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Technical Glitches in Micropropagation
C H A P T E R
13
Technical Glitches in Micropropagation Saurabh Bhatia, Kiran Sharma O U T L I N E 13.1 Introduction
393
13.8 Shoot-tip Necrosis
400
13.2 Microbial Contamination
396
13.9 Habituation
400
13.3 Gaseous Contamination
397
13.4 Cross-culture Contamination
398
13.5 Browning of the Medium or Phenolic Exudation
398
13.6 Absence of Rooting and Acclimatization
399
13.7 Hyperhydricity 13.7.1 The Main Causes of Hyperhydricity in Plant Tissue Culture 13.7.2 Control of Hyperhydricity
399
13.10 Tissue Proliferation 13.10.1 Genetic or Chimeral Variation or Somaclonal Variation 13.10.2 Transient Phenotypic Variation 13.10.3 Epigenetic Variation
401
13.11 Detection of the Contaminants
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References
403
401 402 402
399 400
13.1 INTRODUCTION Contamination is defined as the accidental introduction of undesirable bacterial, fungal, and algal microorganisms and vectors or sources of pathogens into a culture medium. There are various sources of contaminants. Various sterilization procedures and aseptic techniques used for eliminating the contamination problems are highlighted in Fig. 13.1 and Table 13.1. A high concentration of sugar in plant tissue culture media supports the growth of microorganisms Modern Applications of Plant Biotechnology in Pharmaceutical Sciences. http://dx.doi.org/10.1016/B978-0-12-802221-4.00013-3 Copyright © 2015 Elsevier Inc. All rights reserved.
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FIGURE 13.1 Protocols to achieve sterile culture.
like bacteria and fungi. Explant is known to be the major source of contamination. Contaminants that are associated with explant are often more epiphytic than endophytic [1]. Various sterilization protocols are utilized to remove several types of contaminants (Fig. 13.2). When plant is in its native state, the growth of microorganisms is suppressed by the defense mechanism and dry environment. But when plant is brought into in vitro conditions its molecular pathways, which are essential for the production of optimum amounts of secondary metabolites, are disturbed. In addition, plant cell/tissue/organ is exposed to high levels of moist conditions that may favor the growth of microorganisms in the culture medium. These
395
13.1 Introduction
TABLE 13.1 Different Types of Sterilization Strategies Used in Plant Tissue Culture Type of sterilization
Features
Specifications
Sterilization by heat
Heat is the most widely used lethal agent for sterilization.
Dry heat (oven)
170˚C for 120 minutes.
Moist heat (autoclave)
121˚C at 15 lb for 15–20 min.
Sterilization by filtration
A liquid medium or solution that contains microorganisms can be sterilized by passing through a filter.
Filters may have different sizes and pores.
0.2 mm (pore size)
Tyndallization
Fractionated discontinuous method of sterilization.
Media is heated in water bath for 1 h for 3 consecutive days and kept at normal room temperature for two successive instances of boiling.
At 100˚C for 15 min/day up to 3 consecutive days.
Air sterilization
Laminar air flow cabinet equipped with high efficiency particulate air filters (HEPA), blower is used to blow the air through the HEPA filters
A manometer is fitted with the instrument to check the air pressure. Pressure more than 13 bars can choke the filter UV light provides additional sterilization.
HEPA prevents all sorts of microorganisms larger than 0.3 mm with 99% efficiency.
Surface sterilization of the explants
Mercuric chloride Bromine water Sodium hypochlorite Calcium hypochlorite Silver nitrate Hydrogen peroxide Bromine water Antibiotics: tetracycline, ciprofloxacin Ethanol
Sodium hypochlorite can be used for any material.
Time of treatment (5–50 min).
Mercuric chloride is most toxic material. Combinations of antibiotics are beneficial.
microorganisms when inside the medium grow faster than plant cells and multiply up to several fold by depleting carbohydrate sources and producing phytotoxic fermentation products such as ethanol and acetic acid. Plant cells in this starvation state become stressed and produce high levels of secondary metabolites, which are toxic for plant tissue. In addition to microbial contamination there are various other contaminations caused during micropropagation, e.g., chemical, radiation, and gas contamination. However, the frequency of these contaminations is considerably lower than microbial contamination. Browning of the medium or phenolic exudation in response to biotic and abiotic stresses is again a major constraint as this may lead to oxidation of leached phenolic contents causing darkening or browning of the media, which may further block the uptake of nutrients, ultimately leading to death of the explants. Similarly
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FIGURE 13.2 Protocols to sterilize the explants.
absence of rooting and acclimatization, hyperhydricity, shoot-tip necrosis, habituation, and tissue proliferation also suppress the growth of the tissue under in vitro conditions. This chapter describes various types of contamination sources and the alternative strategies to produce a contamination-free environment.
13.2 MICROBIAL CONTAMINATION Microbial contamination is a big problem in plant tissue culture practices. Bacteria, fungi, molds, and yeast are common contaminating microorganisms found in plant tissue culture practices. Microorganisms and their reproductive structures are ubiquitous although their relative abundance may vary considerably with environment and season. Microorganisms are found inside and on the surface of the plants. Microbial contamination of plant tissue culture is due to the high nutrient availability in the almost universally used culture medium. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria [2]. The intracellular pathogenic
13.3 Gaseous contamination
397
bacteria and viruses/viroids may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening of the stock plants with disease and endophyte elimination; pathogen and contaminant screening of established initial cultures; and observation, random sampling, and culture screening for microorganisms in multiplication and stored cultures. The increasing accessibility of both broad-spectrum and specific molecular diagnostics has resulted in advances in multiple pathogen and latent contaminant detection. The hazard analysis critical control point management strategy for tissue culture laboratories is underpinned by staff training in aseptic techniques and good laboratory practices. The ideal procedure for preventing bacterial growth includes the following steps: • • • •
Indexing explants and cultures for contaminants Identifying the source of those contaminants Identifying or characterizing the contaminants Eliminating the contaminating organism with improved cultural practices such as antibiotics (aminoglycosides, quinolones, b-lactams, glycopeptides, polymyxins, macrolides, and lincosamides) [3] or other chemical agent treatment
Bacterial contaminants may affect in vitro plant growth negatively, positively, or not at all. Bacterial growth may be suppressed in plant media by high salts, high sucrose, pH, temperature, tannins in explant sap, and appropriate nutrients not present in tissue culture media, e.g., pseudomonads, xanthomonads, and corynebacteria. In plants, bacteria may be endophytes, which can become pathogenic in vitro (in vitro pathogens = “vitropaths”), and in vivo pathogens, which can become saprophytes in vitro. Bacteria are the most frequent contaminants. They are usually introduced with the explants and may survive even after surface sterilization of the explants because they are in the interior tissues. Bacteria can be recognized by a characteristic ooze. This ooze can be of many colors including white, pink, creamish, and yellow. Fungi may enter cultures on explants or the spore may be airborne. Fungi may be recognized by their fuzzy appearance and they occur in a multitude of colors. Yeast is a common contaminant of plant cultures. Yeast lives on the external surfaces of the plants as well as in the air. Viruses, mycoplasma-like organisms, spiroplasms, and rickettsias are not easily detected but they do form a source of serious contamination in cultures. Several insects are particularly troublesome in plant cultures including ants, thripes, and mites.
