N. L. Biddington

The influence of ethylene in plant tissue culture

Ethylene produced by plant tissues grown in vitro may accumulate in large quantities in the culture vessels, particularly from rapidly growing non-differentiated callus or suspension cultures, and hence is likely to influence growth and development in such systems. Research into this aspect of tissue culture has been sparse, although it has grown recently with the increasing importance of in vitro regeneration. This review deals with the measurement and relevance of the accumulated ethylene, and the influence of both exogenous and endogenous ethylene in the different types of tissue culture systems. The relationships between ethylene and other growth regulators in tissue culture growth and development are also discussed. Although in some cases its influence seems negligible, in many types of tissue culture ethylene may act either as a promoter or inhibitor depending on the species used. Thus ethylene has an important influence on many aspects of in vitro regeneration, but it is also clear that we cannot at present describe a specific role or roles for ethylene in tissue culture which can be applied at a general, species-wide level. If its effects are to be enhanced or diminished in order to improve the efficiency and range of plant tissue culture, then more research is needed to clarify what its fundamental role might be in in vitro growth and development.

The effects of mechanically-induced stress in plants — a review

Mechanically-induced stress (MIS) occurs naturally in plants as the aerial parts are moved, usually by wind, but also by such agents as rain and animals. It can be induced indoors by various actions such as rubbing or bending the stem or shaking or brushing the entire shoot. The most noticeable effect of MIS is a reduction in stem, leaf or petiole length invariably resulting in plants which are smaller and more compact than unstressed controls. However, the response of other variables can often differ between species and there may be either increases or decreases in stem or petiole diameter, root: shoot weight ratio, chlorophyll content or drought resistance. Why species should differ in this way, and what is the endogenous control mechanism for MIS responses, are inanswered questions. Ethylene, which increases as a result of MIS in several species may cause some MIS responses such as increased stem diameter, epinasty or a change in sex expression. However, evidence suggests that MIS retardation of extension growth may equally be due to lower or supraoptimal auxin levels or lower gibberellin levels.The uses in the field of the growth promoter gibberellin or the growth retardant chlormequat chloride (CCC) appear to be examples of respectively reversing or stimulating MIS growth response. MIS may be applied indoors if short compact plants are needed, either for aesthetic purposes as with floral crops, or if hardier and more manageable plants are needed, such as seedlings for transplanting in the field. Much more research is needed to estimate the importance of MIS in the field and to assess how such knowledge may be used to improve crop yield.

Ethylene production during anther culture of Brussels sprouts (Brassica oleracea var gemmifera) and its relationship with factors that affect embryo production

The ethylene inhibitor silver nitrate (AgNO3) is known to overcome the poor response of the Brussels sprouts cultivar Hal to anther culture. Ethylene production by ‘Hal’ anthers after 6 h of culture at 35°C was on average 10- and 20-fold greater than from anthers of the highly responsive cultivars Gower and GA1 x RDF2. The initial 24 h period at 35°C necessary for embryogenesis in anther culture of Brussels sprouts generally reduced ethylene production by the anthers after 6, 24, 48 and 72 h of culture, although the effect was not seen in 2 out of 3 ‘Hal’ experiments until 24 h, and after 6 h was only found with 1 of 3 ‘GA1 x RDF2’ experiments. Embryo production was inhibited by the inclusion of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) or the ethylene-releasing compound, ethephon in the media. Silver nitrate (AgNO3) and the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) promoted embryogenesis but did not substitute for the high temperature treatment. The relevance of ethylene production during anther culture to the effects of genotype and high temperature on anther culture embryogenesis is discussed.

THE ROLE OF CYTOKININS IN THE PHYTOCHROME-MEDIATED GERMINATION OF DORMANT IMBIBED CELERY (APIUM GRAVEOLENS) SEEDS

The dormancy of celery seeds was broken by red light treatment given during imbibition and this effect was reversed by far‐red light. The exact quantitative relationship between the timing and duration of red light treatment and dormancy‐break has not been elucidated. However, the accumulated effect of daily 5 min exposures was greater than a single 5 min exposure on the second day of imbibition. The effects of red light treatment were simulated by treatment with a mixture of the gibberellins A4 and A7 and N6‐‐benzyladenine. A correlation between the requirement for red light and the requirement for exogenous cytokinins in the presence of GA4/7 was demonstrated by using six cultivars with different dormancy characteristics. In order to investigate the role of natural cytokinins in dormancy‐break, quantitative and qualitative changes in cytokinins were measured in celery seeds immediately after red‐light treatments. Rapid increases in n‐butanol‐soluble cytokinins following irradiation were associated with concomitant decreases in water‐soluble cytokinins, suggesting a red light induced cytokinin conversion. Three of the cytokinins present in the n‐butanol fraction of celery seed extracts were chromatographically similar to zeatin, zeatin riboside and N6‐‐A2‐‐isopentenyladenosine (i6Ado). The elution profiles from a PVP column of two others were similar to BA and its riboside. The possibility that these two cytokinins act as specific dormancy‐breaking cytokinins in celery seeds is discussed. There was little evidence of reversal of the cytokinin conversion mechanism by far‐red light exposure.

