MICROPROPAGATION OF CHESTNUT AND CONDITIONS OF MYCORRHIZAL SYNTHESES IN VITRO

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1 Neui Phytol. (\9Sb) 102, 95-\0l 95 MICROPROPAGATION OF CHESTNUT AND CONDITIONS OF MYCORRHIZAL SYNTHESES IN VITRO BY D. G. STRULLU, B. GRELLIER, D. MARCINIAK AND R.LETOUZE Laboratoire de Physiologie Vegetale, Departement Symbioses, CNRS et Universite d'angers, 16 Boulevard Lavoisier, Angers, France {Accepted 29 August 1985) SUMMARY Chestnut (Castanea saliva Mill.) was cloned in vitro. Mycorrhizal syntheses were obtained between cloned plants and Paxillus involutus (Batsch.) Fr. In tbe case studied, MNM nutrient solution did not favour tbe development of tbe bost plants. Tbe composition of an original nutrient solution was determined for tbe mycorrbizal infection of cbestnut in vitro. Tbe mycorrbizas obtained were studied by scanning electron microscopy. Key words: Micropropagation, mycorrbizas, Castanea. INTRODUCTION At present, many mycorrhizal syntheses have been performed in vitro, using axenic plantlets grown from seed and fungi grown in pure culture (Gaie, 1977; Gaie & Heinemann, 1980a, 1980b; Molina, 1981; Chilvers & Gust, 1982; Malajczuk, Molina & Trappe, 1982). The specific techniques of in vitro culture are not generally used for obtaining host plants. Thus, in the associations created, the plant material displays genetic variability. Grellier, Letouze & Strullu (1984) have obtained the mycorrhizal association between Betula pendula (Roth.) and Paxillus involutus (Batsch.) Fr. using cloned seedlings. The beneficial effect of mycorrhizas on the growth of the inoculated plants compared with that of uninoculated control plants was clear. This stimulation was recorded evenly throughout the clone. These results proved the reliability of the method, and encourage its extension to other woodland species that are either rare, or of economic interest. The chestnut, Castanea sativa (Mill.), is an interesting species, both for arboriculture and forestry. Its propagation by traditional methods involves many problems. Thus, several workers have considered its culture in vitro (Vieitez & Vieitez, 1980a, 1980b; Biondi et al., 1981; Rodriguez, 1982; Chevre et al., 1983). The parallel application of mycorrhizal infection to this technique could favour micropropagation of the chestnut. The aim of this paper is to define the conditions for cloning and mycorrhizal infection of this species in vitro. MATERIALS AND METHODS Isolation of fungal symbiont. Fruitbodies of Paxillus involutus (Batsch.) were collected during October 198J near birches in the West of France. The conditions of disinfection and culture were X786/ ^07 $03.00/0 ^^^^ 'T^^ ^^^ Phytologist

2 lh D. G. STRULLU et al. those already described (Grellier et al. 1984). The mycelium was cultured on MNM medium (Marx, 1969). Germination in vitro Chestnut achenes, without prior soaking, were disinfected in 8% calcium hypochlorite for 30 min. The fruits were then rinsed several times in sterile distilled water. The fruit pericarps were removed under aseptic conditions, and further disinfection in calcium hypochlorite was performed for 10 min. After several rinses in sterile distilled water, the amylaceous cotyledons were cut off, and the seedling axes were placed in tubes containing Knop's nutrient solution, diluted by half (Gautheret, 1959). The chestnut seedling axes were transferred under sterile conditions to tubes containing 20 ml of nutrient solution with the following composition: macronutrients of Knop diluted by half; micronutrients of Berthelot (1934); organic substances-calcium panthothenate, 1 mg T^; biotin, 0 01 mg T^; nicotinic acid, lmgl~»; pyridoxine-hcl, lmgl"!; thiamine-hcl, lmgl'^i inositol, 100 mg 1 1; glucose, 10 g T^; agar, 7 g \-\ The ph was adjusted to 57. Culture conditions in the growth chamber were as follows: 16h of light at 23±O-5 C, followed by 8 h of darkness at C, with an irradiance of J 2 ^ Cuttings of axenic seedlings After four weeks in culture, the plantlets were about 10 cm tall and had three or four nodes. Vegetative propagation of this material was performed by culturing cuttings of one or two nodes; the apical extremities were also used. Each plantlet normally gave three cuttings. Media were sought with a view to favour two essential points: mineral nutrition and the rooting of the cuttings. Culture conditions in the growth chamber were the same as those described above. Conditions for mycorrhizal syntheses The protocol of Grellier et al. (1984) was used for mycorrhizal syntheses. Erlenmeyer flasks (500 ml) contained a mixture of Perlite (230 ml), dried peat moss (18 ml) and a nutrient solution (130 ml). The composition of this nutrient solution had to be determined experimentally for the species considered. The Perlite and the peat moss were first autoclaved for 20 min, then a second time under the same conditions, together with the nutrient solution. The rooted chestnut cuttings were transferred under sterile conditions from the tubes to the substrate inoculated with a mycelial suspension. This mycelium was obtained by stirring a culture on liquid MNM medium at 100 rev/min, at room temperature. Electron microscope study After 22 weeks of culture in Erlenmeyer flasks, fragments of mycorrhizal roots were taken, and rapidly freeze-dried. This material was placed on stubs and covered with gold before being subjected to observation by scanning electron microscopy. Preparation of the symbionts RESULTS Culture of the fungus Among several strains grown in the laboratory on MNM medium, one isolate was chosen for syntheses. It possessed the same characters as the reference strain