13.3 GASEOUS CONTAMINATION Apart from the various contaminations caused by microorganisms, gaseous contamination also influences the growth of the culture. Culture ventilation is necessary to facilitate the gaseous exchange with the atmosphere and to allow ethylene to escape from the vessels. This problem can be overcome by using gas permeable filters as lid seals or by replacing the lid or wrapping the open-lidded vessel in gas permeable plastic film [4,5]. There is a wide range of transparent plastic wrapping materials that can be used instead of a conventional
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lid or the film can be wrapped around an open lidded vessel with the provision that the film be adequately permeable to oxygen, carbon dioxide, and ethylene to allow equilibration with the ambient atmosphere. If replacing the lid with a plastic film, the permeability of the film to water vapor should be chosen such that the cultures do not dry out [4,5].
13.4 CROSS-CULTURE CONTAMINATION This generally occurs when a different cell type is inadvertently introduced into the cells that are being cultured. This is a significant problem because the new type of cell could have different morphology and function and react differently to any experimental conditions that are applied. Cross-contamination voids the experiment because results cannot be valid or credible, as the effect of the contaminating cells is unknown.
13.5 BROWNING OF THE MEDIUM OR PHENOLIC EXUDATION Secondary metabolites like phenolic compounds in the plants are produced in response to biotic and abiotic stresses. These are basically involved in the defense mechanism of plants. However, leaching of phenolics contents from the explants of most woody and some herbaceous plants often produce lethal effects for the growing cells or tissues. This problem appears in tissue culture when explants are excised during the preparation of the cultures, which leads to stimulation of phenolic exudation. Oxidation of these exuded phenolic contents causes darkening or browning of the media, which blocks the uptake of nutrients and ultimately leads to death of the explants. Browning did not affect the growth of roots and shoots when explants were cultured in a large volume of medium, but in a small volume it was lethal. Various absorbents and antioxidants can be used to minimize this exudation problem. Alternatives used to minimize this type of problem are: • Frequent subculturing of explants. • Use of antioxidant compounds like ascorbic acid, butylated hydroxyl anisole, or butylated hydroxyl toluene; citric acid may prevent the oxidation of phenolic compounds. • Use of adsorbents (like activated charcoal or PVP) to adsorb polyphenols secreted in the medium. • Culturing in the dark to prevent polyphenolic oxidation as light enhances it. • Soaking in water after excision to reduce the browning effect. • After excision, when the explants are being transferred to nutrient media, the positioning of explants on medium have a considerable effect, e.g., some woody herbaceous plants produce more shoots when placed horizontally instead of vertically on the nutrient medium. • A brief period of culture in liquid medium (3–7 days) to remove phenolics and other substances. • Control of phenolic oxidation by the supplementation of certain compounds to the medium, e.g., adenine sulfate and 300 mg L–1 polyvinylpyrrolidone (PVP). • Sealing the cut ends with paraffin wax to control browning by preventing exudation [6].
13.7 Hyperhydricity
399
13.6 ABSENCE OF ROOTING AND ACCLIMATIZATION Rooting is a critical phase since it depends on the genotype and physiological condition at the time of root induction [7]. This stage is followed by a process called acclimatization and is essential because the in vitro plants are cultivated under heterotrophic conditions [8]. Morphological anomalies, such as nonfunctional stomata, and physiological anomalies, such as a decrease in photosynthesis, might occur during this process [9]. These anomalies interfere with plant survival in the greenhouse or field. Therefore, acclimatization is critical for the transition of micropropagated plants from in vitro cultivation to ex vitro cultivation. The explants can naturally form roots during propagation without the additional rooting stage as with the potato but some species may show root production deficiency. Rooting may be induced by incorporating auxins and carbon in the culture medium. Extent of proper rooting varies species to species. To overcome this, the following critical growth regulators are added: 1-naphthalene acetic acid, indole-3-butyric acid, picloram, 2,4-dichlorophenoxyacetic acid (2,4-D), etc. These in vitro plants are sensitive to the external environment, so maintenance of relatively high humidity is required. Due to the high sensitivity of in vitro plants they should never be transplanted directly to open fields. Acclimatization is a key step to successful production. Tissue cultured plants may develop water stress upon transfer out of the test tube. Plants that do not acclimatize to the greenhouse die quickly. Leaves wilt, shrivel, and fall off. Root tips may turn brown and shrivel. The complete plant may turn brown after several days. Water stress is caused by many anatomical and physiological reasons. The main reason for short-term water loss is the lack of stomatal closure in unacclimatized tissue cultured plants. No worry methods, mist bench/ polytent method, humidification chamber method, are the best solution for acclimatization.
13.7 HYPERHYDRICITY In general terms, swelling/thickening of the tissue, just like callus, is called vitrification or hyperhydricity. In many species, vitrification may be represented by symptoms not visible to the naked eye. It is a physiological malformation that results in excessive hydration, low lignification, poorly developed vascular bundles, impaired stomatal function, and reduced mechanical strength of tissue culture-generated plants [10]. In vitrification, tissue becomes water soaked and translucent, which is mainly caused by excessive water uptake. It is usually controlled by changing agar concentration or source. The consequence is poor regeneration of such plants without intensive greenhouse acclimation for outdoor growth. Additionally, it may also lead to leaf-tip and bud necrosis in some cases, which often leads to loss of apical dominance in the shoots. In general, the main symptoms of hyperhydricity are translucent characteristics signified by a shortage of chlorophyll and high water content [10]. Specifically, the presence of a thin or lack of a cuticular layer, reduced number of palisade cells, irregular stomata, less developed cell wall, and large intracellular spaces in the mesophyll cell layer have been described as some of the anatomic changes associated with hyperhydricity.
