Curbing the menace of contamination in plant tissue culture.
The production of disease free homogenous plants is the main characteristic feature of plant tissue culture. Contamination is caused by the exogenous and endogenous microbes. Possibilities of contamination occur mainly due to two factors. It can be from the explants where, the microbes have the potency to remain latent for a period of time and due to improper handling of the operator (1). Proper identification and knowledge about the microbes are essential to eradicate them from the explants and aseptic handling measures have to be standardized. Several alterations have been followed for the reduction of contamination and cost for economic production of plantlets. Microbial contamination occurring at various levels has been discussed as checkpoints.
Check point--Mother stock
Mother plants or stocks infested with mites, nits, insects and microbes should not be chosen for deriving explants. Juvenile plants are the best source of explants as the level of microbial load is very much reduced in them and these tissues would be free from viral infections (2). Treatment of mother plants with bactericides, fungicides and insecticides may decrease the effect of contamination. Amendment of pesticides reduces significant losses caused by insect pests and other biological agents (3). Apart from these methods following integrated pest management can also reduce the burden of losses caused by the chemicals to the plants and to the environment as being followed by the Food and Agricultural Organization (FAO) in Asia, Near East and West Africa and in few other places (4).
The sterilization protocol for explant varies with different groups of plants. The chemicals used and the treatment time also varies with the nature of the explant. Commercial detergents such as Extran or Tween20 (HiMedia) at a concentration of 1-5% has been recommended as these help in dislodging the microbial spores from the pubescent plants (5,6,7). The commonly used sterilants are 0.1% mercuric chloride, 70% ethanol and 30% Chlorax (6,7,8). Treatment of explants with fungicides such as 1mg/ml concentration of Bavistin (50% carbendazim), 0.2% (w/v) of benomyl and antibiotics such as 1mg/ml concentration of streptomycin and chloramphenicol for half an hour have played a significant role in reduction of microbial contamination (9,10,11). Addition of silver nitrate as surface sterilant has been studied, where the compound had a very minimal inhibitory effect on the in vitro cultures (12). The latest technology that has been incorporated to reduce the deleterious effects caused by microbes is the use of nanoparticles. Silver nanoparticles have been used as a disinfectant (5). Similar use of the same has been reported in surface sterilization of rootstock cultures of hybrid almond at different concentrations, as silver generally possess antimicrobial properties (10,13).
Glasswares and other equipment are autoclaved at 121[degrees]C for 15 psi for 15-20 minutes in laboratories and industries. Chemical treatment of the glasswares is also carried out with sodium hypochlorite (NaOCl). A concentration of 0.002% NaOCl was used for disinfecting non autoclavable vessels for micropropagation of banana, but was not efficient for Agrobacterial cell cultures (14). Similarly 50ppm of NaOCl was used to clean deli containers which had significant cost reduction as the need of autoclaving had been replaced with the use of NaOCl (15).
Check point--Sterilization of the culture media
For preparation of sterile media, the chemicals should be of standard grade. Use of deionized water avoids the interaction of ions between the water and the chemicals. Once prepared, the culture media are autoclaved. Heat liable compounds such as antibiotics, growth regulators and supplements are sterilized through syringe filters of 0.22 [micro]m size and then incorporated into the sterile media. A period of 4-6 days is provided as incubation time to check the microbial growth on the sterile media. Contamination occurring even after practicing these strict protocols can be reduced by incorporation of the following into the media.
1. Use of antibiotic
2. Use of fungicide
3. Use of other chemicals
4. Use of nanoparticles
Use of antibiotic or incorporation of any other chemical compound into the media is a necessity only when the contamination is endogenous. Table.1 depicts the management of contamination in diverse plant groups by the use of biochemical and serological methods. The plant system has a lot of microbial flora associated with it that occurs exogenously and endogenously. The endogenously occurring microbes are commonly referred to as endophytes, remain intact within the plant tissues form the collection date, during the initiation protocols and even during subcultures. These microfloras are less susceptible to any disinfection protocols (16). These endophytes have impact on plants and provide tolerance against biotic and abiotic stresses in field conditions but these are undesirable as they compete for nutrients when cultured under in vitro conditions (17).
