Influence of Microstructured Carbon Materials on Sequoia sempervirens

Influence of Microstructured Carbon Materials on Sequoia sempervirens (D. Don.)

Abstract

The decline of plant species number strongly imposed worldwide, along with the habitat protection and management, the use of ex situ conservation methods based on biotechnologies. The present experimental research has been mainly focused on developing an efficient method for the stimulation of S. sempervirens (D. Don.) Endl. in vitro plant growth by using microstructured carbon materials (CCM), as additives for the optimization of this morphogenetic process. Effects of CCM treatment on S. sempervirens in vitro culture were investigated starting from the hypothesis that CCM can induce effects on the plant culture, similar to those induced by the active charcoal.

The present study used as inoculum source 3 cm long shoots apex from S. sempervirens stock culture. There were tested four experimental mediums represented by variants of MS basal medium (1962) supplemented with 0.2mg/l kinetin, two types of CCM (A and B) and active charcoal (CA). The culture cultivation time was represented by 2 periods of 30 days each one, in controlled conditions of light and temperature. At the end of each growing period, parameters such as fresh weight and seedlings length/experimental treatment were measured. After 60 days the experiments demonstrated no stimulative growth effect of the A, B, CA medium supplement, on S. sempervirens seedlings length as opposed to the control medium (C), on the basis of the measured biological parameters. After the treatment period, there were more significant positive effects on the stimulation of sequoia seedlings growth, using CA medium than A and B medium variants. The result led to conclusion that the possible stimulative effect of A and B microstructured carbon material can be achieved after using CCM in a large variety of concentrations. Looking for the two biological parameters, we can conclude that the effect of the A and B CCM is not toxic, does not inhibit plant growth. The use of new CCM compounds that stimulates biological processes and optimizes biotechnological protocols is an important step both in the study of materials science and in the field of plant biotechnologies.

Key words: Sequoia sempervirens, microstructured carbon materials.

Introduction

It is widely recognized that forests are the most biologically diverse terrestrial ecosystems and that pressure on forest biodiversity continues to increase throughout the world. Around 350 million of the world's poorest people depend almost entirely on forests for their basic needs and 2 billion people depend on wood for cooking and fuel, millions of others depending on trees for food and medicines. Trees are also providing a habitat for many other species and defining the characteristics of forest ecosystems. (Lerche et al.,2002) Information is limited on the distribution and conservation status of tree species. Preliminary surveys undertaken to date suggest that approximately 8,000 tree species are threatened with extinction worldwide. The potential loss of nearly 10 per cent of all tree species is a major conservation issue, requiring international attention and widespread action. The Convention on Biological Diversity (CBD) recognizes that forests are an important repository of biological biodiversity promotes conservation and sustainable use and sets out desirable in situ and ex situ conservation measures. (Thorsell 1997, Sigaty 1977)

Biotechnology encompasses a wide variety of techniques for ex situ conservation such as vegetative propagation. This comprises a broad range of techniques involving manipulation of plant tissue (e.g. sections of stems, leaves, roots, seeds or even cell cultures) which ultimately allows for complete vegetative "re-propagation" of the whole plant, i.e., the production of clonal "varieties" or lines. One of the tree species which imposed in vitro conservation techniques is represented by Sequoia sempervirens (D. Don.) Endl.. This forest community defines the Pacific Coast ecology system and is in danger of being lost or destroyed by logging, development and environmental pollution in this area (IUCN 2002). Sequoia sempervirens (D. Don.) Endl in Romania can be found in few protected areas in Cluj, Arad, Caras Severin, Bihor and Bucharest.

The aim of our work consists in identifying a new and efficient method to stimulate in vitro sequoia plant growth. A possible method, having positive effect on this morphogenetic process, consists in using the microstructured carbon materials (CCM) as additives. The hypothesis for the addition of these compounds in culture medium starts from the premise that CCM can cause within the plant cell culture similar effects as those induced by the active charcoal. In the plant cell culture the active charcoal is used to assure the absorption of organic and inorganic components at the nutritive medium level, the absorption of impurities at the agar level, the absorption of some catabolists with inhibitor effect on growth processes and their liberation which has a stimulative effect. At the same time the active charcoal can assure the optical density necessary for the medium, pH effects, the absorption of compounds secreted during the sterilization from the zaharose degradation and the absorption of some toxic compounds resulted from oxidative and metabolic processes. The same actions promoted by CCM are attributed to their physical, chemical and structural characteristics.

Referat: Ferastrau circular cu masa mobila Referat: Criterii pentru aprecierea rezistentei la foc

Materials and Methods        

The inoculum source of the experiment consists in 2.5-3 cm shoots apex excised from an in vitro stock culture of Sequoia sempervirens obtained by the kindness of Dr. Magdalena Palada from Simeria Forest Research Station. The nutritive medium was represented by a variety of (Murashige 1962, Skoog 1962) basal medium additioned with 0.2 mg/l kinetin as cytokinines.

