Plant Growth Regulation 0 1992 Khmer Academic

11: 173-187, Publishers.

1992. Printed

173

in the Netherlands.

Review

The influence of ethylene in plant tissue culture N.L. Biddington Horticulture Research International,

Wellesbourne, Warwick CV3.5 9EF, UK

Received

1991

3 July

1991; accepted

25 September

Key words: Ethylene, plant tissue culture, plant hormones, review Abstract 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. Abbreviations: ABA, abscisic acid; ACC, l-aminocyclopropane-I-carboxylic acid; AOA, aminooxyacetic acid; ASA, acetylsalicyclic acid; AVG, aminoethoxyvinylglycine; BA, N6 benzylaminopurine; 2,4-D, 2,4dichlorophenoxyacetic acid; DNP, 2,4-dinitrophenol; GA, gibberellin; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; NAA, naphthaleneacetic acid; SAM, S-adenosylmethionine; STS, silver thiosulphate; TIBA, 2,3,5-triidobenzoic acid.

1. Introduction Several aspects of plant biotechnology fuel the ever increasing commitment to plant tissue culture research. There is the need to develop regeneration systems for use in the relatively new techniques of genetic transformation. Tissue culture can provide the system for inserting the genetic information and for raising the novel genotype to an autonomous plant. Anther culture is being adopted as an important aid for plant breeding [29] and the biosynthesis of secondary products from plant cell cultures is the subject of considerable research [46]. In addition, commercial applications of micro-

propagation are increasing [l] and the technique is extending into the more conventional areas of plant production [e.g., 331. Although some tissue culture systems for some species are well described and have become relatively successful, very many are either difficult, or as yet impossible to achieve. Plant tissue culture depends on the external control of morphogenesis by chemical factors, particularly hormones, that act as endogenous regulators of what might be called normal growth and development. Thus the inclusion of auxins and cytokinins in the media is essential for successful tissue culture in the great majority of species [30], and more recently ABA [e.g., 14, 1191 and to a lesser

174 extent GA [83, 851 have proved valuable additions for promoting or improving regeneration. The presence of ethylene in tissue cultures is invariably a consequence of the method, rather than as a deliberate addition to the culture vessel. Because tissue culture by necessity is done within closed containers, gaseous products, such as ethylene, from the plant material may accumulate and the gaseous environment may be very different from that experienced by non-enclosed plants. Thus ethylene is seen as an unwanted contaminant and consequently much of the research on ethylene and tissue culture has centred on understanding how ethylene accumulation might affect regeneration, and the necessity or otherwise of modifying its influence.

2. Regeneration systems Plant tissue culture can take many forms. Plants can be regenerated in vitro via organogenesis, whereby roots and shoots develop from the cultured tissue or via embryogenesis in which plants grow from individual embryos which are formed from the tissue. Regeneration may occur directly from the excised explant or from some other form of cultured tissues such as callus, protoplast or suspension culture. Auxin concentrations in the media are usually critical for organogenesis or embryogenesis. Thus a high auxin with possibly a low cytokinin concentration is usually required for abundant callus growth, whereas for shoot formation low auxin and high cytokinin concentrations are necessary. Auxin alone, or auxin with low cytokinin seem important for root induction, and in tissues that produce somatic embryos, it is usual to transfer unorganised tissue from a high auxin to a low auxin concentration medium, or to medium containing a less potent cytokinin for embryos to form [30]. As well as from vegetative (sporophytic) tissue, plants can be regenerated from gametophytic tissue (immature pollen grains or ovules), a technique used mainly to produce homozygous lines for breeding purposes. Because of the vast numbers of pollen grains in an anther, and hence the large number of potential homozygous lines, anther culture, in which the microspores are cultured within the excised anther, or pollen culture in which

microspores are cultured outside the anther, are invariably preferred to ovule culture, the latter only being used if the former is unsuccessful [29].

3. Measurement of ethylene in tissue culture vessels Although ethylene can easily be detected and quantified by gas chromatography [99], it is of necessity measured outside the plant rather than at or near to its site of action. In order to measure the rate at which ethylene is produced by a plant or by part of a plant it is usually necessary to enclose the plant or excised tissue within a gas-tight container and sample the enclosed atmosphere. Difficulties may arise in relating the findings to ethylene production outside the enclosed system, particularly during long term experiments, and ways of controlling the concentrations of atmospheric gases may be necessary. Measuring ethylene in tissue culture does not present such problems, as it is precisely the accumulation of the gas within the vessel that is of interest. However, although it may be a simple task to measure the ethylene concentration in the culture vessel it may be more difficult to relate this to the rate of ethylene production by the tissue, if this is what is required. The amount of ethylene that will accumulate in the culture vessel will depend not only on the rate of production of ethylene by the plant tissue, but also on the rate it is likely to escape from the vessel. Containers used for tissue culture, even if wrapped with sealing film, may lose ethylene at a fairly rapid rate. Eighty percent of ethylene injected into 30 mm petri dishes, normally used for Brussels sprout anther culture and sealed with Nescofilm to prevent dehydration of the media, was lost from the dishes within 4 h [9]. However, ethylene generated in culture often exceeds leakage, resulting in considerable accumulation [53, 541. Thus following 24 h of anther culture with the Brussels sprouts cultivar Hal, 917 nl ll’ was measured after 24 h in Nescofilm sealed dishes compared to 4 nl l- ’ for unsealed dishes, and 2275 nl 1-l for sealed dishes immersed in saturated ammonium sulphate solution to prevent the escape of ethylene (NL Biddington, unpublished). Often, in order to measure ethylene production from tissue culture, the vessel may be sealed with a ‘gas-tight’ rubber closure, or if this is

