Germination and in vitro multiplication of Helianthemum kahiricum , a threatened plant in Tunisia arid areas

Seeds of Helianthemum kahiricum have an excellent germination rate extending to about 90% within short time not more than four days after scarification (mechanical treatment) and a good protocol of disinfection. A high frequency of sprouting and shoot differentiation was observed in the primary cultures of nodal explants of H. kahiricum on Murashige and Skoog medium (MS) free growth regulators or with less concentration of kinetin (0.5 mg L -1 or 1.0 mg L -1 kin). In vitro proliferated shoot were multiplied rapidly by culture of shoot tips on MS with kinetin (0.5 mg L -1 ) which produced the greatest multiple shoot formation. The kinetin had a positive effect on the multiplication and growth, but a concentration that exceeded 2.0 mg.L -1 decreased the growth. A high frequency of rooting with development of healthy roots was observed from shoots cultured on MS/8 medium hormone-free.

Seeds of Helianthemum kahiricum have an excellent germination rate extending to about 90% within short time not more than four days after scarification (mechanical treatment) and a good protocol of disinfection.A high frequency of sprouting and shoot differentiation was observed in the primary cultures of nodal explants of H. kahiricum on Murashige and Skoog medium (MS) free growth regulators or with less concentration of kinetin (0.5 mg L -1 or 1.0 mg L -1 kin).In vitro proliferated shoot were multiplied rapidly by culture of shoot tips on MS with kinetin (0.5 mg L

INTRODUCTION
Helianthemum kahiricum (called R'guiga or Chaal in Tunisia) (H.kahiricum) is a perennial herb widely found around the Mediterranean basin (Raynaud, 1987).H. kahiricum is an appressed, grey-canescent perennial low shrub that reaches up to 10-25 cm long with many, branched stems.Leaves are 0.5-1.8x 0.15-0.3cm, oblong-Ianceolate, appressed-pubescent, with strongly revolute margins, and acute to obtuse apex.Flowers are with white-villous, violaceous calyx and yellow petals equaling the sepals and are often not opening and arranged in 5-l2-flowered and 1-sided inflorescence.The fruit is an ovoid-globose and hairy capsule with ovoidcompressed, smooth, and brownish.This is a chaméphyte (Escudero et al., 2007), family of Cistaceae, arid regions and semi-arid areas (Perez-Garcia and Gonzalez-Benito, 2006).Despite its ecological and economic interests, this plant is a rare endemic flora of the western basin of the Mediterranean (Escudero et al., 2007), as a result of overgrazing (Aidoud et al., 2006).The size of their population or their range, or both, is

Concentration (mg L
restricted or is greatly diminished.The data indicates that the situation will worsen irreversibly if nothing is done to address this vulnerability.In other words, if the observed situation continues, it will anticipates the disappearance of these species more or less.Among the factors responsible, including loss or degradation of habitat, exploitation of the species, exposure to pollutants, diseases, climate change, overexploitation or other cause result in regression of range or a sustained decline in the number but the population level reaches a critical threshold.The helianthemes have a great pastoral interest.They have a very important role in the fight against desertification and stabilization of vulnerable areas (Diez et al., 2002).In addition, they are involved in the production of desert truffles (Al-Rahmah, 2001;Slama et al., 2006).Desert truffles, known locally as the "terfess" are edible and wild seasonal mushrooms hypogeal (Mandeel et al., 2007).H. kahiricum is considered threatened in an extremely precarious situation.The size of their population or their range, or both, is restricted or is greatly diminished.Thus, to maintain the genetic integrity of clones and conservation of this species, in vitro germination, cultivation microcuttings and stimulation of axillary buds are the most applied in plant micropropagation method.For the species used in this study, in vitro culture seems to be a very interesting alternative for preserving H. kahiricum.Therefore, the purpose of this work was the multiplication of the species by micropropagation highlighting the effect of the composition of the culture medium on the initiation and proliferation of plant.

Plant material
The plant material used consists of H. kahiricum seed from region of Médenine (Benguerdenne: latitude 32°57'09"N, longitude 11°38'26"E, with an arid climate of an average rainfall of 150 mm/year and a sandy soil (Le Floch and Boulos, 2008).The explants which are internodes were taken from 2 months aged mother plants produced in vitro.

