Báo cáo khoa học: "gelling agents on growth, mineral composition and naphthoquinone content of in vitro explants of hybrid walnut tree (Juglans regia x Juglans nigra)"

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Original article Effects of gelling agents on growth, mineral composition and naphthoquinone content of in vitro explants of hybrid walnut tree (Juglans regia x Juglans nigra) C Jay-Allemand E Barbas P Doumas S Chaillou D Cornu 1 INRA, Station d’Amélioration des Arbres Forestiers, 45160 Olivet; 2 INRA, Laboratoire du Métabolisme et de la Nutrition des Plantes, 78000 Versailles, France (Received 15 May 1992; accepted 22 October 1992) Summary — Gelling agents affect growth of walnut in vitro cultured shoots. Gelrite promoted shoot elongation and bud production, whereas agar inhibited growth, induced mature leaf formation and necroses. The 2 gelling agents differed significantly in mineral content. They altered the chemical composition of the medium as well as that of the explants. A pronounced accumulation of Na and several microelements was observed in leaves after 16 d of culture on agar, probably due to a dis- turbance in the K selectivity mechanism and membrane permeability. Moreover, on agar, the level of hydrojuglone glucoside, a marker of juvenility in walnut, decreased drastically in the callus. Mineral element accumulation and decrease of hydrojuglone glucoside were evident after growth inhibition, indicating that they are a result rather than a cause of this inhibition. Lack of growth, mature foliar morphology, Na and microelement accumulation and hydrojuglone glucoside decline support the hy- pothesis that agar accelerates the ageing of in vitro propagated walnut trees. Juglans / micropropagation / gelling agent / mineral composition Résumé — Effets des agents de solidification du milieu de culture sur la croissance, la com- position minérale et la teneur en hydrojuglone glucoside des explants de noyer hybride culti- vés in vitro. Les agents de solidification influent sur la croissance des pousses du noyer cultivées in vitro (fig 1). La gelrite a un effet bénéfique sur l’élongation des explants et la production de bour- geons, tandis que l’agar inhibe la croissance et provoque la maturation des feuilles ou encore des nécroses (tableau I). Les 2 agents de solidification présentent des différences importantes dans leurs teneurs en éléments minéraux (tableau II). Ils altèrent la composition minérale du milieu de cul- ture, comme celle des explants (tableau III). Une accumulation importante de Na et de divers micro- éléments a été observée dans les feuilles après 16 h de culture sur l’agar (fig 2), probablement due aux perturbations du mécanisme de sélectivité de K et de la perméabilité membranaire. De plus sur * Correspondence and reprints
agar, la teneur du cal en hydrojuglone glucoside diminue (fig 5), alors qu’une teneur élevée de ce composé caractérise l’état juvénile chez le noyer. L’accumulation des éléments minéraux (figs 3 et 4) et la diminution de la teneur en hydrojuglone glucoside, interviennent après l’inhibition de la crois- sance, indiquant ainsi qu’il s’agit plutôt d’une conséquence que de la cause de cette inhibition. L’ab- sence de croissance, la formation des feuilles matures, l’accumulation de Na et des microéléments supportent l’hypothèse que l’agar accélère le vieillissement des explants de noyer. Juglans / micropropagation / gélifiant / composition minérale / polyphénol INTRODUCTION Cosmo, 1984; Gershenzon, 1984) and could be used as an indicator of in vitro culture dysfunctioning. Although techniques for micropropaga- The aim of this study was to compare tion of walnut have been species re- the effects of 2 gelling agents, Difco Bacto propagation for fruit produc- ported, mass Agar® (Difco) and Gelrite® (Kelco) on the tion and reforestation remains limited growth of in vitro walnut explants, their due