Tissue culture -Techniques, Applications

Tissue culture

1. Semester V Horticulture, Economic botany, Ethnobotany Tissue culture

2. Plant tissue culture Father of plant tissue culture “Gottlieb Haberlandt ” Historical Background : Gottlieb Haberlandt (1902): Proposed the concept of totipotency . White (1934) : Developed the first synthetic culture medium. Skoog and Miller (1957) : Discovered plant growth regulators.

3. Plant tissue culture Explants : piece of tissue or organ, excised from a plant for culturing in a suitable medium Nutrient medium/culture medium : nutrient mixture in which explants are cultured Calluses : cells undergo division and form masses of actively dividing and undifferentiated parenchymatous cells Embryoids : Diploid somatic cells of calluses differentiation and form non-zygotic embryos Plantlets : Embryoids develop to miniature plants

4. Plant tissue culture – principles Principles of Plant Tissue Culture Totipotency : The ability of a single plant cell to regenerate into a whole plant. Aseptic Conditions : Maintenance of a sterile environment to prevent microbial contamination. Nutrient Media : Formulation of media with essential nutrients, vitamins, and growth regulators. Environmental Control : Regulation of light, temperature, and humidity to optimize growth. Plasticity * Endurance and adaptability of plants in response to change in environmental conditions * enables the subsequent regeneration of the whole plant

5. Totipotency Ability of a single plant cell to develop into an entire plant * Totipotency is the capacity of a single cell to regenerate into a whole organism. In plants, this means that a single cell can give rise to all the different cell types needed to form a complete plant. * All cells not totipotent eg . Tracheids , fibres , sclereids vessel elements bcoz they lack cytoplasm and nucleus Significance * used for genetic modification of plants, production of homozygous diploid plants, somatic hybridization, mutation

6. Types of Plant Tissue Cultures Callus Culture : Growth of undifferentiated cells on a solid medium. Organ Culture : Culture of specific plant organs like roots, shoots, or leaves. Cell Suspension Culture : Culture of plant cells in a liquid medium. Protoplast Culture : Culture of cells without cell walls. Embryo Culture : Culture of isolated embryos to bypass seed dormancy.

7. Plant tissue culture Steps in Plant Tissue Culture Selection and Preparation of Explant : Source : Leaf, stem, root, meristem , or other plant parts. Sterilization : Use of disinfectants like ethanol, sodium hypochlorite. Culture Media Preparation : Basic Components : Macronutrients (N, P, K), micronutrients (Fe, Mn , Zn), vitamins, carbon source (sucrose), agar (for solid media). Growth Regulators : Auxins (e.g., IAA, NAA), cytokinins (e.g., BAP, kinetin), gibberellins. Inoculation : Placement of the sterilized explant on/in the culture medium under aseptic conditions. Incubation : Maintaining cultures in a controlled environment with specific light, temperature, and humidity conditions. Subculturing : Transferring growing cultures to fresh media Regeneration : Inducing shoot and root formation from callus or explants Acclimatization : Gradual adaptation of regenerated plants to external conditions before transferring them to soil.

8. Techniques in Plant Tissue Culture Micropropagation : Rapid multiplication of plants through tissue culture. Stages: Initiation Stage : Establishing aseptic culture from the explant . Multiplication Stage : Repeated subculturing to produce a large number of shoots. Rooting Stage : Inducing root formation in shoots. Acclimatization Stage : Gradual transition of plants to the external environment. Embryo Rescue : Culture of embryos to prevent abortion in hybrids or seedless plants. Somatic Embryogenesis : Development of embryos from somatic or non-reproductive cells. Synthetic Seeds : Encapsulation of somatic embryos or other tissues in a gel for sowing like true seeds. Haploid Production : Production of haploid plants through anther or microspore culture for breeding purposes. Protoplast Fusion : Fusion of protoplasts from different species to create hybrids.

9. Cellular totipotency

10. Cellular totipotency Cellular Totipotency  is the ability of a single  cell  to produce all  cell  types and to organise them into an entire organism when cultured in a suitable culture medium at appropriate temperature and aeration conditions. Spores and Zygote are examples of  totipotent   cells . Demonstrated for the first time by Steward et al (1957) of Cornell University, USA. Explants from phloem of carrot roots placed in coconut milk were used by them. .

11. The method of totipotency is used in : (a) Multiplication of rare plants which reproduce through seeds with great difficulty. (b) Induction and selection of mutants. (c) Rapid multiplication of desired plants. (d) Multiplication of sterile hybrids. (e) Production of virus-free plants. (f) To develop embryos which fail to reach maturity. (g) Shorten the period for development of new varieties. (h) Development of resistance to chemicals like weedicides Cellular totipotency Contn .