Use of antibiotics
Antibiotics which are commonly used in plant tissue culture are tetracycline, streptomycin, vancomycin, rifampicin, gentamycin, cefotaxime etc. These antibiotics are also used in combinations (22). A novel method that has been reported for eliminating bacterial contamination in in vitro propagation of Moss protonema is by the agar embedding system where, antibiotics were added to the agar and embedded on to protonema, thereby reducing the microbial growth due to the continuous contact between the tissues and antibiotics (23). Contamination in Gauda angustifolia Kunth, detected as endophytes have been treated by administration of kanamycin and streptomycin sulphate, where kanamycin at a concentration of 10 [micro]g/ml exhibited best results with no phytotoxicity (7). Similarly incorporation of 80 [micro]M/L of kanamycin in Centella asiatica L., culture medium induced shoot growth and also significantly reduced the bacterial contamination (11). 2-lactam antibiotics such as cefotaxime, carbecillin and timentin were exploited in protoplast cultures of carrot at a range of 100-500mg/L (24). Proper knowledge about the mode of action and the minimum inhibitory concentration level of antibiotics is a prerequisite as malformations and retarded growth of the in vitro grown cultures results if the dosage of the antibiotics is increased (25). The role and mode of action of few antibiotics has been discussed in Table 2.
Usage of fungicides in the culture media
Like bacterial, fungal contamination are also predominantly found in plant tissues as endophytes. These fungal endophytes are isolated and used for the production of phytochemicals which possess anticancerous, antineoplastic, antidepressant and other medicinally important compounds such as taxol (33,34). These endophytes pose threat to plants under in vitro conditions. Their growth can be controlled by use of systemic fungicides. The commonly used fungicide in culture media is Bavistin (50% carbendazim). Bavistin at a range of 150-300 mg/L incorporated into media showed significant reduction in fungal contamination (11). It has antimitotic and antineoplastic activities in fungal cells. It has a similar structure to that of cytokinin (adenine derivatives). Bavistin has been reported to cause shoot proliferation in Stevia rebaudiana cultures 35. Similarly, Dhingani et al, have reported that Bavistin had significant potential to inhibit root rot diseases under field conditions in Cicer arietinum L. caused by Macrophomina phaseolina (Tassi) Goid (36). Figure 1, 2 and 3 brings out a comparison between Bavistin and two cytokinins and also depicts the similarity in the structure among the adenine derivatives.
The use of other fungicides such as ProClin[R]300, mancozeb, thiabendazoles were reported in controlling the contamination of yeast in apple cultures (27). Mancozeb used in a commercial fungicide formulation termed EmCarb (63% mancozeb and 27% carbendazim) had successful results when incorporated at a concentration of 0.5% (w/v) in culture media grown with Asparagus adscendens Roxb (2).
Use of other chemicals
Apart from these antimicrobial compounds, other disinfectants and chemicals are also incorporated into the culture media. In vitro propagation of potato was carried out in a media containing 5-10 ppm of NaOCl (15). Disinfection of contaminants using active chlorine at 0.001% and 0.005% gave similar results to that of the conventional methods (37). Incorporation of active chlorine into the media maintains the stability of heat liable compounds such as Vitamin B and growth regulators (38). Biocides such as Plant Preservative Mixtures[TM] have also been supplemented into the culture media, as it contains a combination of methylisothiazolone (MIT) and chloromethylisothiazolone (CMIT) (31). These compounds inhibit major enzymes produced in the microbial metabolic and energy production pathways (39). Ilex paraguariensis grown with Delcide[TM] at a concentration of 75 [micro]l/L in culture medium resulted in 100% clean cultures without any sign of growth malformations (31).