13.7.1 The Main Causes of Hyperhydricity in Plant Tissue Culture • Oxidative stresses as a result of high salt concentration • The type of explants utilized
400 • • • • • • • • •
13. Technical Glitches in Micropropagation
The concentrations of microelement and hormonal imbalances Low light intensity High relative humidity Gas accumulation in the atmosphere of the jar Length of time intervals between subcultures Number of subcultures Concentration and type of gelling agent High ammonium concentration Evident in liquid culture-grown plants or when there is a low concentration of gelling agent
13.7.2 Control of Hyperhydricity • Monitoring the modified atmosphere of the culture vessels. • Adjusting the relative humidity in the vessel. • Use of gas-permeable membranes to increase exchange of water vapor and other gases such as ethylene with the surrounding environment. • Use of higher concentration of a gelling agent to reduce the risk of hyperhydricity. • Addition of agar hydrolysate. • Use of growth retardants and osmotic agents. • Use of bottom cooling, which allows water to condense on the medium. • Use of cytokinin-meta-topolin (6-(3-hydroxybenzylamino)purine). • Combination of lower cytokinin content and ammonium nitrate in the media. • Use of nitrate or glutamine as the sole nitrogen source. • Decreasing the ratio of ammonium: nitrate in the medium.
13.8 SHOOT-TIP NECROSIS Shoot-tip necrosis can be a major obstruction in the successful propagation of certain species by tissue culture. The symptoms of shoot-tip necrosis are browning and die-back of buds and the youngest leaves [11]. Shoot-tip necrosis occurs in some woody perennial tissue cultures when actively growing shoot tips develop tip die-back. This condition is usually caused by a calcium deficiency in the medium. The first assumption in seeing shoot-tip necrosis is that it is caused by nutrient deficiency [11]. The symptoms of nutrient deficiency of less mobile elements such as calcium (Ca) and boron (B) first appear in the meristematic regions and young leaves whereas symptoms of excessive amounts of these minerals are first observed on the older leaves. However, in in vitro systems, shoot-tip necrosis is caused by a complex set of factors rather than just nutrient deficiency [11].
13.9 HABITUATION Plant tissue culture usually requires sources of the growth hormones auxin and cytokinin for continuous proliferation in culture. In 1942, Gautheret reported that carrot tissue can gradually lose its requirement for exogenous auxin. He called this phenomenon auxin habituation.
13.10 Tissue proliferation
401
It was soon recognized that similar variation can occur for cytokinins and more rarely for some other hormones. Thus, habituation is when a culture continues to develop in the absence of auxin or cytokinin. For example, shoot cultures habituated for cytokinin would continue to produce new shoots on a cytokinin-free medium. Hormone habituation is a phenomenon by which plant cells and tissues lose the requirement of exogenous hormones to sustain cell division and development upon continuous culture [12].
13.10 TISSUE PROLIFERATION Tissue proliferation may be the expression of naturally occurring growths in plants like Rhododendron induced by the tissue culture environment. Variation in micropropagated plants is one of the greatest problems faced by tissue culture during micropropagation. There are many types of variation that cause several problems during tissue proliferation, which can further create a great problem in defining the genotype of new variants [13].
13.10.1 Genetic or Chimeral Variation or Somaclonal Variation Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture. Chromosomal rearrangements are an important source of this variation. Somaclonal variation is not restricted to, but is particularly common in, plants regenerated from callus. The variations can be genotypic or phenotypic, which in the latter case can be either genetic or epigenetic in origin. Typical genetic alterations are: changes in chromosome numbers (polyploidy and aneuploidy), chromosome structure (translocations, deletions, insertions, and duplications), and DNA sequence (base mutations) [13]. Typical epigenetic-related events are gene amplification and gene methylation. If no visual, morphogenic changes are apparent, other plant screening procedures must be applied. There are both benefits and disadvantages to somaclonal variation. The phenomenon of high variability in individuals from plant cell cultures or adventitious shoots is called somaclonal variation [13]. Therefore, it can be defined as the variation that occurs because of genetic mutation caused by in vitro conditions or by chimeral separation. Somaclonal variation is usually undesirable. In some cases, somaclonal variation can lead to new cultivars (e.g., disease resistance, new leave pattern) that may have desirable ornamental characteristics or increased pest resistance. The occurrence of somaclonal variation can be reduced by: • • • •
Avoiding long-term cultures. Using axillary shoot induction systems where possible. Propagating chimeras by other clonal systems. It is well known that increasing numbers of subcultures increase the likelihood of somaclonal variation, so the number of subcultures in micropropagation protocols should be kept to a minimum. • Regularly reinitiating clones from new explants, which might reduce variability over time. • Avoiding 2,4-D in the culture medium, as this hormone is known to introduce variation.
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13.10.2 Transient Phenotypic Variation Plant populations show phenotypic diversity, which may be caused by genetic and epigenetic variation. It has recently been shown that new epigenetic variants are generated at a higher rate than genetic variants and several studies have shown that epigenetic variation can be influenced by the environment. Although the heritability of environmentally induced epigenetic traits has gained increasing interest in past years, it is still not clear whether and to what extent induced epigenetic changes have a role in ecology and evolution. Some reports on model and nonmodel species support the possibility of adaptive epigenetic alleles, indicating that epigenetic variants are subject to natural selection. However, most of these studies rely solely on phenotypic data and no information is available about the underlying mechanisms. Thus, the role of inherited epigenetic variation for plant adaptation is unclear and further investigations are required to gain insights into the significance of epigenetic variation for ecological and evolutionary processes.
13.10.3 Epigenetic Variation In general terms, epigenetics can be defined as the study of changes in gene expression or cellular phenotype, caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: επí-,over, above, outer) genetics. Some of these changes have been shown to be heritable. It refers to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence [13]. Examples of such modifications are DNA methylation and histone modification, both of which serve to regulate gene expression without altering the underlying DNA sequence. In tissue culture, new plants may be generated by outgrowth of axillary buds or by adventitious regeneration. Researchers expected initially that these clonally propagated plants would be exact copies of the parent plant, but frequently aberrant plants were observed. Various causes have been established: • Genetic changes (also referred to as somaclonal variation): changes in the DNA sequence. • Epigenetic variation: long-lasting changes in the expression of the information in the genome. • Chimeral segregation and loss of pathogens in particular viruses. Epigenetic changes are caused by changes in the expression of the information in the DNA brought about by alterations in DNA methylation, in histones, or in both [13]. These modifications may influence gene transcription. Epigenetic changes are often temporary and plants may revert to the normal phenotype relatively easily, but some can be long lasting and may even be transferred during sexual propagation.