3 Mycorrhizal chestnut in vitro 97 Fig. 1. Micropropagation of the chestnut, (a) In vitro development of a chestnut plantlet grown from seed. The cotyledons have been cut off, and the plantlet possesses a strong tap root with secondary ramifications, (b) and (c) Microcuttings in vitro. The piantlet displays several leaves and a thread-like adventitious root (arrows). Paxillus involutus previously described (Grellier et al. 1984). The mycelium had a woolly appearance and was brown in colour. As it grew, brown pigment diffused into the culture medium. Cloning of chestnut Seedlings. The plantlets grown from chestnut seeds developed satisfactorily on the nutrient medium used (see Materials and Methods). They had a tap root which became ramified, and a well-developed aerial system [Fig. l(a)]. Propagation by cuttings from the seedlings. The cuttings taken from these plantlets were propagated in vitro on different media. The following medium was finally chosen. It contains all essential elements: (mineral salts in mgl ' of medium) Macronutrients - NH4NO3, 240; KNO3, 375; KCl, 37-5; MgSO^, 7H2O, 246; Ca(NO3)2, 246; KHgPO^, 136; (NHJaSO^, MicronutrientsnSO4,H2O, 16 9; ZnSO^, 7H2O, 5 7; KI, 015; CuSO^SH^O, 0 25; BO3H3, 12 4;, 0 25; NiCl2,6H2O, 0 25; AICI3, 6H2O, 0 25; Na2MoO4,2H2O, 0 25; 4,,24,4H2O, 0 375; FeSO^, 27 8; NasEDTA, Organic substancescalcium panthothenate, 1 mg \~^; biotin, 0 01 mg T^; nicotinic acid, 1 mg 1 ^; pyridoxine-hcl, 1 mg \'^; thiamine-hcl, 1 mg T^; inositol, 100 mg 1"^ sucrose, 15 g 1~^; agar, 7 g 1~^ The ph was adjusted to 5-7. ANP 102

4 D. G. STRULLU et at. Fig. 2. (a) Appearance of cultures after 22 weeks m the growth chamber. In the foreground, two inoculated in rifro-plants may be observed on the left (M) and in the centre (M), with a control in Ditro-plant (T) on the right. Note the very marked difference in growth, (b) Appearance of mycorrhizas by scanning electron microscopy (arrows). They are irregularly shaped and are covered with a mantle of loosely-tangled hyphae. Note that the extra-matrical network is more strongly-developed at the base of the organ than at the tip. (c) Cross-section of a mycorrhiza observed by scanning electron microscopy. From the outside inwards, observe: free, isolated, slightly ramified hyphae; hyphae clustered in small units of 2 to 5 elements (arrow); a plectenchyma of interlocked hyphae (double arrows) in contact with cortical cells (cc).