175 not possible the tissue is transferred to a vessel sealed in this way. The atmosphere in the container is sampled by inserting the needle of a gastight syringe through the closure, which reseals. However, rubber closures, particularly those made from silicone rubber, are by no means impermeable to ethylene, and some, albeit slight, ethylene loss may need to be considered when they are used [76]. Although sealing culture vessels tightly may allow a better measurement of the rate of ethylene production by the tissue, it does not necessarily measure the accumulation of ethylene under conditions that are normally used for culture. The porosity of the tissue culture vessel to gases may affect growth and development of the culture. Jackson et al. [53, 541 have derived the time in hours for half the ethylene to be lost (tso) from a culture vessel and have used the t,, for comparing and quantifying the ventilation capacities of different vessels sealed in varying ways.

4. Ethylene production by plant tissue cultures Ethylene accumulation in plant tissue culture vessels, first shown by Stewart and Freebairn [ 1091, can be very variable. Gamborg and LaRue [36] measured as much as 890 nmoles g-’ dry wt over a 24 h period in flax suspension culture compared to 5 and 6 nmol in wheat and rice suspension cultures respectively. Thomas and Murashige [ Ill] measured volatiles from several species in tissue culture but they made no direct comparisons between callus and shoot cultures of the same species. Ethylene production showed a very variable pattern with the highest levels in two of the callus cultures. There were large variations between species, with a callus culture of Nicotianum tabacum having over 400 times the concentration of ethylene compared to one of carrot. Although these studies showed that large quantities of ethylene accumulated in culture vessels, particularly during periods of rapid growth, it was not immediately apparent whether or not they had any effect on the tissue culture itself. It is only more recently with the use of compounds that modulate ethylene biosynthesis, action or atmospheric concentration, that some of the effects of endogenous ethylene on growth and development of tissue cultures are being realised.

5. Identifying ethylene effects in plant tissues Ethylene effects can be prevented by potent inhibitors of both ethylene action and biosynthesis [124] or by removing it from the atmosphere by ventilation or by chemicals such as mercuric perchlorate or potassium permanganate [99]. Detailed reviews of ethylene biosynthesis and the use of ethylene inhibitors can be found in the literature [68, 124, 1251. The main agent of control of ethylene biosynthesis in plant tissues is ACC synthase which converts SAM to ACC, the immediate precursor of ethylene. Several chemicals inhibit ethylene biosynthesis; among the most commonly used are AVG and AOA, which inhibit ACC synthase and cobalt ions which inhibit the conversion of ACC to ethylene. No ethylene biosynthesis inhibitor is thought to be specific however. For instance AVG and AOA are inhibitors of pyridoxal phosphate-dependent enzymes of which ACC synthase is one. The silver ion is a very potent inhibitor of ethylene action [7] and has been widely used either as the nitrate or as the more mobile thiosulphate. Norbornadiene, a low boiling point, volatile liquid inhibits ethylene action when used in the gas phase, and as such is particularly useful because it can be applied and removed reversibly. Exposure of tissue cultures to ethylene can be increased by direct introduction of the gas into the surrounding atmosphere or by the addition to the media of the ethylene releasing compound ethephon or the ethylene precursor ACC.

6. Ethylene effects in tissue cultures It is often difficult to divorce unorganised growth from organised growth and development in tissue culture because the latter often appears to proceed at the expense of the former. Other phases of morphogenesis may also be interrelated. Such relationships need to be acknowledged, and the grouping of the following effects of ethylene on different aspects of regeneration under their particular headings are for clarity and are not exclusive. Obviously, understanding what controls the switch from unorganised to organised growth is of prime importance in understanding what controls regeneration.

