Effects of Gamma Irradiation on Seeds Germination, Plantlets Growth and In vitro Antimalarial Activities of Phyllanthus odontadenius Müll Arg

Commissariat General Atomic Energy Regional Center for Nuclear Studies in Kinshasa (CGEA/CREN-K.)/Kinshasa, Division of Life Sciences, Department of Biotechnology and Molecular Biology, DR Congo. Institute of Biomedical Research of the Armed Forces (IRBA), Marseille, France. Department of Biology Faculty Science, Laboratory of Plant Ecology and Systematics. University of Kinshasa, DR Congo. Université Kinshasa, Faculty Science, Department of Biology, Laboratory of Biochemistry of Nutrition and Food, DR Congo. Commissariat General Atomic Energy/Regional Center for Nuclear Studies in Kinshasa (CGEA/CREN-K.), Division of Life Sciences, Department of Microbiology, DR Congo. Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610, Antwerp, Belgium. Faculty of Pharmaceutical Sciences, University of Kinshasa, B.P. 212, Kinshasa XI, DR Congo.


INTRODUCTION
Human malaria is one of the most important health problems in tropical and subtropical regions. The estimated clinical cases for WHO were 216 million in 2010 [1], approximately 40% of world's population were at risk of malaria. Nearly 655,000 died from to malaria disease, mainly children under 5, pregnant women and elderly [1,2,3]. A major obstacle to malaria control is the emergency and spread of antimalarial resistance drugs, and urgent efforts are necessary to identify new classes of antimalarial drugs.
Plants produce more than thousands of different compounds through the secondary metabolism pathways.These secondary metabolites are often the keystone in the interactions between plants and their environment as for example phytoalexins, that are involved in plant defense. The properties of these molecules are used in traditional medicine, but also in modern allopathic medicine through the use of purified or derived components obtained from chemical hemi-synthesis [4].
Plants have been used medicinally throughout history, and the two best conventional antimalarial drugs, artemisinin from Artemisia annua (Asteraceae) and quinine from Cinchona sp (Rubiaceae), are both derived from traditional medicines. Traditional medicine using plant extracts continues to provide health coverage for 80% of the World's population, especially in the developing world [5,6].
The biosynthesis of secondary metabolites is dependent on some extrinsic and intrinsic constraints [7] as some differences in quality and quantity of compounds within the same species are observed depending on regions of the world. The metabolic engineering approach is one of the pathways in the production of secondary metabolites with economic interest value. To that, the metabolic engineering approach consists to modify the plant physiology to make it produce a molecule of economic interest. However, this approach has been facing many difficulties due to the implication of secondary metabolites in many regulations, relatively unknown, contributing to maintain plant homeostasis.
In recent years rapid procedures for obtaining transgenic roots have been developed using Agrobacterium rhizogenes, a soil pathogen which elicits adventitious, genetically (Ri T-DNA) transformed roots [8][9][10][11][12]. As genetic variability is essential for any crop improvement program, the creation and management of genetic variability becomes central base to breeding. Experimentally, induced mutations provide an important source for variability. A variety of ionizing radiation forms, including X-rays, γ-rays, neutrons and ion-beams, have been used as mutagens for mutation breeding in addition to chemical mutagens [13,14].
The damages of cell at radiation were caused often through the formation of reactive oxygen species (ROS). ROS induce oxidative damage of DNA, including strand breaks, base and nucleotide modifications [14]. In general, ionizing radiation predominantly induces the formation of radical by water radiolysis, and these then attacks DNA and produce oxidative damage [17]. The formation of 7,8-dihydro-8-oxo-2'-desoxyguanosine (8-oxo-dG) represents one of the most abundant and best characterized types of oxidative damage [14,17].