12. Factors affecting totipotency Internal factors Genetic Factors : The presence of a complete and undamaged genome and the activation of specific genes. Epigenetic Modifications : DNA methylation , histone modification, and non-coding RNAs that regulate gene expression. Cytoplasmic Factors : Cytoplasmic determinants and proper organelle function. External Factors Plant Growth Regulators : Auxins (cell division), cytokinins (shoot formation), gibberellins (cell elongation), abscisic acid (stress responses), and ethylene (cell differentiation). Nutrient Media : Macronutrients (N, P, K), micronutrients (Fe, Mn , Zn), vitamins, and a carbon source (sucrose). Physical Environment : Optimal light, temperature, humidity, and gas exchange conditions. Sterility and Contamination Control : Aseptic techniques and sterilization to prevent microbial contamination.

13. differentiation

14. In vitro differentiation Plant cells are formed from shoot apex, root apex, and cambium meristems by a process known as differentiation , in which cells are differentiated into distinct structures to fulfill diverse roles in the plant body. During this process, the plant cell wall and protoplasm undergo significant structural modifications. Differentiation occurs in the xylem of vascular plants’ tracheary components. The cells lose their protoplasm, and the cellulose cell walls lignify into secondary cell walls, increasing their flexibility and allowing them to survive high pressure conditions during water transport over longer distances.

15. Dedifferentiation Reversal of mature cell to meristematic cell during callus formation Dedifferentiation of completely differentiated parenchyma cells leads to the production of cork cambium and interfascicular cambium. A dedifferentiated tissue has the potential to function as a meristem , giving rise to a new set of cells. The ability of those cells to differentiate further is influenced by a variety of factors, including genetic and epigenetic differences. This notion is utilised to create a callus in plant tissue culture.

16. Redifferentiation The transformation of undifferentiated callus cells to fully differentiated plant cell Direct regeneration : Indirect regeneration : The cells lose their ability to divide and differentiate once new cells are produced from the dedifferentiated tissues that act as meristems . They eventually mature in order to perform various roles within the plant body. The greatest examples of secondary xylem and secondary phloem to describe the redifferentiation process are secondary xylem and secondary phloem. The secondary xylem on the inside and secondary phloem on the exterior emerge from the dedifferentiated vascular cambium. The secondary phloem and secondary xylem cells lose their capacity to divide and mature into mature cells that perform different activities in the plant body, such as food and water transportation.

17. De-differentiation and re-differentiation Similarities Both processes occur in differentiated cells . Both systems are also important in the genesis and healing of damage. Difference Dedifferentiation is the process by which structures or behaviours that are specialised for a specific function lose that specialisation and become simplified or generalised , whereas Redifferentiation is the process by which a group of previously differentiated cells returns to their original specialised form . 

18. Tissue culture medium

19. Tissue culture medium Liquid or solid preparations of nutrients in which tissues can be cultured Artificial/ synthetic medium : A medium with chemically supplemented constituents eg . MS medium (universally accepted) Murashige and skoog 1962 , B5 medium, White’s medium * Natural medium : Medium contain only natural compounds Basic components in tissue culture medium * Nutrients Macro nutrients (N, P, K, Ca, Mn and S) Micronutrients (Bo, Mo, Cu, Zn, Mg, Fe and chloride) *carbon sources eg . Sucrose, glucose, malt extract, yeast extract *organic supplements *Growth regulators & Hormones eg . Auxin (IAA, NAA, 2, 4-D), cytokinin eg . Adenin , kinetin, zeatin Gibberellic acid

20. Tissue culture medium Contn . Cytokinin : induce cell division, cell differentiation, regulate growth Auxin : promote cell division, callus formation, root differentiation, shoot elongation Gibberellins : promote callus growth and elongation of dwarf plants High auxin to cytokinin imp forcallus initiation, root initiation, embryogenesis Low auxin to cytokinin promote organogenesis and shoot proliferation Amino acid : stimulate cell growth eg . Asparagin , Aspartic acid, glycine glutamine * Solidifying agents eg . Agar ( Gracilaria , gelidum ) *growth factors, vitamins (Vitamin B1 Thiamine, B3 Pantothenic acid, B5 nicotinic acid, B2 Riboflavin, B6 pyridoxin , Ascorbic cid vit C, H biotin, B9 folic acid, B12 cyanocobalamin ) * pH = 5.8 above 7 or below 4.5 cell stop growing and dividing

