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328 The Natural Products Journal, 2012, 2, 328-331

The Effect of Sodium Hypochlorite Solutions on the Viability and In Vitro Regeneration Capacity of the Tissue

Mustafa Yildiz1,*, S. Fatih Özcan2, Cansu T. Kahramanogullari1 and Ege Tuna1

1University of Ankara, Faculty of Agriculture, Department of Field Crops 06110 Diskapi, Ankara, Turkey;

2Cankiri

Karatekin University, Kizilirmak Technical and Business College, Çankiri, Turkey

Abstract: Although there are many chemicals used for sterilization purposes such as ethanol, hydrogen peroxide, bromine water, mercuric chloride, silver nitrate and antibiotics, sodium hypochlorite solutions have been most widely used. Since NaOCl has a strong oxidizing property which makes it highly reactive with amino acids, nucleic acids, amines, and amides, it is highly effective against all kinds of bacteria, fungi, and viruses. The general reaction between amino acids and NaOCl produces the respective aldehyde, NH4Cl and CO2. Thus, during the sterilization process, direct contact of the tissue with NaOCl depending on it’s concentration, application period and temperature may have a hazardous effect on the health of the tissue. In plant tissue culture studies, high-frequency shoot regeneration which is highly affected by tissue health, is a prerequisite for an efficient gene transformation system and a clonal propagation of plants. The most important treatment prior to culture initiation is the sterilization of the tissue. In plant tissue culture, elimination of microorganisms from the tissue has great importance. From one hand, sterilization process aims to eliminate all microorganisms that can easily grow on the tissue; on the other hand, it should guarantee the tissue’s viability and regeneration capacity. This review has focused on the effects of concentration, application period and temperature of NaOCl using for eliminating microorganisms on the viability and in vitro regeneration capacity of the tissue.

Keywords: Sodium hypochlorite, surface-sterilization, tissue viability, regeneration capacity.

INTRODUCTION

A wide range of surface disinfectants, such as ethanol, hydrogen peroxide, bromine water, mercuric chloride, silver nitrate, and antibiotics are used for surface-sterilization; however sodium hypochlorite (NaOCl) has been most widely used. NaOCl is highly effective against all kinds of bacteria, fungi, and viruses [1, 2]. Sodium hypochlorite has a broad-spectrum antimicrobial activity, rapidly killing vegetative and spore-forming bacteria, fungi, protozoa, and viruses [3, 4]. Sodium hypochlorite exerts its antibacterial effect by inducing the irreversible oxidation of sulfhydryl groups of essential bacterial enzymes, resulting in disulfide linkages, with consequent disruption of the metabolic functions of the bacterial cell [3, 5]. Sodium hypochlorite can also have deleterious effects on bacterial DNA, which involve the formation of chlorinated derivatives of nucleotide bases. In addition, NaOCl has been reported to disrupt membrane associated activities [6, 7]. NaOCl has a strong oxidizing property which makes it highly reactive with amino acids [8, 9], nucleic acids [10], amines, and amides [11, 12]. The general reaction between amino acids and NaOCl produces the respective aldehyde, NH4Cl and CO2 [13].

One of the main objectives of plant tissue culture studies is to obtain high-frequency shoot regeneration, which is also a prerequisite for an efficient transformation system and a

*Address correspondence to this author at the University of Ankara, Faculty of Agriculture, Department of Field Crops 06110 Diskapi, Ankara, Turkey; Tel: +90 (312) 5961611; Fax: +90 (312) 3182666; E-mail: myildiz@ankara.edu.tr