In vitro germination
Three experiments were conducted by changing the time and the type of fungicide to optimize better the germination of H. kahiricum seeds on MS medium (Murashige and Skoog, 1962).In all experiments, 20 seeds/replication were soaked in sodium hypochlorite followed by rinsing with distilled water.The seeds were disinfected in 70° alcohol followed by rinsing with sterile distilled water and then applying different fungicides: 1 st experiment (Exp.1): The seeds were treated with benlate (1 g L ˗ 1 ) (10 min), followed by rinsing with distilled water; 2 nd experiment (Exp.2): The seeds were treated with mercuric chloride (1 g L ˗ 1 ) (20 min), followed by rinsing with distilled water; 3 nd experiment (Exp.3): Without fungicide.
After the development of an adequate protocol of seeds disinfection, they were germinated in vials (20 seeds/vial).The germination test was carried out on MS (M0) and MS modified with growth hormones (M1, M2, M3 and M4) (25 repetitions for each test) (Table 1).Incubation takes place in a growth chamber under controlled conditions with an alternating day/night (16:8 h), a relative humidity of 80 and 45%, respectively, and a temperature of 25 ± 2°C for one week.

In vitro multiplication
The vitropousses from in vitro germination were then stripped of their leaves, cut in microcuttings (1 or 2 cm) with 1-2 nodes.Ten explants per vial were transferred to different Culture medium: MS, MS supplemented with growth hormones (C1, C2, C3, C4, C5 and C6) (25 rehearsals for each test), and MS diluted (MS/2, MS/4, MS/6 and MS/8) (25 rehearsals for each test), without hormones (Table 2), with a view follow the morphological attributes such as, bud, the length of the main stem, number of leaves, number of nodes, root length and rooting rate.After their rooting, the vitropousses were transferred into plastic pot filled with sand to ensure their adaptation and possible culture in an experimental plot for acclimation.

Statistical analysis
The results of analysis of variance (ANOVA) of the different parameters were obtained by the software SPSS v.11.5.Multiple comparisons of means and the setting command classes were made by Duncan's test.

In vitro germination
The success of in vitro culture depends on aseptic conditions for the cultivation of plant material used.After incubation, the best germination rate and reduced number of seeds contaminated are generated by the first test disinfection (100%) (Exp.1).The second disinfection test (Exp.2) yielded an average percentage of germinated seeds (32%) while the third disinfection protocol yielded no germination (0%) (Exp.3).Seeds of H. kahiricum have an excellent germination rate; can reach 100% within short time not more than four days after scarification (mechanical treatment) and disinfection (Benlate).Scarification reduces germination time; it can stretch to several months because of the very rigid tegument of seeds.

Effect of growth hormone on seed germination
The seeds of H. kahiricum germinated on culture medium supplemented with growth hormone have present deformation (after germination) which results in vitrification and intense hydration with the formation of callus and necrotic leaves (Figure 1b, c, d, e and f).
Growth hormones have adverse effects on seed germination and thereafter are not effective for the germination of H. kahiricum seeds; while, the seeds on MS without growth hormones present a simple germination (Figure 2a).

Effect of growth hormone on micropropagation of H. kahiricum Multiplication on MS medium without growth hormone
Shoots on MS medium are vigorous with chlorophyll leaves, normal and average percentage of vitrification.Morphological changes of vitropousses during subcultures affected neither leaves nor the appearance of the root.The leaves of plantlets contained the chlorophyll pigments, not necrotic, their size increases a subculture to another and becoming stronger.The stems reached a remarkable stiffness and strength during subcultures (Figure 1g, h and i).In H. kahiricum, it seems that the rooting phase is characterized by the proliferation of shoots on MS without growth hormones.Indeed, the time for obtaining vigorous shoots able to be transplanted is 8 to 12 weeks and it was during this period that shoots rooted (Figure 1j).Thereafter, the rooted vitropousses from the multiplication phase was prepared for the acclimatization stage.

Recovery and proliferation axillary
Axillary buds subcultured on culture medium contains combinational hormones after six weeks of culturing explants bud unlike in the control medium, it takes place after two weeks (Figure 2a).MS has the highest rate of bud break (almost 90%).The effect of growth hormones is manifested by a significant reduction in the bud.The addition of the NAA causes an increase in the rate of bud compared to IBA.This can be explained by the physiological response of the explants on the interaction auxin/cytokinin, and the nature of the substance used for growth.The average shoot length appears to be strongly related to the concentration of hormones (Figure 2b).Using the results of the percentage of bud, it appears that the hormonal combinations recording bud rate reduced compared to the control.The influence of increasing the concentration of kinetin in the medium alone is shown by a significant decrease in the average length of the shoots.