Use of nanoparticles
Nanoparticles have been engineered to eradicate the prevalence of contaminants. Zinc nanoparticles and zinc oxide nanoparticles have been found to possess antimicrobial properties. They have also been found to show no antagonistic activity at a concentration of 200mg/ L in banana cultures (32). Similarly silver nanoparticles were also reported to have the potential to reduce bacterial contamination at a range of 20-100mg/L in tissue cultures of Valeriana officinalis (5). The root stock cultures of an almond hybrid, immersed in a solution containing silver nanoparticles at a concentration of 100mg/L and then cultured in a media containing 150 mg/L of silver nanoparticles were found to have a reduced effect of contamination (10).
Check point--Sub culture and storage
This is the major step in the process of plant tissue culture as it produces multiple plantlets. Leifert et al., have reported that, during sub culturing process contamination occurs due to unsterile air, improper sterilization of the equipment and aseptic handling by the operators (40). Once the sub culturing has been completed the media containing vessels need to be properly sealed, labelled and stored in clean environment with sufficient environmental factors (1). Fumigation of culture rooms with solutions of potassium permanganate and formaldehyde at weekly intervals were carried out for the mass propagation of banana (41). Culture rooms housing the bioreactors for the production of Artemisinin through hairy root cultures were also given similar treatment for reducing the microbial load in the culture rooms (42). Sterility also needs to be maintained when the plantlets are subjected to primary hardening. Ammonium bicarbonate has been used as a fumigating agent for controlling wilt disease caused by a soil borne fungi, Fusarium, which has detrimental effects on major horticultural and food crops (43). Some of the other soil fumigants under use are chloropicrin, dimethyl disulfide, Telone C35 and methyl bromide (44,45).
Microbial hazards cause drastic economic losses in the plant tissue culture industries. Improper handling and non-sterile environment will increase the rate of contamination. Each explant is very precious as they form multiple plantlets. During contamination, competition for the nutrient rich media occurs between plants and microbes leading to the death of plantlets as they are fragile when grown in a controlled environment. This review tries to give an insight into the different levels at which contamination occur and also provides solutions to these problems.
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S. Leelavathy and P. Deepa Sankar *
School of Bio Sciences and Technology, VIT University, Vellore--632 014, India.
(Received: 10 April 2016; accepted: 25 May 2016)
* To whom all correspondence should be addressed.
Tel.: +91 9884048391; E-mail: firstname.lastname@example.org
Caption: Fig. 1.Bavistin
Caption: Fig. 2. Benzyl aminopurine
Caption: Fig. 3. Kinetin
Table 1. Methods of elimination of contamination in plant tissue culture Plant culture Contaminant Detection Boat orchid Odontoglossum ELISA (Cymbidium Sw.) Ringspot Virus Protocorm Apple Rhodotorula Morphological (Malus domestica) slooffiae Axillary bud Physiological, Biochemical characteristic and 26S rDNA sequencing Valerian Xanthomonas sp. Special (Valeriana. laboratory officinalis L) methods Node Grape vine GRSPaV, RT-PCR and (Vitis vinifera) GLRaVl, ELISA Embryo GVA, GLRaV3 Madonna lily Fusarium, Slide culture (Lilium Alternaria, techniques candidum L.) Rhizopus, Node Cylindrocarpon and Aspergillus sp. Bamboo Unidentified Physiological (Phyllostachys sp. bacteria and 16S rDNA and Fargesia sp. gene Node sequencing Lamba Unidentified Gram staining (Curculigo bacteria latifolia Dryand) Rhizome Yerba mate Stenotrophomonas 16S rDNA (Ilex para maltophilia gene analyses, guareinsis analytical Node profile index and biochemical tests Almond hybrid Unidentified Visual (Prunus bacteria and assessment of amygdalus x fungi contamination P. persica) Node Banana Unidentified