176 6.1

Callus growth

Although large doses of exogenous ethylene may inhibit callus growth, e.g. with tobacco [12] and lettuce [128], ethylene may also promote callus growth. Early work showed it enhanced callus production from peach mesocarp tissue [13] and ethephon had a similar effect on cotton ovules [1 lo]. Huxter et al. [51] concluded that although much of the ethylene from non-differentiating tobacco callus is a by-product of rapid growth it probably also stimulates callus growth. However, studies with cloned crown-gall lines of Nicotiana and Lycopersicon with different capacities to synthesise ACC and to convert ACC to ethylene failed to show a correlation between ethyleneproduction and organised growth [73] and the authors concluded from their results that ethylene per se is also unlikely to control crown-gall tumour growth. Four different sunflower lines showed different morphological responses to ACC, although there was a tendency for it to enhance the enlargement of cultured hypocotyl segments and induce the production of friable callus, effects associated with cytokinin and auxin respectively [92]. Work with Brussels sprouts (B. oleracea var. gemmifera) suggests that endogenous ethylene may inhibit callus growth: AgNO, was found to be essential for maintaining callus cultures as well as improving regeneration [121]. 6.2 Shoot production Studies have since shown that although ethylene may promote callus growth it may also inhibit shoot production. Thus callus proliferation was increased in Brassica oleracea hypocotyl cultures by the ethylene precursors SAM and ACC whereas shoot initiation was increased by AVG, CoCl, and AgNO, [103]. Likewise, ACC increased callus production in maize but inhibited subsequent shoot formation [106]. Norbornadiene and AgNOj increased shoot production 12-fold without apparently affecting total callus production. AgNO, promoted shoot regeneration in wheat callus culture, and reversed the inhibitory effect of ethylene and 2,4-D on morphogenesis [84]. It is also enhanced shoot regeneration in callus cultures of nonregenerating or weakly-regenerating callus cultures of Nicotiana plumbaginzjiolia [84]. Ethylene inhibi-

tors enhanced shoot production from cotyledon and seedling explants of various Brassica genotypes [ 19, 201. In Ficus tissue cultures more efficient sealing of the culture vessels resulted in reduced leaf expansion, an effect that was reversed if explants were treated with norbornadiene or the ethylene absorbent ‘Ethysorb’ was placed in the vessel [53]. In some studies, ethylene has been reported to increase shoot formation, an effect that may be greater in the presence of CO,. This is surprising, since CO, is often associated with inhibition of ethylene action 11241. Thus ethylene alone or in combination with COZ substituted for BA or BA with 2,4-D in promoting shoot growth in rice callus [25]. In Pinus radiata the presence of both CO, and ethylene appeared necessary for shoot buds to differentiate on cotyledon explants: removal of either gas from the culture vessel reduced differentiation and removal of both gases inhibited it completely [60]. Bud formation on root explants of Chychorium intybus was increased by ethylene and anaerobosis [61], although it is not known if the effect of the latter was mediated via an effect on ethylene biosynthesis. Ethylene also promoted shoot formation in Digitalis obscura tissues in the presence of IAA and kinetin in experiments using mercuric perchlorate ethylene traps or in which ethylene gas was added to the culture vessels [81]. Contrasting effects of ethylene on shoot formation may not be restricted to differences between species. A pronounced change in tissue sensitivity to ethylene with time has been suggested for Nicotiana tabacum tissue [52]. By exposing the callus to varying amounts of both exogenous and endogenous ethylene it was shown that ethylene inhibited shoot primordia formation early in culture but promoted it at a later stage [52]. Shootforming tobacco callus produced less ethylene [52] and less ACC [42] than non-shoot-forming callus. 6.3 Root production The role of ethylene in rhizogenesis in tissue culture seems to vary with the tissue culture system. In Digitalis obscura in the absence of IAA and kinetin, ethylene promoted root formation [81], and ethephon enhanced the production of rooting callus in wheat leaf explants [43]. In the latter the increased callus production was on the youngest (4th) leaf, the development of which was promoted

177 by ethephon. Ethylene produced by thin cell layer explants of tobacco seemed to be associated with root production. Roots were produced at 25, 30 and 35 “C when ethylene production was relatively high but not at 15 and 40°C when ethylene production was very low [62]. STS prevented root formation but induced the formation of vegetative buds. AgNO, inhibited rooting of Brassica juncea explants and AVG inhibited root elongation in several Brassica genotypes in vitro [20]. In tomato leaf discs cultured in vitro however, exogenous ethylene, and apparently endogenous ethylene, both inhibited IAA-induced root regeneration [23]. In Prunus avium shoot cultures ACC inhibited and AVG promoted root formation. These differing responses to ethylene reflect the variable effects reported for ethylene in the extensive literature on rooting of cuttings [75]. 6.4 Other forms of organogenesis Reports of ethylene involvement in other forms of organogenesis in tissue culture are fairly rare. Ethylene induced inflorescence production in cultured intermode segments of the short day plant Plumbago indica [77], an effect that was subsequently demonstrated with ethephon under non-inductive long days in conventionally grown Plumbago plants [78]. ACC increased and AVG and AgNO, inhibited flower bud formation in thin-layer explants from tobacco pedicels at 7 days but at 14 days AgNO, increased and ethylene inhibited bud formation [108]. Ethylene applied to lettuce pith explants cultured in vitro inhibited xylogenesis at 2nll-‘, a concentration 50-fold less than that necessary to reduce callus growth [128]. However, further work suggested that 2nll-’ ethylene is supraoptimal and that endogenous ethylene plays a positive role in lettuce xylogenesis [74]. Potato shoots produced in vitro from nodes of sprouted tubers grew less well [49, 53, 821 with shortening and thickening of the stems, and tuber production was reduced [50] the more completely the vessels were sealed. Ethephon and sealing had a similar effect as on shoot growth, and normal growth could be established and tuber production improved in sealed vessels by the inclusion of a vial of mercuric perchlorate solution [49, 50, 531. STS also improved growth in both sealed and unsealed boxes and reduced ethylene accumulation in sealed

