Identification and elimination of bacterial contamination during in vitro propagation of Guadua angustifolia Kunth
Harleen Kaur Nadha1, Richa Salwan2, Ramesh Chand Kasana2, Manju Anand3, Anil Sood2
1 Department of Biotechnology and Environmental Sciences, Thapar University, Patiala, Punjab; Division of Biotechnology, CSIR- Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
2 Division of Biotechnology, CSIR- Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India
3 Department of Biotechnology and Environmental Sciences, Thapar University, Patiala, Punjab, India
|Date of Submission||14-Jun-2011|
|Date of Acceptance||25-Jun-2011|
|Date of Web Publication||23-May-2012|
Scientist G and Head, Division of Biotechnology, CSIR- Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh - 176 061
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Guadua angustifolia Kunth is a very important bamboo species with significant utility in pharmaceutical, paper, charcoal, and construction industries. Microbial contamination is a major problem encountered during establishment of in vitro cultures of Guadua. Objective: This study has been designed to analyze the identity of contaminating bacteria and to develop the strategy to eliminate them during micropropagation of Guadua. Materials and Methods: We isolated and consequently analyzed partial sequence analysis of the 16S rRNA gene to identify two contaminating bacteria as (1) Pantoea agglomerans and (2) Pantoea ananatis. In addition, we also performed antibiotic sensitivity testing on these bacterial isolates. Results: We identified kanamycin and streptomycin sulfate as potentially useful antibiotics in eliminating the contaminating bacteria. We grew shoots on multiplication medium containing BAP (2 mg/l) and adenine sulfate (10 mg/l) supplemented with kanamycin (10 μg/ml) for 10 days and transferred them to fresh medium without antibiotics and found that bacterial growth was inhibited. Moreover, we observed intensive formation of high-quality shoots. Streptomycin sulfate also inhibited bacterial growth but at higher concentration. We also demonstrated that shoots grown in streptomycin sulfate tended to be shorter and had yellow leaves. Conclusion: Thus, we have developed a novel strategy to identify and inhibit intriguing microbial contaminations of (1) Pantoea agglomerans and (2) Pantoea ananatis during establishment of in vitro cultures of Guadua. This would improve in vitro establishment of an important bamboo, Guadua angustifolia Kunth for large scale propagation.
Keywords: 16S rRNA gene sequencing, bacterial contamination, Guadua angustifolia Kunth, in vitro propagation
|How to cite this article:|
Nadha HK, Salwan R, Kasana RC, Anand M, Sood A. Identification and elimination of bacterial contamination during in vitro propagation of Guadua angustifolia Kunth. Phcog Mag 2012;8:93-7
|How to cite this URL:|
Nadha HK, Salwan R, Kasana RC, Anand M, Sood A. Identification and elimination of bacterial contamination during in vitro propagation of Guadua angustifolia Kunth. Phcog Mag [serial online] 2012 [cited 2021 Jul 25];8:93-7. Available from: http://www.phcog.com/text.asp?2012/8/30/93/96547
| Introduction|| |
Guadua angustifolia Kunth is one of the three largest and most important bamboo species in the world. Bamboo tar oil, recovered as a secondary product during the carbonization process, has significant medicinal value due to its antibiotic and antioxidant activities. Guadua also have a great potential to fix atmospheric carbon dioxide. Due to its versatility, lightness, flexibility, endurance, hardness, strength, climatic adaptability, seismic-resistance, rapid growth, and easy handling, it is widely employed in pharmaceutical, paper, charcoal, and construction industries.
Bamboo culms are traditionally harvested from natural forests, but overexploitation has led to rapid depletion of their natural vegetative strands. As a result, most of the area under tropical rain forests and biodiversity has vanished and millions of hectares have been transformed into pastures and croplands. Therefore, there is a great concern about the conservation of bamboo's natural populations and thus need to develop novel propagation methodologies for new plantations and re-establishment of cleared strands.  The traditional propagation method by "offsets" limits the number of propagules. Moreover, the use of nodal segments for propagation is cumbersome and labor intensive for large-scale establishment of bamboo plantations.  Due to profound difficulties in the conventional propagation of bamboos, it is imperative to adapt alternative methods for rapid multiplication, and therefore micropropagation offers a feasible alternative.
Our attempts to obtain aseptic in vitro cultures using explant of Guadua from glasshouse-grown plants and to optimize a micropropagation procedure were hindered by persistent appearance of bacterial contamination in the cultures. Bacterial contamination in tissue culture is well documented,  and the failure of surface sterilization procedures to produce aseptic cultures is a major problem with woody plants.  Different experimental procedures including chemical sterilization and antibiotics have been used at various levels of success to minimize or eliminate such contamination. However, the type, concentration, and duration of antibiotic treatment vary for different plant tissue cultures. Therefore, it is pertinent to optimize antibiotic treatment strategy before its use. ,
The growth medium selected for in vitro propagation also serves as a good source of nutrients for microbial growth. These microbes further compete adversely with plants for nutrients.  The presence of microbes or latent infections in these plant cultures usually results in increased culture mortality, variable growth, tissue necrosis, reduced shoot proliferation, and reduced rooting. 