to problems such as high transfer mineral content and the typical naphthoqui- frequency, latent contamination, low multi- none content associated with the above- plication and rooting rates (Driver and mentioned factors. Kuniyuki, 1984; McGranahan et al, 1988; Cornu and Jay-Allemand, 1989; Revilla et al, 1989). MATERIALS AND METHODS As result of their physical and chemi- a cal properties, gelling agents influence growth (Lee et al, 1986; Cornu and Jay- growth conditions Tissue culture and Allemand, 1989) and organogenesis (Titel et al, 1987; Koda et al, 1988). It has been Walnut explants were obtained by micropropa- shown that agar gels and their aqueous gation of an embryonic axis of hybrid walnut (Ju- extracts contain several cations (Kordan, glans regia x Juglans nigra) according to the 1988) which are available to plant tissues technique described by Jay-Allemand and Cor- (Kordan, 1980, 1981).Organic impurities, nu (1986). Shoots obtained from elongated buds absorbing in the same UV wavelength as of nodal segments were subcultured in 750-ml phenols are also present (Scherer et al, jars containing 125 ml of media solidified by Gel- rite. The medium was the same as the DKW me- 1988). dium (Driver and Kuniyuki, 1984) except for the Walnut tissues containa great amount microelements which were (in μM): H 200; , BO 3 of major polyphenol of wal- A polyphenols. MnSO 200; ZnSO 74; Kl, 10; Na O, 2 ·H 4 O, 2 ·7H 4 2 nut, identified as the hydrojuglone gluco- MoO 2, CuSO 2; CoCl 2. O, O, 2 2 O, ·2H ·SH ·6H 442 side, is found in significant quantities at The pH was adjusted to 6 prior to autoclaving. the onset of growth in juvenile shoots and Each vessel contained 6 explants. Cultures were is therefore considered to be a biochemi- maintained in a growth chamber under a 16-h photoperiod with day and night temperatures of cal marker of walnut juvenility and rejuve- 25 ± 1 °C and 22 ± 1 °C respectively under cool- nation (Jay-Allemand et al, 1990). The lev- white fluorescent lamps at 75 μE m s After -2 -1 . el of this compound is also found to excision of the callus, 8-month-old explants, decrease with foliar ageing (Cline and subcultured on Gelrite every 14 d, were trans- Neely, 1984). Moreover, accumulation of ferred to a medium which was solidified either phenolic compounds was associated with by 0.6% (w:v) Difco Bacto agar (Difco) or by 0.2% (w:v) Gelrite (Kelco). deficient mineral nutrition and stress (Di-
min and 100% B isocratically for 5 min; the flow Determination of mineral content rate was 1 ml min Peaks were recorded at . -1 250 nm. The naphthoquinones hydrojuglone glu- A digestion method was used for inorganic cat- coside and juglone were characterized by their ion analysis. Approximately 50 mg of dried spectrum. Results were expressed in μmol g -1 leaves (at 80 °C, over 48 h) were mineralized by DW of 6-methoxyflavone (internal standard). the consecutive addition of 1 ml concentrated ni- Quantitative variations due to the extraction and tric acid and 1 ml concentrated hyperchloric analysis method were determined from 6 repli- acid. Organic matter was totally digested by cates (extracts) of the same dry matter. The co- heating. The solution was evaporated to dry- efficient of variation did not exceed 6%. ness and the ash was taken up in 10 ml hydro- chloric acid (0.1 N). Analysis of Ca, K, Na, P, Fe, Mg, Mn, Zn, Cu, and B was performed by RESULTS coupled plasma emission spectrometry. The same method was used on the gelling agents for the determination of their mineral content. Growth Analysis of polyphenols Gelrite where- shoot elongation promoted agar strongly inhibited elongation of ex- as plants (fig 1).Lack of elongation was ap- Phenolic compounds were extracted and puri- fied according to the method adapted to walnut parent on agar, while explants cultured on by Jay-Allemand et al (1988). Twenty mg Gelrite continue to grow until the end of