5 Mycorrhizal chestnut in vitro This medium induced regular rhizogenesis and provided satisfactory mineral nutrition [Fig l(b) and (c)]. It also proved favourable to the growth of the fungus, and was chosen for the mycorrhizal syntheses. Mycorrhizal syntheses The application of the fungal symbiont in the form of a liquid suspension enabled the substrate to be rapidly colonized. After 9 to 10 weeks in culture, considerable differences were observed between inoculated and uninoculated microcuttings. These differences became more evident during the weeks that followed. After 22 weeks in culture, a study of the root system revealed the existence of mycorrhizas, possessing a fungal mantle surrounding the secondary roots, and the beginning of the formation of a Hartig net. The association between Castanea sativa/paxillus involutus, made in vitro from rooted microcuttings of the same age, led to a stimulation of the growth of the inoculated seedlings, as a whole, compared to that of the uninoculated control plants of the same clone. This stimulation was obtained for four different clones [Fig. 2(a)]. Scanning electron microscopical study The morphology of the root system of the chestnut is modified by the presence of the fungus around the roots. Many rounded apices appear. Short mycorrhizal roots are twisted and irregularly ramified; they are completely surrounded by the fungal mantle. On the surface of this mantle is a slack network of isodiametric hyphae, extended toward the exterior by an extra-matrical network, more strongly developed at the base of the mycorrhiza than at the apex [Fig. 2(b)]. In cross-section, the fungal mantle displays, on the edge, free, ramified hyphae, and, in contact with cortical cells, hyphae clustered in small groups [Fig. 2(c)]. Gradually, a plectenchyma with interlocked hyphae is formed. In older mycorrhizas, this plectenchyma consists of 10 to 15 layers of fungal cells. From the deepest layers of the mantle, certain hyphae penetrate between the cortical cells of the root, givmg rise to a Hartig net. So the mycorrhizas are similar to those formed under normal conditions and to those formed in vitro with seedlings (Grellier et al. 1984). DISCUSSION The Castanea sativa/paxillus involutus association is a new example illustratmg the use of cloned plants for mycorrhizal experiments developed by Strullu, Grellier & Letouze (1984). For this association, as for that formed by Betula pendula and Paxillus involutus, in vitro culture techniques were used for the host plants. Rooted clones of birch and chestnut were obtained before performing the syntheses. Grellier et al. (1984) have already emphasized that in vitro cloning limits genetic variability. The concept of mycorrhizas and cloned plants has another meaning. Mycorrhizal syntheses performed in vitro using plants grown from seed create diverse, non-reproducible combinations. If seeds are used different genomes are introduced in each combination. However, if the host plants are cloned, the same genome is always introduced, and the combination is reproducible, which is an advantage when monitoring effectiveness. The protocol used for the Betula pendula/paxillus involutus association was also used for the chestnut. However, the nutrient solution applied to the Perlite and peat moss mixture was modified to suit the requirements of this species. Prior culture of the fungus in MNM liquid medium did not prevent the very rapid 4-2

ioo D. G. S T R U L L U et al. colonization of the new medium. The possibility of the growth of the fungus alone, on the chestnut medium chosen for the synthesis, was also investigated.

