In vitro surface sterilization of the shoot tips of Bougainvillea spectabilis Willd

In vitro studies were carried out to investigate the effects of different sterilants for the surface sterilization of the shoot tips of Bougainvillea spectabilis Willd. Three sterilints i.e. HgCl2, NaOCl, CaOCl2 were selected to test their surface sterilization ability of the of Bougainvillea spectabilis Willd. The sterilants were for observing their surface sterilization potential in different concentrations and duration. Silver Chloride, HgCl2 (0.05%) for 3 minutes was found as the most effective sterilant with maximum survival percentage of 91.6% followed by HgCl2 (0.05%) for 5 minutes with 75% survival. The minimum survival (8.33%) was observed with 4% Calcium hypochlorite for 5 minutes.


Introduction
Bougainvillea (Bougainvillea spectabilis Willd.)belongs to the Dicot family, Nyctaginaceae.The plant is a shrub, tree or vine, which can reach over 30 feet high.Its leaves are large, simple and mostly ovate [1, 2].They may show rippling along the edges and hair can be seen underneath.Bracts are usually in shades of red, shades of dark pink or shades of purple.Thorns are large and may be curved.Flowers are cream coloured.Its habitat is dense and the bracts appear up and down the branches.During the dry season or in a cool spell, flowering is triggered.The root system is very fragile and doesn't form a good root ball [3].Bougainvillea has many adaptations to its climate and environment.It lines in a climate from subtropical to dense forests where it can cling and grab onto others plants to reach the sunlight.It can tolerate semi-shaded areas and can also tolerate full sun.It thrives in full sun.At least 5 h a day of full sunlight is the minimum light required for a good bloom.More hours of direct sun is better, the plant does not like to live with a lot of water.The plant has earned a reputation as to be the pride of gardens among the naturalists and ornamentalists due to its wide range of habitats, prolonged flowering seasons and variety of flower colours.It also has a wide range of uses in landscaping.Various concentrations and combinations of sterilants has been reported and used in preliminary trials in vitro on tissue of juvenile and adult trees explant of guava to see the effect of free infestation explant and also on its growth.Micro-propagation of Bougainvillea by shoot tip culture has been successful [5, 6, 11].Taking into account, the economical and ornamental value of Bougainvillea, the aim of this study was to improve the micro-propagation through shoot tip culture.

Materials and methods
Bougainvillea spectabilis Willd.was selected for the present studies.Plant materials were collected from vigorously growing plants.1cm long shoot tips were treated with 3 drops of Zip as a detergent and then washed by running tap water for ½ an hour to remove dust particles.The explants were surface sterilized with 70% ethanol for 30 seconds [9] followed by various sterilants in different concentrations and exposure time.0.05% and 0.25% Mercuric Chloride for 1, 3 and 5 minutes, 2% and 4% Sodium Hypochlorite (NaOCl) and Calcium Hypochlorite (CaOCl2) for 1, 3 and 5 minutes were used according to the procedure of [11].Then the shoot tips were rinsed three times with sterile distilled water in laminar flow bench to remove sterilants.All sterilization manipulations were carried out in laminar airflow hood.Experiment was arranged in a randomized complete block design with three replications per treatment, each with 20 explants.The data were recorded after 5-7 weeks and statistically analyzed by using Duncan's Multiple Range Test [11], to check the level of significance between the treatments.These sterile were then grown MS nutrient media [8] under different hormones as mentioned [1].

Results and discussion
Application of different sterilants for surface sterilization on the shoot tips showed that the shoot tips treated with 0.05% HgCl2 for 3 minutes (Figure 1C) showed maximum survival (91.6%) while 0.05% and 0.25% HgCl2 for 5 and 3 minutes respectively gave (75%) survival.2% NaOCl for 5 minutes and 1 minute gave 68.75% and 64.58% survival of shoot tips respectively (Figure 1B).2% CaOCl2 for 1 minute (Figure 1C) gave 30% survival of the shoot tips.Minimum survival of 8.33% was observed when the shoot tips were treated with 4% CaOCl2 for 5 minutes (Figure 1C).Among the three sterilants used, CaOCl2 was found the worst for the sterilization of bougainvillea shoot tips.The explants treated with this sterilant did not show any growth after sterilization.Surface sterilization of explants is an important preliminary step of in-vitro techniques.The explant is usually contaminated with dust and various microbial colonies and their removal from the explant is a pre-requisite for the in-vitro culturing [11].We used three different types of sterilants for the sterilization of shoot tips and found that HgCl2 as the best sterilant giving higher percentage of the explant survival (Table 1 and Figure 1).The results are in line with those of [5, 10] who carried out surface sterilization with 0.05% solution of HgCl2 for 5 minutes followed by 3 washing with sterilized distilled water.0.1% HgCl2 for five minutes in combination with 0.1% Sodium lauryl sulphate is the best sterilant for the surface sterilization of nodal explants of 22 cultivars [3].HGCl2 is an effective sterilant for potato [2].It is suggested that HgCl2 is very strong and highly toxic for the microbial colonies even in very low concentrations and shorter durations.HgCl2 may not only be harmful to the microbes but also may be the tissues of explant as we observe the blackening of the shoot tips.2% NaOCl for 5 minutes was found as the second best sterilant with 68.75% survival (Figure 1B).NaOCl is reported as one of the effective surface sterilant for the cotyledon explant of Arachis hypogea [6].This sterilant was not only effective for sterilization but also showed better growth of the explants after sterilization in contrast to HgCl2.2% NaOCl is used for sterilization of shoot tips of Bougainvillea but the time of exposure is not known [4].

Conclusion
It is concluded that HgCl2 is effective sterilant for the shoot tips explant of Bougainvillea; however, the growth after the sterilization is negatively affected.On the other hand, NaOCl is less effective than HgCl2 but nevertheless maximum growth of shoot tips was observed after sterilization.CaOCl2 was found not only least effective sterilant but also having lethal effects on the shoot tips.

Figure 1 .
Figure 1.Percentage survival rates of the shoot tips under different conditions In vitro propagation needs effective sterilization of the explants.The explant taken from field grown plant are very difficult in tissue culture because of infestation with microorganisms [5].