the freeze-dried material of leaf, stem or callus were experiment (d 16). Morphological changes extracted with acetone/water (80/20, v/v) at 4 °C also observed. Agar led to fully ex- were by sonication for 30 min. Supernatants were col- panded leaves but the formation of new lected after centrifugation and solvents were limited. On Gelrite solidified leaves was evaporated in vacuo (Speed-vac). The phenolic medium, the leaves smaller, bright compounds were separated by high perfor- were mance liquid chromatography using a C18 re- green in color and new leaves were regu- versed phase column: lichrospher 5 μm 100 CH- larly formed. After 2 subcultures (32 d), 18/11 (Merck), 250 x 4.6 mm; solvent A was shoots cultured on Gelrite produced more aqueous acetic acid (1%, v/v) and B methanol/ buds than those on agar and the fresh acetronitrile (50/50, v/v); the elution gradient weight of callus formed at the end of the 15-40% B in A for 20 min, 40-60% B in A was stem was greater (table I). Leaf discolora- for the next 5 min, then 60-100% B in A for 3
tion, leaf abscission and episodic explant 3-fold higher in the medium solidified was resulted from of by agar than in the medium solidified by repetitive necroses use gelrite (table II) and K/Na ratio was 3-fold agar. lower with agar than Gelrite (table III). Mineral content of gelling agents Mineral content of leaves The 2 gelling agents presented major dif- ferences in mineral content (table II). Gel- The mineral content of the leaves varied according to the gelling agent and the peri- rite contained a higher amount of Ca and Mg and K (4-fold) and Fe than agar. Agar od of time in culture. A significant accumu- contained 2-fold more Na than Gelrite. lation of Na was found in the leaves of the explants cultured on agar. After 16 d of cul- Since all the mineral elements are practi- ture the concentration of Na in leaves was cally available in the capillaries of the gels (Debergh, 1983), it is expected that the 2 3-fold higher than in those explants cul- gelling agents alter the composition of the tured on Gelrite (fig 2). Explants cultured media in proportion to the quantities (0.6% on Gelrite had a higher final (16th d) con- centration of K and Mg, but only the in- agar and 0.2% Gelrite, w:v) required for medium solidification (table II). Thus, agar crease in Mg was significant (fig 3). Differ- adds a greater amount of Na, P, Mn, Cu ences in K and Na concentrations in the and B than Gelrite does. The latter adds a leaves led to a lower K/Na ratio in the greater amount of Fe and K. The Na/K ra- leaves on agar than on Gelrite (table III). tio is strongly modified: Na concentration However, the amount of K+Na remained
in both More- tor for fruit trees comparable gelling agents. (Martin-Prevel et al, over, the K/Na ratio in the leaves was simi- 1984). Natrophobic plants have effective lar to that of the solidified media. Total P mechanisms for blocking sodium transport was the only macroelement with a foliar parts of plants, in order to the upper to concentration which was significantly high- avoid its detrimental effect on the fine er on agar than on Gelrite at the end of the structure of chlorophyll. However, replace- subculture ment of K by Na may occur in senescing (fig 3). leaves even in natrophobic plants (Marsch- minor differences were found in Only ner, 1986). Such a replacement probably microelement concentrations in leaves un- occurred in walnut explants grown in agar til the 8th d of culture. However, at the end since the K/Na ratio changed while the K + of 16 d of culture a much higher concen- Na amount remained constant (table III). tration of microelements Mn, Cu, Fe, B, Al This suggests a substitution of K by Na, and Zn was observed in leaves of explants probably due to a disturbance in the K se- growing on agar (fig 4). lectivity mechanisms. Van Steveninck (1978) showed that exogenously applied