Phyllanthus odontadenius Müll. Arg. (Synonym: Phyllanthus bequaertii Robyns & Lawalrée)
is present in all the coastal countries of West Africa from Guinea-Bissau to the Congo, and from Rwanda, Kenya and Uganda to southern Africa. In Ghana, the leaves are eaten to get the hiccups. In Rwanda, the stem part extract is used to treat diarrhea and cholera. The alcohol crude extracts of leaves and stems have used to calme diarrhea induced castor oil in mice. A water extract of the entire plant inhibited the DNA polymerase of hepadnaviruses [18], 19]. [20] Reported that plant part unspecified used to treat earache in the northern Nigeria. The common names geron-tsuntsaye, gero itache, ebógi and ebó zùnmaggi are used for P. amarus, P. odontadenius, P. pentandrus, probably P. urinaria and probably also some noo-Phyllanthus species.
Some author published results on Phyllanthus species which ones by the error appellation were called Phyllanthus niruri that would be probably P. odontadenius or P. amarus. For that, they reported that P. niruri contains primarily lignans, flavonoids, alkaloids, phthalic acid and tannins [21]. It has been widely used in treating a number of traditional ailments [20], [21,22] and has demonstrated In vitro antibacterial actions against Staphylococcus, Micrococcus, and Pasteurella bacteria as well as In vivo and In vitro anti-malaria properties.
Ion beams and γ-rays are utilized as models of high-linear energy transfer (LET) and low-LET radiation, respectively, to study the biological effects from various radiations. It is known that high-LET radiations tend to confer greater biological effects (such as cell-killing and mutagenesis) than low-LET radiations [17]. Results obtained by [14] indicate that the transversion G (guanine) to T (Thymine) was greatly induced by γ-rays exposure. [23] reported that γ-rays were the most efficient ionizing radiation for creating mutants in plants as they can induce high mutation numbers in plants.
The aim of the present study was to monitor the effects of γ-rays ( 137 Cs) on the production of active secondary metabolites in P. odontadenius aerials parts in order to amplify those with In vitro antimalarial activity.

Plant material
The plant material used for harvesting fruits was identified by the senior Assistant research Anthony KIKUFI, Laboratory of Systematic Botany and Plant Ecology, Department of Biology (Faculty of Science). The seeds of P. odontadenius were used such as study material.

Irradiation of seeds
Seeds of P. odontadenius obtained from drying fruits harvested on the Kinshasa university site were irradiated with gamma rays from Cesium 137 (Cs 137 ) source in the Conservatome Lisa I Irradiator at the Department of Biochemistry, General Atomic Energy Commission (GAEC). The dose rate was 1.21 Gy/min [24,25] and the treatments ranged from 0 to 300 Grays (Gy). The first generation of irradiated seeds was designed as M1.

In vitro seeds germination
The irradiated material was germinated in Petri dishes with 3 replications. M1 seeds were disinfected with 70% (v/v) ethanol for 1 min, sterilized with 0.125% (w/v) HgCl 2 for 3 min, and washed with sterile distilled water. They were then handled with gibberellic acid (GA 3 ) 200 mg/L for 4 h and finally drained before being cultivated on modified Murashige and Skoog (MS) basal media without sucrose or growth regulators and supplemented with 0.8% agar [26,27]. The pH of the media was adjusted to 5.6 before autoclaving at 121ºC for 15 min. Cultures were incubated at 25±1ºC under fluorescent light with 16h photoperiod. Percentage of seeds or the germination rate for each dose was determined by the equation before.
Where n: number of germinated seeds and N: the number of seeds in de Petri dish.

In vitro and in situ seedling transfer
After 2 months, M1 plantlets from Petri dishes were transferred to tubes containing MS basal media with 3% sucrose, without growth regulators supplemented with 0.8% agar for In vitro culture or in polyethylene bags containing 300 g of soil for in situ growth (Chaves, 2006). Bags were then buried in 3/4 in the ground in randomized complete block (RCB) design with 3 replications [28][29][30][31]. The plantlets placed in situ were watered three times a week, the odd days, with the same amount of water (20L per plot 5 dm/6dm).

