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Introduction to Plant Tissue Culture

This document provides an introduction to plant tissue culture. It describes how plant parts can be grown in vitro in artificial nutrient media. The key aspects covered are the types of explants used, applications of plant tissue culture, essential operations, techniques like callus and suspension cultures, steps involved like preparation of media, sterilization, incubation, and acclimatization of plants. Common components of nutrient media and roles of plant hormones are also summarized.

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Plant Tissue Culture•
Introduction to PLANT TISSUE CULTURE Dr SV Suresh Kumar Professor of Pharmacognosy CES College of Pharmacy, Kurnool Andhra Pradesh
Plant Tissue Culture  Growth of plants parts in vitro in an artificial nutrient media.  Cultivation of medicinal plants in vitro in an artificial nutrient media.
 Micro propagation of plants in vitro under sterile conditions to produce the clones of the plants.  Laboratory technique of growing, culturing, maintaining the cells, tissues, organs in vitro. EXPLANTS Cell,Tissue, or Organs-preferably more rapidly growing tissue or tissue at an early stage of development.
Applications of PTC  Production of useful components in higher levels irrespective of climatic conditions.  Diosgenin from callus cultures of Discorea floribunda  Solasodine form callus cultures of Solanum khasianum.  To produce plants free from microbes.  Temperate or tropical of any kind can be cultivated to get specific metabolites.  Synthesis of compounds, difficult to produce by synthetic means-plant specific compounds.  Production of uniform BIOMASS.
 For studying Biotransformation reactions  Low cost precursors can be converted to high cost secondary metabolites.  Less potent compounds can be converted to highly potent compounds.
 For studying various biogenetic reactions.  Production of Immobilized cells.  Production of high producing strains by genetic manipulations.  Production of novel compounds.  Nor sanguinarine-benzephenanthridine alkaloid from Papaver somniferum  3-methyl lucidine –Anthraquinone from Digitalis lanata  Peniculides-sesquiterpenes form Andrographis paniculata.
Essential operations consists of- * removal of cells from their normal environment. * proper explantation on to substrate * subsequent nourishment by nutrient medium. Techniques of cultures: 1. Static/callus culture 2. Suspension culture.
Callus culture:  When explant is explanted on to nutrient medium and subjected for incubation, there occurs growth of mass of undifferianted cells known as callus.  Performed in solid medium  Results in dedifferentiation changes .  Performed in dark.
 Manipulation of auxins/cytokinins ratio-Organogenesis.  Rhizogenesis and caulogenesis.  Callus culture are used to initiate suspension cultures.  Compact and friable cultures.
Suspension cultures:  In suspension cultures there is an active proliferation of callus into individual cells which get suspended or dispersed in the liquid medium.  Relatively large inoculum should be used so that the released cell numbers build up quickly i.e usually with cell density of 9-15 x 103 cells/ml.  Cell suspensions can be maintained relatively simply as batch cultures in conical flasks.They are continually cultured by repeated sub culturing into fresh medium.
 When the cells reaches to stationary phase sub culturing is done.  Friability of compact callus can be increased by  Repeated sub culturing  Culturing on semi solid medium  Manipulation of media contents by the addition of 2,4-D,Yeast extract, malt extract etc.
Steps involved in PTC:  Preparation of nutrient media  Sterilization of nutrient media  Preparation of explants  Explantation of explants  Incubation  Acclimatization of plants and transfer to pots.
Preparation of nutrient medium- In consists of selection of the media components and preparation of the media.  Inorganic nutrients:  Macro elements- more than 0.5 mmol/l- N,P,K,Ca, Mg, S  Micro elements- less than 0.5 mmol/l- Fe, Zn, Mn, Co, Mo, Cu, B, I.  Nitrogen should be given in the form of Ammonium nitrate or Ammonium salts (combination preferred).  Potassium should be given in the form of KCl, KNo3, KH2PO4.  Fe and Zn should be in the form of EDTA chelates • Vitamins: - Most important vitamin isThiamine (B1) -0.1 to 10 mg/l. - others includes Pyridoxine (B6), Calcium pantothenate (B5) and Niacin (B3).
 Carbon source:  The important carbon source in culture medium is carbohydrates and mainly 2-5% sucrose.  Others include maltose and galactose.  Amino acids:  Until unless the nitrogen is supplied through the inorganic source, the addition of amino acids is not necessary.  However cysteine is added to prevent the oxidation of phenolics and there by blackening of the tissues.  Combination of glutamine and arginine is added to use in protoplast culture.  Activated charcoal:  0.5 to 3% acid washed activated charcoal is added to the media has both favourable and unfavourable effects.