boxes [82]. It was suggested that the superior growth in the unsealed boxes, compared to that in the sealed ones, was because of ethylene accumulation in the latter, although ethylene was not measured in the unsealed ones. Hypertrophy of the lenticels in potato tubers produced in vitro also resulted from ethylene accumulation in the culture vessel [53]. 6.5 Somatic embryogenesis As with shoot regeneration ethylene generally appears to inhibit somatic embryogenesis. Both ethylene added to the culture vessel atmosphere [ 1121 or ethephon added to the medium [ 112, 1221 inhibited carrot somatic embryogenesis. Ethephon caused much greater inhibition than the free gas, presumably because, at the concentrations used, it resulted in the tissue being exposed to a much higher concentration of ethylene [ 1121. However, it also appeared that a non-volatile as well as volatile component from the ethephon inhibited embryo production [112]. Although the inhibition caused by the addition of Sppm ethylene gas was slight, the concentration was sufficient to produce severe epinasty in the developing plantlets, suggesting that ethylene produced by the tissue would have had little or no effect on embryogenesis, no epinasty having been reported in the untreated controls. More recently however, the ethylene biosynthesis inhibitors CoCl,, NiCl,, salicyclic acid and acetylsalicylic acid [94,95] were shown to inhibit ethylene production and promote embryo formation in carrot cell cultures, suggesting that embryo production was promoted by reduced ethylene biosynthesis. AgNO, also promoted embryogenesis in the same system [96]. In Picea abies (Norway spruce) ethylene production was much higher in non-embryolic callus cultures than in embryogenic ones [120]. The authors suggested that the apparent association of a high rate of ethylene evolution with unorganised callus growth in Norway spruce might be indicative of a more general relationship between high ethylene production and a lack of morphogenic potential in conifer cell cultures. They cited the fact that loblolly pine suspension cultures that had never demonstrated any potential for morphogenesis also evolved considerable amounts of ethylene [79]. Non-embryogenic suspension cultures of Picea glauca (white spruce) also

178 accumulated higher levels of ethylene than embryogenie cultures. Cell growth and formation of proembryos was inhibited by sealing the flasks with serum caps or incubating the cultures with high levels of ethylene and CO2 [59]. The production of embryogenic calli in Hevea brasiliensis could be increased either by avoiding the accumulation of ethylene in the culture vessel, removing ethylene with mercuric perchlorate, inhibiting ethylene production with AOA or by the addition of AgNO, to the medium [3]. Sunflower hypocotyl callus produced more embryos from light-grown seedlings than from dark-grown ones, although treating dark-grown seedlings with AVG 4 days prior to culture and reducing ethylene synthesis increased subsequent regeneration to that from light grown seedlings [91]. However, light grown seedlings produced slightly more ethylene than dark grown ones, which suggested that different rates of ethylene production in light and dark were not the cause of the differences in regeneration from seedlings grown in the two environments. ACC applied to the seedlings inhibited AVG-induced promotion but for only the first 4 days after the AVG treatment, after which time it became ineffective. It was suggested that differences between light and dark were due to differences in sensitivity to ethylene, and that sensitivity became less with time [91]. In Medicago sativa it was not possible to demonstrate a relationship between ethylene and somatic embryogenesis using inhibitors of ethylene biosynthesis. Salicyclic acid inhibited somatic embryo production; CoCl, and NiCl,, while not affecting embryo induction, strongly retarded embryo formation [70, 711. Other ethylene biosynthesis inhibitors, namely AVG, AOA and DNP also inhibited somatic embryo formation at concentrations that had no effect on tissue growth [71]. Although CoCl, and NiCl, inhibited ethylene biosynthesis, AVG, AOA and DNP did not affect ethylene production and salicyclic acid even increased it [70, 711. Whether cobalt or nickel ions, which are non specific inhibitors of ethylene biosynthesis, retarded embryo formation by lowering ethylene levels or by some other effect on metabolism is not known. This work demonstrates the need for caution in interpreting results from inhibitors of ethylene production, and the necessity to check that they are indeed blocking biosynthesis. Meijer [70] found only small, but possibly important