The two microstructured carbon compounds (A and B notations) that have been tested proceed from the Institute ICPE - Advanced Researches. A compound represents expanded graphite obtained from an intercalated compound with H2S04, with a surface area of 158 m2/g. B compound represents expanded graphite obtained from FeCl3 with a surface area of 10 m2/g. The A microstructured carbon compound is mainly micro- and mezoporous, while B compound is microporous. They have been included in the growth medium in concentrations of 2g/l, after medium sterilization.

During the research, different experimental variants have been analyzed. (Table 1)

All the medium variants were solidified using a concentration of 8% agar. 10 ml of medium was equally allocated for each Sigma tube.

Culture conditions were represented by two successive growth periods that took place in controlled conditions consisting in a temperature of 240Cą 20C, 1500 lux cold fluorescent light and16 hours light/day photoperiod, each growth period lasting 30 days.

At the end of the each 30 days of the growing interval biological parameters, such as fresh mass and length, corresponding to each seedling/experimental medium variant were estimated.

The biological parameters were determined for each plant/each medium variant at 30, respectively 60 days of the growing period. The significance of the differences between experimental variants was estimated using student test.

Results

After the in vitro multiplication of the stock culture (Fig.1) and the inoculation of explants represented by 3 cm shoot apex on all the tested experimental variants (Fig.2), mass increase was significant emphasized (at least double) for each cycle of 30 days. (Fig.3)

The analysis performed after the 30 days interval of growing, pointed out that as opposed to the control, neither A or B compounds nor active charcoal (CA), did not influence significantly the evolution of the two determined parameters (fresh mass and seedlings length).(Fig.3a)

After 60 days of growing S. sempervirens seedlings in different medium variants supplemented with A, B compounds and active charcoal, it can be established that only the active charcoal influenced positively and significantly the fresh mass parameter, as opposed to the control and A, B compounds.(Fig. 3a)

Regarding the seedlings length, after 60 days of growing, a favorable influence of the active charcoal (CA) was recorded compared only to the A and B experimental variants and not to the Control variant. (Figure 3b)

Different results for the two biological parameters studied were obtained for each substance tested by comparing the medium values using student test (Figure 4). The results of this test estimate that A and B compounds make no significant difference in comparison to the Control. Instead, the active charcoal makes significant differences in comparison to the Control and to the other two compounds (A and B). (Figure 4)

Discussion

The previous experiment achieved with CCM on Vitis vinifera callus culture has demonstrated a stimulativ in vitro effect of these compounds on the cell proliferation and the anthocyanin biosynthesis. (Cogalniceanu G. et al., 2006) These results represent the starting point for the in vitro testing the influence of graphite intercalated compounds on growing processing at Sequoia sempervirens. The figures of the present experiment allowed us to ascertain that the specific response of the biological system to the treatment with microstructured carbon materials depends on the species and the experimental system used. Thus different types of biological material and processes showed a high variability in their responses. Probably the in vitro culture systems are intermediated by specific mechanisms, different from those corresponding to the active charcoal. So, regarding the Sequoia sempervirens the positive effects are modest and these can be obvious only after a growing time on medium with different concentrations of A and B compounds. It is important to mention that in all cases studied by us, these compounds are not toxic and they are not modifying the normal pattern of cytodifferentiation and morphogenesis. For this reason further, studies on the effects induced by the graphite intercalated compounds on different experimental biological materials must be continued and represent an important step both in the study of materials and in the field of biotechnologies.

Conclusions

The results of the current experiment lead to the conclusion that some possible stimulative effects of microstructured carbon compounds on biological parameters can be distinguished only after testing more concentrations of microstructured carbon materials. From this point of view it will be useful to test these compounds on growing periods longer than 60 days and to use more variants of these compounds concentration.

The results of the student test indicate that the two microstructured carbon compounds are not toxic, do not inhibit the normal morphogenetic processes and the possible effects at the level of in vitro culture are intermediated by specific mechanisms, different from those corresponding to the active charcoal.

Finally, we consider that the obtained data are promising and that this research direction should be further developed.

Bibliography

1.Cogalniceanu G., Hristea G., Brezeanu A., Optimizarea biosintezei de antociani in cultura de calus long-term de Vitis vinifera L. prin utilizarea unor compusi de intercalare grafitici, 2006. Analele SNBC, vol.X., (pp. 438-443).

2. IUCN, 2002. IUCN Red List of Threatened Species.  http://www.redlist.org/.

3. Murashige T. Skoog F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant., no.15, (pp.473-497).

4. Lerche C., Iversen P. A., Sigaud, P., 2002. Forest Genetic Resources No. 30. FAO, Rome, Italy.

5. Thorsell J., Sigaty T., 1997. A Global Overview Of Forest Protected Areas On The World Heritage List, Natural Heritage Programme, IUCN Gland, Switzerland.

Tables and figures

Table 1. Types of medium variants used in the experiment.

Figure 1. The in vitro multiplication of Sequoia sempervirens stock culture.

Figure 2. Explants inoculation on the tested experimental medium variants.

Figure 3a. Fresh mass dynamic of S. sempervirens seedlings after 30 and 60 days growth on nutritive medium supplemented with A, B, CA or on control (C).

Figure 3b S. sempervirens seedlings length dynamic after 30 and 60 days growth on nutritive medium supplemented with A, B, CA or on control.

Figure 4. Comparative average values using student test (a=0,05)