INTRODUCTION TO PLANT TISSUE CULTURE: It is the process of producing plants from tissue of the desired plant in an artificialnutrient medium under controlled environment. The plants so grown would be exactly similar to the mother plant in all aspects. The science of plant tissue culture takes its roots from path breaking research inbotany like discovery of cell followed by propounding of cell theory. In1839, Schleiden and Schwann proposed that cell is the basic unit of organisms.They visualized that cell is capable of autonomy and therefore it should be possiblefor each cell if given an environment to regenerate into whole plant. Based on thispremise, in 1902, a German physiologist, Gottlieb Haberlandt developed theconcept of in vitro cell culture. He isolated single fully differentiated individualplant cells from different plant species like palisade cells from leaves of Laminumpurpureum, glandular hair of Pulmonaria and pith cells from petioles of Eicchorniacrassiples etc and was first to culture them in Knop’s salt solution enriched withglucose. In his cultures, cells increased in size, accumulated starch but failed todivide. Therefore, Haberlandt’s prediction failed that the cultured plant cells couldgrow, divide and develop into embryo and then to whole plant. This potential of acell is known as totipotency, a term coined by Steward in 1968.
STERILIZATION TECHNIQUES: Sterilization Methods Used in Tissue CultureLaboratory - All thematerials, e.g., vessels, instruments, medium, plantmaterial, etc., used in culture work must be freedfrom microbes. This is achieved by one of thefollowing approaches:(i) dry heat treatment,(ii) flame sterilization,(iii) autoclaving,(iv) filter sterilization,(v) wiping with 70% ethanol, and(vi) surface sterilization.
EXPERIMENTS:Protocol.1- Tissue Culture Media PreparationPRINCIPLE:Murashige and Skoog medium or (MSO or MS0 (MS-zero)) is a plantgrowth medium used in the laboratories for cultivation of plant cellculture. MSO was invented by plant scientists Toshio Murashige andFolke K. Skoog during Murashiges search for a new plant growthregulator. It is the most commonly used medium in plant tissueculture experiments. A series of experiments demonstrated thatvarying the levels of these nutrients enhanced growth substantiallyover existing formulations. It was determined that nitrogen inparticular enhanced growth of tobacco in tissue culture.
Protocol-2- Explant Preparation and SurfaceSterilizationPRINCIPLE:Surface sterilization treatments applied on the explants were:Dipping in Ethyl alcohol 70% for 5 minutes (mins). (T1)Dipping in Chlorox 3% (commercial sodium hypochlorite, activeingredients 5.2%) for 20 mins. (plus Tween 20). (T2)Dipping in undiluted Chlorox for 20 mins. (plus Tween 20). (T3)Dipping in Ethyl alcohol 70% for 5 mins. then in Chlorox 3% (plusTween 20) for 20 mins. (T4)dipping in Ethyl alcohol 70% for 5 mins. then in Mercuricchloride 0.3% for 5 mins. (T5) After each treatment explantswere rinsed three times in autoclaved distilled water.
Protocol-3-EMBRYO CULTURE:PRINCIPLE:Organogenesis is the formation of individual organs such as shoots and roots eitherdirectly on the explant in which pre-formed meristem are lacking or the meristemsdevelop de novo from callus. Although callus is an actively growing undifferentiatedmass of cells, differentiation can take place at random, but may be associated withcentres of morphogenesis, which can give rise to organs such as shoots, roots andembryos. To induce shoot organogenesis from the callus, concentration of growthregulators is varied in the medium. Cytokinins such as BAP and kinetin promoteshoot organogenesis.RESULT:
Protocol-4- Culture of Anther for Production ofAndrogenic HaploidsPRINCIPLE:Anther and Microscope Culture - One of the very popular methods for production ofhaploids is through culturing anthers or microspores on artificial culture medium.This leads to the growth of microspores into saprophytes. After the initial reports ofsuccessful production of haploids from anther culture in Datura (Guha andMaheshwari, 1966, 1967), haploids have been obtained in more than 150 speciesbelonging to 23 families of angiosperms (Maheshwari et a1., 1980). These include awide variety of economically important species.More often, anthers rather than microspores are cultured, since the extraction andculture methods for microspores differ and have been successful only in a few species(Datura inoxia, Nicotiana sylvestris, N. tabacum, Oryza sativa, etc.).
Protocol-5-Meristem culturePRINCIPLE:Organogenesis is the formation of individual organs such asshoots and roots either directly on the explant in whichpre-formed meristem are lacking or the meristemsdevelop de novo from callus. Although callus is an activelygrowing undifferentiated mass of cells, differentiation cantake place at random, but may be associated with centresof morphogenesis, which can give rise to organs such asshoots, roots and embryos. To induce shootorganogenesis from the callus, concentration of growthregulators is varied in the medium. Cytokinins such asBAP and kinetin promote shoot organogenesis.