13.11 DETECTION OF THE CONTAMINANTS Contaminants may be introduced with the explants or during manipulation in the laboratory. They may express themselves immediately or remain latent for long periods of time. Eyeball determination method (visual determination method), stock plant indexing (taking part of the plant and transferring to media that are specific for bacteria and fungi, e.g., potato
REFERENCES 403
TABLE 13.2 Identification and Characterization of Bacterial Contaminants Test
Procedure
Reference
Biochemical tests
Gram stain, motility, gelatinase, oxidase, and oxidation/ fermentation
[20]
Bergey’s Manual of Systematic Bacteriology
Descriptions of genera and species, which are helpful for identifying bacteria
[21]
Tetrazolium dye test
Reduction of tetrazolium dye in response to cellular respiration and results are compared with standard database
[22]
The API identification system
Carbon source utilization test: relies on visual detection of the test bacterium
[23]
Fatty acid analysis profiles test
Fatty acid methyl esters with those of known organisms
[24]
Probe test
DNA probes and 16S rRNA use PCR amplification and probes for known sequences
[25]
TRADITIONAL TESTS
CONTEMPORARY TESTS
dextrose agar and NB broth), semiselective media, and polymerase chain reaction (PCR)based molecular techniques are usually used for the detection of the contaminants. Hazard analysis critical control point systems are used in commercial plant tissue culture laboratories. Several detection strategies are highlighted in Fig. 13.1. Visual inspection of the medium may provide evidence of some contaminants but it is not adequate for slow growing bacteria, e.g., endophytes, or those bacteria that do not grow on plant tissue culture media [14]. There are various screening methods reported for the identification of many contaminants [14–17]. Some bacteria require specialized media and some contaminants can be easily detected by screening on two or three commercially available bacteriological media [18,19]. For indexing microbes, plant tissue can be inoculated into liquid and agar solidified yeast extract glucose, Sabouraud glucose, and AC media and incubated for 3 weeks at 30°C. This procedure is reported for indexing microbes in woody plant species. This can be further utilized by growing explants in a liquid culture system at pH 6.9. There might be chances of increase in expression of pathogens under greenhouse conditions. Therefore, such cultured batches should be separately placed to avoid multiplication of contaminants among healthy propagates. Various strategies for identification and characterization of bacterial contaminants are highlighted in Table 13.2.
References [1] Fogh Jørgen, Holmgren NB, Ludovici PP. A review of cell culture contaminants. In Vitro 1971;7(1):26–41. [2] Cassells AC. Problems in tissue culture: culture contamination. In: Debergh PC, Zimmerman RH, editors. Micropropagation technology and application. Dordrecht, Netherlands: Kluwer Academic Publishers; 1991. p. 31–44. [3] Falkiner FR. The criteria for choosing an antibiotic for control of bacteria in plant tissue culture. Newsletter International Association for Plant Tissue Culture 1990;60:13–23. [4] Marino G, Altan AD, Biavati B. The effect of bacterial contamination on the growth and gas evolution of in vitro cultured apricot shoots. In Vitro Plant 1996;32(1):51–6.
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[5] Odutayo OI, Amusa NA, Okutade OO, Ogunsanwo YR. Sources of microbial contamination in tissue culture laboratories in southwestern Nigeria. Afr J Agr Res 2007;2(3):067–72. [6] Bhat SR, Chandel KP. A novel technique to overcome browning in tissue culture. Plant Cell Rep 1991;10 (6-7):358–61. [7] Martins L, Pedrotti EL. Enraizamento in vitro e ex vitro dos porta-enxertos de macieira M7, M9 e Marubakaido. Rev Bras Frutic 2001;23(1):11–6. [8] Hazarika BN. Acclimatization of tissue-cultured plants. Curr Sci 2003;85(12):1704–12. [9] Rogalski M, Moraes LKA, Felisbino C, Crestani L, Guerra MP, Silva AL. Aclimatização de porta-enxertos de Prunus sp. micropropagados. Rev Bras Frutic 2003;25(2):279–81. [10] Kevers C, Franck T, Strasser RJ, Dommes J, Gaspar T. Hyperhydricity of micropropagated shoots: a typically stress-induced change of physiological state. Plant Cell Tiss Org Cult 2004;77(2):181–91. [11] Bairu MW, Stirk WA, Staden JV. Factors contributing to in vitro shoot-tip necrosis and their physiological interactions. Plant Cell Tiss Org Cult 2009;98(3):239–48. [12] Meins F. Habituation: heritable variation in the requirement of cultured plant cells for hormones. Annu Rev Genet 1989;23:395–408. [13] Neelakandan AK, Wang K. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. Plant Cell Rep 2012;31:597–620. [14] Kane ME. Indexing explants and cultures to maintain clean stock. In Vitro 1995;31:25A. [15] Reed BM, Buckley PM, DeWilde TN. Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cell Dev Biol 1995;31:53–7. [16] Leifen C, Camotla H, Wailes WM. Effect of combinations of antibiotics on micro-propagated Clematis, Delphinium, Hosta, Iris, and Photinia. Plant Cell Tiss Org Cult 1992;29:153–60. [17] Holland MA, Polacco JC. PPFMs and or her covert contaminants: is there more to plant physiology than just plant? Annu Rev Plant Physiol Mol Biol 1994;45:197–209. [18] George KL, Falkinham JO. Selective medium for the isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum. Can J Microbiol 1986;32:10–4. [19] Gunson HE, Spencer-Phillips PTN. Latent bacterial infections: epiphytes and endophytes as contaminants of micropropagated plants. In: Nicholas JR, editor. Physiology, growth and development of plants in culture. Dordrecht, Netherlands: Kluwer Academic Publishers; 1994. p. 379–96. [20] Klement Z, Rudolph K, Sands DC, editors. Methods in phytobacteriology. Budapest: Akademiai Kiado; 1990. [21] Krieg NR, Holt JG, editors. Bergey’s manual of systematic bacteriology, Vol. I. Baltimore: Williams and Wilkens; 1984. [22] Jones JB, Chase AR, Harris GK. Evaluation of the Biolog GN MicroPlate system for identification of some plantpathogenic bacteria. Plant Dis 1993;77:553–8. [23] Leifert C, Waites WM, Nicholas JR. Bacterial contamination of micropropagated plant tissue cultures. J Appl Bact 1989;67:353–61. [24] Chase AR, Stall RE, Hodge NC, Jones JB. Characterization of Xanthomonas campestris strains from aroids using physiological, pathological, and fatty acid analysis. Phytopathology 1992;82:754–9. [25] KJijn N, Weerkamp AH, deVos WM. Identification of mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16s rRNA and specific DNA probes. Appl Environ Microbiol 1991;57:3390–3.
