A REVIEW ON COST EFFECTIVE APPROACHES IN DIFFERENT STAGES OF COMMERCIAL MICROPROPAGATION TECHNOLOGY
Shirish Patidar
Assistant Professor, School of Agriculture, Renaissance University, Indore (M.P)
Abstract - Micropropagation is an artificial technique for fast multiplication of plants in a very short period using the tissue or cell culture techniques during a controlled atmosphere. Initially, the technique of micropropagation for large-scale production of plants was utilized primarily to decorative plants only however currently it's been extended to regarding a thousand plant species as well as numerous vegetable and fruit crops like potato, strawberry, oil palm, banana, etc., meditative and aromatic plants and trees The need for affordable plant tissue culture systems, applicable for micropropagation and in vitro conservation of plant genetic resources, has been stressed emphasized the large-scale application of such technology in developing countries. The most important advantage of micropropagation is that the very high multiplication rates. Therefore, themajor disadvantage of this method is that the comparatively higher cost concerned as compared to different plant propagation ways. The main objective of the review is to create awareness among the utilization of cost-efficient alternates in different stages of micropropagation technology utilizing the community to create the technologies easy.
Keywords: Micropropagation, large-scale production, cost effective, In Vitro, Alternatives.
1 INTRODUCTION
Micropropagation is a artificial technique for fast multiplication of plants in a very short period using the tissue or cell culture techniques during a controlled atmosphere. The plant is propagated by both asexual (by vegetative propagation) and sexual (through seed) suggests that. Reproduction involves the fusion of gametes that result in the production of seeds with a high level of heterogeneousness. In micropropagation, plants are cloned. They are propagated either by asexual suggests that of replica or vegetative propagation, each ways in which involve the assembly of genetically identical plants by multiplying one individual underneath in vitro conditions. The natural propagation of plants is done once the aim is to grow plants in your vegetable garden. You can simply by cutting, grafting, or budding. However, if there are large-scale industrial desires, they need to produce plants by the micropropagation. Initially, the technique of micropropagation for large-scale production of plants was utilized primarily to decorative plants only (Ammirato et al., 1983), however currently it's been extended to regarding a thousand plant species as well as numerous vegetable and fruit crops like potato, strawberry, oil palm, banana, etc., meditative and aromatic plants and trees (Bajaj, 1986; Bajaj et al., 1988).
Micropropagation is the production of rapid multiplying stock material of relative plants, using plant tissue culture ways. Micropropagation could be a vegetative propagation of plant in the sterile conditions. Small amount of tissue (explants) used for micropropagation, that is not possible with typical technique, so the technique is extremely valuable explants is limited and we can produce disease-free plants by excluding disease- causing organisms throughout the propagation cycle. The most important advantage of micropropagation is that the very high multiplication rates. Therefore, this system is extremely fitted to speedy multiplication of rare genotypes, and of plants having rare genotypes. Some plants with terribly little seeds, together with most orchids and other medicinal plants. The major disadvantage of this method is that the comparatively higher cost concerned as compared to different plant propagation ways
The need for affordable plant tissue culture systems, applicable for micropropagation and in vitro conservation of plant genetic resources, has been stressed emphasized the large-scale application of such technology in developing countries. The utilization of chemicals like carbon sources, gelling agents, inorganic and organic supplements, and growth regulators in culture media, make this method costly. Sucrose is typically used as a supply of carbon and agar because the gelling agent, and along they constitute the foremost pricy components of the culture media. The amount of knowledge of cost-efficient propagation of plants is not common and has slowed down the domestication
of the many industrial plants. The inexpensive propagation of plants could be an answer for the domestication and conservation of native and vulnerable plant species.
Micropropagation usually involves the subsequent distinct stages: 0- pre- propagation step or selection and pre-treatment of suitable plants., I. Initiation of cultures, II. Shoot multiplication, III. Rooting of in vitro full-grown shoots, and IV. Acclimatization.
different plant tissue culturing components are, namely, nutrients/ media chemicals (plant growth hormones, vitamins, and minerals nutrients), plant materials, instrumentation (culture containers, autoclave, laminar flow, instruments used for micropropagation, pH meter, etc), and also the infrastructures (media preparation, inoculation, growth and hardening rooms) and all these factors are subjected to play vital roles in price reduction as noted by Ganapathi et al. (1995). The main objective of the review is to create awareness among the utilization of cost-efficient alternates in different stages of micropropagation technology utilizing the community to create the technologies easy.