differences between ethylene evolution from tissue cultures of 2 genotypes of M. sativa, one highly embryogenic and the other virtually nonembryogenic. Ethylene production initially increased rapidly, peaked after 10 days and then declined sharply, the decline being greater when the cultures were transferred to hormone-free, embryoinduction medium. This decline was greater in the embryogenic culture. It is possible that the lower ethylene levels in the embryogenic genotype may have been related to greater embryogenic competence. In contrast to all the above findings, low concentrations of ethephon (0.01-l .Omg 1-l) increased embryogenesis in citrus ovular callus, although higher concentrations proved inhibitory [57]. Ethylene may also have promotive effects on embryogenesis that are to some extent indirect. Thus with Zea mays ethylene inhibitors enhanced the production of callus suitable for establishing embryogenic suspension cultures, as well as improving the regenerative capacity of the callus [114; 1151. Thus as with shoot formation, ethylene may have positive effects on somatic embryogenesis in certain species. 6.6 Suspension culture The early work of Gamborg and LaRue [36, 371 and MacKenzie and Street [66] suggested that the high concentrations of ethylene that accumulated in the atmosphere of cell suspension culture vessels during the exponential phase of growth had little positive or negative effect on growth. Although ethylene concentrations above 1Oppm were found in suspension cultures of Acer pseudoplatanus there was no evidence to suggest that its presence was inhibitory to growth [66]. Neither did ethephon stimulate growth. Gamborg and LaRue [37] also found that 10% ethylene, lOOO-fold greater than that which normally accumulated in the culture vessels, had very little effect on the growth of Ruta, Rosa and wheat cell suspension cultures. Jackson et al. [54] have questioned the methodology used by the latter to investigate the effects of a hormone that is potent at sub-ppm levels. However, Sauerbrey et al. [loll in a much more recent study, using AVG and other inhibitors of ethylene biosynthesis and action in 2,4-D-dependent sunflower suspension cultures, concluded that the

179

rapid rise in ethylene production, which occurred mainly during the exponential growth phase did not play a role in initiating and maintaining cell division, and that ethylene was a byproduct of the rapid growth. Although AVG and several other compounds inhibited both growth and ethylene production, ethylene biosynthesis was more sensitive to AVG than was growth, suggesting that there was no relationship between the two. Also, ethephon did not overcome the AVG inhibition, and although ACC partially reversed the AVG effect, this appeared to be related to a reduction in AVG uptake caused by the simultaneous application of the ACC. There are however other reports indicating that ethylene does have inhibitory effects on plant cell suspension cultures. Removal of ethylene with mercuric perchlorate showed that ethylene accumulating in culture vessels inhibited greening and growth of spinach cell suspension cultures [26]. Kumar et al. [59] also showed that growth of cell suspension cultures of Picea glauca and multiplication of proembryos were reduced if the culture flasks were sealed with gastight serum caps as compared to foam bungs. Measurements of ethylene and CO, in the flasks and treatment of the cultures with enhanced levels of ethylene or CO,, suggested that accumulation of the two gases in the sealed flasks caused the reduction in growth and proembryo formation. In contrast to the above reports, growth promotion by ethylene of suspension cultures has been shown with tobacco cells treated with ethylene dissolved in water [35]. Another potentially useful effect has been shown with soybean cultures. Synchronising growth and cell division in suspension cultures is important for fundamental studies. Flushing soybean suspension cultures with 3% ethylene for 3 h every 36 h, particularly if it was followed by 3 h of 3% CO, and 30 h of aeration, induced partial synchrony in the cultures [24]. This effect was presumed to be a consequence of an arrest of the cell cycle in two zones of the interphase. Although ethylene alone enhanced growth slightly, a greater effect was obtained using the above gassing regime. 6.7 Anther

culture

The few experiments

on the role of ethylene in

anther culture suggest that the effects of ethylene on microspore embryogenesis depend very much on the species, or even the cultivar. In barley anther culture the response to ethylene appeared to be genotype-dependent [22]: ethephon or ACC increased embryogenesis in two cultivars whose anthers had the lowest concentrations of ACC and produced ethylene the most slowly, whereas embryogenesis was enhanced by the ethylene synthesis inhibitor putrescine in a third cultivar whose anthers contained the highest amounts of ACC and produced ethylene the most rapidly. In Brussels sprouts (B. oleracea, var. gemmifera) anther culture, ethephon and ACC inhibited embryo production [9] and AgNO, promoted it, particularly with non- or poorly-responsive cultivars [IO]. Ethylene production by the anthers of a Brussels sprouts cultivar which responded poorly in anther culture was 20-fold higher than in a highly responsive cultivar after 6 h of culture, suggesting that the low embryogenic responses of some Brussels sprout cultivars may result from high rates of ethylene production by the anthers [9]. Brassica anthers usually require a period of elevated temperature at the start of the culture period if embryos are to be produced [8] and high temperature is known to inhibit ethylene biosynthesis in other tissues [17, 1261. Although ethylene production was reduced following a 35 ‘C treatment in Brussels sprouts anthers, AVG failed to promote embryo production in the absence of a high temperature treatment. This suggests that the hightemperature induction of embryogenesis is mediated through factors other than, or in addition to, reduced ethylene biosynthesis. The effect of ethylene in N. tabacum anther culture appeared more equivocal [28]. Removal of ethylene from the culture vessel atmosphere could either enhance or retard embryo induction, embryo survival and the number of plantlets produced, depending on the size of the vessel and the age of the anthers. Although AgNO, only increased embryo induction slightly the comparison was made with a very high yielding control. However, Horner et al. [47] reported that removal of ethylene produced during anther culture of Nicotiana neither promoted nor inhibited embryogenesis, although their interpretation of their data has been questioned [28]. Positive responses to ethylene were seen in anther culture of Solanum carolinese [88] and Datura

