INTRODUCTION

Cyanobacteria, the only oxygenic photosynthetic bacte- ria, are interested due to their ability to grow in aquatic ecosystem. They are a group of prokaryotic organisms exhibiting the general characteristics of Gram-nega- tive bacteria. They are unique among the prokaryotes

ARTICLE INFORMATION:

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Received 1st June, 2015

Accepted after revision 30th June, 2015 BBRC Print ISSN: 0974-6455

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because of their capacity to carry out oxygenic pho- tosynthesis. Cyanobacteria can be found in terrestrial and aquatic environment as well as in symbiosis with bryophyta, pteridophyte, gymnosperm, angiosperm and fungi (Rai, 1990). A study by Komarek and Anagnos- tidis, (1989) shows that more than 50% strains in culture collections are misidentified. The diversity of cyanobac-

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teria has focused attention of researchers to realize their untapped applied and environmental potential.

The North-East region of India has been described as a biodiversity hot spot harboring different kinds of flora and fauna unique to this region. The semitropical climatic condition augmented with high annual rainfall has played a crucial role in enhancing the biodiversity richness that supports luxuriant growth of cyanobacte- ria. Little information is available about the occurrences of cyanobacteria in Loktak Lake and adjoining areas (Tiwari and Singh, 2005; Ojit et al., 2015). To cope up with this cell cloning can apply for larger sale research endeavors for various applications by manipulating cells with cloning. Cell cloning (also called reproduc- tive cloning) refers to the procedure of creating a new multicellular organism, genetically identical to another. In essence this form of cloning is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place. Asexual reproduction is a naturally occurring phenomenon in many species of cyanobacte- ria. Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium. However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.

The least-expensive methods of single-cell isola- tion is plating on solid medium. Plating of phyto- plankton has been restricted to the taxa that are suit- able for this approach (Andersen and Kawachi, 2005). It is routine culture technique for some chlorophytes, such as Chlamydomonas, Chlorella and desmids as well as euglenophytes (Harris, 1989; Galloway, 1990). Dia- toms and cyanophytes are also among the most com- monly plated of phytoplankton taxa (Galloway, 1990; Garduno et al., 1996; Watanabe et al., 1998). Wall-less algae are especially problematic because growth on a surface (i.e. at the solid-gaseous interface) exposes such cells to physical demands that are beyond their capa- bilities to withstand. The physical stresses imposed on a wall-less cell can be alleviated by plating such cells in an embedded fashion, where the cells are included within the medium before the plates are poured. Such a pour-plating approach has been applied successfully to several species of freshwater cyanophytes (Watanabe et al., 1998) and the wall-less protoplasts of macro- phytes (Fuller et al., 1993; Junxue et al., 2003). Typical concentrations of gelling agents for pour-plating are in the 0.4%-0.6% range (Watanabe et al., 1998; Junxue et al., 2003). Collection, identification and formulation of quick techniques for cloning of identical cell of cyano- bacterial strains from habitats of Loktak Lake, a wetland

of international importance under Ramsar convention (1990) was the aim of present study.

MATERIAL AND METHODS

STUDY SITES AND SAMPLING

The soil and algal samples were collected from Loktak Lake and adjoining areas, Imphal, Manipur; samples comprising all kinds of soil and algal mats with the help of trowel and spatula in sterilized polyethylene bags. The water sample of the lake was also collected separately in pre sterilized plastic bottles and brought to laboratory for examination.

ISOLATION AND PURIFICATION OF

CYANOBACTERIA

All samples were initially examined under a light micro- scope for the presence of cyanobacteria and a small por- tion of each sample was inoculated in 50 ml sterilized liquid nutrient BG-11 medium (Stanier et al., 1971) for culture enrichment. The isolated and purified cyano- bacterial strains were maintained in liquid medium and stored in culture cabinet with the light intensity of 14/10h light/dark cycle at temperature 28±1ºC.

IDENTIFICATION OF CYANOBACTERIA

All cyanobacterial isolates were observed under light microscope and identified following the taxonomic descriptions in monographs of Desikachary (1959). Morphological taxonomic features included trichomes, thickness, shape, size and position akinetes/ heterocyst. Images captured using a Carl Zeiss Microscope.

PROTOCOL FOR CLONING OF CYANOBACTERIA

Algal cell growth

The studied strains were cultured in the defined medium of BG-11 in sterile Ehrlenmeyer flasks. All chemicals were of reagent grade. Standard conditions used for liq- uid cultures were as follows: 19°C, 50 μE m-2s-1 photon flux density on a 14:10 light:dark photoperiod. Culture conditions for studied strains are presented in table 1.

CELL COUNTING

Homogenized algal cultures from exponential phase were taken. Dropped of 20μl in haemocytometer bed make sure that coverslip put at appropriate place in heamacytometer before loading of algal samples. Cell densities were assayed using a Touff 0.100mm brightline Haemacytometer. Observed in compound microscope

starting from lower magnification i.e., 4x, 10x,40x and then calculated the no. of cells as per structure men- tioned below from grid of haemacytometer.

