Types Of Fungal Contamination In Plant Tissue Culture Pdf

Types of fungal contamination in plant tissue culture pdf

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. This study has been designed to analyze the identity of contaminating bacteria and to develop the strategy to eliminate them during micropropagation of Guadua.

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.

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In addition, we also- performed antibiotic sensitivity testing on these bacterial isolates. We identified kanamycin and streptomycin sulfate as potentially useful antibiotics in eliminating the contaminating bacteria. 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.

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. 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.

On the basis of the life span of culture, the cell lines are categorized into two types:

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.

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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.

Moreover, the use of nodal segments for propagation is cumbersome and labor intensive for large-scale establishment of bamboo plantations. 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,[ 3 ] and the failure of surface sterilization procedures to produce aseptic cultures is a major problem with woody plants.

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.

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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.

Types of fungal contamination in plant tissue culture pdf

We used nodal segments measuring 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.

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The pH was adjusted to 5. 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. Pure cultures of these bacteria were obtained by picking up the colonies and streaking them onto the fresh medium.

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.

Types of fungal contamination in plant tissue culture pdf

To test their effectiveness in eliminating bacterial contamination, the antibiotics were added to the multiplication medium, i. 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.

Types of fungal contamination in plant tissue culture pdf

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.

Subculturing without contamination

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.

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.

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. Both bacteria are gram negative and closely related. 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. It is commonly isolated from plant surfaces, seeds, fruits namely mandarin oranges , and animal or human feces. Bacterial contamination in the region around shoot base in the agar medium.

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The bacteria appeared as creamish white growth around the base of shoots in the agar gelled medium after 20 days of inoculation. 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.

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. The addition of kanamycin grossly inhibited the bacterial growth while allowing the formation of high-quality Guadua shoots [ Figure 2 ]. Moreover, the shoot number and the quality of Guadua were also reduced.

Types of fungal contamination in plant tissue culture pdf

Such inhibition of shoot growth by streptomycin has also been noted during micropropagation of Pelargonium. The effect of various antibiotics on the growth of bacteria and Guadua angustifolia Kunth. The growth of healthy G. 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.

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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 ].

Acclimatized plants of G. Plantlets were transferred to plastic pots containing sand in polytunnels and covered with jars to maintain high relative humidity. 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. Streptomycin sulfate, at higher concentration, also inhibited bacterial growth during micropropagation of G.

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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. Source of Support: Nil.

Problems in tissue culture: culture contamination

Conflict of Interest: None declared. National Center for Biotechnology Information , U. Journal List Pharmacogn Mag v. Pharmacogn Mag. Author information Article notes Copyright and License information Disclaimer.

Contamination in Tissue Culture

Address for correspondence: Dr. E-mail: moc. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3. This article has been cited by other articles in PMC. Abstract Background: Guadua angustifolia Kunth is a very important bamboo species with significant utility in pharmaceutical, paper, charcoal, and construction industries.

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. Results: We identified kanamycin and streptomycin sulfate as potentially useful antibiotics in eliminating the contaminating bacteria.

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. 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.

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.

Types of fungal contamination in plant tissue culture pdf

Open in a separate window. Figure 1.