abscissic acid could induce Na selectivity Naphthoquinone content even in K selective genotypes of beetroot slices. This stress-related regulator of Regarding the content of hydrojuglone glu- growth has also been mentioned as a stim- coside and juglone in leaves, stem and ulator of membrane permeability (Penon, callus, significant differences were found 1982; Marschner, 1986), and is involved in in leaves, stem or callus in shoots depend- the senescing process. Altered membrane ing on the gelling agent. During the first 8 permeability could also explain the exces- d of culture, hydrojuglone glucoside con- sive accumulation of the microelements in leaves of Gelrite-cultured explants tent in the leaves of explants cultured in agar, higher than that of agar (cultured ex- was which was observed after 16 d of culture. In the callus of explants plants) (fig 5a). cultured in agar, the amount of this com- It is unclear whether growth of walnut pound decreased to the lowest level deter- explants, which are Na-sensitive (Heller, mined (fig 5c). On the same gel, the 1981),was restricted by the accumulated amount of this polyphenol increased only amount of Na. It seems that accumulation in the explant stem (fig 5b). Juglone of Na or microelements is not the primary showed similar fluctuations in leaves and of this inhibition because significant cause callus (figs 5d, f) on both gelling agents, accumulation of these elements does not while a significant accumulation of this occur before growth inhibition takes place. compound was observed in the stem of It has been shown that reduced growth the explants cultured in agar (fig 5e). is accompanied by decreasing amounts of the hydrojuglone glucoside in walnut annu- al shoots during senescence (Jay- DISCUSSION Allemand et al, 1989). Growth decline ac- companied by decrease in hydrojuglone Considerable differences were observed in content of callus was also ob- glucoside growth, mineral and phenolic content of served on in vitro cultured explants as a re- explants growing on the same medium but sult of agar used. The same compound solidified by 2 different gelling agents. was found to decrease drastically in callus Na is considered unnecessary ele- of explants grown in Gelrite after 28 d of an ment for and even a toxic fac- glycophytes culture (data not shown). However, the
the presence of juglone and hydrojuglone reasons and the mechanisms of hydroju- glucoside in black walnut. Phytopathology glone glucoside decline are still unknown. 74, 185-188 It is possible that juglone was released Cornu D, Jay-Allemand C (1989) Micropropaga- from hydrojuglone glucoside after chemical tion of hybrid walnut trees (Juglans nigra x or enzymatic hydrolysis. Juglone is an Juglans regia) through culture and multiplica- aglycone (oxidized form) which has been tion of embryos. Ann Sci For 46 suppl, 113- correlated with growth inhibition in other 116 species (Ficher, 1978). PC (1983) Effects of agar brand and Debergh Most of the biochemical differences ob- concentration on the tissue culture medium. Physiol Plant 59, 270-276 served in tissues growing on the 2 gelling DiCosmo F (1984) Stress and secondary metab- agents were evident after 8 d of culture, olism in cultured plant cells. In: Phytochemi- suggesting that they were a result rather cal Adaptations to Stress. Proc 23rd Annu than a cause of growth inhibition. Scherer Meet Phytochem Soc N Amer (Timmerman et al (1988) pointed out that no significant BN, Steeling C, Loewus F, eds) Rec Adv differences exist between agar and Gelrite Phytochem 18, 97-176 in water potential, osmolality and water ac- Driver J, Kuniyuki A (1984) In vitro propagation tivity, whereas significant differences are of paradox walnut rootstock. HortSci 19, 507- found in diffusion behavior of cationic 509 dyes. So it is possible that growth inhibition Ficher RF (1978) Juglone inhibits pine growth could be related to