21. Types of tissue culture media MS medium – Murashige and skoog (1962) B5 medium – Gamborg et al (1968) SH medium – Schenk and Hildebrandt (1972) Nitsch’s medium - Nitsch and Nitsch (1969) Solid and Liquid media Choice depend on purpose of culture/species/plant variety Solid medium : Addition of gelling agents; enhance viscosity of medium Semi-solid medium : close contact b/t explant tissue and medium (More preferrable ) Liquid medium : cost effective, time saving, ideal for mass propagation cell suspension culture and bioreactors Disadv : cause asphyxiation in explants due to immersion Soln : partially immersed allowing aeration

22. Murashige and Skoog medium

23. Preparation of medium For 1 L MS medium 10ml stock solution of Nitrate, sulphate , halide and Na Fe EDTA is used Appropriate amnt of vitamin stock added Sucrose 20-30g/L pH adjusted to 5.4-5.8 Vessel containing medium is autoclaved (15 min)at 120 C Agar is added and allow the medium to solidify Using stock Stock solutions are mixed made up to 1000ml pH adjusted to 5.5-6 Sterilize before the addition of growth regulators Keep culture tubes in dark and cool place to minimize degradation Medium is transferred to Laminar air flow

24. Aseptic techniques in in vitro culture Contamination consist of impurities, chemicals or pathogens. Make the cultured tissues unfit for further development of healthy plantlets Contaminants; bacteria and fungi Sterilization In tissue culture, sterilization refers to the process of eliminating all forms of microbial life, including bacteria, fungi, viruses, and spores, from the surfaces of explants, culture media, and equipment used in the tissue culture procedure. Different methods: 1) Dry heat technique 5) laminar air flow 2) Flame sterilization 6) Alcohol 3) Autoclaving 7) Surface sterilization 4) Filter sterilization

25. 1) Dry heat technique Sterilization of empty glasswares , teflon , plastic wares and instruments Oven at 160-180 ºC - 3 hrs Glass bead sterilizers – 300 ºC – metallic instrument 2) Flame sterilization * Sterilization of instruments (Forceps, scalpels, needles) : dipping in alcohol followed by flaming Frequently done 3) Autoclaving Culture vessel with or without medium Autoclave: 121 ºC at 15 psi for 15-40 min 4) Filter sterilization Sterilization of thermolabile growth regulators such as vitamins, GA3, ABA by passing the solution through a membrane filter pore size less than 0.4µ

26. 5) Laminar airflow sterilization Create aseptic working space By blowing filter sterilized air through an enclosed space open on one side Laminar air flow cabinet Ideal, convenient, reliable technique 6) Wiping with 70% alcohol * Sterilize the platform of laminar air flow cabinet, walls, ceilings, doors and windows of culture related rooms and hands 7) Surface sterilization Plant materials Sterilizing agent Eg : calcium hypochlorite(9-10%), sodium hypochlorite (2%), mercuric chloride (0.1-1%). Silver nitrate (1%), bromine water (1-2%), Hydrogen peroxide (10-12%), antibiotics, HgCL 2

27. Sterilization of laboratories, culture rooms and transfer areas Walls, doors, windows & floors Washing with lotion and detergents Spraying or wiping with desired disinfectant (20% sodium hypochlorite, 90% ethyl alcohol, lysol ) UV radiation Sterilization of culture vessels and instruments Hot air oven : 160º C to 180 º C -2-4 hrs Autoclave : 15-20 min 121 º C :15 psi Flame sterilization : Metalic instruments: 95% ethyl alcohol : flaming and cooling Glasswares : soaked in chromic acid solution-24hrs Sterilization of nutrient media Two common method: Autoclaving and filter sterilization Autoclaving and filter sterilization Sterilization of plant materials Running tap water & surface sterilization

28. Working principle of Laminar Air Flow Carefully enclosed bench It provides a sterile working environment by using a constant, unidirectional flow of filtered air. HEPA Filters : High-efficiency particulate air (HEPA) filter particles larger than 0.3µ Speed of air blow 1.8 km per hour – blower motor 10-150 min UV Lamp, Fluorescent lamp Unidirectional Air Flow Principle: a contamination-free environment by directing a continuous flow of filtered air over the work surface.

29. Principle of Autoclave An autoclave is a device that works on the principle of moist heat sterilisation , wherein saturated steam is generated under pressure in order to kill microorganisms such as bacteria, viruses, and even heat-resistant endospores from various types of instruments. This is done by heating the instruments within the device to temperatures surpassing the boiling point of water.