PLATING METHOD

Cell cloning of cyanobacteria followed the protocol of Lakeman et al., (2007). A stock of 1% (w/v) OmniPur standard agarose (OmniPur ® Agarose PCR Plus Merck Millipore), was prepared in BG-11 medium and then melted and sterilized by autoclaving. After autoclaving the melted solution was held at 70°C in a water bath until needed. A suspension of cells with known cell density was prepared for plating in BG-11 medium 100ml in 250ml conical flask. Added 1% agarose stock to achieve a final concentration of agarose from 0.08% to 0.16% w/v. Total volume (100ml) consisted of cells, growth medium and agarose stock. One third (18.3 ml) of this plating mix was quickly poured into each of three petridishes (Tarsons petridish Cat no: 460090-90MM) at room temperature. All the plates were sealed with Parafilm ® (M250 Lot.no.: 0000067604 Himedia, India) to reduce evaporation. Incu- bate the plates at 28±1°C and 4-5 Klux light intensity 14:10 light: dark period and were grown until colonies were scorable within 15 days. Plates were grown until colonies were scorable, usually within 10-21 days. Colo- nies were counted using a microscope with a bright-field/ dark-field base. Colonies are low enough, the whole plate was counted and five fields per plate were chosen at ran- dom and using a 10mm square counting grid, between 20 and 200 colonies were counted per field.

RESULTS AND DISCUSSION

CHARACTERISTICS OF THE LOKTAK LAKE

The Loktak Lake is located at an altitude of 750 M which has been declared as a Ramsar site due to its biological,

Thingujam Devi et al.

cultural and geographical values. It is situated between 24º 25´N to 24º 40´N latitude and 93º 45´E to 93º 55´E longitude in the southern part of the Imphal valley of Manipur, India (Fig 1). The lake is famous for its float- ing mats of vegetative locally called as Phumdis. Loktak Lake is associated with a number of smallest fresh water lakes and swamps from a major wetland of the state.

ALGAL CELL PLATING

Fourteen (14) strains belong from three genera were tasted and studied for their ability to form colonies in growth medium that was solidified by agarose had a large effect on plating success (Table 1 Fig 1). Best and most consistent results were obtained using OmniPur brand agarose, which had a maximum plating effi- ciency of 80%. Colonial growth of the studied strains was achieved when cells were poured with agarose- medium. Plating results showed good in 0.07 % aga- rose concentration perfectly solidified. This concentra- tion was determined to be optimal for supporting the studied strains colony formation while maintaining sufficient structural integrity in the agarose medium to allow routine laboratory use. Microscopic observation of each strains showed that cell division was not obstructed with formation of complete daughter cells. The agarose apparently restricted the movement of the daughter cells so that an indeterminate colony was formed (Photoplate 1, 2, 3).

The cells in the colonies appeared on agarose plate pigmentation appeared unchanged. All the studied strains formed colonies reasonably well at 0.07% of aga- rose except Cylindrospermum sp. BTA1113 were formed very less colonies only in 15th day growth it showed col- onies (Photoplate 3). Density of agarose allowed robust colonial growth and remained discrete. As we have seen with the colonies released from agarose that the non motile cells of the strains reported here lead us to believe

CYANOBACTERIA OF CLONING CELL QUICK FOR PROTOCOL STANDARDIZED 18

OMMUNICATIONSC ESEARCHR IOTECHNOLOGYB IOSCIENCEB

Table 1: Habitat, isolation and maintenance of cyanobacterial strains investigated for cell cloning

.al et Devi Thingujam

that we have replicated a naturally occurring phenom- enon. In natural habitats both colonies and aggregated cells formation occurred. Cloning is relatively easy for continuous cell lines difficult in primary cultures never- theless possible a serious limitation is senescence clon- ing of attached cells carried out in petridishes multiwall plates flasks not hard to distinguish individual colonies cloning in suspension accomplished by seeding cells in a gel (agar).

Viscous solution with agar underlay viscous matrix ensures that daughter cells remain in colony. Nos- toc sp.BTA 60, Nostoc commune BTA67, Calothrix sp. BTA73, Nostoc sp. BTA80, Nostoc muscorum BTA950, Anabaena sp. BTA964 and Nostoc sp. BTA1097 formed quick colonies very efficiently from 5th days. Not all algal strains were amenable to plating. Cylindrospermun sp. BTA1113 was failed to form colonies very less growth were noted and cells died within days. We suspect that their lack of cell wall, cause the agarose matrix to be too restrictive to allow growth.

ACKNOWLEDGEMENTS

The lead author (Indrama) acknowledges the financial support provided by DST, Govt. of India vide order No. SR/WOS-A/LS-45/2013. We express our sincere thanks to the Director of IBSD, Imphal for providing laboratory facilities and required infrastructure.

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