In vitro mycorrhization of micropropagated Helianthemum almeriense plantlets with Terfezia claveryi ( desert truffle )

The mycorrhizal synthesis of Terfezia claveryi with micropropagated plantlets of Helianthemum almeriense was carried out in vitro on modified Modified MelinNorkrans (MMN) agar medium with pH 8.0. The mycorrhization rate was about 80% after 12 weeks. T. claveryi formed ectendomycorrhizas without hyphal mantle. The effect of the fungus on in vitro rooting was also studied. T. claveryi did not enhance in vitro rooting of microcuttings of H. almeriense. In vitro survival of the plantlets was the limiting step and mycorrhizal inoculation appeared to improve the survival rate of rooted plantlets. The effect of the mineral composition and pH of the medium on survival are discussed.


Introduction
Helianthemum almeriense Pan belongs to the Cis- taceae and is one of the most abundant shrubs of the semi-arid areas in Spain.This species, which is of great interest for reafforestation, establishes ecto-endomycorrhizae with Ascomycetes such us Terfezia sp. and Balsamia sp. (desert truffles).
Terfezia claveryi is a hypogeous fungus found in marl-gypsum soils under H. almeriense and in marl calcareous and sandy soils under H. alm- eriense and H. lavandulifolium in the region of Murcia (Honrubia et al. 1992) .This fungus, called "turma" in Spanish semi-arid zones, is well adapted to the xeric conditions of the Southern Mediterranean area, where it is prized and is of marked gastronomic and economic importance.
We report here the first in vitro mycorrhiza-lion of micropropagated H. almeriense plantlets with Terfezia claveryi.

Plant material
H. almeriense plants were micropropagated fol- lowing the procedure described by Morte and  Honrubia (1992).After 4 weeks, plantlets for experiments were collected from the rooting me- dium of Murashige and Skoog (1962) with salt strength diluted to 1:4, without auxins.At inoculation, each rooted plantlet had a shoot length of about 3 cm, and four to five nodal seg- ments with two leaves each.The average number of roots per plantlet was three.They were un- ramified, with lengths ranging from 1 to 1.5 cm.These plantlets were called R+ to differentiate them from unrooted explants with the same shoot characteristics called R-.
Cultivation conditions throughout the process of micropropagation were 25 ± 2°C, 40 nmol.rrr 2 .s - Growlux fluorescent light and 16-h photoperiod.These culture conditions were maintained for the mycorrhizal synthesis experiments, too.

Experiments
Two kinds of substrate were used for the in vitro inoculation of H. almeriense with T. claveryi: 1) A sterilized mixture of vermiculite: peat (2:1, v/v) watered with MMN liquid medium with 2.5 g.l -1 of glucose and pH 8.0.Five pieces of agar, 0.25 cm 2 in surface, with mycelium of T. claveryi grown on a modified MMN (MMN nutrients less malt and P0 4 H(NH 4 )J liquid medi- um with 2.5 g.l ' of sucrose were used as inocu- lum.The rooted plantlets obtained on the MS/4 medium were cultivated with the mycelium and 100 ml of substrate in Erlenmeyer flasks of 250 ml .The experiments were repeated three times with 20 inoculated and 20 control plantlets each time.
2) Modified MMN medium with 8 g.l ' of agar and 2.5 g.l -1 of glucose, pH 8.0.Pieces of agar, 0.25 cm 2 in surface, with mycelium of T. claveryi grown on MMN medium (15 g.l -1 of agar and pH 8.0) were used as inoculum.To facilitate the inoculation process and to see the effect of this fungus on rooting, rooted plantlets (R+) and un- rooted microcuttings (R-) were inoculated with two pieces of agar with mycelium per tube.The tubes had a diameter of 2.5 cm and were 20 cm long.25 ml of medium per tube was used (Fig. 1).The experiments were repeated twice.In vitro system for inoculating micropropagated H. almeriense plantlets with T. cla veryi, on a modified MMN medium. (Photo: M.A. Morte)  with 15 plantlets or explants being used for each treatment each time.
Fungal colonization was assessed on cleared and stained root samples (Phillips and Hayman  1970).The percentage of root infection was esti- mated according to the grid-line intersect method (Giovannetti and Mosse 1980).

Results and discussion
Experiment 1 The mycelium grew and had colonized the whole substratum after just four weeks.Plantlets elon- gation was clearly visible after the same period of time.However, no mycorrhization was ob- served 2, 4 and 6 months after inoculation.The survival rate of the plantlets after 24 weeks in vitro was almost the same (about 80%) for both the control and inoculated plantlets.
We consider it probable that plantlets did not form mycorrhizas because the substrate used was very rich in nutrients.

