidly dividing callus mass (2). Loss of structural integrity is correspondingly associated with an alteration of physiology that is subsequently reflected in the production of an altered metabolism. Therefore, a meaningful study of fruit development using callus derived from fruit tissues is often not possible. Recently, we studied the parameters involved in the maintenance of citrus fruit tissue integrity (2). In this paper, the culture of isolated fruit tissues, as well as half and whole fruit culture, is demonstrated using the lemon fruit (Fig. 1-3).

Flower organ culture.

A variety of factors contribute to flower induction in nature. These same factors are assumed to be responsible for in vitro flowering (I). Flower formation in tissue culture has been observed in several plant species, and arises from a variety of explant sources (2). However, the factors responsible for flowering in culture have not been extensively studied (I). Reasons for studying flower formation in tissue culture can be summarized as follows:

Clonal propagation of orchids.

This chapter will deal with methods of clonal propagation for members of the two major morphological groups of orchids. The first group, sympodials, includes such genera as Cymbidium, Cattleya, Dendrobium, and Oncidium. They are characterized by a multi-branchingrhizome that can supply an abundance of axillary shoots for use as explants. They were among the first orchids to be successfully propagated, and techniques for their in vitro initiation (i.e., establishment) and subsequent proliferation are well established (1-8). The second group, monopodials, include Phaluenopsis and Vanda, and are characterized by a single, unbranched axis of growth that possesses few readily available axillary shoots for use as explants. Significantly different in their morphologies, the two groups require different approaches to explant selection and subsequent culturing. The successful large scale micropropagation of monopodials is, in fact, a relatively recent achievement (9): the culmination of a wide variety of studies using different media compositions and supplements (10-18).

Micromachines to automate plant tissue culture.

This chapter is concerned with the use of mechanical and electronic techniques (i. e., the Automated Plant Culture System [APB]) to aid in the sterile cultivation of plants for the purpose of prolonging culture life and/ or increasing yields over that obtained from conventional technology (bd1,bd2). The use of machines to facilitate the culturing of plants in vitro is common (e. g., shakers, roller bottle apparatuses, incubators, magnetic stirrers, and roller drums). However, current techniques employed in plant tissue culture (i. e., agar or liquid culture), developed about 30 yr ago, are highly labor intensive because of the requirement for numerous hand-plant interactions in the culturing of plants in a sterile environment. Culture of any plant in vitro requires periodic transferring to fresh medium. Nutrient medium is either exhausted and/or altered by plant cultures, thus requiring the constant transfers to fresh medium (usually every 4-8 wk or sooner). The system presented in this chapter is meant to reduce dependence on labor through substitution of hand-plant interactions with machine-plant interactions via automatic medium replenishment (Fig. 1). The APCS culture vessel is, therefore, large enough to accommodate the increasing plant culture size associated with long-term growing of plants in a single culture vessel.

Automation of the surface sterilization system procedure.

Reliable methods for obtaining sterile explants (i. e., that part of the parent plant introduced to in vitro conditions) are critical in tissue culture. Normally, explants are soaked in disinfectants in order to eliminate the coating layer of microorganisms ubiquitously found on plants. This chapter is concerned with the use of mechanical and electronic techniques (i. e., an automated Surface Sterilization System [SSS]) to aid in the sterile establishment of explants. The automated SSS reduces labor input and increases the effectiveness and reproducibility of the surface sterilization treatment (1) (see Fig. 1). Numerous disinfectant types and techniques have been developed to surface sterilize plants (2). However, all procedures can be summarized as follows.

Isolation of Pathogenic Bacillus circulans from Callus Cultures and Healthy Offshoots of Date Palm (Phoenix dactylifera L.).

Pure cultures of a gram-negative, rod-shaped bacterium, which produced endospores after 3 to 5 days on solid medium, were isolated exclusively from tissue cultures of the date palm Phoenix dactylifera L. Electron microscopic examination of thin sections of the bacteria revealed the bilayer membrane typical of gramnegative bacteria and confirmed the nature of the spores as true endospores. Biochemical and physiological tests indicated that the bacteria were Bacillus circulans. B. circulans was consistently isolated from the internal tissues, including the meristem, of apparently healthy offshoots of date palm. When meristem and embryo callus tissue culture samples were injected with B. circulans isolated from similar tissue culture samples and from offshoots, the majority of the isolates produced a rapid, destructive soft rot of the tissues.

Effects of cryogenic treatment on plantlet production from frozen and unfrozen date palm callus.

Embryogenic date palm (Phoenix dactylifera L. var. Medjool) callus cultures were treated with a cryoprotective mixture of polyethylene glycol (Carbowax 6000), glucose, and dimethylsulfoxide (10%/8%/10%, w/v); treated with the mixture, frozen to -196 degrees C, and then thawed; or left untreated. Growth subsequent to treatment was measured as fresh weight increase and as the number of embryos produced during 18 weeks of culture. The growth of calli that were frozen and thawed, compared to the other treatments, was greatly inhibited during the first 9 weeks of culture. This inhibition disappeared in subcultured tissue. In all treatments, cultures initiated plantlets after 9 weeks. Enzyme polymorphism, for five gene-associated enzyme systems including alcohol dehydrogenase, esterase, peroxidase, phosphoglucomutase, and phosphoglucoisomerase, was analyzed in leaves of regenerated plantlets by using starch gel electrophoresis for separation. Isozyme patterns were similar for all treatments.

Repression of asexual embryogenesis in vitro by some plant growth regulators.

A 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 and Daucus carota L. 'Queen Anne's 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 of C. 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 in Citrus 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.

Effects of ethephon, ethylene, and 2,4-dichlorophenoxyacetic Acid on asexual embryogenesis in vitro.

Asexual embryogenesis in Daucus carota L. ;Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 mul/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution.