PHYSIOL. PLANT. 53: 181-187, Copeiihsigen 1981 Mass propagation of globe artichoke (Cynara scolymus): Evaluation of different hypotheses to overcome vitrification with special reference to water potential p. Debergb, Y. Harbaoui and R. Lemenr Debergh, P., Harbaoui, Y. and Lemeur, R. 1981. Mass propagation of globe artichoke {Cynara scolymus}: Evaluation of different hypotheses to overcome vitrification with special reference to water potential. - Physiol. Plant. 53: 181-187. In search of a technique for rapid clonal propagation and sanitation of Cynara scotymus L. we have been confronted with the problem of vitrification. We only succeeded in overcoming this problem by raising the agar concentration of our medium to 1.1% instead of 0.6%. By using the Cbardakov-method and direct measurement of the water potential with a thermocouple psychrometei we were able to prove that this result was attributable to the matric potential. Additional key-words: Agar. P. Debergh and Y. Harbaoui, Laboratorium voor Tuinbouwplantenleeli; R. Lemeur, Laboralorium voor Plantecologie, Rijksuniversiteil Gent, Coupure Links 533, 9000 Gent, Belgium. Y. Harbaoui, (present address) Institut National Agronomique (I.N.A.T.}, Avenue Charles NicoUe 43, Tunis, Tunisia. and very pronounced elongation-growth and reach lengths which are 5 to 6 times greater than those of In search of a technique for rapid clonal propagation normal leaves; and finally, the original green leaves beand sanitation of globe artichoke {Cynara scolymus L.) come almost translucent and later necrotic. (Harbaoui and Debergh 1980), we have been conMicroscopical observations revealed that vitrified fronted with a frequently occurring (different personal leaves do not have palisade ttssue, but only spongy communications) but never or only vaguely described mesophyll. (Quoirin and Lepoivre 1977, Boxus et al. 1978, 1979; This phenomenon has been observed in each stage of Zuccherelli 1979) physiological phenomenon for which the ctilture: initiation, elongation and propagation. we use the name "vitrification". In this study we review the different hypotheses A few examples of other plants in which we have which might be put forward to overcome this problem. observed vitrification are: Allium porrum L. (leek), At the same time, a solution to eliminate vitrification in Apium graveolens L. (celery), Brassica oleracea L. var. our tissue culture system of globe artichoke is prebotrytis L. (catiliflower), Dianthus caryophyltus L. (car- sented. nation), Ficus lyrata Warb., Malus spp., Pinus spp., Prunus spp., Raphanus sativus L., Sequoia semperviA bbrevialions rerts (Lamb.) Endl. BA 6-benzyladenine For glohe artichoke the symptoms during the process BM basic medium CCC 2-chloroethyl-trimethyl-ammoniumchloride of vitrification could be described as follows: the GA3 gibberellic acid leafblade and the petiole become humid; especially very IAA ii3dole-3-acetic acid young leaves in the centre of the rosette become very 2iP 6(2'-isopentenylaminopudne) turgescent; small turgescent leaves manifest a stidden K 6-furfurylaminopurine Introduction Received 8 January, 1981; revised 5 March, 1981; finalized 10 May, 1981 PlijsW. Plant. 53, 1981 0031-9317/81/100181-07 $ 03.00/0 © 1981 Physiologia Piantarum 181 M W Wfi Wj Wni S TIBA The media were sterilized in an autoclave at an overpressure of 0.5 kg for 30 min. Gibberellic acid was filter-sterilized and added after autoclaving. mannitol water potential osmotic potential pressure potential matric potential sucrose 2,3,5-triiodobenzoic acid Material and methods For each experiment we have scxired the results of a minimum of twelve containers per treatment and each experiment was repeated at least twice. For the initiation and development stage and for the propagation stage test ttibes ( 0 25 tnm, length 150 mm) and glass jars (content 250' ml,, 0 60 tnm) were respectively used. For the sake of clarity some details, particularly those relating to the experimental validation of a hypothesis, will be described and discussed in the section "Results". Cultural conditions All cultures were maintained in a growth chamber. Artificial tight was provided by parallel fluorescent tubes installed above and below the cultures. The luminous intensity was 2000 lux being the eqtiivalent of 31.4 |.iE m~^ s~' of photosynthetic active radiation. The atnbient temperature varied between 2] and 25°C. Humidity was not controlled. Water potential measurement In order to evaluate the water potential of our media, the technique developed by Chardakov (Salisbury and Ross 1969) was used first. The method was slightly modified compared to the original technique. Two sets of test tubes each containing 20 ml of the graded solutions of known concentraPlant material Since 1977 we have been using in our experitnents a tions were prepared. In one set we introduced a cylinder clone of Cynara scolymus L. cv. Violet d'Huyeres, of agar (10 mm high, 0 8 mm) and a drop of a concenselected at S.A.M. (Station d'Appui Manouba, la Man- trated methylene blue solution (this does not signifiouba, Tunisia) by Zitouni. The plants were cultivated in cantly change the osmotic potential). Ample time was the south mediterranean climate of Tunisia and also in allowed for equilibration. Then a small drop of the colthe temperate climate of Belgium. This clone was pro- oured test solution was introduced with a fine syringe in the middle of the control solution. pagated vegetatively by off-shoots. The cultures were initiated with apical buds containA second series of water potential measurements was ing a few leaf primordia, approximately 0.5—0.8 mm in performed with a thermocouple psychrometer (type height. Wescor, model HR-33 with C-52 sample chamber). The sterilization procedure is the same as the one Samples of the ctilture media were left in the sealed described by Harbaoui and Debergh (1980), with al- chamber for 10 min, so that reasonable vapour equilibrium between the sample and the air in the chamber was cohol omitted. For the subcultures the explant comprises a single obtained before measurement. For liquid media the sample chamber was loaded with a filter paper disk ( 0 6 shoot or a cluster of a few buds and shoots. mm) wetted with the test solution. The solid agar media were prepared in small aluminium containers, which Culture medium The basic medium (BM) contained: Murashige's & had been moulded in the sample holder. The psychroSkoog's macro salts and FeEDTA-solution (1962), meter was used in the dew point mode. The water poNitsch's & Nitsch's micro salts (1969), thiamine • FICl tential of the medium was determined after applying a 10 s cooling current. The dew point depression was re0.4 mg/l, myo-inositol 100 mg/l, sucrose 20 g/1. For solid media Difco agar was added in different corded on a strip chart recorder (see Bristow and De concentrations. For liqtiid cultures the bottom of the Jager 1980). The dew point psychrometer was calibracontainer was filled with cotton. After adding the hor- ted against NaCl standards in bars at 20°C. mones the pH was adjusted to 5.0 for liqtiid media and 5.8 for solid media. The hormonal additives for each stage of the culture Results (Harbaoui and Debergh 1980) are presented in Table 1. After 3 years of experimentation we can conclude that the provenance (Tunisia or Belgium), and the Tab. t. Hormonal additives (in mg/I) for each of the stages of physiological stage (developmental stage of the mother plant at the tnoment of inoculation) do not interfere the culture. with vitrification. Additive IAA GA3 2iP 182 Initiation 1 0.025 1 Development Propagation 1 0 20 Temperature treatment Boxus effl/.(1978) were able to reduce the frequency of vitrification in their culttires of Pruntis and Malus spp. by a 3 to 4 weeks stay in a cold room (3-4''C). During Physiol. Plant. 53, ISgJ this treatment BA was omitted from the medium and the leaves were removed (Druart, 1978 personal communication), Similar treatments with globe artichoke did not give positive results; all the material was completely necrotic at the end of the treatment. Air volume Thomas and Murashige (1979a, b) stated that volatile compounds, particularly concentrations of ethanol, are related to some organogenic reactions of the plant material. In series of experiments we tried to detect if the concentration of volatile compounds could affect vitrification. Comparable explants as used for propagation were inoculated in equal volumes of liquid medium, but with differing air volumes in each type of container. Each container received 50 ml of culture medium; the volume of the containers being 250, 500 and 1000 ml. The type of lid was the same for each container, viz. Rotisac-plastic (Reynold films Inc.). The opening of the different containers was approximately the same. Notwithstanding the volume of the containers the vitrification was comparable (in time and in frequency) in each of them and reached an average of more than 50% of the shoots produced. Consistency of the medium A change in consistency of a medium can control differentiation (White 1939) or influence the propagation (Miller and Murashige 1976). We did not observe differences in differentiation between liquid and solid (agar 0.6%) media of the same composition, but the propagation ratio was higher in liquid than in solid media, viz. ±4/3. The frequency of vitrification was comparable (more than 50%) in both cases, all the other characteristics being the same. Changes in the formulation of the major elements Quoirin and Lepoivre (1977) reduced the frequency of vitrification in their cultures of Prunus by replacing the original Murashige & Skoog's macro salts formulation (1962) by one containing no chlorine ions, that is, the medium of Lepoivre (Quoirin et al 1977). The same modification of our basal medium was withotit effect, neither negative nor positive, on growth in general and vitrification in particular. In analogy with treatments to overcome water core in lettuce, we raised in our basal medium on the one hand the concentration of NO3~ (Van Oijen 1979) and on the other hand the concentration of Ca^"^ (Benoit 1980). Ca^+ was supplied as NajCaEDTA (1, 2 or 3 times 1119 mg/l) with and without correction of the Cl" concentration with HCl. In all treatments with NajCaEDTA the cultures became 100% necrotic. As usual control showed more than 50% of vitriflcation. Modifications of the level of Physiol. Planl. 53, 1981 NH4NO3 or addition of urea were without positive effect as well. Anti-gibbereliins One of the characteristics of vitrified plants of globe artichoke are elongated leaves with a very pronounced midrib. It has been observed that gibberellic acid can also induce similar phenomenon (Thimann 1977). To test the probability of a causal relationship between vitrification and the activity of endogenous gibberellic acid, we tested CCC, an anti-gibberellin. Mother plants were treated with CCC either as foliar sprays (0, 2000 and 4000 mg/l, 1 to 6 times with intervals of 3 weeks) or incorporated in the culture medium (0, 0.200, 2 mg/l a.s.). At regular intervals CCC-treated-plants were cultured in vitro, starting with shoot apices. None of the treatments improved the results from the point of view of vitrification, but there was a higher degree of expiants with a quicker reaction at the initiation. Cytokinin effects James (1978, personal communication) formulated the hypothesis that vitrification could be the result of the accumulation of some remnants of the catabolism of synthetic cytokinins. This idea also is plausible for globe artichoke as we have stated that the frequency of vitrification is higher with the synthetic cytokinin BA (up to 100%). To check possible dilution of hypothetically responsible remnants, non-vitrified-expiants were maintained on media without cytokinin for three successive passages. We also investigated the possibility to adsorb this hypothetical product by adding 0.3% charcoal (autoclaved separately) to the medium without cytokinins; this treatment was also used on three successive subcultures. The same trend of losses (>50%) was maintained at each subculture, with or without charcoal in the medium. Auxin effect Due to the influence of auxin the mechanical properties of cell walls change; they become much more plastic (Thimann 1977). As one of the characteristics of vitrified leaves is the occurrence of ceils filled with water, an approach to overcome vitrification consisted in trying to change the plasticity of the cell wall by tising anti-auxins. The incorporation of TIBA (200 mg/l) in the propagation mediurn did not improve the results. Osmotic requirements From the results of Thorpe's group (Brown etal 1979, Thorpe and Brown 1980) it is clear that the water potential of a tissue culture system is a determinating factor in differentiation. The treatments entimerated in Table 2 were applied to change the osmotic equilibrium of the culture system 183 Tab. 2. Influence of the addition of different concentrations of carbohydrates (sucrose or mannitol) or agar (Difco-Bacto) on the frequency of vitrification and on the propagation ratio. The cytokinin content is 20 mg/i 2iP. The explant is an isolated shoot IO—20 mm in height. The prop,agation ratio is the number of shoots and macroscopically visible buds after 5 weeks of culture. Values followed by the same letter do not differ at P £ 0.05. Addition BM BM + BM + BM + BM + BM + BM + BM + BM + BM + BM + BM + BM + BM + % of vitrified shoots 60-90 40-80 40-80 40-80 40-80 75-100 40-70 40-70 40-70 40-70 sucrose 29 mM sucrose 58 mM mannitol 29 mM mannitol 58 mM agar 0.6% agar 0.6% + sucrose 29 mM agar 0.6% + sucrose 58 mM agar 0.6% + mannitol 29 mM agar 0.6% + mannitol 58 mM agar 0.9% agar 1.1% agar 1.5% agar 2% 20 0 0 0 Propagation ratio .3.75 a 2.8 2.8 2.8 2.8 3.3 3.3 3.3 3.3 3.3 1.9 1.5 1.2 1 b b b b ab ab ab ab ab c cd d d Tab. 3. Comparison of the water potentia! (Chardakov-metliod) of the basic culture medium with different agar (%} aod carbohydrate concentrations (mM). The water potential of the control soiution in which the dropiet of the coloured test solution remained nearly floating at the level of introduction, after 20 h of equilibration, ss indicated by a black dot. Centered dots correspond with full equilibrium (no upward nor downward movement of the coloured droplet in the control solution duie to equal osmotic potentials in both soiutions), Non-centered dots correspond with a situation where the osmotic potential of the coloured droplet is intermediate to the potential of two successive control solutions (slight upward or downward movement). Test solution Agar % Carbohydrates mM Control solution Sugar, mM 0.6 0 0.8 0 t.t 0 , S:29 0.6 S:58 M:29 Mi58 S:29+M:29 W,b,ar 50 -1.21 75 -1.81 100 -2.42 120 -2.90 140 -3.39 160 -3.87 and to control the effect on vitrification. The propagation ratio was also scored. There is no significant difference in propagation ratio between BM and BM -I- agar 0.6%. Addition of sucrose or mannitol (29 tnM and 58 tnM) to the BM does change the propagation xatio itj a significant way. However, this effect was not observed by adding sticrose or tnannitol to the BM + agar 0.6%. Notwithstanding increasing levels of carbohydrate, the percentage of vitrified shoots remains high. A lower incidence of vitrifications is obtained only by raising the agar level above 0.6%. At a concentration of 1.1% and 184 1.5 0 higher the vitrification disappeared completely. These treatments, however, lower the propagation ratio in a significant way and its vaitie was only 1.5 for the optimal concentration of 1.1% Difeo Bacto agar. As there was a clearcut difference between the effect of the carbohydrates and the agar we proceeded with the determination of the water potential of otir culture media with both the Cbardakov-method and the thermocouple psychrometer. Table 3 lists the results of the water potential measuretnents, with tbe Ghardakov-method after 20 h ol equilibration of the agar cylinders in the test solution. Phjsiiil. Plant. 53', 1981 Tab. 4. The influence of different cytokinins on the propagation ratio. The explaot was a duster of 3-4 small shoots (5-10 mm) and the medium contained BM + Difco Bacto Agar 1.1% + sucrose 2% + IAA 1 mg/l. The propagation ratio is defined as the number of shoots and macroscopically visible buds after 5 weeks, divided by the number of shoots and buds at the start of the experiments. Cytokinin treattnent, mg/l 2ii' BA K I 5 20 30 2.5 5 7 2.5 5 10 20 K2.5 + 2iP2-10 Propagation ratio Quality evaluation 1.2 2,2 2.7 2.3 one dominant shoot, tbe others are inhibited; leaves are stunted, especially with 30 mg 3.43 3.41 2.95 all the organs are fasciated, there is a dose-response reation; with increasing concentrations callus becomes more prominent 2.2 with more than 2.5 mg/l the cultures are dwarfed, there is no elongation growth 3.13 2.64 2.58 2.46-2.75 good propagation ratio and equal development of normal shoots Increasing the equilibration time up to 96 h did not change the results. For the BM with different agar concentrations we obtained a value between —1.2 and —1.8 bar. There is only a slow increase of the measured valties with increasing agar concentrations. For the BM + agar 0.6% and different concentrations of sucrose and/or mannitol the values are variable. With sucrose we observed lower (more negative) values than with mannitol,, and a combination of both is intermediate. For sticrose and mannitol the.values decrease with increasing concentrations. K 2.5 mg/l yields numbers of shoots of good quality and a propagation ratio of 2.2, but the development of the buds to shoots is unequ,al. Hence this treatment is not optimal. Higher K concentrations cause extreme dwarfing of the organs without any elongation growth. A combination of 2iP and K allowed us to combine the characteristics of both individual products: a good elongation and an acceptable propagation. Discussion Cytokinin experiments to improve the propagation ratioBased on the results of the different hypotheses tested These experitnents were interpreted as a function of the to overcome vitrification in our in vitro propagation propagation system proposed by Debergh and Maene system of artichoke, we can conclude that the following (1981), i.e. the prodtiction of cuttings in vitro which can factors do not influence this phenomenon: be rooted in vivo. For this reason we aimed at producing temperature treatment - cold storage at 3-4°C; propagules that can be either maintained in the propa- air-volume — different volumes of culture atmosphere gation cycle or that may be tjsed for the production of (250-1000 ml); cuttings. For artichoke the ideal propagule can be iden- consistency of the medium - without or with agar 0.6%; tified as a cluster of shootlets 5-10 mm in height. changes in the formulation of the major elements As the increase in agar concentration lowered the modification in the level of Ct, NOj^ Ca^+; propagation ratio drastically, experiments were set up anti-gibberellins — CCC on the motherplant or in the to improve this situation. The treatments and data of culture medium; those experiments are given in Table 4. cytokinin effect - repeated subculture on media without On an agar (1.1%) ttiedium, 2iP (1 to 30 mg/I) causes cytokinin or culture on media without cytokinin but the development of one dominating shoot. This with charcoal (0.3%); phenomenon is not pronounced in liquid media. auxin-effect - incorporation of TIBA (200 mg/l) in the Moreover the other shoots and buds are inhibited and medium. stunted. There is a dose response reaction on propagaRegarding the air-volume experiments,, we can only tion up to 20 tng/l, but no further development of the conclude that vitrification is not related to emanation shoots is obtained. and concentration of volatile products in the culture With BA included in the medium the propagation atmosphere. However, we can neither prove nor refute ratio is highest but all the organs are fasciated. There is the possibility that volatile compounds are produced a dose response reaction with increasing concentrations endogenously and have an effect on the cells prior to of BA for the fasciation as wall as for the development being evolved and becoming part of the ctilture atmosphere. ofxallus at the base of the expiant. 13 Physiol. Planl, S3, 1981 185 The only hypothesis which proved to be valuable was that related to the water potential of the culture system. From our data in Table 2 it is clear that carbohydrates do not have a regulatory function on vitrification of globe artichoke, but the agar cotjcentration has. Thorpe et al. (Thorpe and Meier 1972, Ross and Thorpe 1973, Thorpe 1974) have proven that carbohydrates are necessary for the high energy-requiring processes of meristemoid and shoot primordia formation, but they also emphasized the osmoregulatory role of part of the carbohydrate content on the differentiation of buds (Thorpe and Murashige 1970, Thorpe 1977, Brown et al. 1979). In 1980 Thorpe and Brown came to the conclusion that shoot-forming callus maintained more negative water potentials and osmotic potentials and more positive pressure potentials than non-shoot forming callus. Increasing carbohydrate concentrations did not improve the propagation ratio in artichoke. On the contrary it was lowered. This could be due to the fact that Thorpe et al. worked with a system producing adventitious buds, while we used only axillary bud development. Both processes might be influenced in a different way by increasing carbohydrate concentrations. Furthermore, tobacco and artichoke might behave differently. The effect of agar concentration was first checked with the Chardakov-method (Tab. 3). This method was developed for systems which have semi-permeable membranes, i.e. systems with kinetic action. Our system is a non-kinetic matrice, where water is held in a capillary or diffuse meshwork (Hammel and Scholander 1976). Although this method is not very sensitive, it allowed us to obtain some good indications, Agar and carbohydrates behave in a completely different way in the Chardakov-method. For the different agar concentrations we obtain a valtie between —1.2 and -1.8 bar after 20 h equilibration. With carbohydrates we obtain more negative values, and those values vary as a function of their concentration or their combination. Comparable doses of sucrose and mannitol give different values in this modified Chardakov-method: with sucrose the values are lower than with mannitol. This indicates that in our system, sucrose is a better osmoregulator than mannitol. The Chardakov-method does not show a dose response reaction for different agar concentrations. On the other hand, the method revealed the infltience of varying concentrations of different carbohydrates on the water potential (Tab. 3). Direct measurements of the total water potential with the dew point psychrometer did not confirm these first findings. On the contrary, a clearcut relationship of ^ with agar concentrations was established (Fig. 1). Similar results have been reported by Brown et at (1979). Indeed, Figure 7 of their publication allows one to obtain a linear relationship between the agar concentrations and the Water poterttials after 28 days of culture. Thus carbohydrates and. agar 186 WATER POTENTIAL 1 bar ) Y y : &.M15 + 2.9545X + R^: 0.9835 OIFCO BACTO AGAR CONCENTRATION Fig. 1. Tbe effect of agar concentrations on the water potential of tbe basic medium. The data points are averages of at least three roeasurements witb the Wescor dew point psychrometer. Apparently, there is a very strong correlation between tbe agar concentration and total water potential (R' = O'.98,85). influence the water potential of the media, but apparently not the same component. The reconciliation of the apparently contrasting findings between the two methods can be found in the equation expressing the total water potential: W = W. -I- "Pp + ip^ (Salisbury and Ross 1969). In an agar medium the pressure potential does not exist, and hence can be omitted from the equation. By definition, the osmotic potential is that part of the water potential that is attributed to solutes (in our case the carbohydrates) and the matric potential is the contribution of the waterbinding colloids and surfaces. Accordingly sucrose and mannitol — tissue penetrating osmotic agents - influence the osmotic potential of the medium. The concentration of agar - a non tissue penetrating agent - to the water potential is not measured by Chardakov's tnethod. Apparently, the method is not suitable to determine the water potential component due to the agar gel matrice. However, the direct W measurement, which deterttiines the sum of the osmotic and matric component, revealed a clear agar concentration effect. The conclusion is that agar is responsible for the matric component of the medium water potential. As the only way.to overcome vitrification in tissue-cultured artichoke consisted in raising the agar content of the medium, we may conclude that the matric potential is responsible for this phenomenon. However, a very negative side-effect of raising the agar concentration is the drastic lowering of the propagation ratio. The most effective cytokinin treatment in liquid medium, i.e. 2iP 20 mg/I,, is less effective on Physiol, Plant. 53, 1981 media with agar concentrations higher than 0.6% and the propagation ratio is reduced to 1 for a concentration of 2%. One could speculate that with raising the agar concentration the availability of cytokinin is reduced, because of the fact that the propagation ratio is equally reduced to 1 on a medium without cytokinin. From the data of Table 4 it is clear that none of the cytokinine used alone give propagules of good quality: with 2iP a kind of apical dominance occurs, BA causes fasciation, and K dwarfs the organs. A combination of K 2.5 mg/l and 2iP 2 to 7 mg/l allows the re-establishment of an acceptable propagation ratio and the obtention of shoots of good and equal quality. Miller, L. R. & Murashige, T, 1976. Tissue culture propagation of tropical foliage plants. - I n Vitro 12, 12: 797-813. Murashige, T. 1974, Plant propagation through tissue cultures. - Annu. Rev. Plant Physiol. 