Protocol-6- Meristem tip culture for production ofVirus –free Plants Theme: Shoot apical meristem lies in the shoot tip beyond theyoungest leaf or first leaf primordium ; it measures upto about 100 µm indiameter and 250 µm in length. Thus a shoot-tip of 100-500 11m wouldcontain 1-3 leaf primordia in addition to the apical meristem.Inpractice, shoot-tips of up to 1 mm are used when the objective is viruselimination. Shoot-tip culture is widely used for rapid clonal propagationfor which much larger, e.g., 5-10 mm, explants are used. Therefore, mostcases of meristem culture are essentially shoot-tip cultures. Nodalexplants of various sizes are also commonly employed for rapid clonalpropagation. When the objective is vegetative propagation, the size ofshoot-tip used for culture is not important. The upper few millimeters(ca.5-6mm) in a shoot apex is considered to be free from virus in thoseplants which are systematically infected. Due to active cell division (fasterthan virus multiplication), absence of vascular connection and high auxinconcentration the shoot meristem remains virus free. If the meristem tipis used as an explant for propagation, virus free plats can be obtained.
Protocol-7- Induction of SomaticEmbryogenesis (Monocot and Dicot System) In somatic embryogenesis the embryo arises from somatic cells, tissue or organs under in vitroconditions. Somatic embryo is a bipolar structure and has no vascular connection with thematernal cultured explant. The somatic embryos are functionally equivalent to zygotic embryosbut the process of embryogeny is different. It induces four developmental phases i.e.0, 1, 2, and3 phases.Phase 0: In phase 0 the competent single cells (state 0 cells) form embryogenic cell clusters(state 1 cells) in the presence of auxin. During this phase the cell cluster formed from singlecells gains the ability to develop into embryos when auxin (a PGR used to induce somaticembryogenesis) is removed from the medium giving rise to state 1 clusters. Phase 1: Phase 1 is induced by transfer of state 1 cell clusters on to auxin free medium. In thisphase the cell cluster proliferate slowly and undifferentiately. Phase 2: In phase 2, rapid cell division occurs in certain parts of cell clusters leading toformation of globular embryos.Phase 3: In phase 3, globular embryos develop into plantlets via heart shaped, torpedo shapedand cotyledonary stage embryos.Somatic embryos could be induced either directly from the explant tissue in the absence ofcallus formation (direct somatic embryogenesis) or via the callus from the explant (Indirectsomatic embryogenesis). Embryogenic cells are small, isodiametric in shape, filled with densecytoplasm and have a conspicuous nucleus. In comparison to this, on-embryogenic cells arerelatively large, vacuolated and lack dense cytoplasm.
Protocol-8- Protoplast Isolation, Culture andRegenerationPRINCIPLE:Mechanical Method of Isolation of Protoplast - Inmechanical method, cells are kept in a suitable plasmolyticum(in plasmolysed cells, protoplasts shrink away from cell wall)and cut with a fine knife, so that protoplasts are released fromcells cut through the cell wall, when the tissue is againdeplasmolysed. This method is suitable for isolation ofprotoplasts from vacuolated cells (e.g. onionbulbs, scales, radish roots). However, this method gives pooryield of protoplasts and is not suitable for isolating protoplastfrom meristematic and less vacuolated cells. The mechanicalmethod, though, was used as early as 1892, is now only rarelyused for isolation of protoplasts.
Protocol-9- Microculture Chamber Technique forSingle Cell IsolationPRINCIPLE: Single Cell Culture - Establishment of a single cell culture provides anexcellent opportunity to investigate the properties and potentialities ofplant cells.Such studies contribute to our understanding of the interrelationships andcomplementary influences of cells in multicellular organisms. Severalworkers have successfully isolated single cell division and even raisedcomplete plants from single cell cultures.Using cell cultures in studies designed to describe the pathways of cellularmetabolism was another aspect that initially attracted the attention ofplant biologists. It was soon realized that single cell systems have greatpotential for crop improvement. The microculture chamber technique was first developed by Jones et al.(1960) and later was used by Vasil and Hildebrandt (1965) after somemodifications. This method consists of culturing 30-50μ/ of mediumcontaining one or more protoplasts on a microscope slide enclosed by acover glass resting on two other cover glasses placed on either side of thedrop. The cultures are sealed with sterile paraffin oil and incubated in lightat 23-25º C.