30. Working Principle The device works by the use of inwards flow of air through one or more HEPA filters to create a particulate-free environment. The air is taken through a filtration system and then exhausted across the work surface as a part of the laminar flow of the air. The air first passes through the filter pad or pre-filter that allows a streamline flow of air into the cabinet. Next, the blower or fan directs the air towards the HEPA filters. The HEPA filters then trap the bacteria, fungi and other particulate materials so that the air moving out of it is particulate-free air. Some of the effluent air then passes through perforation present at the bottom rear end of the cabinet, but most of it passes over the working bench while coming out of the cabinet towards the face of the operator. The laminar flow hood is enclosed on the sides, and constant positive air pressure is maintained to prevent the intrusion of contaminated external air into the cabinet.

31. Working principle of Autoclave pressurized steam The high temperature and pressure effectively kill all forms of microbial life, including spores, which are often resistant to other sterilization methods. Steam Penetration : The high-pressure steam penetrates materials and kills microorganisms by denaturing their proteins and disrupting cell membranes.

32. Preparation of explants Surface sterilization running tap water for 10-30 minutes few drops of a mild detergent 70% ethanol for 30 seconds to 1 minute sodium hypochlorite (commonly household bleach diluted to 10-20%). 10-20 minutes Hydrogen peroxide (3-6%) Mercuric chloride (0.1-0.2%) Rinse the explants thoroughly with sterile distilled water 3-4 times to remove any residual sterilant . (Crucial step)

33. Inoculation * Using sterile forceps, carefully place the explants onto the surface of the culture medium. Ensure that the explants make good contact with the medium for optimal nutrient absorption. For some applications, explants may be partially embedded in the medium to enhance stability and nutrient uptake. Talking and sneezing should be avoided Neck or mouth of culture container should be flamed Seal the culture vessels to prevent contamination while allowing gas exchange. Common methods include using parafilm , plastic wrap, or specific culture vessel lids. * Label each culture vessel with relevant information, such as the date, explant type, and experimental conditions.

34. Incubation Place the inoculated culture vessels in an appropriate growth environment, typically a growth chamber or incubator. Maintain optimal conditions for light, temperature, and humidity based on the specific requirements of the plant species and the desired growth outcome. Temperatures - 20-25°C. A photoperiod of 16 hours of light and 8 hours of darkness. Light intensity typically ranges from 30-50 µmol m⁻² s⁻¹. High humidity (70-80%) within the culture vessels is usually maintained to prevent desiccation of explants. Adequate gas exchange

35. Subculturing Transferring growing tissues or cells to fresh culture medium to maintain healthy growth and proliferation Transfer Process: Using sterile forceps and a scalpel, carefully excise the explants or tissues to be subcultured . Transfer the excised tissues to fresh culture medium, ensuring proper orientation and placement for optimal growth. Minimize the exposure of cultures and media to the air to reduce the risk of contamination. Frequency of Subculturing The frequency of subculturing depends on the growth rate of the culture and the type of tissue being cultured. Rapidly growing cultures may require subculturing every 2-4 weeks. Slower-growing cultures may be subcultured every 4-8 weeks. Keep detailed records of subculturing activities, including dates, medium composition, and any observations of growth and development.

36. Micropropagation Micropropagation is a technique used in plant tissue culture to rapidly produce large numbers of genetically identical plants. Clonal propagation Clone Five major or stages Stage 0 : Mother plant selection Stage 1: Establishment of Aseptic culture Stage 2: Multiplication of shoots Stage 3: In vitro rooting Stage 4: Transplantation or hardening

37. Phases of micropropagation

38. Stage 0 : Mother plant selection Preliminary step Choose healthy, disease-free plant material. Common explants include shoot tips, nodal segments, leaves, and meristems . Clean the explants thoroughly and perform surface sterilization to eliminate contaminants. Stage 1: Establishment of Aseptic culture Initial stage of micropropagation Initiation and establishment of aseptic cultures Four steps 1) Explant isolation - separation of explants from selected stock plant 2) Explant preparation - Surface sterilization using ethyl alcohol, mercuric chloride, sodium hypochlorite 3)Inoculation – introduction of sterilized explant to suitable culture medium 4) Establishment of cultures : maintain aseptic conditions, provide essential nutrients and growth factors