13
Technical Glitches in Micropropagation Saurabh Bhatia, Kiran Sharma O U T L I N E 13.1 Introduction
393
13.8 Shoot-tip Necrosis
400
13.2 Microbial Contamination
396
13.9 Habituation
400
13.3 Gaseous Contamination
397
13.4 Cross-culture Contamination
398
13.5 Browning of the Medium or Phenolic Exudation
398
13.6 Absence of Rooting and Acclimatization
399
13.7 Hyperhydricity 13.7.1 The Main Causes of Hyperhydricity in Plant Tissue Culture 13.7.2 Control of Hyperhydricity
399
13.10 Tissue Proliferation 13.10.1 Genetic or Chimeral Variation or Somaclonal Variation 13.10.2 Transient Phenotypic Variation 13.10.3 Epigenetic Variation
401
13.11 Detection of the Contaminants
402
References
403
401 402 402
399 400
13.1 INTRODUCTION Contamination is defined as the accidental introduction of undesirable bacterial, fungal, and algal microorganisms and vectors or sources of pathogens into a culture medium. There are various sources of contaminants. Various sterilization procedures and aseptic techniques used for eliminating the contamination problems are highlighted in Fig. 13.1 and Table 13.1. A high concentration of sugar in plant tissue culture media supports the growth of microorganisms Modern Applications of Plant Biotechnology in Pharmaceutical Sciences. http://dx.doi.org/10.1016/B978-0-12-802221-4.00013-3 Copyright © 2015 Elsevier Inc. All rights reserved.
393
394
13. Technical Glitches in Micropropagation
FIGURE 13.1 Protocols to achieve sterile culture.
like bacteria and fungi. Explant is known to be the major source of contamination. Contaminants that are associated with explant are often more epiphytic than endophytic [1]. Various sterilization protocols are utilized to remove several types of contaminants (Fig. 13.2). When plant is in its native state, the growth of microorganisms is suppressed by the defense mechanism and dry environment. But when plant is brought into in vitro conditions its molecular pathways, which are essential for the production of optimum amounts of secondary metabolites, are disturbed. In addition, plant cell/tissue/organ is exposed to high levels of moist conditions that may favor the growth of microorganisms in the culture medium. These
395
13.1 Introduction
TABLE 13.1 Different Types of Sterilization Strategies Used in Plant Tissue Culture Type of sterilization
Features
Specifications
Sterilization by heat
Heat is the most widely used lethal agent for sterilization.
Dry heat (oven)
170˚C for 120 minutes.
Moist heat (autoclave)
121˚C at 15 lb for 15–20 min.
Sterilization by filtration
A liquid medium or solution that contains microorganisms can be sterilized by passing through a filter.
Filters may have different sizes and pores.
0.2 mm (pore size)
Tyndallization
Fractionated discontinuous method of sterilization.
Media is heated in water bath for 1 h for 3 consecutive days and kept at normal room temperature for two successive instances of boiling.
At 100˚C for 15 min/day up to 3 consecutive days.
Air sterilization
Laminar air flow cabinet equipped with high efficiency particulate air filters (HEPA), blower is used to blow the air through the HEPA filters
A manometer is fitted with the instrument to check the air pressure. Pressure more than 13 bars can choke the filter UV light provides additional sterilization.
HEPA prevents all sorts of microorganisms larger than 0.3 mm with 99% efficiency.
Surface sterilization of the explants
Mercuric chloride Bromine water Sodium hypochlorite Calcium hypochlorite Silver nitrate Hydrogen peroxide Bromine water Antibiotics: tetracycline, ciprofloxacin Ethanol
Sodium hypochlorite can be used for any material.
Time of treatment (5–50 min).
Mercuric chloride is most toxic material. Combinations of antibiotics are beneficial.
microorganisms when inside the medium grow faster than plant cells and multiply up to several fold by depleting carbohydrate sources and producing phytotoxic fermentation products such as ethanol and acetic acid. Plant cells in this starvation state become stressed and produce high levels of secondary metabolites, which are toxic for plant tissue. In addition to microbial contamination there are various other contaminations caused during micropropagation, e.g., chemical, radiation, and gas contamination. However, the frequency of these contaminations is considerably lower than microbial contamination. Browning of the medium or phenolic exudation in response to biotic and abiotic stresses is again a major constraint as this may lead to oxidation of leached phenolic contents causing darkening or browning of the media, which may further block the uptake of nutrients, ultimately leading to death of the explants. Similarly
396
13. Technical Glitches in Micropropagation
FIGURE 13.2 Protocols to sterilize the explants.
absence of rooting and acclimatization, hyperhydricity, shoot-tip necrosis, habituation, and tissue proliferation also suppress the growth of the tissue under in vitro conditions. This chapter describes various types of contamination sources and the alternative strategies to produce a contamination-free environment.
13.2 MICROBIAL CONTAMINATION Microbial contamination is a big problem in plant tissue culture practices. Bacteria, fungi, molds, and yeast are common contaminating microorganisms found in plant tissue culture practices. Microorganisms and their reproductive structures are ubiquitous although their relative abundance may vary considerably with environment and season. Microorganisms are found inside and on the surface of the plants. Microbial contamination of plant tissue culture is due to the high nutrient availability in the almost universally used culture medium. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria [2]. The intracellular pathogenic
13.3 Gaseous contamination
397
bacteria and viruses/viroids may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening of the stock plants with disease and endophyte elimination; pathogen and contaminant screening of established initial cultures; and observation, random sampling, and culture screening for microorganisms in multiplication and stored cultures. The increasing accessibility of both broad-spectrum and specific molecular diagnostics has resulted in advances in multiple pathogen and latent contaminant detection. The hazard analysis critical control point management strategy for tissue culture laboratories is underpinned by staff training in aseptic techniques and good laboratory practices. The ideal procedure for preventing bacterial growth includes the following steps: • • • •
Indexing explants and cultures for contaminants Identifying the source of those contaminants Identifying or characterizing the contaminants Eliminating the contaminating organism with improved cultural practices such as antibiotics (aminoglycosides, quinolones, b-lactams, glycopeptides, polymyxins, macrolides, and lincosamides) [3] or other chemical agent treatment
Bacterial contaminants may affect in vitro plant growth negatively, positively, or not at all. Bacterial growth may be suppressed in plant media by high salts, high sucrose, pH, temperature, tannins in explant sap, and appropriate nutrients not present in tissue culture media, e.g., pseudomonads, xanthomonads, and corynebacteria. In plants, bacteria may be endophytes, which can become pathogenic in vitro (in vitro pathogens = “vitropaths”), and in vivo pathogens, which can become saprophytes in vitro. Bacteria are the most frequent contaminants. They are usually introduced with the explants and may survive even after surface sterilization of the explants because they are in the interior tissues. Bacteria can be recognized by a characteristic ooze. This ooze can be of many colors including white, pink, creamish, and yellow. Fungi may enter cultures on explants or the spore may be airborne. Fungi may be recognized by their fuzzy appearance and they occur in a multitude of colors. Yeast is a common contaminant of plant cultures. Yeast lives on the external surfaces of the plants as well as in the air. Viruses, mycoplasma-like organisms, spiroplasms, and rickettsias are not easily detected but they do form a source of serious contamination in cultures. Several insects are particularly troublesome in plant cultures including ants, thripes, and mites.