We observed bacterial contamination in micropropagation of Guadua angustifolia. The contaminants were evident at the culture establishment stage and resulted in the loss of plants when bacteria overgrew the explants. In the present study, we have characterized two bacteria from Guadua shoot cultures, and determined effects of various antibiotics on these bacteria without adversely affecting the health of in vitro grown plant material.
| Materials and Methods|| |
We used nodal segments measuring 2-4 cm cm in length from 4-year-old potted plants for initiating aseptic cultures. Briefly, we subjected explants to repeated washings after removal of leaf sheaths. This would remove all the adhering dust particles and microbes from the surface. The explants were then cleaned with a liquid detergent (Tween 20-HIMEDIA, Mumbai, India) followed by treatment with a suitable fungicide (e.g., Bavistin, 0.2%). Under sterile conditions in a laminar air flow bench, these explants were sterilized with 70% ethanol (v/v) and soaked in 0.04% HgCl 2 .
Initiation of aseptic cultures
The sterilized explants were placed vertically in test tubes containing the MS (Murashige and Skoog's) medium  supplemented with BAP (6-Benzylaminopurine, 2mg/l), sucrose (2%), and agar (Murashige and Skoog 1962). The pH was adjusted to 5.7 prior to autoclaving. The cultures were incubated at a photosynthetic photon flux density (PPFD) of 70 ± 5 μmol/m 2 /s from cool, white, fluorescent lamps at 25 ± 2°C. Moreover, the day length was maintained at 16 hours in a 24-hour light/dark cycle.
Isolation and identification of bacteria
Bacterial growth appeared as a cloudy zone in the agar medium around the shoot base within 20 days invariably in all the cultures. The contaminating bacteria were isolated by placing material with loop from visibly contaminated culture directly on the LB medium. After incubation at 28 o C for 24 hours, two types of colonies were observed. Pure cultures of these bacteria were obtained by picking up the colonies and streaking them onto the fresh medium. These cultures were further maintained in glycerol stock at −80 o C.
Antibiotic treatment of plants
The luria agar plates containing different antibiotics like kanamycin, carbenicillin, ampicillin, rifampicin, and streptomycin sulfate were inoculated with isolated bacterial contaminants for antibiotic sensitivity screening.
We selected two antibiotics, kanamycin, and streptomycin sulfate, on the basis of their effectiveness in antibiotic sensitivity testing. To test their effectiveness in eliminating bacterial contamination, the antibiotics were added to the multiplication medium, i.e., the liquid MS medium containing BAP (2 mg/l) and adenine sulfate (10 mg/l) in the following dosages: 0, 5, 10, 15, 25, 40, 50 g/ml alone and in combinations. Contaminated explants were then dipped in this medium for 10 days. Controls (plant tissue grown in the multiplication medium without antibiotics) were also included with each experiment. After 10 days of the antibiotic treatment, the physical conditions of plants were noted again and then placed in the liquid multiplication medium without any antibiotic. Shoots with no detectable signs of bacterial contamination were individually transferred onto the fresh medium without antibiotics and subcultured every 3 weeks. Growth rate and plant appearance were monitored to determine whether the antibiotics had any phytotoxic effects on plants during the multiplication and rooting phase.
16S rRNA gene sequencing
We isolated bacterial DNA from pure culture and performed PCR amplification of almost the entire length of 16S rRNA gene fragment. We used following primers 5′-AGAGCTTTGATCATGGCTCAGA-3′ and 5′-GTTACCTTGTTACGACTT-3′ to amplify 8 to28 and 1493 to 1510 parts of 16S rRNA gene of Escherichia More Details coli and are useful for amplifying the 16S rRNA gene from various kinds of bacteria. The PCR was performed and analyzed on an agarose gel as described earlier. 
The 16S rRNA gene of bacteria was further sequenced to analyze its identity. Briefly, the amplified 16S rRNA gene was purified from the agarose gel using a Nucleospin Extract II kit. The PCR-purified product was directly used for nucleotide sequencing of the gene by using a Big Dye R Terminator Cycle sequencing kit (Applied Biosystems). To identify bacteria, preliminary searches in the NCBI database were performed with BLASTIN program (http://www.ncbi.nlm.nih.gov/BLAST/, NCBI, Bethesda, MD, USA).
| Results and Discussion|| |
The bacterial contamination encountered during in vitro propagation of plants is a major bottleneck which obstructs successful experimentation and establishment of aseptic cultures. Serious attempts have been made to develop procedures for eliminating these bacterial contaminants through (1) rigorous manipulation of environmental and nutritional factors or (2) treatment with antibiotics.  The association of bacteria with in vitro cultures of different crop plants, like watermelon, grape, banana, papaya, and capsicum has been encountered. This has been the cause of decline in the performance of cultures, degeneration of long-term maintained stocks, and lack of reproducibility of tissue culture protocols. ,,,
We utilized 16S rRNA gene sequence analysis to identify bacterial contaminants [Figure 1] in Guadua angustifolia Kunth . These contaminants were found to be highly similar to Pantoea agglomerans (NCBI # FR872702) and Pantoea ananatis (NCBI # FR872704). Both bacteria are gram negative and closely related. , P. ananatis is a common epiphyte. It infects both monocotyledonous and dicotyledonous plants. It also occurs endophytically in hosts where it has been reported to cause disease symptoms. Apart from being associated with plants as an epiphyte, pathogen, or symbiont, it also occupies diverse and unusual ecological niches where it may function as a saprophyte. P. agglomerans is known to be an opportunistic pathogen in the immunocompromised, causing wound, blood, and urinary tract infections. It is commonly isolated from plant surfaces, seeds, fruits (namely mandarin oranges), and animal or human feces.
