Abstract: Plant tissue culture is the finest method to propagate in large scale and to protect the rare, endangered and important plant species. It is in urgent to concentrate on conservation of rare, endangered medicinal and commercially important plant species. But the success rate of propagation in this method is attenuate in particular with a few medicinal plants. Also the researchers are facing lot of troubles with propagation of plant tissues and acclimatization of in vitro raised plants in the natural habitat. There are many reasons behind these problems. This review covered all that difficulties right from laboratory construction up to field adaptation of tissue cultured plants along with remedies to elucidate all the complications in this technique.
INTRODUCTION
To satisfy the global demand in the field of agriculture for producing more food crops and plant based medicine, it is in urgent to conserve the agricultural, economical, rare and endangered plants. They had high medicinal and ecological impact and should be propagated widely, even it was a failure in the conventional methods. Plant tissue culture is an important tool to propagate the plants in large scale through the eminent way in the short1. Culture of plant and various parts in the aseptic condition with the concept of totipotency2-4. A special media fortified with inorganic nutrients, vitamins, carbohydrates and environmental factors are added in vitro condition5. Cell totipotentiality and cellular plasticity is the major physiological principle behind the plant tissue culture. Cell plasticity responses for the division and differentiation capacity of the culture cells6. The ability of the single cell to transform into a whole plant alike as the mother plant7. The propagation method should be under the controlled environment, hence there is absence of seasonal effect. Though the plants are cultivating in the large scale, plants will be genetically uniform, true to type8. In plant tissue culture, plants are grown in the nutrient media, where the nutrients required for the plants are added and also had a space for microbes, because of having rich nutrients. Microbial growth in the media is due to improper sterilization and ill procedures. Contamination in tissue culture is not universal to find the exact reason but implies perfection in every step leads to reduce the contamination menace in plant tissue9. Contamination may be physiological and pathological and the contaminants in the culture are bacteria, fungi and yeast10. For a successful culture of plant tissue needs to provide aseptic condition, selection of plant tissue free from microbes, proper sterilization, appropriate nutrition in tissue culture media, taking right measures in the browning and somaclonal variation. Hardening in the green house and acclimatization to the field conditions is the final problem in the in vitro raised plants.
PROBLEMS AND ITS REMEDY
Selection of explants: Explant selection is the initial step in the plant tissue culture, any part of the plant can be selected as explants. It should be from the healthy mother plant, possibility for better growth was determined by position and age of the explants11. If the explants are collected from the green house condition is considered as the better one and the rate of survivable capacity will be higher. Survivability of the culture was increased, where the explants were collected in the dry spell of the year12. Shoot tip and internodes were found as the best source of explants for sterilization and better growth13.
Sterilization of media and culture vessel: The most important step in tissue and organ culture techniques is sterilization14. Contamination occurs through improper sterilization of glassware and vessels. It is controlled by chemical and physical methods, dry heat, wet heat, chemical and ultra filtration. Media should be autoclaved for 15 min at 121°C, which kills the microbes on the culture vessel15,16. Glassware’s which are not able to autoclave properly are sterilized with the help of sodium hypochlorite with 0.002% concentration and heat sterilized with hot air oven is better for the glassware sterilization17.
Explant sterilization: Surface sterilization of the explants is the essential step in the plant tissue culture18. If explants taken from the external environment was exposed to microbial contamination will leads to the mortality of the plant tissue19. Microbial contamination in the external part of the explants can be sterilized with the running water and chemical substances, includes ethanol, sodium hypochlorite, mercuric chloride and plant preservative mixtures etc. Ethanol is the strong sterilizing agent but it was phytotoxic to the plant cells. High concentration of ethanol and mercuric chloride affects the growth of the plant in the culture12. There is the evidence for plant preservative mixture is a best sterilizing agent for the culture of Cestrum nouturnum L.20. For the successful propagation of Andrographis paniculata, fungicide, bleaching powder, amoxicillin antibiotic, carbendazim and mercuric chloride were used in the surface sterilization. Mercuric chloride at the concentration of 0.1% solution treated for 2 min showed the best result in number of plant species. Calcium hypochlorite in the concentration of 5% for 20 min shows a better result in the propagation of Ziziphus Spina-christi (L.) Desf21. Mercuric chloride provides better result in the explants sterilization of Pinellia ternate (Thunb.) Breit22. Effective sterilization method may vary with the nature of the explants. Even if the explants taken from the different part of the same plant, their effective sterilizing agent was varied in the Salacia chinensis L.23.