clonal propagation of plants. The introduction of foreign genes coding agronomically important traits into plant cells has no meaning unless transgenic plants are regenerated from the genetically modified cell(s). Explant health is the main factor determining regeneration capacity. Viability and age of explant, and the tissue source from which the explant is excised are very important for high-frequency shoot regeneration [14]. The most important treatment prior to culture initiation is perhaps surface-sterilization of the tissue. Since in vitro conditions provide bacteria and fungi with an optimal growth environment, unsuccessful sterilization hinders the progress of tissue culture studies (Fig. 1). On the one hand, sterilization of the tissue aims to eliminate all microorganisms that can easily grow in vitro conditions; on the other hand, it should guarantee the explant’s viability and regeneration capacity which are known to be affected by the concentration, application period [15] and temperature [14] of NaOCl. Since direct contact of explant with disinfectant during the sterilization process may have a severe effect on regeneration capacity of the tissue [14], using aseptic tissues as source of explant is highly recommended [16, 17]. This review was prepared to discuss the adverse effects of sodium hypochlorite solutions on the viability and regeneration capacity of the tissue.

EFFECT OF NaOCl CONCENTRATION AND

APPLICATION PERIOD

Seed germination, seedling growth and the viability of the tissue were negatively affected by NaOCl at high concentrations [14, 18, 19] while it is uneffective for sterilization of tissues at low concentrations. The negative

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NaOCl Effects on the Tissue The Natural Products Journal, 2012, Vol. 2, No. 4 329

effects of NaOCl concentration became more severe with increasing application period.

Since regeneration capacity of the tissue is negatively affected by higher concentrations and longer application periods of disinfectants [15, 20], sterilization process under in vitro conditions should aim to use the lowest concentration of disinfectant for the shortest time.

It was reported that seed germination, seedling growth, hypocotyl and root lengths of Linum usitatissimum L. which has been used as a medicinal plant for thousands of years and components of the plant such as lignans and �-linolenic acid have been used in many drugs [21, 22], all decreased significantly by higher concentrations of NaOCl.

In another study aiming to evaluate the effects of NaOCl solutions used for sterilization on seed germination and seedling growth in Lathyrus chrysanthus Boiss., the best results were obtained from 3.75% NaOCl concentration and 15 min. application period for all parameters examined [23]. Seedborne contamination increased gradually by decreasing concentrations and application periods of NaOCl below 3.75% and 15 min. Dramatic decreases were observed at 5.00% NaOCl concentration in all cases. At this concentration, NaOCl showed deleterious effect on the embryo of the seed. Seed germination decreased to 65.18% when NaOCl concentration increased to 5.00% from 3.75% at 15 min. application period. Seedling growth from seeds sterilized with 3.75% NaOCl concentration for 15 min. were observed growing faster than that of sterilized with other concentrations and application periods of NaOCl. By increasing NaOCl concentration to 5.00%, seedling length decrased to 3.45 cm from 3.90 cm. Higher fresh and dry weights obtained from 3.75% NaOCl concentration and 15 min. application period could be attributed to an increase in the absorption of water and other components from the medium via high tissue metabolic activity as reported by Dale [24] to cell division and new material synthesis [25]. Higher results in seedlings grown from seeds sterilized with 3.75% NaOCl concentration for 15 min. could be caused by higher tissue water content as reported that in vitro explant growth and plantlet establishment have been affected significantly by tissue water content [26].

Emerson [27] has reported that there is a close relationship between photosynthesis and chlorophyll content

which affects photosynthesis directly and its amount changes under stress conditions [28-30]. Moreover, chlorophyll content of leaf is accepted as an indicator of photosynthetic capacity [31-33] which shows tissue metabolic activity. Telci et al. [23] have reported that in the study for disinfection of Lathyrus chrysanthus Boiss. seeds under in vitro conditions, seedlings grown from seeds sterilized with 3.75% NaOCl for 15 min. were more vital and well-grown. Similarly, the highest values with respect to chlorophyll a, chlorophyll b and total chlorophyll contents were obtained from 3.75% NaOCl and 15 min. application period. Analogy between lower chlorophyll contents and lower results related to seed germination and seedling growth percentages, seedling and root lengths, seedling fresh and dry weights clearly indicated that concentrations and application periods of NaOCl over 3.75% and 15 min. affected seed germination and seedling growth negatively.