2 ALTERNATIVE APPROACHES IN STAGE- 0
The pre-propagation stage (also known as stage 0) needs proper maintenance of the mother plants within the greenhouse under disease- and insect-free conditions with minimal dust.
Clean enclosed areas, glasshouses, plastic tunnels, and net-covered tunnels, offer prime quality explants supply plants with lowest infection. Collection of plant material for clonal propagation should be done when applicable pretreatment of the mother plants with fungicides and pesticides to reduce contamination in the in vitro cultures. This improves growth and multiplication rates of in vitro cultures. The explants, obtained from the green house found to be simple to surface sterilize than field fully grown plants in term of the key drawback of contamination, it reduces the maintenance cost (Sudipta et al., 2011)
3 ALTERNATIVE APPROACHES IN STAGE- I
This stage refers to the inoculation of the explants on sterile medium to initiate sterile culture. Initiation of explants is that the very initiative in micropropagation. a good clean explant, once established in an sterile condition, may be increased many times; therefore, explants initiation in an sterile condition should be considered a important step in micropropagation. More than usually, explants fail to establish and grow, not due to the shortage of an acceptable medium however because of contamination. The explants are transferred to in vitro atmosphere, free from microorganism contaminants. For the preparation of medium and sterilization method we need use some alternatives.
3.1 Low Cost Washing and Sterilizing Operations
The washing cost of containers can be reduced by washing them manually instead of using costly machines and then they can be dried in the sun. The autoclaves used for sterilization operations are very expensive so these can be replaced by pressure cookers by placing a wire mesh at their base (Ahloowalia et al., 2002) Contamination are not detected when media and equipments were sterilized using a pressure cooker. Costly aluminium foil is generally used for wrapping the instruments before sterilization which can be substituted by autoclavable stainless steel containers.
3.2 Low Cost Gelling Agent Alternatives
Agar was introduced as a gelling agent over one hundred years past and since then it has been extensively used as for microbic and plant structure culture media (Babbar and religion, 2006). it is helpful for the aim of culturing because of its stability, high clarity, nontoxic nature and resistance to its metabolism. in the recent past many attempts are created to seem for suitable substrata that would possibly replace agar in substance due to doubts about its inertness and non-toxic nature, concern of over-exploration of its sources and specially, the high cost of tissue culture grade agar (Babbar and jain, 1998: Babbar and jain, 2006). it is the most costly constituent of plant tissue culture media and it is reported that agar, that is sometimes added to increase media consistency contributes 70th of the media prices. in the recent past agarose (Johanson, 1988) alginates, gelrite, isubgol , xanthan gum, gum (Babbar et al., 2005) starch are used with affordable success
3.3 Low Cost Nutrient Alternatives
Plant tissue culture media contains macronutrients: nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S) for better growth. The important micronutrients for plant tissue growth include iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu) and molybdenum (Mo). Among all the micronutrients iron is usually the most critical of all the micronutrients.
Locally available fertilizers in appropriate concentrations can be used as low cost source of nutrients for tissue culture. The conventional sources of MS media can be replaced by mixed nutrients containing both macro and micronutrients. For the micropropagation of cassava, fully substituted media with commercially available nutrients (Hydro Agri’s fertilizer) was used resulting in reducing cost by 93.1% as compared to the traditional media (Santana et al., 2009). Different kinds of fertilizers at different concentrations were tried which resulted in cost reduction of 24.4 percent for the medium prepared (Escobar et al., 2005). The possibility of using locally available fertilizers as alternative nutrient sources for cassava micropropagation was evaluated (Ogero et al., 2012) and a low cost protocol for cassava tissue culture using Easygro vegetative fertilizer as an alternative source for conventional MS salts was also developed (Ogero et al., 2012).
The effect of using low cost macronutrient substitutes- Ammonium fertilizers, potassium fertilizers and epsom salt on in vitro regeneration of sweet potato has also been studied (Mvuria et al., 2014). It was found that the Epsom salt substituted media, performed better in regeneration in term of leaves and nodes formed compared to the conventional media, while in other substitutes significant differences were not detected. It was observed that the fertilizer based medium was able to stimulate root formation in potato in vitro micropropagation culture just as much as the MS medium and in terms of plant height, no significant difference was recorded (Bihnchang-Ngwa et al., 2018).