Experiment 2
Rooting: In comparison with plantlets rooted on MS/4 me- dium (Morte and Honrubia 1992), the rooting rate of explants within the first 4 weeks of cul- Fig. 2. Longitudinal section of infected root; the hyphae form coils that fill the host cells, showing a bead shape.40 x. (Photo; M.A. Morte)   Fig. 3. Cross-section showing absence of hyphal mantle, and inter-and intracellular infections affecting only the cortical cells.40 x. (Photo; M.A, Morte)  ture on MMN was low (Fig. 4) .In vitro rooting of explant is a complex process that can be influ- enced by many factors (Pierik 1987).One of the factors causing the low rooting rate in our exper- iments may have been the carbon source.It has been firmly established that sugar is necessary for adventitious root formation (Pierik 1987).The kinds of carbohydrates and their concentrations also influence rooting (Moncousin et al. 1992,  Uosukainen 1992, Caboni et al. 1992).Generally, sucrose and glucose, both at low concentra- tions (10-20 g.F 1 ), are the most effective carbon sources for rooting (Chalupa 1977, Cheng and  Voqui 1977, Timmis and Ritchie 1984).Our experiments suggest that the kind of sugar (glucose) and the low concentration (2.5 g.l -1 ) used were not conducive to rooting.Also mineral nu- trients may have affected rooting, since the MMNmodified medium we used contains few mineral nutrients, and they are at lower concentrations than in the MS medium.
In this specific, and probably not optimal, nu- trient context, the presence of the mycorrhizal fungus T. claveryi did not improve rooting of the explants (Fig. 4).lAA release of T. claveryi was not investigated but, if present lAA production under our experimental conditions but if any, this did not enhance in vitro rooting of the H. alm- eriense cuttings.

Mycorrhization
The mycelium grew from the surface of the inoc- ulum into the substrate within 4 weeks.This myc- elial growth into the agar may have been due either to the fact that Terfezia is a hypogeous fungus or to the different agar concentrations (8 g.l -1 in the tube and 15 g.l -1 for the pieces of inoculum).It allowed the fungus to colonize the root system very well.
Until the 4 lh week, no mycorrhization was ob- served, only some infection points were visible.After 8 weeks, the mycorrhization rate was 35% and it increased to 80% after 12 weeks (Fig. 5).The newly produced roots of R-M+ microcut- tings were very short (0.1-0.3 cm), with an aver- age of five roots/explant, and did not form myc- orrhizas.
Using non-micropropagated plants of Helian- themum guttatum and a substrate of perlite wa- tered with a nutrient solution, Fortas and Chevalier (1992) obtained mycorrhizas of Terfezia under axenic conditions.Roth-Bejerano et al.  (1990) also obtained mycorrhizas with seedlings of Helianthemum sessiliflorum and Terfezia le- onis in a half-strength Hoagland's solution with agar and activated charcoal.
The morphology of the mycorrhizas formed in our experiments was similar to that of the ecten- domycorrhizas described by Fortas and Cheva- lier (1992).Both the extramatricial and intercel-Fig.4. Rooting percentage of H. almeriense microcut- tings grown on MS/4 and MMN media and inoculated with T. claveryi.Fig. 5.In vitro mycorrhization percentages of R+ M+ plantlets.lular hyphae were moniliform or beadlike.The intracellular hyphae formed coils which filled the whole lumen (Fig. 2).
Hyphal mantle was absent from some sections of the mycorrhizas (Fig. 3).Only some hyphae growing along the surface of the roots were ob- served.The infection was inter-and intracellular, affecting the outer layers of cortical cells.The epidermis, endodermis and central cylinder were uninfected.Similar features characterized the my- corrhizas formed under gnotoxenic conditions by seedlings of H. almeriense inoculated with spo- ral suspension of T. claveryi in an autoclaved soil (Cano et al. 1991).
Inoculation of T. claveryi improved in vitro survival of rooted plantlets (Fig. 6).However, this positive effect did not prevent apical necro- sis and leaf fall at 6 weeks.This indicates that the MMN medium is not adequate.In comparison with MS medium, MMN is a nutrient-poor medium.Moreover, its high pH ( 8) is known to inhibit plant development in vitro (Pierik 1987).
In conclusion, the results of mycorrhizal synthesis have shown that it is possible to obtain in vitro mycorrhizal symbiosis between micropropagated plantlets of H. almeriense and T. claveryi on a modified MMN medium.Our further re- search will concentrate on developing a modified MS medium in order to obtain better plant growth and mycorrhization in vitro, and on studies of post vitro development of mycorrhizal H. alm- eriense plantlets.Fig. 6.In vitro survival percentages of mycorrhizal (R+M+) (R-M+) and non mycorrhizal (R+M-) (R-M-) plantlets or microcuttings grown on an MMN modified medium.