 Organic supplements:  Protein hydrolysates like casein hydrolysates; yeast extract, malt extract, Coconut milk, etc.  Used to enhance the growth rate of cells.  Its uses is discouraged as is difficult to get reproducible results.  Solidifying agents:  Non essential component of the medium.  0.8 to 1% agar, 0.4 % agarose, 10% gelatin, 0.1 to 0.25 % gelrite etc.  Antibiotics:  Not included in the medium as they inhibit the cell division.  If necessary streptomycin, kanamicin are used.
 Plant hormones/Growth regulators: In addition to the nutrients it is generally necessary to add one or ore growth substances such as auxins, cytokinins and gibberellins, to support good growth of tissues and organs.  The auxins commonly used in tissue culture for root differentiation and includes.. IBA (Indole-3-butyric acid), NAA (naphthalene acetic acid), NOA (naphthoxy acetic acid), 2,4D (Dichloro phenoxy acetic acid) 2,4,5-T (Tricholorophenoxy acetic acid), PCPA (P chloro phenoxy acetic acid), Pilcoram (4-amino 3,5,6 trichloro picolinic acid). Etc.
• The cytokinins are concerned with cell division, shoot differentiation etc. More commonly used are, BAP (benzyl amino purine), iso pentenyl adenine (IPA) and kinetin (Furfurylamino purine).  The gibberellins  GA3 (Gibberellic acid) is common one.  However they are used rarely to stimulate normal development of plantlets.  Some commercially available media includes: Murashige skoog media, Heller’s medium Nitsch’s medium White’s medium etc
 Preparation of medium: All the chemicals are dissolved in distilled water, the stock solution of vitamins, micro nutrients and growth hormones are added and then the pH is adjusted between 5 to 6.
Sterilization of the media:  The medium is transferred in to flasks and stoppered with cotton plugs, and autoclaved at 120°C to 121°C for 15 to 40 min.  Pressure used is 15 psi or 1.06 kg/cm2 .  The thermo labile substances can alternatively sterilized by filtration sterilization using membrane filters having pour size of not more than 0.2 microns. Eg: Millipore and Seitz filters.  The flasks are removed for cooling as early as possible and stored at 10°C prior to use.
Preparation of explants:  Irrespective of the plant part used as an explant, the explants are cleaned with a liquid detergent in running water and surface sterilized with disinfectant solutions like  sodium hypochlorite 0.5 to 5%  Calcium hypochlorite 9 to 10%  Hydrogen peroxide 10 to 12 %  Bromine water 2%  Mercuric chloride 0.1 to 1%  Benzalkonium chloride 0.01 to 0.1%  Ethanol 75 to 95% etc.  Aseptic explants can also be obtained from aseptic seedling developed from inoculated seeds. For this, seeds are surface sterilized with 0.1% mercuric chloride, rinsed in sterile distilled water and inoculated on basal medium to obtain aseptic seedling in vitro.
Explanation of explants:  The transfer of explants onto the sterilized nutrient culture medium taken in culture vessels is called inoculation/explantation.  The inoculation of the explant is carried out in aseptic environment i.e in the laminar flow chamber.
Incubation:  The culture vessels with inoculated explants are incubated under controlled conditions of temperature, illumination and humidity.  The cultures are incubated for 3 to 4 weeks during which, the cells of the explant absorb the nutrients, grow and undergo repeated divisions to produce a proliferating undifferentiated mass of cells known as callus or produces shoots or roots directly.  The explant or callus cultured on different combinations of auxins and cytokinins will produce shoots or roots, called as organogenesis.  High proportion of auxin and low proportion of cytokinin induce root development from callus called and is known as rhizogenesis.  Low proportion of auxin and high proportion of cytokinin in the medium induce shoot development from the callus and is called as caulogenesis.
 Alternatively, embryo like structures develop from callus and this phenomenon is known as embryogenesis (Somatic embryogenesis).  The embryos like structures which develop from callus are called embryoids. Sometimes the explant also produces the embryoids directly without callus production.  Since these embryoids develop from somatic tissue they are referred to as somatic embryos.The somatic embryos are transferred to other culture media for development into complete plants.
Acclimatization:  The plants regenerated via organogenesis or embryogenesis need to be acclimatized before their transfer to pots.  The plants are washed gently to remove the culture media and planted in plastic pots containing soil-rite (material consists of coconut shells and other organic material).  The pots are covered with polythene bags and maintained in the laboratory at room temperature for 1-2 weeks.When plants appear strong and healthy the bag is removed and transferred to a regular pot containing a mixture of soil and manure.