180 in which embryo yields were reduced by inhibitors of ethylene action or biosynthesis and increased by ethephon or precursors of ethylene biosynthesis.

mentel[4]

6.8 Protoplasts

There are few reports of ethylene effects in protoplast production or regeneration. However, STS improved protoplast yield and callus production from the protoplasts when applied to donor potato shoot cultures or the protoplasts themselves [82]. ASA, an inhibitor of ethylene biosynthesis [64] and STS both reduced ethylene production from donor plants and also during protoplast release when included in the maceration fluid [82]. STS also enhanced the transient expression of an alien gene for chloramphenicol acetyltransferase in the protoplasts. ASA also promoted colony formation from protoplasts isolated from embryogenic suspension cultures of maize [ 161. However, salicylic acid had no effect on colony formation, and it was suggested that enhancement of colony formation by acetylsalicylic acid was not related to ethylene production, although no measurements of ethylene concentrations were made [16]. 6.9 Secondary

product

biosynthesis

Suspension and immobilised cell cultures offer the potential for commercial production of secondary products [46]. Because of the large quantities of ethylene that can accumulate in the atmosphere of cell culture vessels, an understanding of ethylene effects on the biosynthesis of such products would seem important. Ethephon has been shown to promote alkaloid production in both CoDa arabica and Thalictrum rugosum cell suspension cultures [21], and it was suggested that because the biochemical pathways in the two species differed widely, ethylene may be useful for stimulating secondary product biosynthesis in other systems. However the production of sanguinarine by Papaver somniferum cell cultures appeared not to be influenced by ethylene [107]. Further studies are obviously necessary. 6.10

VitriJication

Vitrification

is a serious physiological

disorder

which affects tissues propagated in vitro, producing a waterlogged, translucent appearance associated with a lack of lignification and hence a lack of cell wall rigidity [55]. It seems to be related to a number of factors associated with the culture medium [see references in [69]. A role for ethylene in vitrification is uncertain, although it has been suggested that certain stress conditions result in a burst of ethylene biosynthesis by the explant which, in the absence of subculturing, subsequently inhibits further ethylene production, and it is the low ethylene production which results in the reduced lignification [55]. However ethylene production remained higher in vitrified compared to non-vitrified carnation tissue if subcultured daily and added ethylene did not induce vitrification and ethylene biosynthesis inhibitors did not prevent it [56]. This suggests that changes in ethylene production associated with vitrifying conditions do not affect the vitrification process. More recently experiments with apple plants failed to implicate ethylene in vitrification but showed that cytokinin (BA) could induce the condition [80]. 6.11

Chlorophyll

biosynthesis

Ethylene accumulating in culture vessels has been shown to inhibit chlorophyll production in spinach cell suspension cultures [26] and Magnolia shootlets cultured in vitro [27]. 6.12

Long

term efsects of ethylene

in tissue culture

Most studies have concerned the short term effects of ethylene on a specific tissue or an individual aspect of growth in vitro. Some mineral components of tissue culture media have been shown to affect ethylene biosynthesis, such as cobalt [127], calcium and magnesium [18, 651 and phosphate [ 1051 and hence tissue culture media may influence the growth and development of plants in vitro by affecting ethylene biosynthesis. Bartolo and Macey [5] grew tissue cultures in the absence of cobalt, which is included in most media at a concentration of 0.025 mg 1-l (0.105 PM), and which is commonly used as an ethylene biosynthesis inhibitor [124]. Absence of cobalt resulted in fewer plantlets and abnormal leaf structures in Brassica oleracea (broccoli), and reduced viability and increased leaf abnormalities in PassiJiora mollissima Bailey,

181 whereas in Saintpaulia ionantha Wendl, there was an increase in growth rate and plantlet numbers in the absence of cobalt. The authors suggested that increased ethylene biosynthesis by the tissues in the absence of cobalt may have accounted for the differences in the cultures, although they did point out that inhibition of ethylene biosynthesis in plant tissues requires concentrations of cobalt 100 times greater than that used in tissue culture media. However, such information has come from relatively short exposures to cobalt, and over long periods lower concentrations may be effective. Ethylene concentrations were not measured in these experiments. In another study on the effect of ethylene on overall growth and development Lentini et al. [63] grew rapid-cycling Brassica campestris from seed to maturity in vitro in sealed containers. Ethylene accumulation was associated with inhibition of development, including fewer and smaller leaves and abortive flowers or no floral buds. Plants treated with norbornadiene or grown in vented vessels developed normally to maturity. Although these experiments [63], did not involve regeneration as such, the growing of the plants under ‘in vitro’ conditions has obvious relevance to tissue culture.

7. Relationships between ethylene and other biologically active compounds Successful in vitro regeneration nearly always depends on the use of hormones, usually auxins and/or cytokinins, at some stage in the culture. Hence most effects of ethylene on tissue cultures might be said to involve some interaction with other hormones. Ethylene biosynthesis can be affected by other growth regulating compounds and ethylene can influence the levels of hormones and other biologically active compounds within the plant. CO, can also inhibit or delay many ethylene responses [ 1241. 7.1 Auxin The most commonly studied relationship is of the effect of auxin on ethylene biosynthesis. Auxin has long been known to stimulate ethylene biosynthesis and it appears that many auxin effects are mediated by this promotion of ethylene production [125].