Biochemical (Musa bacteria and fungi and gram sapientum L.) staining and Sucker lactophenol cotton blue staining Carrot Unidentified Antibiotic (Daucus bacteria sensitivity carota L.) screening Protoplast Bamboo Pantoea sp Antibiotic (Guadua sensitivity angustifolia screening and Kunth) Node 16S rRNA gene sequencing 16S rRNA Mosses Paenibacillus gene (Ceratodon taichungensis, sequencing purpureus and P. humicus, Physcomitrella Bacillus patens) megaterium Protonema Plant culture Treatment Boat orchid Ribavirin (Cymbidium Sw.) (VIRAZOLE[R]) at Protocorm 35ppm along with media Apple Mancozeb (15mg/L) (Malus domestica) thiabendazoles Axillary bud (40mg/L), silver nitrate (588 [micro]M) and Silveret77(0.01%) Valerian Nano silver (Valeriana. solutions at 25, 50 officinalis L) and 100mg/L were Node used before and after sterilization Grape vine -- (Vitis vinifera) Embryo Madonna lily Benomyl with (Lilium Nystatin candidum L.) (100 mg[l.sup.-1]) after Node surface sterilization Bamboo -- (Phyllostachys sp. and Fargesia sp. Node Lamba Chloramphenicol, (Curculigo steptomycin and latifolia Dryand) Bavistin(0.1%) Rhizome Yerba mate 0.75ml/L of (Ilex para Delcide[TM] TG and guareinsis other isothiazolone Node biocides Almond hybrid Nano silver solution (Prunus at 100ppm as amygdalus x immersion solution P. persica) and along with Node media Banana Zinc and zinc oxide (Musa nanoparticles(100 sapientum L.) mg/L) Sucker Carrot [??]-lactam (Daucus antibiotics carota L.) Protoplast Bamboo Streptomycin and (Guadua kanamycin (10[micro]g/ml) angustifolia along with media Kunth) Node for ten days Mosses Vancomycin at (Ceratodon 50 [micro]g/ml in purpureus and embedded agar Physcomitrella block patens) Protonema Plant culture Significance Boat orchid Early and reliable (Cymbidium Sw.) indexing of in vitro Protocorm grown plantlets to eradicate virus (26) Apple Increase in (Malus domestica) decontamination Axillary bud was observed when compounds were added into the media (27) Valerian Nano silver had no (Valeriana. detrimental effects officinalis L) on the cultured Node plantlets (5) Grape vine Somatic (Vitis vinifera) embryogenesis was Embryo efficient against phloem limited grapevine virus (28) Madonna lily Effective sterilization (Lilium using candidum L.) chemotherapeutic Node substances provided higher percentage of decontamination (29) Bamboo 15 out of 18 species (Phyllostachys sp. of bacterial and Fargesia sp. endophytes were Node reported for the first time (30) Lamba Pretreatment for 9 (Curculigo hours significantly latifolia Dryand) reduced Rhizome contamination (9) Yerba mate (Ilex para Bactericidal activity guareinsis in transpiration Node stream was reported using isothiazolone biocides (31) Almond hybrid Optimum (Prunus concentration of amygdalus x 100ppm, was P. persica) observed to contain Node microbe growth (10) Banana (Musa Reduction in sapientum L.) contaminations were Sucker observed at 200mg/L without affecting the physiological and morphological processes (32) Carrot Inhibited the (Daucus biosynthesis of carota L.) peptidoglycan (24) Protoplast Bamboo Kanamycin had best (Guadua resultants but angustifolia streptomycin Kunth) Node resulted in yellow and stunted plantlets (7) Mosses Contamination (Ceratodon reduced gradually, as purpureus and the tissues were in Physcomitrella constant contact with patens) antibiotic (23) Protonema Table 2. Mode of action of few antibiotics against bacteria S.No Antibiotic Mechanism of Action Reference 1 Vancomycin Inhibits synthesis of peptidoglycan 18 in cell membrane 2 Cefotaxime Inhibits bacterial cell wall 19 synthesis 3 Rifampicin Inhibits bacterial RNA polymerase 20 4 Tetracycline Prevents the attachment of aminoacyl 21 rRNA to ribosomal acceptor (A) site 5 Streptomycin Prevents the initiation of protein 7 synthesis 6 Kanamycin Inhibits translocation during 7 protein synthesis
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|Author:||Leelavathy, S.; Sankar, P. Deepa|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Sep 1, 2016|
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