Effect of ultra violet-c radiation in explants sterilization: The explants are allowed to ultraviolet-C radiation for 5 min and surface sterilization for about 10 min, acts as the better way to reduce the external contamination in Solanum tuberosum. The intensity in the UV-C radiation can deactivate the DNA in the pathogen24. But there may be a chance to damage the plant molecules especially with plant DNA.
Role of endophytic organism in contamination: Epiphytic microbes in the explants were removed by surface sterilization but the occurrence of the endophytic microbes in the explants leads to contamination in the culture25,26. The concentration of sterilizing the agents depends upon the plant explants may vary from one to another.
Medium preparation, nutrients and hormonal imbalance: Media is the place where the explants are inoculated to grow and the success of the tissue culture depends upon the selection of medium. Most commonly used medium is MS by Murashige and Skoog5. The right nutritional medium consists of inorganic salts, organic supplements, vitamins, growth regulators and carbon source27. Agar is used as a gelling agent in the culture medium28. Right pH is required for the medium and optimum pH of the nutrient medium is maintained properly (5.6-5.8). There may be a lack of uptake of nutrient from the medium when it is highly alkaline or acidic29. Plant growth hormones are naturally synthesized by the plant but there is need to add some external growth hormones for the better growth and to enrich the metabolite synthesis, particularly plant bioactive molecules30. Plant growth hormones are auxins, cytokinins and gibberellins. Some of the major auxins includes indole-3-acetic acid, indole-3-butyric acid, 2, 4 dichlorophenoxyacetic acid, naphthalene acetic acid, 2, 4, 5-trichlorophenoxyacetic acid and naphthoxyacetic acid, which helps in the cell division, elongation and root differentiation. Cytokinins like benzyl amino purine, isopentenyl adenine, kinetin, 4-hydroxyl-3-methyl-trans-2-butenylaminopurine plays a vital role in the shoot induction, development and proliferation. Gibberellins help in flowering and elongation29. Thiamine is considered as the essential vitamin in the medium. Glucose or sucrose is considered as the carbon source at the concentration of 2-4%. Liquid and semi solid medium is better for the successful propagation of plant species. There is a need of relative humidity in the medium31 about 70-75%. Humidity in the culture vessel also plays a vital role in the growth of the plant32.
Contamination: Contamination is the common problem in the in vitro culture. To determine the source of contamination is tedious. Endophytic microbial contamination was not possible to stop with the surface sterilization; still it is a big problem for number of plant species. It may cause severe problem to the culture especially the growing plant tissue33. Microbes which are resistant to surface sterilization and the endophytic microbes cause drastic problem to the culture34. Laboratory must be maintained in sterile condition with good ambience which is the root cause of causing contamination35. The data were presented and documented in the Table 1 and 2 for future reference which includes the common bacteria and fungi are involving in contaminating the culture of different plant tissues.
The occurrence of microbes in the culture varies with the plant species and the zone of geographical distribution49. Aseptic condition in the in vitro culture is the best way to eradicate contamination.
Construction of tissue culture laboratory: Even the construction and structural deformities of the laboratory can cause problems in the cultures with possible contamination. The well designed and planned laboratory with washing room, sterile room, growth culture room, store room and hardening area is quite mandatory for successful cultures. There should be a well-designed and proper construction of various rooms, which may have less chance of contamination. The wash room and sterile room are distant from each other, sterile and growth room has to be kept close to each other to reduce the various sources of contamination.