13.3 GASEOUS CONTAMINATION Apart from the various contaminations caused by microorganisms, gaseous contamination also influences the growth of the culture. Culture ventilation is necessary to facilitate the gaseous exchange with the atmosphere and to allow ethylene to escape from the vessels. This problem can be overcome by using gas permeable filters as lid seals or by replacing the lid or wrapping the open-lidded vessel in gas permeable plastic film [4,5]. There is a wide range of transparent plastic wrapping materials that can be used instead of a conventional
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lid or the film can be wrapped around an open lidded vessel with the provision that the film be adequately permeable to oxygen, carbon dioxide, and ethylene to allow equilibration with the ambient atmosphere. If replacing the lid with a plastic film, the permeability of the film to water vapor should be chosen such that the cultures do not dry out [4,5].
13.4 CROSS-CULTURE CONTAMINATION This generally occurs when a different cell type is inadvertently introduced into the cells that are being cultured. This is a significant problem because the new type of cell could have different morphology and function and react differently to any experimental conditions that are applied. Cross-contamination voids the experiment because results cannot be valid or credible, as the effect of the contaminating cells is unknown.
13.5 BROWNING OF THE MEDIUM OR PHENOLIC EXUDATION Secondary metabolites like phenolic compounds in the plants are produced in response to biotic and abiotic stresses. These are basically involved in the defense mechanism of plants. However, leaching of phenolics contents from the explants of most woody and some herbaceous plants often produce lethal effects for the growing cells or tissues. This problem appears in tissue culture when explants are excised during the preparation of the cultures, which leads to stimulation of phenolic exudation. Oxidation of these exuded phenolic contents causes darkening or browning of the media, which blocks the uptake of nutrients and ultimately leads to death of the explants. Browning did not affect the growth of roots and shoots when explants were cultured in a large volume of medium, but in a small volume it was lethal. Various absorbents and antioxidants can be used to minimize this exudation problem. Alternatives used to minimize this type of problem are: • Frequent subculturing of explants. • Use of antioxidant compounds like ascorbic acid, butylated hydroxyl anisole, or butylated hydroxyl toluene; citric acid may prevent the oxidation of phenolic compounds. • Use of adsorbents (like activated charcoal or PVP) to adsorb polyphenols secreted in the medium. • Culturing in the dark to prevent polyphenolic oxidation as light enhances it. • Soaking in water after excision to reduce the browning effect. • After excision, when the explants are being transferred to nutrient media, the positioning of explants on medium have a considerable effect, e.g., some woody herbaceous plants produce more shoots when placed horizontally instead of vertically on the nutrient medium. • A brief period of culture in liquid medium (3–7 days) to remove phenolics and other substances. • Control of phenolic oxidation by the supplementation of certain compounds to the medium, e.g., adenine sulfate and 300 mg L–1 polyvinylpyrrolidone (PVP). • Sealing the cut ends with paraffin wax to control browning by preventing exudation [6].
13.7 Hyperhydricity
399
13.6 ABSENCE OF ROOTING AND ACCLIMATIZATION Rooting is a critical phase since it depends on the genotype and physiological condition at the time of root induction [7]. This stage is followed by a process called acclimatization and is essential because the in vitro plants are cultivated under heterotrophic conditions [8]. Morphological anomalies, such as nonfunctional stomata, and physiological anomalies, such as a decrease in photosynthesis, might occur during this process [9]. These anomalies interfere with plant survival in the greenhouse or field. Therefore, acclimatization is critical for the transition of micropropagated plants from in vitro cultivation to ex vitro cultivation. The explants can naturally form roots during propagation without the additional rooting stage as with the potato but some species may show root production deficiency. Rooting may be induced by incorporating auxins and carbon in the culture medium. Extent of proper rooting varies species to species. To overcome this, the following critical growth regulators are added: 1-naphthalene acetic acid, indole-3-butyric acid, picloram, 2,4-dichlorophenoxyacetic acid (2,4-D), etc. These in vitro plants are sensitive to the external environment, so maintenance of relatively high humidity is required. Due to the high sensitivity of in vitro plants they should never be transplanted directly to open fields. Acclimatization is a key step to successful production. Tissue cultured plants may develop water stress upon transfer out of the test tube. Plants that do not acclimatize to the greenhouse die quickly. Leaves wilt, shrivel, and fall off. Root tips may turn brown and shrivel. The complete plant may turn brown after several days. Water stress is caused by many anatomical and physiological reasons. The main reason for short-term water loss is the lack of stomatal closure in unacclimatized tissue cultured plants. No worry methods, mist bench/ polytent method, humidification chamber method, are the best solution for acclimatization.
13.7 HYPERHYDRICITY In general terms, swelling/thickening of the tissue, just like callus, is called vitrification or hyperhydricity. In many species, vitrification may be represented by symptoms not visible to the naked eye. It is a physiological malformation that results in excessive hydration, low lignification, poorly developed vascular bundles, impaired stomatal function, and reduced mechanical strength of tissue culture-generated plants [10]. In vitrification, tissue becomes water soaked and translucent, which is mainly caused by excessive water uptake. It is usually controlled by changing agar concentration or source. The consequence is poor regeneration of such plants without intensive greenhouse acclimation for outdoor growth. Additionally, it may also lead to leaf-tip and bud necrosis in some cases, which often leads to loss of apical dominance in the shoots. In general, the main symptoms of hyperhydricity are translucent characteristics signified by a shortage of chlorophyll and high water content [10]. Specifically, the presence of a thin or lack of a cuticular layer, reduced number of palisade cells, irregular stomata, less developed cell wall, and large intracellular spaces in the mesophyll cell layer have been described as some of the anatomic changes associated with hyperhydricity.