Protocol-10- Encapsulation of somatic embryos / shoot buds forProduction of Synthetic seedsPRINCIPLE:Synthetic seeds are prepared by encapsulating the somatic embryos obtained fromtissue culture in a protective jelly capsule, which is usually prepared with sodiumalginate. From the synthetic seeds whole plant can be recovered under in vitro,greenhouse and field conditions. Alternatively, shoot buds, conservation andmaintenance of rare and threatened species.Synthetic Seeds - In the conventional plant tissue culture for clonal propagation,storage and transportation of propagules for transplantation is a major problem.To overcome this problem, in recent years the concept of synthetic or artificial seedshas become popular, where somatic embryos are encapsulated in a suitable matrix(e.g. sodium alginate), along with substances like mycorrhizae, insecticides,fungicides and herbicides. In. India, this technique of synthetic seeds is beingstandardized and practiced for sandalwood and mulberry at BARC (Bombay) underthe leadership of Dr. P.S. Rao. Synthetic seeds have many advantages including thefollowing:(i) they can be stored upto a year without loss of viability;(ii) they are easy to handle, and useful as units of delivery;(iii) they can be directly sown in the soil like natural seeds and do not needhardening in greenhouse.The only limitation of synthetic seeds, is the high cost of their production. However,this may go down in future, so that these synthetic seeds will become popular at thecommercial scale in due course of time.
Protocol-11- Establishment of Cell SuspensionCulturePRINCIPLE: Suspension Culture Growth and Subculture - Cell suspensions areclonally maintained by the routine transfer (subculture) of cells in the earlystationary phase to a fresh medium. During the incubation period thebiomass of the suspension cultures increases due to cell division and cellenlargement. This continues for a limited period since the viability of cellsin suspension after the stationary phase decreases due to the exhaustion ofsome factors or the accumulation of toxic substances in the medium. At thisstage an aliquot of the cell suspension with uniformly dispersed free cellsand cell aggregates is transferred to afresh liquid medium of the originalcomposition. The timing of a subculture is very important. Types of Suspension Cultures - There are mainly two types of suspensioncultures, batch cultures and continuous cultures. Batch cultures aremaintained by propagating a small aliquot of the inoculum in the movingliquid medium and transferring it to a fresh medium at regular intervals.Generally cell suspensions are grown in flasks (100-250 ml) containing 20-75ml of the culture medium: The biomass growth in batch cultures follows afixed pattern.
Protocol-12- Culture of single Cells (Bergmann’s cell platingtechnique) PRINCIPLE: PRINCIPLE: In this technique, free cell are suspended in a liquid medium (if cell aggregates arethere, the culture is filtered), and a culture medium with agar (0.6-1%) is cooled andmaintained at 35ºC in a water bath. Equal volumes of these liquid and agar media aremixed and rapidly spread in a Petri dish, so that cells are evenly distributed in a thinlayer, after solidification. The Petri dishes are sealed with parafilm and examined withinverted microscope to mark single cells (marking is done on outer surface of thedish). The Plates are incubated in dark at 25°C and cell colonies developing frommarked single cells are used to obtain single cell cultures. Various other methods (e.g.filter paper raft technique; microchamber technique) have also been developed togrow individual cells (Bhojwani and Razdan, 1983)In this technique, free cell are suspended in a liquid medium (if cell aggregates arethere, the culture is filtered), and a culture medium with agar (0.6-1%) is cooled andmaintained at 35ºC in a water bath. Equal volumes of these liquid and agar media aremixed and rapidly spread in a Petri dish, so that cells are evenly distributed in a thinlayer, after solidification. The Petri dishes are sealed with parafilm and examined withinverted microscope to mark single cells (marking is done on outer surface of thedish). The Plates are incubated in dark at 25°C and cell colonies developing frommarked single cells are used to obtain single cell cultures. Various other methods (e.g.filter paper raft technique; microchamber technique) have also been developed togrow individual cells (Bhojwani and Razdan, 1983)
Protocol-13- Maintenance of cell linePRINCIPLE:Maintenance of Cultures Cell Lines - When a primary culture produced ona substrate or in suspension has increased to the extent that all the availablesubstrate is occupied or the medium largely consumed, there arises theneed to subculture it. From a very heterogeneous primary culturecontaining many types of cells derived from the original tissue, during subculturing (passages or transfer) a more homogeneous cell line emerges.The culture now called a cell line can be propagated, characterized andstored. The potential increase in cell number and uniformity of cells, openup a much wider range of possibilities. The term cell line implies thepresence of several cell lineages either similar or distinct. Among these celllineages, if a particular cell lineage has specific properties, which areidentified in bulk of the cells of that culture, it is described then as a cellstrain. A "cell line" or cell strain may be finite or continuous, dependingupon whether it has limited culture life spans or it is immortal in culture.Finite cell lines grow upto 20-80 population doublings before I extinction.Some commonly used cell lines and cell strains and a comparison of theproperties of mite and continuous cell lines is presented