3.4 Alternatives to Carbon Sources
Sucrose is that the most commonly used carbon supply in the small propagation of plants.
Sucrose adds considerably to the media price. Household sugar and alternative sugar (sugar cubes) sources may be used to reduce the value of the medium (Thorpe et al., 2007) .Sucrose made from cane sugar and contains 99.98% sucrose and 0.01% reducing sugar.
house sugar (crystalline sugar) made from cane sweetener treated with SO2 (sulfitation) or co2 (carbonation) and contain 96-97% sucrose and 0.75-1% reducing sugar. Sugar cubes made up of grains of refined crystalline sugar, and contain 99.5% sucrose and 0.03%
reducing sugar. it is considered to be higher quality than crystalline sugar (Tyagi et al., 2007)
3.5 Alternatives of distilled water
Water is that the main element of all plant tissue culture media. Usually in tissue culture research, distilled or doubled distilled and de-ionized water is employed. distilled water made through electrical distillation is expensive. In some cases, different water sources may be used to lower the price of the medium. Distilled water was replaced by tap autoclaved water. If tap water is free from heavy metals and contaminants, it may be substituted for distilled water (Sharifi et al., 2010).
4 ALTERNATIVE APPROACHES IN STAGE- II
Stage- II is the propagation phase in that the explants are cultivated on the suitable media for multiplication of shoots. The first goal is to achieve propagation without losing the genetic stability. Repeated culture of axillary and adventitious shoots, cutting with nodes, somatic embryos and different organs from Stage- I lead to multiplication of propagules in large numbers. The propagules made at this stage can be additional used for multiplication by their repeated culture. Generally it is necessary to subculture the in vitro derived shoots onto totally different media for elongation. All the media or sterilization technique connected approaches will use same as stage-I; however Culture bottles were additionally replaced by jam jars (Gitonga et al., 2010)
5 ALTERNATIVE APPROACHES IN STAGE- III
The in vitro shoots obtained at Stage II are rooted to produce complete plants. If the proliferated material consists of bud-like structures (e.g. orchids) or clumps of shoots (banana, pineapple), they must be separated when rooting and not before. several plants (e.g. banana, pineapple, roses, potato, chrysanthemum, strawberry, mint, many grasses and lots of more) can be rooted on half-strength-MS (Murashige and Skoog, 1962) medium with none growth-regulators good sturdy well-rooted plants are essential for high survival throughout commutation and later transfer to soil. This stage is labor intensive and pricey.
For this stage we are able to use liquid media eliminates the necessity of agar. Suspension cultures while not gelling agents are normally used for culturing callus, cell clusters, buds and somatic embryos. Suspension systems permit larger contact between the explants and the medium. Moreover, agitation of such media reduces the diffusion gradient in the nutrient supply. The harmful metabolites exuding from the tissues are distributed effectively and also help for healthy root proliferation (Thorpe et al., 2007).
6 ALTERNATIVE APPROACHES IN STAGE- IV
At this stage, the in vitro micropropagated plants are weaned and hardened. This can be the final stage of the tissue culture operation after which the micropropagated plantlets are prepared for transfer to the greenhouse. Steps are taken to grow individual plantlets capable of completing photosynthesis. The hardening of the tissue-cultured plantlets is completed step by step from high to low humidity and from low light intensity to high intensity conditions. If grown on solid medium, most of the agar can be removed gently by removal with water. Plants is completed left in shade for 3 to 6 days wherever diffused natural light conditions them to the new atmosphere. The plants are then transferred to an acceptable substrate (sand, peat, compost, etc.), and step by step hardened. Affordable choices embody the employment of plastic domes or tunnels, which reduces the natural light intensity and maintains high ratio throughout the hardening method. If the plants are still joined together after maturation, these should be planted as bunches in the soil and separated once 6 to 8 weeks of growth. In alternative approach Plantlets were successfully acclimatized using rice husks then transplanted into the potted soil within the shade net 83% of the plants survived during the acclimation procedure when rice husks were used compared to 80th using standard approach (use of vermiculite). Rice husks that are available and free of charge in the rice growing areas therefore, be used as an alternate resource throughout acclimatization to reduce price
7 CONCLUSIONS
The scientific survey has recommended that, in vitro propagation of plant will develop at low price relatively modern technology. In this review, we have tried to present the outstanding and the recent findings on the inexpensive strategies used in the various stages of micropropagation.
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