INFLUENCE OF MEDIUM AND GROWTH REGULATORS ON CALLOGENESIS OF QUINOA ( CHENOPODIUM QUINOA WILLD . ) AND EFFECT OF HYDROUS STRESS INDUCED BY P . E . G 6000 ON THE CALLUS

The induction and growth of quinoa’s callus depend on several factors, including the culture medium and the nature of the growth hormone and its dose. In effect, the best callogenesis rates were obtained with the media MS and B5 with respect to the media WHITE and KNOP the callogenesis is too low or zero. The best combination used was 0.2 BA+2.4 D give well-developed callus. To obtain water-stress resistant cell lines, the effect of water stress induced by polyethylene glycol (P. E. G 6000) on the growth, osmotic potential and metabolic parameter of Chenopodium quinoa callus was studied. Applied water stress showed a reduction in the growth of stressed callus compared to the control. The presence of PEG in the culture medium caused a decrease in the content of fresh matter as well as the dry matter content compared to the control. Water stress also significantly affected the water parameters of calluses. The chlorophyll a, b and carotenoids content decreased, but this decrease is not too pronounced.


INTRODUCTION
The in vitro culture is a very recent technique since it was developed only at the beginning of this century by GAUTHRET [1], who was among the first to cultivate tissues, coming from cambial cells of different trees (poplar, maple, willow), on agar medium under aseptic conditions.Today, under the term "in vitro culture" is hiding much diversified fields and techniques, which strongly developed and specialized since thirty years.In the vegetable field, in vitro culture methods apply to a very diversified material (protoplasts, tissues, organs, whole plants), to achieve oriented objectives, either towards research or towards industrial production [2].
On the other hand, plants can be regenerated from callus tissues of various explants by dedifferentiation prompted by exogenous growth regulators.Regeneration from callus is by organogenesis or somatic embryogenesis [3].The present study was conducted with an aim of finding the influence of medium and growth regulators on callogenesis of quinoa (Chenopodium quinoa Willd.) and effect of hydrous stress induced by P. E. G 6000 on the callus.

Experimental details
The experiments were carried out in the in vitro culture unit at the Arid Area Institute (IRA), located at el FJE Medenine (33 °03' N; 10 °38' E) in south-east Tunisia.The quinoa's calluses are initiated from stems (fragments of internodes; 5 cm long) of the cultivar Q-37 originated from Chile at the rate of ten explants × three repetitions per test.
For the sterilization of the plant material, we applied the most used method, which is proposed by GAUTHRET [4], by putting it in a solution of sodium hypochlorite 50% (NaClO) for 30 min, followed by five rinses with sterile distilled water during five minutes each, to remove traces of bleach.Then we have put fragments, in 70% alcohol for five minutes, five rinsing with sterile water of five minutes each one to remove all traces of alcohol.This is done under a laminar flow hood.
The transplanting material (scalpels, tongs, petri-dishes, etc.) is cleaned and then autoclaved in a vapor phase at 120°C for 20 min.The culture mediums are also sterilized by autoclaving.The pH's medium is adjustets to 5,5-5,8 with a solution of NaOH (1N).
During the manipulation, each tool is rinsed in 70 alcohol.Finally, the cultures are placed in an air-conditioned chamber at 25±1°C, with "Phillips-40 W" tubes, guaranteeing an illumination of 2000-2500lux.The photoperiod is 16 h of light a day.
In this study we try to find the most favorable nutritional conditions for the callogenesis of quinoa's explants.For this, we have chosen the following mediums: MS [5], B5 [6], KNOP and WHITE [7] (table 1).The main constituents of these media are ionized water and mineral salts, which are divided into two groups: macroelements (N, P, K, S, Mg, Ca) and microelements (Fe, B, Mn, Zn, Cu, Co, Mo, I).The source of carbon is sucrose and in these media is found vitamins, amino acids and gelling agent.
After two months of cultivation the following measures were made: • Percentage of callogenesis with simple counting; • Diameter of the calluses using a sliding caliper; • Color and aspect of calluses.