Role of antibiotics: Usage of antibiotics plays an essential role in the elimination and inhibition of microbial contamination50,51. By adding appropriate amount of antibiotics like Streptomycin, Tetracycline, Vancomycin, Rifampicin, Gentamicin, Cefotaxime in the medium may eradicate the contamination in the culture17,3,52,53. Benomyl and Streptomycin were added in the medium provides better culture of nodal explants54. In the in vitro cultivation of potato, Streptomycin is considered as the better antibiotic to reduce contamination and to enhance the growth of the plant tissue42. Use of nano-particles in the culture medium can reduce the contamination, like nano-silver and titanium dioxide were having anti-microbial activity55.
Culture types in plant tissue culture: Propagation in the culture media had different forms of callus cultures like cell suspension culture, protoplast culture, embryo culture, anther culture and pollen culture.
| Table 1: | Bacterial contamination in plant tissue culture |
| Table 2: | Fungal contamination in plant tissue culture |
Each type is preferred for various aspects in the culture of plants. Cell suspension culture is mainly used to enhance the quantity of plant secondary metabolites56. And the embryo culture is succeeded by efficient germplasm hybrids57.
Browning of explant: Oxidation of phenols within the tissue leads to browning of explants. It may found in the in vitro culture of woody plants. Browning reduces the cell division and explant regeneration capacity, may leads to the failure in the plant tissue culture. Use of absorbent and antioxidants is the finest way to reduce the browning in the in vitro propagation. There are some evidences recorded, ascorbic acid reduces the oxidized substrates and hence it prevents the browning efficiently58. Browning leads to darken the medium and incompatible for cell cultures. Potassium citrate is variably better antioxidant in the culture of Musa pardisiaca59. Rate of browning may vary and depends upon the season and also upon the presence of plant secondary metabolites. If the explants collected during the months December and March, the low level of browning was observed due to poor production of secondary metabolites60. The best way of removal of secondary products from the explants is to collect explants at earlier morning before sunrise (the plant do not start the photosynthesis before sunrise) and immerse the explants in the water immediately after collection as it is a best solvent to elucidate the secondary compound61.
As per my experience, one of the major problems associated with tissue culture is browning of explants due to oxidation of phenolic compounds released from the cut end of the explants by poly-phenoloxidases, peroxidases, as this led to cell death. Propensity to browning was anti-oxidant specific and the presence of an anti-oxidant greatly minimized the browning rate. To overcome phenolic exudation, CA, AC and PVP were investigated and activated charcoal found to be the best62. Standardization of surface sterilization is need for prevention of phenolic browning and microbial contaminations of the explants63.
Somaclonal variation: The genetic change may occur in the in vitro raised plants is termed as somaclonal variation64. The genotypic nature of the plant, growth regulators, frequency of sub culturing, proliferation rate of tissues are the reasons for somaclonal variations65. Factors that influence the genetic variability are mutations, natural selection and migration has rich genetic variations in in vitro cultures1. But for commercially producing plants like Casuarinas, it is not necessary to concentrate on somaclonal variations.
Contamination in sub-culture: Sub-culture is the process to multiply the plantlets in vitro. Aseptic conditions are essential to maintain and regulate the sub-culturing of plant species. After sub-culturing the cultured vessels are stored in better environment with proper identification and much needed aseptic conditions17.
Hardening and acclimatization: Hardening refers to transfer of the explants from in vitro to the green house. During primary hardening soil fumigants are used for sterility. Dimethyl disulfide, methyl bromide, chloropicrin are frequently used as fumigants17. Use of farm yard manure in the ratio of 1:4 provides, better results in the hardening66. Successful propagation of plants depends upon the field survival. So the process of plant tissue culture is based upon the varied plant species31,67. But the mangrove plant somewhat difficult to propagate by in vitro techniques due to its high content of phenolics68.
CONCLUSION
Widespread of medicinal and economically important plants have been recorded in India as well as in the World, though some plant species were still in critical condition. Conservation of plants is the main aim in the plant tissue culture. This paper is widely focused on the problems that occur during plant tissue culture. Also an amicable solution was made by enabling the previous records along with my fifteen years’ of exposure and experience in the same field have documented. Utmost care in every step may lead to success in the field of plant tissue culture and conservation of valuable and rare plant population.