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Curbing the menace of contamination in plant tissue culture.

Plant tissue culture is a field of biotechnology where, quite a large number of plantlets are produced from a single cell. This makes use of less sophisticated methods for commercial production of plantlets in large numbers. Introduction of molecular techniques in this field has improved its scope and applications. Plant tissue culture plays a vital role in production of plants which takes many years to flower and also where the seeds remain dormant. The thrust areas include the production of plantlets where vegetative propagation is cumbersome, the production of secondary metabolites from cell suspensions and for conduction of new trials of mutation induction through plant cell lines.

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).

Check point--Explants

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).

Check point--Equipment

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).

CONCLUSION

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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(41.) Shankar, C., Shiv, P., Balaji, D., Sathish S. Mass Propagation of Banana (Musa sp.) cv. Grand Naine through Direct Organogenesis by Benzyl Adenine Purine and Kinetin. Journal of Academia and Industrial Research., 2014; 3(2): 92-97.

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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: pdeepasankar@vit.ac.in

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
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Date:Sep 1, 2016
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