Seedling growth and Plantlets survival
The length of vitro plants was shouted using a numeric apparatus PENTAX Optio w30 WATERPROOF and measured with Optimas6 software. The number of leaves per plant was counted manually.
The survival rate of plants was determined with following formula: Survival rate (%) = (number of the surviving plants x 100)/number of transferred plants The number of mutants (albina, xantha and viridis) observed following the irradiation treatment in M1 was determined and the mutation frequency (Msd) was calculated as follows [28]: Msd = [number of mutant (albina + xantha + viridis) x 100]/N with N is the total number of seedlings obtained for a dose.
Parameters such as collar diameter shoot length, number of branches for the selected M1 plants were measured after four months of culturing. The length of plants was performed using a lathe measuring 50 cm. The collar diameter was measured using Slot-foot Digital CALIPER 150 mm (6'') and the number of branches was measured manually. Fresh Biomass and dried biomass for aerial parts after plants harvest were measured using a balance DENVER APX-100.

Culturing of Second Generation Seeds
The second generation (M2) was obtained from In vitro culture of M1 seeds using the same methods as for irradiated seeds. After In vitro germination, 50 plants of each irradiation dose treatment were sowing in row and plants were watered also three times a week. The same parameters such as diameter of collar, shoot length and branches number were determined. Fresh and dried biomass for aerial parts of M2 plants were equally measured after four months plants harvest.

In vitro Antimalarial Activity
Antimalarial activity assays were performed at the National Institute of Biomedical Research (NIBR) in Kinshasa/Gombe, DR Congo and at the UMR-MD3 laboratory, Institute of the Biomedical Research of the French Army -Antenna Marseille, France.

Effects of plant drugs on clinical isolates of P. falciparum
The stock solutions were 200µg/ml for M1 and 125µg/ml for M2 extracts. These solutions were prepared in 1% DMSO and diluted in two fold to have test concentration. Clinical isolates of P. falciparum were obtained from symptomatic malaria children (0-5 years) with high parasitaemia and who did not receive antimalarial treatment in the three weeks preceding the diagnosis at Maternity Hospital of the Sisters of Kindele, Mont-Ngafula, Kinshasa. Venous blood samples (4ml) were collected in tubes containing 1% heparin, and centrifuged for 5 min at 3000 rpm to separate the plasma and the erythrocytes. 1ml of erythrocytes were mixed with 9 ml of RPMI 1640 containing 25mM HEPES, 25mM sodium bicarbonate and 10% of pooled human serum. After homogenization, 50µl of the suspension were distributed in each well of a spot plate containing decreasing concentrations (125 to 0µg/ml) of M1 or M2 extracts [32].
Plates were then maintained at 37ºC in a humid atmosphere containing 5% CO 2 . Quinine was used as control. After 48h-incubation, thin smears were made and stained with GIEMSA 5% and parasitaemia were determined with a Zeiss Primo Star microscope (GmbH/Germany) [33,34]. Inhibition of parasitaemia (percent) was calculated as following: Inhibition (%) = (A -B/A) x 100, where A is the parasitaemia in the negative control and B, the parasitaemia in the treated plates bucket. The IC 50 of each sample was obtained using the dose-response curves.
The in vitro susceptibility to extracts was determined by measurement of [ 3 H] hypoxanthine incorporation into parasite nucleic acids using the method of [36]. Chloroquine was used as control. Extracts and chloroquine were placed in 96 wells microplates and serial dilutions were made in RPMI medium (final concentration ranging from 0-100µg/ml for extracts). Synchronous culture with parasitaemia of 0.8% and 1.5% final hematocrit were distributed. Parasite growth was assessed by adding 1µCi of [3H] hypoxanthine (specific activity of 14.1 Ci/mmol; Perkin-Elmer, Courtaboeuf, France) to each well at time zero. The plates were incubated for 42 h at 37ºC in an atmosphere of 10% O 2 , 5% CO 2 and 85% N 2 and then frozen at -80ºC. After thawing, the content of each well was collected onto filter plates (Filter Mate Cell Harvester, Perkin-Elmer) and 25µl of scintillation fluid (Microscint O, Perkin-Elmer) was added to each well.
The level of parasite incorporation of radioactivity (in counts per minute) was measured with a scintillation counter (Top Count, Perkin-Elmer). Antimalarial activity was determined as concentration of drugs inducing 50% of growth inhibition (IC 50 ) by nonlinear regression analysis from the dose-response relationship as fitted by ICEstimator software [37].