events at the interface under certain moisture regimes. Soil Sci Soc of the solidified medium and the basal part Am J 42, 801-803 of the explants. These events could be a Gershenzon J (1984) Changes in the levels of plant secondary metabolites under water and differential diffusion behavior of excreted nutrient stress. In: Phytochemical Adapta- substances in the 2 gelling agents, a reten- tions to Stress. Proc 23rd Annu Meet Phyto- tion of growth substances by the agar, or chem Soc N Amer (Timmerman BN, Steeling even a breakdown of callus cells related to C, Loewus F, eds) Rec Adv Phytochem 18, one or both of the above events. 273-303 Inhibition of formation of mature growth, Heller R (1984) Nutrition. In: Physiologie Végé- leaves, K substitution by Na, excessive tale. Masson, Paris, 244 ac- cumulation of microelements and hydroju- Jay-Allemand C, Cornu D (1986) Culture in vitro d’embryons isolés de noyer commun (Ju- glone glucoside decline combine to sup- glans regia L). Ann Sci For 43, 189-1292 port the hypothesis that agar accelerates Jay-Allemand C, Capelli P, Bruant B, Cornu D the ageing of in vitro propagated walnut (1988) Variabilité clonale in vitro des noyers explants. hybrides (Juglans nigra x Juglans regia). Re- lation avec le contenu polyphénolique des pousses. In: CEE Agric, 2 Colloque Noyer- e ACKNOWLEDGMENT Noisetier. Bordeaux, 18-20 September 1988, 79-87 Jay-Allemand C, Drouet A, Ouaras A, Cornu D the financial E Barbas gratefully acknowledges (1989) Polyphenolic and enzymatic charac- support provided by the EEC. terization of ageing and rejuvenation of wal- nut trees (Juglans nigra x Juglans regia): re- lationship to growth. Ann Sci For 46 suppl, REFERENCES 190-193 Jay-Allemand C, Keravis G, Lancellin JM, Cornu D, Macheix JJ (1990) Naphthoquinones and Neely D (1984) Relationship between Clines S, flavonols associated with walnut rejuvenation juvenile-leaf resistance to anthracnose and
and involved in different physiological pro- Plantes Tempérées et Tropicales. Lavoisier, cesses. Plant Physiol (suppl) 93, 147 Paris, 9-74 Koda T, Ichi T, Yamagishi H, Yoshikawa H Mc-Granahan G, Leslie CA, Driver JA (1988) In (1988) Effects of phytohormones and gelling vitro propagation of mature Persian walnut agents on plant regeneration from proto- cultivars. HortSci 23, 220 plasts of red cabbage. Agric Biol Chem 52, (1982) Substances de croissance. In: Penon P 2337-2340 Physiologie Vegetale: II. Croissance et Déve- Kordan HA (1980) Impatiens holstii pollen ger- loppement (Mazliak, ed) Hermann, Paris, 15- mination on an aqueous agar extract. Experi- 125 mentia 36, 1 058 Majada J, Rodriguez R (1989) Wal- Revilla MA, Kordan HA (1981) Impatiens holstii pollen ger- nut (Juglans regia L) micropropagation. Ann mination nutrient agars. Plant Sci Lett on non Sci For (suppl 46) 149-151 23, 157-160 Lippert H, Wolff G (1988) Scherer PA, Muller E, Kordan HA (1988) Inorganic ions present in analysis of agar and Gelrite im- Multielement commercial agars. Biochem Physiol Pflanz purities investigated by inductively coupled 183, 355-359 plasma emission spectrometry as well as physical properties of tissue culture media Lee N, Wetzstein H, Sommer H (1986) The ef- prepared with agar or the gellan gum Gelrite. fect of agar vs liquid medium on rooting in Acta Hortic 226, 655-658 tissue cultured sweetgum. HortSci 21, 317- 318 Titel C, Ehwald R, Zoglauer K, Hellmig A (1987) A parenchymatic medium solidifier for plant Marschner H (1986) Nutritional physiology. In: in vitro culture. Plant Cell Rep 6, 473-475 Mineral Nutrition of Higher Plants. Academic Press, London, 3-407 Van Steveninck R (1978) Control of ion trans- port in plant storage tissue slices. In: Bio- Martin-Prevel P, Gagnard J, Gautier P (1984) chemistry of Wounded Plant Tissues (Kahl La nutrition végétale. In: L’Analyse Végétale G, ed) Walter de Gruyter, Berlin, 503-541 dans le Contrôle de l’Alimentation des