|Figure 1: Bacterial contamination in the region around shoot base in the agar medium.The bacteria appeared as creamish white growth around the base of shoots in the agar gelled medium after 20 days of inoculation|
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Many bacteria grow slowly or not at all in media used in plant culture, thus escaping detection until considerable time and materials have been invested.  The ideal approach is to use antibacterial substances
(e.g., antibiotics) but it has met with varying degrees of success. , In many cases, antibiotics have been found to be phytotoxic at high concentrations enough to destroy all contaminants. , The lack of descriptive information and antibiotic susceptibilities of a large number of plant-associated bacteria further complicate the use of antibiotics.  Therefore, the characterization and identification of plant-associated bacteria can lead to more successful antibacterial therapies. 
Our antibiotic sensitivity testing revealed kanamycin and streptomycin sulfate as the most effective antibiotic against the contaminating bacteria [Table 1]. The kanamycin was least phytotoxic during micropropogation of G. angustifolia. The shoot tips were grown for 10 days on the multiplication medium containing the kanamycin (10 μg/ml). The addition of kanamycin grossly inhibited the bacterial growth while allowing the formation of high-quality Guadua shoots [Figure 2]. In contrast, streptomycin was effective at reducing bacterial growth in tissue culture at higher concentrations (15 μg/ml). Moreover, the shoot number and the quality of Guadua were also reduced. Such inhibition of shoot growth by streptomycin has also been noted during micropropagation of Pelargonium. 
|Figure 2: The growth of healthy G. angustifolia shoots in the multiplication medium after treatment with kanamycin. The treatment of shoots with kanamycin (10 μg/ml) grossly inhibited the bacterial growth without affecting their quality and shoot numbers|
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|Table 1: The effect of various antibiotics on the growth of bacteria and Guadua angustifolia Kunth|
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Kanamycin interacts with the 30S subunit of prokaryotic ribosomes. It induces substantial amount of mistranslation and indirectly inhibits translocation during protein synthesis. Streptomycin binds to the S12 protein of the 30S subunit of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This prevents initiation of protein synthesis and leads to death of microbial cells. It may also inhibit protein synthesis in chloroplasts and mitochondria in plant tissues, and thus resulting in small and yellow leaves.
Traditionally, combinations of antibiotics have been used against bacterial contaminants in plant tissue culture. , The combinations of two or more antibiotics for eliminating bacterial contaminants are very well recommended. , However, we interestingly found kanamycin (10 μg/ml) as very effective in eliminating bacterial contaminants with least phytotoxicity. Such shoots with no detectable signs of bacterial contamination were transferred onto the fresh multiplication medium without antibiotic after every 3 weeks. The multiplication rate of shoots treated with antibiotic was similar to that of healthy plants. These shoots were able to produce healthy roots in the same multiplication medium without addition of auxin. These plants were successfully hardened under green house conditions [Figure 3].
|Figure 3: Acclimatized plants of G. angustifolia. Plantlets were transferred to plastic pots containing sand in polytunnels and covered with jars to maintain high relative humidity. After 1 month of hardening, these plants demonstrated 90% survival when transferred to pots containing 1:1:1 mixture of soil, sand and manure|
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| Conclusion|| |
The common problem of bacterial growth around in vitro shoots in Guadua angustifolia tissue culture is due to (1) Pantoea agglomerans and (2) Pantoea ananatis. This can be resolved through treatment of G. angustifolia shoots with kanamycin for 10 days. No phytotoxicity appeared when shoots were treated with kanamycin (10 μg/ml) and the multiplication rate of treated G. angustifolia shoots was found to be similar to that of healthy plants. Streptomycin sulfate, at higher concentration, also inhibited bacterial growth during micropropagation of G. angustifolia. In addition, shoots grown in streptomycin sulfate tended to be shorter and have stunted leaves. Thus, our study provides us with a technique to identify and resolve bacterial contamination of (1) Pantoea agglomerans and (2) Pantoea ananatis during in vitro culture of G. angustifolia.
| Acknowledgements|| |
The authors are grateful to the Council of Scientific and Industrial Research, New Delhi, for Senior Research Fellowship to Harleen Kaur Nadha and also to Dr PS Ahuja, Director, CSIR- IHBT, Palampur for providing all the facilities for carrying out this piece of research work. The manuscript has been vetted by CSIR-IHBT (IHBT Publication # 3188).
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[Figure 1], [Figure 2], [Figure 3]