Preparation of crude extracts
10g of dried plant material were macerated separately with ethanol and dichloromethane (300 ml each) for 24h. Each mixture was filtered and dried at 45ºC for 72h. The aqueous extract was prepared by mixing 10g of dried plant material with 300 ml distilled water. The mixture was boiled at 100ºC for 15 min, cooled, filtered and dried at 45ºC for 72h.

Statistical analysis
Data were subjected to Analysis of Variance (Anova) using MSTAT-C Software (Borzouei et al. [9] and compared to the software Statistica with General Linear and LSD test (Least Significant Difference) in order to identify differences between treatments. Means of different treatments were separated with LSD at 5% level of probability.

Effects of Gamma-Irradiation ( 137 Cs) on the Growth of P. odontadenius
Results obtained in this work were mentioned in Figs. 1 (a-d), 2 (a-e) and 3 (a and b) and in the Tables 1 to 5.

Seeds Germination and Growth of In vitro P. odontadenius seedlings
The Table 1 exhibiting the effects of Gamma-Ray (Cs 137 ) on In vitro irradiated seeds germination and seedling growth, shown that rate of In vitro seeds germination of M1 plants decreases with increasing irradiation doses. The highest germination rate (43.85±11.17%) was observed for the control (0Gy), while the lowest germination rate (7.06±1.22%) was observed for 225Gy irradiation treatment. A small effect on In vitro seeds germination was observed at doses of 25, 50, 75 and 100Gy ( Table 1).
The reduction of the emergence in M1 plants was higher after gamma-irradiation at 225Gy (83.90% reduction when compared with the control) ( Table 1). Low reduction of emergence was found following irradiation at 25, 50 and 75Gy (19.77%, 19.73% and 16.33%, respectively). Following 100Gy irradiation treatment, the reduction of the emergence of M1 plant was above 30% ( Table 1).
The size and the number of leaves seedlings from In vitro cultured M1 were higher than those of the control (0Gy) ( Table 1). The size of leaves measured for the control plant was significantly different from that of M1 plants obtained after irradiation treatment from 150Gy to 300Gy. The number of leaves calculated for the control plant was significantly different from that of M1 plants obtained after irradiation treatment at 300Gy.

Growth of in situ P. odontadenius plants
Results on growth of P. odontadenius plants in situ were presented in Table 2 exhibiting Length, Collar diameter, seeds number per plant or seeds Weight (mg) and biomass per plants.
For plants which grew in situ, the size and collar diameter (14.01±5.12cm and 2.68±1.07 mm, respectively) of M1 plants obtained after seeds irradiation at 50Gy dose were significantly higher than those observed for the plant control (9.34±4.38 cm and 2.01±1.09 mm, respectively) and for seeds irradiated at 300Gy (3.60±0.28 cm and 1.10±0.14 mm, respectively).
In P. odontadenius control plants, the number of seeds per plant was 234±58 weighting 70.32±17.30mg. The largest number of seeds per plant was observed at 225Gy with 715±182 seeds per plant weighting 214.69±54.72 mg while the most important quantity of biomass was obtained at 50Gy (29.60±9.19 g) and the lowest at 300Gy (2.00±0g).

Morphological effects on P. odontadenius plants in situ
Phenotypic effects were also observed on P. odontadenius plants growing in the field. In particular, we observed changes on leaves of P. odontadenius plants whose seeds were irradiated at 50, 100, 125 and 225Gy. In particular, at dose of 100Gy, the change of plant leaf in purple color profoundly affects the individual plant who dies (Fig.1c). These effects are reflected to the roots which would furnish themselves too specific phenotypic traits (Fig.2  cand Fig. 2d).
Color defects in the roots are appeared blackish accompanied by white roots or blackish simply for plants obtained with seeds irradiated at 100Gy (c and d). 50Gy (b) has very long roots exceeding the size of the aerial part (Fig. 2). The frequency of mutations differs significantly depending on the doses used. The mutation frequency was the highest at 225Gy with 22.22%, followed by 100Gy with 13.33%. Frequencies at doses of 50 and 125Gy were respectively 8.33% and 9.09%. These percentage changes are an indication of what may be the changes in M2.       (Fig. 3a and Table 6). For crude extracts from M2 plants ( Fig. 3b and Table 6), values ranged between 1.00±0.22µg/ml (200Gy) to 5.96±0.91 µg/ml (75Gy). The lowest antimalarial activity was thus observed for M1 and M2 crude extracts obtained after seeds irradiation at 75Gy.