39. Medium: Usually, Murashige and Skoog (MS) medium or similar, supplemented with appropriate hormones (e.g., cytokinins for shoot induction). Aseptic Conditions: Conduct all operations in a laminar flow hood to maintain sterility. Two major problems : Microbial contamination Phenolic exudation Solutions : Sterilization of explants -Add charcoal & antioxidant chemicals ( polyphenol pyrrolidene & ascorbic acid

40. Stage 2 multiplication of shoots Longest phase of micropropagation and somatic embryogenesis 10-36 months ; large no. of sub-cultures Stage II repeated several times until sufficient branches and root sprouting are formed Involve shoot multiplication Four steps Callus mediated shoot proliferation Apical or axillary shoot proliferation Adventitious Shoot proliferation Direct somatic embryogenesis

41. 1) Callus mediated shoot proliferation Accomplished by callus culture Formation of callus tissue followed by the induction of shoots from this undifferentiated mass. Callus derived from protoplast, cell tissue, organs Auxin favour callus induction Maintained for several weeks by subculturing once in every four weeks The medium typically contains high levels of auxins (e.g., 2,4-Dichlorophenoxyacetic acid (2,4-D)) to promote callus formation. Maintain the cultures in a controlled environment with optimal temperature (around 25°C) and light conditions (usually in darkness or low light). Once a substantial callus mass is formed, transfer the callus to a shoot induction medium. This medium usually has a higher concentration of cytokinins (e.g., Benzylaminopurine (BAP)) and a lower concentration of auxins .

42. * Shoots begin to emerge from the callus tissue.Regularly subculture the proliferating shoots to fresh medium to sustain growth and increase shoot numbers. * Excise individual shoots and transfer them to a rooting medium.The rooting medium typically contains auxins like Indole-3-butyric acid (IBA) or Naphthaleneacetic acid (NAA). Advantages Enables the rapid multiplication of plants Disease Elimination Genetic uniformity Drawbacks Progresive decline and ultimate loss of regeneration potential of callus cells Callus may exhibits genetic variations and may result in morphological variations

43. 2) Apical bud Axillary bud proliferation The apical bud is the growing tip of a plant shoot where active cell division occurs. It is responsible for the vertical growth of the plant. Axillary buds are found in the leaf axils, the angle between the leaf and the stem. These buds can give rise to lateral shoots. Procedure: Choose healthy shoot tips or apical buds from the donor plant. Clean the explants thoroughly and surface sterilize Place the sterilized apical buds onto an appropriate culture medium The medium should contain cytokinins (e.g., benzylaminopurine (BAP)) to promote shoot proliferation. Subculture the newly formed shoots onto fresh medium at regular intervals to increase the number of shoots. Maintain optimal environmental conditions such as light, temperature, and humidity.

44. Axillary bud proliferation Achieved in three principle ways 1) Meristem culture 2) Shoot tip culture 3) Single node culture

45. 1) Meristem culture - micropropagation using meristem - apical dome with one/two leaf primordia - Meristem dome of totipotent cell - Explant 0.1-0.5mm from terminal bud - virus free plant

46. 2) Shoot tip culture Explant : shoot tip 7cm long Shoot apex and leaves V shaped cut stem Cytokinin Shoots are transferred to rooted plantlets

47. 3) Single node culture Meristem located in leaf axil It will not develop in vivo due to apical dominance Single nodes containing axillary bud are isolated from seedlings or mature plants Cultured in cytokinin rich medium Nodal explant - shoots-root-plant Mass propagation

48. 3) Adventitious Shoot proliferation Shoot proliferation is from induction and multiplication of adventitious meristems of explant through organogenesis or embryogenesis without callus mediated regeneration Induced from non- meristematic tissues, such as leaves, stems, or roots. They maintain genetic uniformity which is important for the production of ornamentals

49. 4) Direct somatic embryogenesis Direct somatic embryogenesis is a plant tissue culture technique where somatic (non-reproductive) cells develop directly into embryos without an intervening callus phase. First induced in suspension culture (Stewart et al., 1958) callus culture ( Reinert 1959) Embryoids : Somatic and non-zygotic embryo Three-step procedure - Induction of embryogenesis - Development of the embryo - maturation Somatic embryos have abnormal features with three or more bell-shaped cotyledons

50. Direct somatic embryogenesis - It involves the development of the embryos in a direct way from the cells of the explants, such as the cells of the immature embryos. - Here, there is no intermediary stage (like the formation of the callus). Indirect somatic embryogenesis - It includes the formation of somatic embryos by reiterating numerous cycles of cell divisions . - It includes intermediary steps of growth of the callus , and hence the process includes multiple steps. -The cells which do not carry the pre- embryogenic determined cells are caused to differentiate for the formation of the embryo by revealing different treatments. - The cells modify into IEDs (induced embryogenic pre-determined cells).