13.7.1 The Main Causes of Hyperhydricity in Plant Tissue Culture • Oxidative stresses as a result of high salt concentration • The type of explants utilized
400 • • • • • • • • •
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The concentrations of microelement and hormonal imbalances Low light intensity High relative humidity Gas accumulation in the atmosphere of the jar Length of time intervals between subcultures Number of subcultures Concentration and type of gelling agent High ammonium concentration Evident in liquid culture-grown plants or when there is a low concentration of gelling agent
13.7.2 Control of Hyperhydricity • Monitoring the modified atmosphere of the culture vessels. • Adjusting the relative humidity in the vessel. • Use of gas-permeable membranes to increase exchange of water vapor and other gases such as ethylene with the surrounding environment. • Use of higher concentration of a gelling agent to reduce the risk of hyperhydricity. • Addition of agar hydrolysate. • Use of growth retardants and osmotic agents. • Use of bottom cooling, which allows water to condense on the medium. • Use of cytokinin-meta-topolin (6-(3-hydroxybenzylamino)purine). • Combination of lower cytokinin content and ammonium nitrate in the media. • Use of nitrate or glutamine as the sole nitrogen source. • Decreasing the ratio of ammonium: nitrate in the medium.
13.8 SHOOT-TIP NECROSIS Shoot-tip necrosis can be a major obstruction in the successful propagation of certain species by tissue culture. The symptoms of shoot-tip necrosis are browning and die-back of buds and the youngest leaves [11]. Shoot-tip necrosis occurs in some woody perennial tissue cultures when actively growing shoot tips develop tip die-back. This condition is usually caused by a calcium deficiency in the medium. The first assumption in seeing shoot-tip necrosis is that it is caused by nutrient deficiency [11]. The symptoms of nutrient deficiency of less mobile elements such as calcium (Ca) and boron (B) first appear in the meristematic regions and young leaves whereas symptoms of excessive amounts of these minerals are first observed on the older leaves. However, in in vitro systems, shoot-tip necrosis is caused by a complex set of factors rather than just nutrient deficiency [11].
13.9 HABITUATION Plant tissue culture usually requires sources of the growth hormones auxin and cytokinin for continuous proliferation in culture. In 1942, Gautheret reported that carrot tissue can gradually lose its requirement for exogenous auxin. He called this phenomenon auxin habituation.
13.10 Tissue proliferation
401
It was soon recognized that similar variation can occur for cytokinins and more rarely for some other hormones. Thus, habituation is when a culture continues to develop in the absence of auxin or cytokinin. For example, shoot cultures habituated for cytokinin would continue to produce new shoots on a cytokinin-free medium. Hormone habituation is a phenomenon by which plant cells and tissues lose the requirement of exogenous hormones to sustain cell division and development upon continuous culture [12].
13.10 TISSUE PROLIFERATION Tissue proliferation may be the expression of naturally occurring growths in plants like Rhododendron induced by the tissue culture environment. Variation in micropropagated plants is one of the greatest problems faced by tissue culture during micropropagation. There are many types of variation that cause several problems during tissue proliferation, which can further create a great problem in defining the genotype of new variants [13].
13.10.1 Genetic or Chimeral Variation or Somaclonal Variation Somaclonal variation is the variation seen in plants that have been produced by plant tissue culture. Chromosomal rearrangements are an important source of this variation. Somaclonal variation is not restricted to, but is particularly common in, plants regenerated from callus. The variations can be genotypic or phenotypic, which in the latter case can be either genetic or epigenetic in origin. Typical genetic alterations are: changes in chromosome numbers (polyploidy and aneuploidy), chromosome structure (translocations, deletions, insertions, and duplications), and DNA sequence (base mutations) [13]. Typical epigenetic-related events are gene amplification and gene methylation. If no visual, morphogenic changes are apparent, other plant screening procedures must be applied. There are both benefits and disadvantages to somaclonal variation. The phenomenon of high variability in individuals from plant cell cultures or adventitious shoots is called somaclonal variation [13]. Therefore, it can be defined as the variation that occurs because of genetic mutation caused by in vitro conditions or by chimeral separation. Somaclonal variation is usually undesirable. In some cases, somaclonal variation can lead to new cultivars (e.g., disease resistance, new leave pattern) that may have desirable ornamental characteristics or increased pest resistance. The occurrence of somaclonal variation can be reduced by: • • • •
Avoiding long-term cultures. Using axillary shoot induction systems where possible. Propagating chimeras by other clonal systems. It is well known that increasing numbers of subcultures increase the likelihood of somaclonal variation, so the number of subcultures in micropropagation protocols should be kept to a minimum. • Regularly reinitiating clones from new explants, which might reduce variability over time. • Avoiding 2,4-D in the culture medium, as this hormone is known to introduce variation.
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13.10.2 Transient Phenotypic Variation Plant populations show phenotypic diversity, which may be caused by genetic and epigenetic variation. It has recently been shown that new epigenetic variants are generated at a higher rate than genetic variants and several studies have shown that epigenetic variation can be influenced by the environment. Although the heritability of environmentally induced epigenetic traits has gained increasing interest in past years, it is still not clear whether and to what extent induced epigenetic changes have a role in ecology and evolution. Some reports on model and nonmodel species support the possibility of adaptive epigenetic alleles, indicating that epigenetic variants are subject to natural selection. However, most of these studies rely solely on phenotypic data and no information is available about the underlying mechanisms. Thus, the role of inherited epigenetic variation for plant adaptation is unclear and further investigations are required to gain insights into the significance of epigenetic variation for ecological and evolutionary processes.
13.10.3 Epigenetic Variation In general terms, epigenetics can be defined as the study of changes in gene expression or cellular phenotype, caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: επí-,over, above, outer) genetics. Some of these changes have been shown to be heritable. It refers to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence [13]. Examples of such modifications are DNA methylation and histone modification, both of which serve to regulate gene expression without altering the underlying DNA sequence. In tissue culture, new plants may be generated by outgrowth of axillary buds or by adventitious regeneration. Researchers expected initially that these clonally propagated plants would be exact copies of the parent plant, but frequently aberrant plants were observed. Various causes have been established: • Genetic changes (also referred to as somaclonal variation): changes in the DNA sequence. • Epigenetic variation: long-lasting changes in the expression of the information in the genome. • Chimeral segregation and loss of pathogens in particular viruses. Epigenetic changes are caused by changes in the expression of the information in the DNA brought about by alterations in DNA methylation, in histones, or in both [13]. These modifications may influence gene transcription. Epigenetic changes are often temporary and plants may revert to the normal phenotype relatively easily, but some can be long lasting and may even be transferred during sexual propagation.