Phytochemical analysis of P. odontadenius
Phytochemical analysis indicates that different types or chemical groups of secondary metabolites are more present in the second generation than in the first one (Tables 4 and 5).
Except for tannins, anthocyanins and polyphenols that were detected in M1 and M2 plants control and the absence of anthraquinones in M1 and M2 control plants and those obtained after irradiation of seeds in any samples, the presence of other major chemical groups in both M1 and M2 generations varied depending of irradiation treatment. Alkaloids were not detected in M1 plants but were detected in M2 to plants from irradiated seeds at 25, 50 and 75Gy. By contrast, saponins were found only in M1 plants obtained after irradiation at 25, 50, 75, 100, 150 and 250Gy. Finally, compounds like free quinones, flavonoids, terpenes and steroids were more found in M2 than in M1 (Tables 4 and 5).

DISCUSSION
On isolates P. falciparum, all extracts M1 from irradiated seeds have higher antimalarial activity against P. falciparum with the exception of those of control with 6.95µg/ml who exceeded 5µg/ml and has moderate antiplasmodiale activity [32]. In M2, except the control (0Gy) and 75Gy those have IC 50 exceeding 5µg/ml with10.45±1.18 and 5.96±0.91µg/ml and were presented moderate antimalarial activity, all extracts from irradiated seeds presented higher antimalarial activities against P. falciparum with their IC 50 low than 5µg/ml.
Absence or presence of one or another group of secondary metabolites in samples from M2 seeds could be explained by changes at the molecular level that would be induced by the energy of radiation given during the irradiation of the seeds. Some genes are sensitive and others were no sensitive. Certain genes would be sensible and the others never because of the sequence reparations on DNA damages or the presence of redundant genes can be explain this phenomena [44][45][46]. It knows that punctual mutation request two respective cycles of replication for their definitive fixation. Their effectiveness was possible in the second cycle of replication in the moment that the incorrect pair of DNA produce definitive change in the sister molecules [47].
From the above, the results obtained in this work confirm the hypothesis that secondary metabolites are stimulated by both abiotic (irradiation and various chemical compounds) and biotic elicitors groups (glycoproteins, molecules derived from microorganisms and polysaccharides) [23]. The gamma rays interact with atoms and molecules to create free radicals which are capable of altering the major components of plant cells. These radicals affect morphology, anatomy, biochemistry and physiology of the plant according to the irradiation dose.
From 100Gy dose, there is presumption of probable and obvious mutations or changes because emergencies reduction from 100Gy were upper 30% [28]. Our results are consistent with those of [39] who obtained a high frequency of mutations with durum wheat (Cicer arietinum L.) to 100Gy for Sofu cultivar.
Studies have shown that parameters such as the rate of seed germination, shoot length, root and seeding are affected by irradiation and chemical mutagen. For example, [48] showed that the rate of seed germination and plant height (stem or root) decreased when seeds of rice (Oryza sativa L.) were treated with gamma radiation and sodium chloride (NaCl). These observations were confirmed by [49] on Jatropha curcas L. using mutagens (EMS and gamma rays). [50] Reported that sodium azide (NaN 3 ) affects the rate of seed germination, shoot length, root and delays germination. All the parameters decrease with increasing doses of irradiation or with increasing chemical mutagen concentrations. Then, as the survival of mature plants depends on the nature and extent of chromosomal damage [51], the increasing frequency of chromosomal damage associated with increased doses may be responsible for poor germination and reduced size and survival of plants.
Doses causing an emergency reduction of seeds or a reduction in size of plants by over 30% compared to the control are considered to be high for a large-scale program for mutation of plants with high probability to obtain mutants [28].
Survival rate of plants is significantly reduced and the lowest rate (10%) was observed at 300Gy irradiation doses. Witness presented 100% survival of plants followed those of 125Gy and 175Gy 46.67% to 46.15%.
Effects of gamma irradiation varied according to whether they could be positive, increasing an existing character for such dose, or negative, decrease or disappear to a character that existed for such another dose. [52] Show that the best plant survival of chickpea (Cicer arietinum L.) was observed at 180Gy. [53] Observed that Survival of carrot plants from seed in their study was reduced by >50% at the 10-and 20-krad doses. The decrease in survival of irradiated plant material resulting in a physiological imbalance at various events affecting vital cellular macromolecules. [54] Noted that the lower survival rate of plants after gamma irradiation is attributed to the destruction of auxin.
In contrast to the stimulation of the size observed with increasing doses by [55] for two varieties of chickpea (Cicer arietinum L.), P. odontadenius showed in this study, a decrease in size in vitro and in situ, in the second generation, when the plants are subjected to a continuous shade. Average plant height of P. odontadenius showed some higher values regardless of the growth rates in situ. Our results are consistent with those obtained by [56] in the analysis of the effects of sodium azide (NaN 3 ) on growth and yield of Eruca sativa L. for the 30th and 45th day. Comparing our results with those obtained by the same authors in the 90th day, they do not correspond to those found with E. sativa.