ABA promotion of ethylene production in anther culture of Brussels sprouts (Brassica oleracea var. gemmifera) and its relevance to embryogenesis

Abscisic acid (ABA) inhibited embryogenesis in anther culture of Brussels sprouts. This was accompanied by enhanced ethylene production during the first half of the anther culture period followed by a reduction in ethylene during the latter half, when compared to anthers not treated with ABA. The enhancement of ethylene production by ABA 6 h and 48 h after the start of the culture period was counteracted by the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG). Both AVG and the ethylene antagonist AgNO3 removed much of the ABA inhibition of embryogenesis, suggesting that at least part of the ABA effect on embryo production is mediated through increased ethylene biosynthesis. ABA promotion of ethylene production was reduced by high temperature: less ethylene evolved from ABA-treated anthers following a 24 h treatment at 35°C than from ABA-treated anthers incubated continuously at 25°C. A high temperature treatment such as this is invariably necessary for embryogenesis in Brussels sprouts anther culture.

The effects of gibberellic acid, fluridone, abscisic acid and paclobutrazol on anther culture of Brussels sprouts

Both gibberellic acid (GA3) and fluridone, a non-specific inhibitor of ABA biosynthesis, promoted embryo production in anther cultures of Brussels sprouts cv. Hal, but not in cv. Gower. Abscisic acid (ABA) and the gibberellin-biosynthesis inhibitor paclobutrazol inhibited embryo production in both cultivars.

Variations in response to high temperature treatments in anther culture of Brussels sprouts

The level, time of application and duration of the high temperature treatment necessary for embryo production from Brussels sprouts anther culture were examined. The effects of 29, 32, 35, and 38°C given for 24 h immediately following removal of the anthers from the bud, were tested on different cultivars, on different plants within the cultivars and on different occasions for each plant. Most embryos were produced following 32 and 35°C, very few following 30°C and none following 38°C. Although there was a tendency for some cultivars to respond better to one or other of the two more favourable temperatures, this varied considerably between individual plants. Plant to plant variation was also seen in the overall level of the response, although responsiveness tended to decline with successive samplings of the same plant. Experiments with cultivars Hal and Gower suggested that high temperature was required for at least 12 h after anther removal, but beyond that time the optimum period varied from plant to plant. If the excised anthers were held at 25°C for 16 h or more with Hal or 24 h or more with Gower before being exposed to the high temperature treatment, embrogenesis tended to be reduced. It is suggested that apparent non-responsiveness in anther culture may result to a large extent from the specific conditions that are used during the anther culture process.

A comparison of the effects of mechanically-induced stress, ethephon and silver thiosulphate on the growth of cauliflower seedlings

Mechanically-induced stress (MIS) was applied to cauliflower seedlings by brushing with paper for 1.5 minutes each day. With the possible exception of the effect on leaf 1 thickness, none of the growth responses induced by MIS were significantly nullified by spraying the seedlings with the ethylene inhibitor, silver thiosulphate (STS). The ethylene-releasing compound, ethephon, induced some changes in cauliflower growth similar to those caused by MIS, such as reduced shoot and leaf 1 weight, a reduction in cotyledon and leaf 1 area, and an increase in the thickness of leaves 1 and 2. However, other effects of ethephon were different from those of brushing. Petiole length and diameter and the weight and area of leaf 2 were reduced by brushing but generally increased by ethephon. STS reversed, at least partially, most of the ethephon effects on growth, thus demonstrating that it acts as an inhibitor of ethylene action in cauliflower.The results are discussed in relation to the possible endogenous control of MIS growth responses by endogenous ethylene and auxin.

Evidence for the accumulation of a germination inhibitor during progressive thermoinhibition of seeds of celery (Apium graveolens L.)

The germination of seeds of celery (Apium graveolens L.) becomes progressively thermoinhibited on incubation in the dark at high temperatures, the inhibitory temperature being dependent on the cultivar used. In two high-dormancy cultivars of celery, the production of germination inhibitors in seeds incubated in the dark at 26°C gradually increased over a 7-day period. Inhibitor production was measured by incubating seeds of the low-dormancy cultivar Florida 683 in homogenates of the thermoinhibited seeds of the high-dormancy cultivars and recording germination either in the light or with the gibberellins A4 and A7 (GA4/7) in the dark. Most Florida 683 seeds which failed to germinate in the homogenates after 15 days were induced to germinate by addition of N6-benzyladenine (BA). The presence of BA in addition to GA4/7 throughout incubation in the dark completely overcame the inhibitory effects of homogenates. This indicates that thermoinhibition of celery seeds is associated with the accumulation of a germination inhibitor which interacts with cytokinins. This does not appear to be abscisic acid (ABA) since ABA levels in thermoinhibited seeds were lower than in untreated seeds and did not increase with duration of high temperature treatment.

High temperature enhances ethylene promotion of anther filament growth in Brussels sprouts (Brassica oleracea var. gemmifera)

A study was made of the effect of high temperature on the growth response of Brussels sprout filaments to ethylene. Filaments with or without the anthers attached were incubated continuously at 25 °C or 35 °C for 7 days or for 2 days at 35 °C followed by 5 days at 25 °C. Growth was reduced during both 35 °C treatments compared to that of filaments at continuous 25 °C. Ethylene had little effect on filament growth at continuous 25 °C, whereas with treatment for either 2 or 7 days at 35 °C ethylene promoted filament growth considerably. Thus ethylene effectively overcame the growth inhibition induced by the 35 °C treatment.High temperature treatments reduced ethylene production from filaments alone, and from filaments with anthers attached. The ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) and the ethylene action inhibitor AgNO3 enhanced filament growth at 25 °C but had little or no effect at 35 °C. The relevance of temperature to ethylene sensitivity is discussed in relation to filament growth and to other plant processes in general.