EFFECT OF NaOCl TEMPERATURE

It was firstly reported that besides concentration and application period, temperature of NaOCl is also one of the most important factors affecting in vitro seed germination, seedling growth and regeneration capacity [14]. At the 2.00% NaOCl concentration using for surface-sterilization of flax (Linum usitatissimum L.) seeds, when the temperature of NaOCl was set below 10°C, bacterial and fungal contamination was observed. However, increases in NaOCl temperature above 10°C resulted in dramatic decreases in seed germination, seedling growth, hypocotyl and root lengths. Decreases in all parameters in NaOCl temperature above 10°C could be the fact that disinfection activity of

Fig. (1). Fungi grown under in vitro conditions.

Fig. (2). In vitro seedling growth from Linum usitatissimum L. seeds sterilized with 2.00% NaOCl at temperatures of (a) 10ºC, (b) 20ºC, and (c) 30ºC.

330 The Natural Products Journal, 2012, Vol. 2, No. 4 Yildiz et al.

NaOCl increases [34] and disinfectant penetrates more easily through the seed coat [35]. Higher NaOCl temperatures resulted in morphologically abnormal seedlings with stunted hypocotyls and roots (Fig. 2).

Telci et al. [23] have reported that higher results in Lathyrus chrysanthus Boiss. regarding stomatas and cells were obtained from seedlings grown from seeds sterilized with 3.75% NaOCl at 35°C for 15 min. High cell number at 35°C NaOCl temperature could be based on high cell division while lower cell number at 45°C NaOCl temperature could be explained by inhibited cell division under stress

conditions [36] (Fig. 3). Quarrie and Jones [37] and Cutler [38] have reported that the number of stomata and cell size were less in the leaves of stressed plants. At higher temperatures, NaOCl easily reacted with amino acids of the seed and consequently high level of CO2 came out as noted by Kantouch and Abdel-Fattah [9]. This increased CO2 could be an explaination of fewer stomata on leaves as a plant response [39]. According to the findings of McElwain and Chaloner [40], rising CO2 concentration caused a decrease in both stomatal density and stomatal index. Results from the study of Telci et al. [23] showed that rising CO2 concentration caused a decrease in both stomatal density and stomatal index at 45°C NaOCl temperature. Lower stomata width at 45°C disinfectant temperature could be attributed to closed stomatas due to high CO2 concentration and low water content as reported by Solomon [41]. On the other hand, high stomata widths at 35°C NaOCl temperature could be attributed to open stomatas under low CO2 concentration and high water content [23].

Increasing NaOCl temperature reduced regeneration capacity in Linum usitatissimum L. significantly [14]. The highest results in regeneration and shoot number per explant were obtained from 10°C NaOCl temperature. At higher temperatures above 10°C, substantial decreases in regeneration capacity of the tissue were observed. Shoot number per hypocotyl obtained from the seeds sterilized with 2.00% NaOCl decreased to 7.38 at 30°C while it was 23.40 at 10°C disinfectant temperature.

CONCLUSION

Sodium hypochlorite has been widely used in many areas such as medicine, public health and plant tissue culture to eliminate microorganisms causing contamination. However, in the sterilization process of the tissues, the concentration, application period and temperature of NaOCl solutions are closely related to each other and they should be considered together. Direct contact of the tissue with disinfectant during the sterilization process may have a severe effect on the viability and regeneration capacity depending on concentration, temperature and application periods of disinfectant [14, 15]. In addition to this common knowledge, not only seed germination and seedling growth are directly affected by sterilization process, but also regeneration capacity of explants and health of regenerated shoots are indirectly influenced significantly in plant tissue culture studies. That means NaOCl affects the success of in vitro studies, from seed germination and seedling establishment to regeneration capacity of the tissue and recovery of plantlets.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS.

Declared none.

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Fig. (3). Stomata and cell from the upper leaf surface of Lathyrus

chrysanthus Boiss. seedlings grown from seeds surface disinfected with 3.75% NaOCl at temperatures of (a) 25°C, (b) 35°C and (c) 45°C for 15 min. bar = 30 �m.

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Received: May 26, 2011 Revised: January 23, 2012 Accepted: April 10, 2012