13.11 DETECTION OF THE CONTAMINANTS Contaminants may be introduced with the explants or during manipulation in the laboratory. They may express themselves immediately or remain latent for long periods of time. Eyeball determination method (visual determination method), stock plant indexing (taking part of the plant and transferring to media that are specific for bacteria and fungi, e.g., potato
REFERENCES 403
TABLE 13.2 Identification and Characterization of Bacterial Contaminants Test
Procedure
Reference
Biochemical tests
Gram stain, motility, gelatinase, oxidase, and oxidation/ fermentation
[20]
Bergey’s Manual of Systematic Bacteriology
Descriptions of genera and species, which are helpful for identifying bacteria
[21]
Tetrazolium dye test
Reduction of tetrazolium dye in response to cellular respiration and results are compared with standard database
[22]
The API identification system
Carbon source utilization test: relies on visual detection of the test bacterium
[23]
Fatty acid analysis profiles test
Fatty acid methyl esters with those of known organisms
[24]
Probe test
DNA probes and 16S rRNA use PCR amplification and probes for known sequences
[25]
TRADITIONAL TESTS
CONTEMPORARY TESTS
dextrose agar and NB broth), semiselective media, and polymerase chain reaction (PCR)based molecular techniques are usually used for the detection of the contaminants. Hazard analysis critical control point systems are used in commercial plant tissue culture laboratories. Several detection strategies are highlighted in Fig. 13.1. Visual inspection of the medium may provide evidence of some contaminants but it is not adequate for slow growing bacteria, e.g., endophytes, or those bacteria that do not grow on plant tissue culture media [14]. There are various screening methods reported for the identification of many contaminants [14–17]. Some bacteria require specialized media and some contaminants can be easily detected by screening on two or three commercially available bacteriological media [18,19]. For indexing microbes, plant tissue can be inoculated into liquid and agar solidified yeast extract glucose, Sabouraud glucose, and AC media and incubated for 3 weeks at 30°C. This procedure is reported for indexing microbes in woody plant species. This can be further utilized by growing explants in a liquid culture system at pH 6.9. There might be chances of increase in expression of pathogens under greenhouse conditions. Therefore, such cultured batches should be separately placed to avoid multiplication of contaminants among healthy propagates. Various strategies for identification and characterization of bacterial contaminants are highlighted in Table 13.2.
References [1] Fogh Jørgen, Holmgren NB, Ludovici PP. A review of cell culture contaminants. In Vitro 1971;7(1):26–41. [2] Cassells AC. Problems in tissue culture: culture contamination. In: Debergh PC, Zimmerman RH, editors. Micropropagation technology and application. Dordrecht, Netherlands: Kluwer Academic Publishers; 1991. p. 31–44. [3] Falkiner FR. The criteria for choosing an antibiotic for control of bacteria in plant tissue culture. Newsletter International Association for Plant Tissue Culture 1990;60:13–23. [4] Marino G, Altan AD, Biavati B. The effect of bacterial contamination on the growth and gas evolution of in vitro cultured apricot shoots. In Vitro Plant 1996;32(1):51–6.
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[5] Odutayo OI, Amusa NA, Okutade OO, Ogunsanwo YR. Sources of microbial contamination in tissue culture laboratories in southwestern Nigeria. Afr J Agr Res 2007;2(3):067–72. [6] Bhat SR, Chandel KP. A novel technique to overcome browning in tissue culture. Plant Cell Rep 1991;10 (6-7):358–61. [7] Martins L, Pedrotti EL. Enraizamento in vitro e ex vitro dos porta-enxertos de macieira M7, M9 e Marubakaido. Rev Bras Frutic 2001;23(1):11–6. [8] Hazarika BN. Acclimatization of tissue-cultured plants. Curr Sci 2003;85(12):1704–12. [9] Rogalski M, Moraes LKA, Felisbino C, Crestani L, Guerra MP, Silva AL. Aclimatização de porta-enxertos de Prunus sp. micropropagados. Rev Bras Frutic 2003;25(2):279–81. [10] Kevers C, Franck T, Strasser RJ, Dommes J, Gaspar T. Hyperhydricity of micropropagated shoots: a typically stress-induced change of physiological state. Plant Cell Tiss Org Cult 2004;77(2):181–91. [11] Bairu MW, Stirk WA, Staden JV. Factors contributing to in vitro shoot-tip necrosis and their physiological interactions. Plant Cell Tiss Org Cult 2009;98(3):239–48. [12] Meins F. Habituation: heritable variation in the requirement of cultured plant cells for hormones. Annu Rev Genet 1989;23:395–408. [13] Neelakandan AK, Wang K. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. Plant Cell Rep 2012;31:597–620. [14] Kane ME. Indexing explants and cultures to maintain clean stock. In Vitro 1995;31:25A. [15] Reed BM, Buckley PM, DeWilde TN. Detection and eradication of endophytic bacteria from micropropagated mint plants. In Vitro Cell Dev Biol 1995;31:53–7. [16] Leifen C, Camotla H, Wailes WM. Effect of combinations of antibiotics on micro-propagated Clematis, Delphinium, Hosta, Iris, and Photinia. Plant Cell Tiss Org Cult 1992;29:153–60. [17] Holland MA, Polacco JC. PPFMs and or her covert contaminants: is there more to plant physiology than just plant? Annu Rev Plant Physiol Mol Biol 1994;45:197–209. [18] George KL, Falkinham JO. Selective medium for the isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum. Can J Microbiol 1986;32:10–4. [19] Gunson HE, Spencer-Phillips PTN. Latent bacterial infections: epiphytes and endophytes as contaminants of micropropagated plants. In: Nicholas JR, editor. Physiology, growth and development of plants in culture. Dordrecht, Netherlands: Kluwer Academic Publishers; 1994. p. 379–96. [20] Klement Z, Rudolph K, Sands DC, editors. Methods in phytobacteriology. Budapest: Akademiai Kiado; 1990. [21] Krieg NR, Holt JG, editors. Bergey’s manual of systematic bacteriology, Vol. I. Baltimore: Williams and Wilkens; 1984. [22] Jones JB, Chase AR, Harris GK. Evaluation of the Biolog GN MicroPlate system for identification of some plantpathogenic bacteria. Plant Dis 1993;77:553–8. [23] Leifert C, Waites WM, Nicholas JR. Bacterial contamination of micropropagated plant tissue cultures. J Appl Bact 1989;67:353–61. [24] Chase AR, Stall RE, Hodge NC, Jones JB. Characterization of Xanthomonas campestris strains from aroids using physiological, pathological, and fatty acid analysis. Phytopathology 1992;82:754–9. [25] KJijn N, Weerkamp AH, deVos WM. Identification of mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16s rRNA and specific DNA probes. Appl Environ Microbiol 1991;57:3390–3.