25: 135-166. - & Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures, - Physiol. Piant. 15: 473-497. Nitsch, J. P. & Nitscb, C. 1969. Haploid plants from pollen grains. - Science 163: 85—87, Ouoirin, M. & Lepoivre, P. 1977. Etude de milieux adaptes aux cultures io vitro de Prunus. - Acta Hortic. 78: 437-442. - Lepoivre, Ph, & Boxas, Ph. 1977. Un premier bilan de 10 annees de recherches sur les cultures de meristemes et la multiplication in vitro de fruitiers ligoeux. - Compte rendu des recherches 1976-1977 et rapports de synthese: 93-117. Ross, M. K. & Thorpe, T. A. 1973. Physiological gradients and shoot initiation in tobacco calius cultures. - Plant Cell Acknowledgements — We are grateful to Prof, G. Boesman, tr. Physio!. 14: 473-480, M. Flamee, Ir. L. Maene, Ir. H. Verlodt, Ir. B. Zitouni for Salisbury, F. B. & Ross, C. 1969. Plant Physiology. valuable advice and help, and to Prof, I. impens for supplying Wadsworth. Publishing Co. Inc. Belmont California. the Wescor. Thimann, K. V. 1977. Hormone Action in the Whole Life of Plants, - The University of Massachusetts Press. Amherst, pp. 448, Thomas, D. des S. & Murashige, T. 1979a. Volatile emissions References of plant tissue culture. I, Identification of the major components. - In Vitro 15 (9): 649-653. Benoit, F, 1980. Tecbnisch verslag, Comite voor onderzoek op 1979b, Idem. II. Effects of the auxin 2,4-D on producgroentegewassen. — Sint Katelijne Waver, Belgium, tion of volatile in callus culture. -Ibid. 15 (9): 654—658. Boxus, P., Druart, P. & Brasseur, E. 1978. Activiteitsverslag. Thorpe, T. A. 1974. Carbohydrate availability and shoot forRijkscentrum voor Landbouwkundig Onderzoek, mation in tobacco callus cultures. — Physio!. Plant. 30: Gembloux, 124. 77-81. 1979. Idem. -Ibid., 116. - 1977. Carbohydrate metabolism and sboot formation in Bristow, K. L. & De Jager, P, M., 1980. Leaf water potential tobacco callus. - Meded. Fac. Landbouwwetenschappen, measurements using a strip chart recorder with the leaf Rijksuniv, Gent 42: 1681-1689. psychrometer. - Agric. Meteorol. 22: 149-152. - & Brown, D. C. W. 1980. Osmotic relations during shoot Brown, D. W, C, Leung, D, W. M. & Thorpe, T, A., 1979. formation in tobacco callus. - In Vitro 16 (3): 217. Osmotic requirement for sboot formation in tobacco cal- & Meier, D. D. 1972, Starch metabolism, respiration and lus. - Physiol, Plant, 46: 3 6 ^ 1 , sboot formation in tobacco callus cultures. — Physiol. Plant. Debergh, P. & Harbaoui, Y. 1978. In vitro propagation of 27: 365-369. globe artichoke (Cynara scolymus): Primary results. — In - & Murashige, T. 1970. Some histological changes underPropagation of Higher Plants through Tissue Culture. A lying shoot initiation in tobacco cultures. — Can. I. Bot. 48: Bridge between Research and Application. Symposium 277-285. Proceedings University of Tennessee Knoxville, pp. Van Oijen, 1. 1979. Met extra stlkstof minder risico. - De 250-251. Tuinderij 19, 21: 50-51. - & Maene, L. 1981, A scheme for commercial propagation of ornamental plants by tissue culture. - Sci, Hortic. 14 Welvaert, W. & Van Vaerenbergh, J. 1981. Rechercbes sur la degenerescence de I'artichaut en Tunisie. - 3e Congres (4): 335-345. International sur I'artichaut. Baris 1979 (in the press). Hammel, H. T. & Scholatider, P. F. 1976. Osmosis and Tensile Solvent. — Springer-Verlag. Berlin, Heidelberg, New York. White, P. R. 1939. Controlled differentiation in a plant tissue culture. - Bull. Torrey Bot. Club 66: 507-513, Harbaoui, Y. & Debergb, F. 1980. Multiplication in vitro de Zuccherelli, G. 1979. Moltiplicazione in vitro del portainnesti clones selectiotmes d'artichaut (Cynara scolymus L.). - In clonali del pesco, — Frutticoltura 41 (2): 15-20. Application de la culture in vitro a I'amelioration des plantes potageres. Eucarpia-meeting, Versailles (France) 1-7. Lattanzio, V. & Morone, J. 1978. Artichoke active principles determination during the plant growing season. — Atti Soc. ital. Sci. Nat. Musi Civ. Stor. Nat. Milano 119 (3-4): 329-340. Edited by C.H.B. 13* Physiol. Plant. 53, 1981 187