Effect of water stress on callus
To test the effect of hydrous stress on callogeninis of quinoa's tissues, we have used Polyethylene Glycol 6000 (P.E. G) which is a polymer of chemical formula COCH2(C2H4O)n.The various levels of water potential were obtained by the formula established by MICHEL and KAUFMAN [8], this equation relates the hydric potential (ΨH), the concentration of P. E. G and the temperature: The solutions of P. E. G prepared corresponds to the values of the following water potentials: 0 bar (T0);-1 bar (T1);-2 bars (T2).
To avoid the influence of the effect of the medium on the results, the calluses initiated from internodes are growing on the medium MS+0.2 mg/l BA with ten calus × three repetitions for each treatment.
In order to detect the effect of water stress on the morphological, hydric and metabolic characters of calluses; various parameters were measured after two months of culture (table 2).

Data analysis
All data were statistically elaborated using analysis of variance (ANOVA), followed by means separation using S-N-K's multiple range t-test at P<0,05.All calculations were performed with the help of the PASW statistics 18.

Effect of hydrous stress on callogenesis
There is a difference between the average diameters of the control calluses and the stressed ones, this difference is accentuated with the concentration of the medium with P. E. G.A reduction of 26% and 60%, respectively, for the water stress-1bar and -2bars (fig.6).The analysis of the results indicates that the production of fresh matter is clearly affected by water restriction.During the culture, the difference is in favor of the control and the reduction is highly significant (p<0.01) of the order of 70% for-1bar and 84% for-2 bars (fig.7).
The analysis of this fig.
shows that, in the absence of water stress, calluses have the highest dry matter content (0,064g), whereas in the presence of P. E. G 6000, a highly significant decrease was recorded for stressed ones (p<0.01)(fig.8).
Water stress has negatively affected the moisture content, which is further reduced when P. E. G concentrations are high.This is confirmed by the ANOVA data (p<0,01).A maximal average (13 ml/g) was registered under unstressed medium.The water content is reduced to 70% under the water regime T2 (fig.9).The results of this fig.
show that RWC was significantly affected calluses subjected to water stress (P<0.01).Under (-2) bars regime, a reduction of 55% was registered in comparison with the control calluses.
The chlorophyll content showed a significant (p<0.01)decrease.The response of calluses to water stress is proportional to the intensity of the water deficit (fig.11).This decrease does not lead to 13% for T1 treatment, while it is more pronounced under T2 stress in the order of 42% compared to controls.The examination of the fig.12 shows that the chlorophyll b content is closely related to the applied water regime.Analysis of the data shows a highly significant reduction (p<0,01).In fact the recorded contents are 1,48±0,07 μg.g - 1 ; 1,23±0,07μg.g -1 and 0,89±0,07μg.g -1 respectively for T0, T1 and T2.

The effect of medium and growth regulators on callogenesis
The monitoring of callogenesis rate and callus's diameters revealed significant differences between the culture mediums favoring both MS and B5 (p<0,01).This difference can be explained by the effect of the variation of mineral elements that constitute them.The difference is essentially due to the nitrogen and potassium contents [12].Taking into account the nitrophilic tendency of most Chenopodiaceae [13], the presence of sufficient nitrate in MS (1900 mg/l) and B5 (2500 mg/l) medium favored cellular development of quinoa's explants.
Contrariwise, the KNOP and WHITE mediums are poor of potassium nitrate (KNO3) that's why they showed very low levels of callogenesis.Similar results have been proven by Radosvich and Paupardin [14] with axillary budding of quinoa proving that for a potassium nitrate content equal to 2700 mg/l, the development of axillary buds had increased.
Also, the beneficial effect of K and Mg ions has been reported by several authors [15,16,17].These ions are also known to promote plant growth.CABECHE [18] mentioned the very important role of the nitrogen/carbohydrate ratio in the control of the biosynthesis of growth regulators in undifferentiated tissues.The callus grown on the KNOP medium, marked by iron deficiency, are characterized by a light green color, which explains why exogenous iron can be involved in the synthesis of the structural elements of chloroplasts, thus directly influencing the synthesis of chlorophyll [14].It can thus be said that the composition of the in vitro culture base medium plays a very important role in the callogenesis of the quinoa explants.The effect of the medium results from the whole of the interactions of the different elements which compose it.Some of them stimulate in vitro development processes, while others have less influence on development [12,19,20].On the other hand, the influence of cytokins on callogenesis is poorly documented.Some authors [21] consider that the addition of cytokins in the induction medium causes, at low or high doses, browning of the tissues which is harmful to the formation of callus.For the coconut tree, Verdeil et al. [22] notes that they are not essential for callogenesis.These results are contrary to our observations.Indeed, for quinoa, the presence of cytokinin BA in culture media MS and B5 gave very high callogenesis rates of the order of 100%.Our results are in line with those of Branton and Blakes [23], which show that cytokins sometimes stimulate callogenesis.
During the experience, we had also noticed that development of callus and their consistency varied with the hormonal composition used.Callus formed on MS and B5 media with the presence of 2,4D+0,2 mg/l BA have significant development of diameter and they are friable, similar results have been demonstrated by the work of EISA et al. [24] on quinoa.Callus formed with the presence of ANA are smaller and compact.An improvement in callogenesis rates is observed by the addition of auxin in the culture medium.Several studies have showed that the use of auxin induces triggering or induction of callus in a large number of botanical species: the oil palm [25]; Atriplex halimus [26,27,28]; banana and plantain [29]; Phoenix dactylifera L. [30].One of the functions of auxin is to stimulate the mitotic activity of the cambial tissues, a property widely exploited in tissue culture in vitro [31,32].
Other studies have demonstrated the positive effect of combining cytokinin (BAP or kinetin) with auxin (AIA, ANA, AIB).According to Ramirez et al. [33], Mtimet [34], Lemhamdi [35], and Mhatre et al. [36], the combination of an auxin with a cytokin promotes an increase in the number of shoots per plant.For this reason, Chatibi et al.
[37] and Chatibi [38] reported that addition of growth regulators is essential for in vitro multiplication of ligneous plants and that axillary bud neoformation varies with the nature and combination of growth regulators used.Masmoudi [39], working on the callogenesis of the date palm, variety Arichi, has also shown that, although it is essential, a toxic effect of high concentration of 2,4-D (10 mg/l) on callus manifested by an early senescence of explants.
Finally, the growth regulators used in these experiments (BA, 2,4-D and ANA) and their concentrations are able to modify the "register" of cell totipotency and the externalization of the potential for organogenesis in the explants of quinoa.