In vitro control plantlets showed higher mean values for the rate of seed germination, rate of seedling survival and for the seedling average number of leaves than of all average rates of third parameters for plantlets from irradiated seeds. Parameters of P. odontadenius plant transferred in situ showed high average values of height, number of seeds per plant, collar diameter and biomass average for plants from irradiated seeds indiscriminately compared to control plants. Many authors agree that observed characteristics are also dependent on environmental factors [30,57] because the phenotype is the sum of factors genetic and environmental.
The effects of size and collar diameter have been reported previously by [58] on the primary and mutagenic effects of gamma irradiation on A. thaliana seeds. These observations confirm the theory that the first generation generally have a higher rate of heritable traits than the following for which the expression of these traits results from genetic and external conditions [59,30,57].
However, the quantity of biomass per plant and fertility tend to decrease as the radiation dose increases. The decrease in biomass could be explained by disturbances in the synthesis of chlorophyll, reliable molecular index for the assessment of genetic effects and gas exchange at the plant [60,61,54] explains that the abnormal leaf growth (biomass) is due to disruption of pigments (e.g., chlorophyll), the chromosome aberration and inhibition of mitotic division.
These disturbances are responsible for some of the negative effects expected by the treated plant material to gamma radiation [62][63][64]. They include changes in cell structure and metabolism of the plant, for example, dilatation of thylakoid membranes, and alteration of photosynthesis or modulation antioxidant systems and accumulation of phenolic compounds [65].
Effects of mutation were more pronounced in M1 than of M2 generation comparing to the control. Results obtained by [66]  [67] Confirm that seeds M1 of beans (Cyamopsis tetragonoloba (L.) Taub) produced more lethal and effects of pollen sterility while in M2, it are more thoroughly, the presence of various viable chlorophyll mutations. However, mutagenic effects increase both M1 in M2 with increasing doses of mutagenic [13]. [68] Show that sodium azide (NaN 3 ) gives more pronounced effects at 0.5mM concentration than 0.1mM only when the rice (Oryza sativa L.) is soaked in sodium azide. These results confirm the hypothesis that effects of irradiation are more pronounced in the first generation compared to the second [67,66].
[69] Give four main groups of secondary metabolites in plants including terpenoids for the first group, phenolic compounds for the second group, Saponins, cardiotonic heterosids and cyanogenic heterosids and glucosinolates for the third group and alkaloids the fourth. These four groups are found in varying amounts in plants of P. odontadenius derived from irradiated seeds.
Flavonoids and other phenolic compounds (tannins, free quinones, anthocyanins and polyphenols) were prominently reported previously. [70] Reported that phenolic compounds have antioxidant activities, allowing them to protect plants against damage caused by radiations. Flavonoid synthesis can be explained by the activity of phenylalanine aminolyase (PAL), which, as a response to radiation (gamma and UV) in alleviating the damage caused by it [23].
With the presence of flavonoids in all samples of M2, our results confirm those of Lois (1994) cited by [23] which showed that irradiation of the Arabet plant (Arabidopsis thaliana of the Brassicacaceae family) increased the level of flavonoids which are accumulated in the aerial parts of the plant. Besides flavonoids, our results show that the level of alkaloids, free quinones, terpenes and steroids and anthocyanins increased.
Presence of alkaloids in M2 and their absence to M1 generation could be explained by the effect of gamma radiation on cellular proteins that release amino acids such as lysine, phenylalanine, tyrosine, tryptophan and ornithine which are the starting point for the biosynthesis of alkaloids [69]. It could be the same even for terpenoids and steroids derived from acetyl-CoA pathway, this latter come from to a disorder of the Embden-Meyerof way.
Our results on antimalarial activity obtained in this work, M1 and M2 appear low values of IC 50 in all, to say that high antimalarial activities, compared to those obtained by Soh et al.
(2009) on the antimalarial activity of P. niruri (certainly P. odontadenius or P. amarus) collected at three different sites in the Democratic Republic of Congo (DRC). The best antimalarial activity was the one with the aqueous extracts from stems of Kisantu's P. niruri with IC 50 value of 11± 2µg/ml, whereas we obtained IC 50 <11 except for doses of 25, 50, 75, 100 and 125Gy in M2.
In vitro antimalarial activities from crude extracts of P. odontadenius with high doses of gamma rays can be explained by the increase in total phenolics and specific compounds such as coumarin, p-coumaric acid and salicylic acid unlike what [71] found on the biosynthesis of phenolic compounds in seedlings cilantro gamma irradiated at low doses.
Control M1 crude extract showed good antiplasmodial activity on P. falciparum chloroquinoresistant exceeding all samples. This exceptional antiplasmodial activity can be explained by original compounds found in P. odontadenius or by phenolic compounds produced during storage which give place on enzymatique reactions [72,73]. It known that effects of mutation were more pronounced in M1 than of M2 generation comparing to the control [66].