6 ioo D. G. S T R U L L U et al. colonization of the new medium. The possibility of the growth of the fungus alone, on the chestnut medium chosen for the synthesis, was also investigated. Thus, ordinary MNM medium can be used for culturing and maintaining the strains. The change to a specific synthesis medium does not appear to cause any problems. For the birch and the chestnut, mycorrhizal infection by Paxillus involutus is revealed in the same way: presence of a mycorrhizal root system and stimulation of the growth of mycorrhizal plants compared to non-mycorrhizal controls from the same clones. For the chestnut, cloned plants have an added advantage. The plantlets grown from seeds have a strong main root, and rapidly-growing secondary roots. This makes in vitro mycorrhiza formation difi[icult in necessarily restricted spaces. Under the experimental conditions used here, the development of the adventitious roots obtained is homogeneous and limited. This method therefore favours the in vitro formation of complexes in woodland trees whose seedlings possess a large main root. Mycorrhiza formation on natural complexes of angiosperms and gymnosperms has already been described (Strullu & Gerault, 1977; Strullu & Gourret, 1980). It is revealed by the gradual development of a fungal mantle and a Hartig net. Tissue senescence is centripetally oriented. In vitro, the sequence described for the Betula pendula/paxillus involutus association using seedlings and clonal plants (Grellier et al. 1984) is confirmed for Castanea sativa with the same fungal symbiont. It may be noted that the figures for birch (Grellier et al. 1984) and Figure 2c of the present paper, for chestnut, are very similar. However, associations involving other symbionts should be studied, since work by Nylund and Unestam (1982) has shown a different sequence on Picea roots. Mycorrhiza formation is revealed by stimulation of the growth of mycorrhizal plants compared to non-mycorrhizal control plants from the same clone. Fungal isolates from the same species have significantly different effects on a given clone (Grellier, et al. 1984). With chestnut, the response to a symbiont was significant for the four clones studied. This result was also obtained in birch (Strullu, Grellier & Letouze, unpublished results). Current experiments should enable the response of different clones to a selected isolate to be compared. REFERENCES (1934). Nouvelles remarques d'ordre chimique sur le choix des milieux de culture naturels et sur la maniere de formuler les milieux synthetiques. Bulletin Societe Chimique Bioloeique «</,, BERTHELOT, A. BiONDi, S., CANCIANI, L. D E PAOLI, G. & BAGNI, N. (1981). Shoot formation from bud cultures of mature chestnut. In: Colloque Internationale sur la Culture in vitro Des Essences ForestieresAFOCEL, pp IUFRO, Fontainbleau. CHEVRE, A. M., G I L L, S. S., MOURAS, A., SALESSES, G. (1983). In vitro vegetative multiplication of chestnut. Journal of Horticultural Science, 58, CHILVERS, G. A. & GUST, L. W. (1982). The development of mycorrhizal populations on pot-grown seedlings of Eucalyptus st-johnii R. T. Bak. New Phytologist, 90, GAIE, W. (1977). Mycorrhization comparee, en milieu controle, de Betula pendula par Pisolithus arhizus, Scleroderma citrinum et Paxillus involutus. Bulletin Recherches Agronomiques Gembloux, 12, ' GAIE, W. & HEINEMANN, P. (1980a). Synthese et morphologie de l'ectomycorhize Betula pendula/astraeus hygrometricus. Bulletin Societe Royale Botanique Belgique, 113, GAIE, W. & HEINEMANN, P. (1980b). Mycorhization du genre Betula par Pisolithus arhizus en conditions axeniques. Bulletin Jar din Botanique National Belgique, 50, GAUTHERET, R. J. (1959). La Culture des Tissus Vegetaux: Techniques et Realisations. Masson et Cie, Paris. GRELLIER, B., LETOUZE, R., STRULLU, D. G. (1984). Micropropagation of birch and mycorrhizal formation in vitro. New Phytologist, 97, MALAJCZUK, N., MOLINA, R. & TRAPPE, J. (1982). Ectomycorrhizal formation in Eucalyptus.1. Pure culture synthesis, host specificity and mycorrhizal compatibility with Pinus radiata New Phvtoloeist s..

7 Mycorrhizal chestnut in vitro ioi M.ARX, D. H. (1969). The influence of ectotrophic mycorrhizal fungi on the resistance of fire roots to pathogenic infections.l Antagonism of nnycorrhizal fungi to root pathogenic and soil bacteria. Phytopathology, 59, MOLINA, R. (1981). Ectomycorrhizal specificity in the genus Alnus. Canadian Journal of Botany, 59, NYLUND, J. E. & UNESTAM, T. (1982). Structure and physiology of ectomycorrhizae. L The process of mycorrhiza formation in Norway spruce in vitro. New Phytologist, 91, RODRIGUEZ, R. (1982). In vitro propagation of Castanea sativa through meristem tip culture. HortScience, 17, STRULLU, D. G. & GERAULT, A. (1977). Etude des ectomycorhizes a Basidiomycete et a Ascomycete de Betula pubescens en microscopie electronique. Comptes rendus Academie des Sciences Paris, 284, STRULLU, D. G. & GOURRET, J. P. (1980). Donnees ultrastructurales sur l'integration cellulaire de quelques parasites ou symbiotes de plantes.il Champignons mycorhiziens. Bulletin Societe Botanique France, 111, STRULLU, D. G., GRELLIER, B. & LETOUZE, R. (1984). Micropropagation et mycorhization in vttro: concepts et realisation. Comptes rendus Academie Agriculture, 11, VIEITEZ, A. M. & VIEITEZ, E. (1980a). Plantlet formation from embryonic tissue of chestnut grown in vitro. Physiologia Plantarum, 50, VIEITEZ, A. M. & VIEITEZ, M. L. (1980b). Culture of chestnut shoots from buds in vitro. Journal of Horticultural Science, 55,

Working with Mycorrhizas in Forestry and Agriculture

Working with Mycorrhizas in Forestry and Agriculture SUB Gdttingen 206 384661 Mark Brundrett, Neale Bougher, Bernie Dell, Tim Grove and Nick Malajczuk CONTENTS Chapter I. INTRODUCTION 1.1. MYCORRHIZAL