The effect of hydrous stress on callogenesis
The addition of increasing concentrations of P. E. G 6000 to MS medium induced a very significant diminution of the morphological, water and physiological parameters of callus.Indeed, this reduction is all the more important as the concentration of P. E. G increases.These results show that P. E. G 6000 can be used to create in vitro water stress.Its role as a factor that induces water stress has been proved by different authors [40,41,42].
High concentrations of P. E. G 6000 severely inhibited callus growth.Dehydration of stressed callus may be primarily due to the direct effect of P. E. G 6000 and secondly to osmotic adjustment by accumulation of organic solutes and minerals [43].Such behavior has been frequently reported in woody or herbaceous species and it's considered as an adaptive response to drought [44].When stress is not tolerated by the plant, both fresh and dry yields are reduced, this has been shown also by the work of Trought and Drew [45].
The analysis of the water parameters of the callus, can describe in a global way the water status in response to the hydric stress undergone, as well as to give the level of turgor at the cellular level [46].In our study, the effect of PEG induced a substantial decrease in water content (WC) and relative water content (RWC).This reduction is relatively small in comparison with the high concentration of P. E. G in the culture medium.Our results are consistent with those reported in Sesuvium portulacastrum [47] and Aeluropus lagopidesi [48].

The work of Malin et al.
[49] and Diaz-Perez et al. [50] suggest that genotypes achieve high RWC, despite water stress, are tolerant.Also, hydrous stress had a negative effect on the levels of chlorophyll pigments.There was a gradual decrease, proportional to the intensity of the water stress, of chlorophyll a, b and carotenoid contents.Our results corroborate those of Tahri et al.

CONCLUSION
The objectives of this work were to develop techniques of micro propagation by callogenesis of chenopodium quinoa as well as the determination of the behavior of the callus against water stress by addition of P. E. G.At the end of this work the following conclusions can be drawn: -the most favorable mediums for the callogenesis of quinoa explants are MS and B5; -the best hormonal combination giving the best results is 0,2BA+0,5(2,4D); -the effect of water stress on callus is reflected by a decrease in diameter, fresh and dry matter content, water and chlorophyll pigment content; this reaction is a mechanism of resistance to drought stress of quinoa at the cellular level.27.Benrebiha

Fig. 11 :Fig. 12 :Fig. 13 :
Fig. 11: Effect of water stress on callus's chlorophyll a content [51] and Scheirs and Bruyn [52].It should be noted that the presence of selective agents in the medium may increase the probability of obtaining tolerant plants [53].Tolerant plants have been obtained after in vitro selection by P. E. G of durum wheat [54, 55], rice [56, 57] and sorghum [58] and leading to genetic drift [59].