CONCLUSION
The investigations carried out Gamma-irradiation of P. odontadenius seeds induced reduction of parameters such as percentage of seeds germination ( Synthesis of secondary metabolites increase in the second generation compared to the first one with a more important synthesis in phenolic compounds. The In vitro antiplasmodial activity on the clinical isolates P. falciparum (6.95±0.64 µg/ml to 1.00±0.22 µg/ml; 10.45±1.18µg/ml to 1.00±0.05µg/ml) or P. falciparum K1 (4.08±1.49µg/ml to 0.92±0.91 µg/ml; 9.68±2.21µg/ml to 3.91±2.69µg/ml) showed low antimalarial activities from M1 and M2 controls (0 Gy) than that of extracts from treated plants.
The highest In vitro antiplasmodial activity was observed at 100Gy while for M2 crudeextract, the best antimalarial activities were observed at 50, 175, 200 and 225Gy. The high inhibitory effects of crude extracts plants from treated seeds have justified the usefulness of gamma-irradiation in the increasing production of secondary metabolite against malaria in the Word particularly in DRC.
We need to test, however, whether the plant extracts from these doses could also provide high antimalarial activities and lower toxicity on human cells. These studies are essential for plant breeding of P. odontadenius in the antimalarial activities improving.
Professor Daniel PARZY (Institut de Recherche Biomédicale des Armées (IRBA), Marseille/France) for the analysis of in vitro antiplasmodiale activities by microdilution isotopic method; Dr. Patrick DOUMAS (SupAgro-Montpellier/France) for the software Statistica with General Linear and LSD test; CGEA/CREN-K. for the assistance for the realization of this work.