The relationship between auxin and ethylene production in tissue cultures has received little attention despite the requirements of most types of culture for auxin in some form. What evidence there is suggests that auxins in tissue culture enhance ethylene production, but the relationship between the resultant increase in ethylene biosynthesis and growth and development appears more difficult to define. Thus although 2,4-D enhanced ethylene production in suspension cultures of Acer pseudoplatanus [66] it seemed that this effect was independent of its effect on culture growth. In sunflower cell suspension cultures a greater concentration of 2,4-D was required to increase ethylene production than was necessary to enhance growth, again suggesting that the two effects were independent [loll. In carrot callus cultures the inhibition of embryogenesis by 2,4-D was also unrelated to ethylene evolution, although surprisingly ethylene production fell slightly in the presence of the auxin [ 1121. Further evidence that a link between ethylene production and auxin effects on tissue culture may be tenuous, can be seen with results from tobacco callus. Grady and Basham [42] suggested that the production of less ethylene [52] and ACC [42] from shoot-forming tobacco callus compared to nonshoot-forming callus was because of the absence of auxin (NAA) in the medium of the shoot-forming tissue. However, although Grady and Basham induced shoot formation and increased ethylene production by removing NAA from the medium, Huxter et al. [52] did not alter the auxin concentration but changed the composition of the medium in other ways, including increasing the cytokinin (kinetin) content. In Prunus avium shoot cultures ACC inhibited and AVG promoted root formation whereas IBA promoted both root formation and ethylene biosynthesis [l I], indicating that the IBA effect on root induction was unrelated to its effect on ethylene synthesis. Although the above reports did not establish a link between auxin effects on tissue culture and ethylene biosynthesis, others have suggested that such a relationship may exist. In NAA-induced flower bud production in thin-layer explants from tobacco pedicels a negative link was suggested [log]. Although NAA increased ethylene production, ethylene reduced flower bud formation and made the tissue less sensitive to NAA, whereas the NAA effect was enhanced by AgN03. The

182 conclusion drawn was that under normal culture conditions (i.e., with just NAA) the regenerative capacity of the tissue was submaximal as a result of NAA-promoted ethylene biosynthesis. A more obvious positive relationship between auxinpromoted ethylene biosynthesis and tissue culture response was seen in anther culture of Solanum carolinense, in which different effects of 2,4-D and IAA appeared to be related to different rates of ethylene production by the tissue in response to the two auxins [86,87, 88, 891. When cultured on IAA, which stimulated ethylene production [88], the microspores formed embryos [87] but when cultured on media containing 2,4-D ethylene did not accumulate [89] and callus was formed [86]. As well as auxin stimulating ethylene production, ethylene has been reported to reduce endogenous auxin concentrations probably by conjugation of IAA to form indole-3-acetylaspartic acid and/or decarboxylation [see references in 971. Ethylene also inhibits polar auxin transport [39]. Evidence for ethylene effects on auxin concentrations or movement in tissue cultures is sparse. Both ethephon and ethylene, albeit at high concentrations, reduced the growth of habituated cultures of N. tabacum var Xanthi, effects that were reversed by NAA [12]. The results suggest that addition of NAA compensated for a reduced availability of endogenous auxin that resulted from the ethylene treatment. Although no effect of NAA on ethylene production was found (no ethylene could be measured in the culture vessels not treated with ethephon, either in the presence or absence of NAA), later work [58] showed ethylene in habituated cultures of Xanthi tobacco with large increases in auxin (2,4-D or IAA) treated tissues. The effects of ethylene on adventitious bud formation in vitro from bulb-scale explants of Lilium speciosum appear to result from effects on polar auxin transport [116, 1171. Ethylene applied on the 3rd or 7th day of culture promoted bud formation and suppressed the predominantly basipetal polarity of the regeneration sites. The promotion by ethylene was not as great as that by NAA. There was a slight synergism between the two hormones and it was suggested that ethylene increased the tissue sensitivity to NAA. AVG inhibited bud formation and its effect was reversed both by ACC and the auxin-transport inhibitor TIBA. It was suggested that inhibition of polar auxin transport