Average number of nodes
The combination of auxin and cytokinin in the culture medium indicates that the increase in the kinetin medium, alone or in combination with auxin results is in a decrease in the average number of nodes relative to the control (Figure 2c).At a concentration of 0.5 mg L -1 Kin (C1), the average number of nodes reduced to about 20% whereas for 1.0 mg L -1 Kin (C4) it was in the order of 25%.In addition, 0.5 mg L -1 kin combined with 1.0 mg L -1 NAA (C3) gave a greater than 50% reduction compared to the control and the combination C2 (0.5mg L -1 kin + 1.0 mg L -1 IBA).It seems that the addition of growth regulators had a regressive effect on the average number of nodes.

Rooting
The cuttings rooted after three weeks in MS, then after a month we observed the emergence of some very harsh roots of short length on media supplemented with growth regulators.It appears that the observed formation of    callus at the base of the explants and at the location of sections inhibited rooting.Cuttings made on media containing no auxin (C1 and C4) showed highest rooting percentage.It also turns out that the addition of auxin in the medium does not improve rooting.Whatever the amount of kin in the medium, we find that IBA promoted rooting with a rate of 16% and the NAA with a rate of 21%.The rooting percentage and mean root lengths evolved in a proportional manner (Figure 2e).

Effect of ratio auxin/cytokinin on explants of H. kahiricum
The auxin/cytokinin ratio determines the physiological functioning of the explants, the percentage of callus is quite high compared to the control for all hormonal combinations (Table 3).However, the rate of rooting and sprouting of control are significantly higher compared to other treatments.The ratios of auxin/cytokinin about 2 (1:0.5 mg L -1 ) promote rooting, indeed, for the combination C2 (1:0.5 mg L -1 ) rooting percentage was 41% against a rate of 16% for the combination C5 (1:2 mg L -1 ).The C5 (1:0.5 mg L -1 ) promote axillary bud (62%), while C3 (1:0.5 mg L -1 ) promoted it by 22%.However, the dilution does not influence the rate of bud, but it manifests itself on the strength of axillary shoots.There is also a strong ability of budding in vitro H. kahiricum associated with its proliferation of axillary explants in the presence of all medium (Table 4).

Acclimatization
The survival rate of plantlets is influenced by the nature of the substrate.The best survival rate of seedlings (63%) was recorded on a substrate perlite.

DISCUSSION
H. kahiricum is considered threatened in an extremely precarious situation.Micropropagation of this plant was an attempt conducted from nodal explants of plantlets in vitro to preserve/conserve this species by the micropropagation technique which aims to safeguard biodiversity; thereby developing an appropriate protocol for disinfecting seeds which was necessary to achieve aseptic cultures.Seed germination after strong disinfection has the minimal contamination and a high rate of seeds germinated on MS medium without hormone.It appears that H. kahiricum does not require growth hormone for its germination.Hence, the study was based on regeneration by in vitro proliferation of axillary buds of H. kahiricum by the use of growth hormones in order to improve the multiplication.Microcuttings grown on MS medium without hormone presents the highest rate of budding and rooting.It seems that this plant has high potential to multiply on a hormone-free MS medium and the bud of the axillary shoots is easy without going through an induction medium.Growth substances (auxins and cytokinins)

Figure 2 .
Figure 2. (a) Action of culture medium on the percentage of axillary buds of H. kahiricum, (b) action of media on the average length of H. kahiricum shoots, (c) action of media on average number of nodes, (d) effect of media on the average root length (cm) of H. kahiricum explants, (e) effect of media on rooting rate of H. kahiricum plantlets.

Table 2 .
Composition of culture medium with growth hormone (auxin and cytokinin) for the multiplication of Helianthemum kahiricum explants.

Table 3 .
Change in callus, bud break and rooting with hormone combinations of auxin/cytokinin in seedlings of two months old Helianthemum kahiricum from explants.

Table 4 .
Effect of MS dilute of the attributes morphological seedling of Helianthemum kahiricum aged two months from explants.
b Means with different letters are significantly different at threshold p<0.05 (Duncan test).