51. Stage 3: In vitro rooting Auxin Treatment : Explants are cultured on a medium with a higher concentration of auxins to initiate root formation. Environmental Conditions : Temperature, light, and humidity are carefully controlled. Typically, 24-28°C with a 16-hour photoperiod is optimal for many species. Root Development : Root Initiation : The formation of root primordia at the base of the explants. Root Elongation : After initiation, the roots begin to elongate and develop secondary roots. Transplantation to Rooting Medium : Transfer : Shoots with initiated roots are transferred to a rooting medium with lower or no auxin concentration to promote further root growth and hardening. Acclimatization Phase : Gradual reduction in humidity and adjustment to ambient light conditions to prepare for transfer to soil. Hardening and Acclimatization : Transfer to Soil or Potting Mix : After sufficient root development, plantlets are transferred to soil or a potting mix. Controlled Environment : Initial transfer to a greenhouse or a misting chamber to reduce transplant shock. Gradual Acclimatization : Gradual exposure to external conditions to enhance survival and growth .

52. Factors Affecting In Vitro Rooting Type of Auxin : Different species respond differently to various auxins . Commonly used auxins include Indole-3-Acetic Acid (IAA), Indole-3-Butyric Acid (IBA), and Naphthalene Acetic Acid (NAA). IBA is often preferred for its effectiveness in root induction with minimal negative effects on shoot growth. Concentration of Auxin : The concentration of auxin in the medium is critical. Too high a concentration can inhibit root growth, while too low a concentration may be ineffective. Culture Medium Composition : Besides hormones, the nutrient composition of the medium affects rooting. A balanced supply of macronutrients and micronutrients is essential. Environmental Conditions : Light : Generally, a 16-hour photoperiod is beneficial. Temperature : Optimal temperature ranges between 24-28°C. Humidity : High humidity is required initially, which is gradually reduced during acclimatization. Genotypic Variation : Different species and even different cultivars within a species can have varied rooting responses.

53. Stage 4: Transplantation or hardening Transfer the plants from a tissue culture environment to a greenhouse providing the same environment in which it was cultured in the lab. You need to transfer the plantlets to a potting mix, irrigated with inorganic nutrient solution. A variety of potting mixes are available nowadays including peat, vermiculite, soil, sand, etc. You can also keep the culture vessels with loose lids for a few days in the greenhouse. You can leave the plantlets in shade for 3-6 days under diffused natural light. This would help them to establish under new environment. According to scientists, acclimatization should occur in 2-3 phases where you gradually expose plantlets first to greenhouse and then to field conditions.

54. In order to adapt plants from high to low humidity, you need to keep them in shade with loose plugs for a week or two. Then you should transfer them to pots containing sterile soil and sand mixture. You can cover these pots with polybags . You can also precondition your rooted plantlets in different sucrose solutions (20- 30g/L of concentrations) before transferring to a potting mixture. This would increase the growth of shoots. In several studies, it is also suggested to add anti- transpirants like ' paclobutrazol ' in the rooting medium. This helps plantlets to have normal funtioning of stomata, thickened roots and also reduces wilting. You need to customize these steps for each plant variety based on the plant's growth requirements.

55. Advantages and Disadvantages of micropropagation

56. Advantages Small amount of source required Rapid and cost effective multiplication Rapid multiplication Carried out all year around Produce infection free plants Relatively short time Long term storage of germplasm Disease free plants Storage handling and transport easy and convinenient Disadvantages Perfect and flawless technology Capital investment and maintenance cost Chances for contamination Progressive decline in growth rate and multiplication of shoot : vitrification Possibility of loss of plantlets during hardening and transfer

57. Applications of  Micropropagation Plant tissue in small amounts is sufficient for the production of millions of clones in a year using  micropropagation . An alternative method of vegetative propagation for mass propagation is offered through  micropropagation . Plants in large numbers can be produced in a short period. Any particular variety may be produced in large quantities and the time to develop new varieties is reduced by 50%. Large amounts of plants can be maintained in small spaces. This helps to save endangered species and the storage of germplasm . The  micropropagation  method produces plants free of diseases. Increased yield of plants and increased vigor in floriculture species are achieved. The  micropropagation  technique is also useful for seed production Production of important plant-based phytopharmaceuticals .