by ethylene or TIBA resulted in an accumulation of intracellular auxin resulting in enhanced bud production over the whole explant [116]. 7.2 Other hormones Although there are many reports of ethylene production being influenced by other hormones [40, 68, 90, 1041 there is little information relating to such effects on tissue culture. Both ABA and ethylene induced callus formation in cultured buds of Shamouti orange (Citrus sinensis) [41]. ABA increased ethylene biosynthesis and ABA-induced callus formation was inhibited by rhizobitoxine, an inhibitor of ethylene biosynthesis, and it was concluded that the effect of ABA on callus formation was mediated via ethylene [41]. Both ABA and ethephon also stimulated embryogenesis in ovular callus from Shamouti orange although in that study ethylene production was not measured [57]. The rapid evolution of ethylene by cell suspension cultures prompted Sauerbrey et al. [ 1021 to suggest that they should provide an appropriate tool for analysing the effects of biologically active compounds on cellular ethylene biosynthesis: hence they used sunflower cell suspension cultures to study the inhibition of ethylene biosynthesis by various growth retardants. 7.3 Polyamines Polyamines, of which spermidine and spermine and their diamine precursor putrescine are the most common in plants, are not usually classed as plant hormones, although they do appear to act as endogenous growth regulators [38]. Endogenous polyamines have been implicated in the control of organogenesis and embryogenesis in vitro [15, 31, 341. Meijer and Simmonds [72] working with Medicago sativa embryogenesis suggested that their importance lay not in the control of embryogenesis, but rather in a more general reprogramming of cells in new patterns of development, which would encompass regeneration as a whole. Polyamines and ethylene inhibit each other’s biosynthesis, and their endogenous concentrations tend to be inversely related, possibly because both have SAM as a common precursor [see references in 381. Thus any effect of ethylene on tissue culture needs to take account of possible polyamine effects and vice

183 versa. Very few such studies have been done however. In barley anther culture inhibition of ethylene biosynthesis by putrescine appeared to be related to its effects on embryogenesis [22]. Also, the activity of arginine decarboxlase, a key enzyme of the polyamine biosynthesis pathway, was reduced in carrot suspension cultures by ethephon, which inhibits carrot somatic embryogenesis, and was stimulated by AgNO, which promotes embryogenesis [96]. It was suggested that ethylene may inhibit embryogenesis by reducing polyamine biosynthesis. Biondi et al [I I] showed that ethylene inhibited and that polyamines appeared to play a positive role in root production in Prunus avium shoot cultures, although the reciprocal relationship between the two biosynthetic pathways was not particularly evident. In Brassica hypocotyl explants however ethylene precursors and putrescine and spermidine enhanced callus production and the polyamines increased ethylene biosynthesis, evidence which did not support the ‘competition for SAM’ hypothesis [103]. Both ethylene and polyamines have been studied in somatic embryogenesis of Medicago sativa [70,72] although no relationship between the two was reported. 7.4 Brassinosteroids

Brassinosteroids, which were first identified in Brassica napus pollen and have since been found in other tissues and organs act synergistically with auxin as potent growth promoters [67], although their possible role in plant growth and development remains to be elucidated. There are few reports of brassinosteroid effects in tissue culture. They increased carrot cell enlargement but not cell division [6,98] whereas they inhibited the growth of transformed N. tabacum callus cultures [93]. Brassinosteroids stimulate ethylene biosynthesis [2], but this has not been studied in tissue culture. 7.5 co,

Because CO, can inhibit or delay many ethylene responses [124], it would seem likely that its presence in tissue culture vessels might prevent some of the effects resulting from ethylene accumulation. However there appears little evidence of this being so. The promotion of shoot formation in vitro by CO, and ethylene together [25, 601, has already

been discussed (see 6.2), and inhibition of leaf expansion in Ficus by ethylene seemed to be unaffected by the presence of CO, evolved from the tissue [53]. 7.6 Ethylene-regulated

ethylene

biosynthesis

As well as its effects of other growth regulators, ethylene can regulate its own biosynthesis, either stimulating it or inhibiting it [125]. Such effects will presumably be of importance in controlling ethylene concentrations in tissue culture vessels, although this aspect of ethylene physiology, as it might relate to tissue culture, rarely seems to have been considered. The possible relationship between a low rate of ethylene biosynthesis resulting from an initial burst of ethylene production and vitrification has been mentioned previously (6.10).

8. Conclusions It seems that ethylene has many varied effects on tissue culture growth and development, and its production by the tissues and accumulation in the culture vessels needs to be taken into account in several tissue culture systems. The fact that ethylene may often have diverse, and indeed opposite effects in similar systems, demonstrates how far away we are from measuring what the primary effect of ethylene is at the molecular and biochemical level. However, molecular biology techniques are now available that will lead to the identification and cloning of genes that control ethylene biosynthesis [45, 100, 1181 and will enable studies to be made of ethylene effects on gene expression. Such research, coupled with work on identifying ethylene receptor proteins [44], will ultimately provide a much greater understanding of how ethylene acts, and explain many of the anomalies. However, it may still be possible to account for many of the differences at the whole tissue physiology level. Ethylene production rates and concentrations may be critical and may have varying effects on different stages of tissue culture growth and development. Also very little account has been taken of possible interactions with the many variables that characterise different tissue culture experiments, such as media composition, metabolites produced by the tissues, including

184 volatiles such as CO*, ethane, acetaldehyde and ethanol [ 111, 1131, or environmental factors such as light or temperature, both of which are known to affect ethylene biosynthesis [17, 32, 48, 1261, and may also affect tissue response to ethylene [123]. More research is needed to understand the diverse ways in which ethylene has been reported to influence in vitro growth and development, in order that such knowledge can aid in a more rational approach to improving the efficiency and range of plant tissue culture.

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