2 How can we obtain better Crops ? selectionbreedinghybridizationcloninggraftingradiation mutagenesischemical mutagenesisgene splicinggenomics/gene expressiontissue culture
3 Plant cell & tissue culture In 1905, first attempt was made to culture monocot cells on artificial medium, but did not succeed in producing plant from cultured cellsDuring 1930s, roots and shoots were successfully cultured from cells on artificial nutrient medium… thus the beginning of “organ culture”Since then, remarkable achievements are made in culturing plants from tissues, cells, anthers, pollens, protoplasts and embryos.During 1980s, emergence of genetic engineering technology has further paved the way of crop improvement by transferring genes from unrelated species to plant cells.. Leading to transgenic crops or genetically modified plant species.
4 Basic terms Cell culture Culturing whole plants cells, tissues and organs on a suitable nutrient medium.Organ CultureIn vitro culture of whole plants from roots tips, shoot apices, and embryos etc. is called organ culture.PlasticityAbility to be shaped or formed.Ability of differentiated cells to undergo trans-differentiationMany of the processes involved in plant growth and development adapt to environment.Allows plants to alter their metabolism, growth and development to best suit their environment.Abilities to initiate cell division from almost any tissue of plant and to regenerate lost organs or undergo different developmental pathways in response to particular stimuli.
5 TotipotencyAbility of a single cell to divide and produce all the differentiated cells in an organism.Totipotent cells formed during sexual and asexual reproduction include spores and zygotes.For example, a plant cutting or callus can be used to grow an entire plant.When plant cells and tissues are cultured in vitro they generally exhibit a very high degree of plasticity, which allows one type of tissue or organ to be initiated from another type.This regeneration of whole organisms depends upon the concept that all plant cells can, given the correct stimuli, express the total genetic potential of the parent plant.
6 Explant Callus Protoplasts Regeneration Any excised portion of the plant such as leaf, stem, root etc. that is used for culturing a whole plant.CallusUndifferentiated and unorganized mass of cells.ProtoplastsThese are naked plant cells from which the cell wall has been removed by treatment with an enzyme cellulase.RegenerationIt is a process in which cells are made to divide and differentiate to gave rise to a whole plant.
7 Basic TechniqueCollection of explants (such as seedlings, buds, stems, leaf, immature embryos etc.)Sterilization of the material by dilute solution of surface – sterilants, e.g. sodium hypochlorite, hydrogen peroxide, alcohol etc.Removal of surface – sterilants by washing with sterile distilled water.Transferring the material to sterile glass tube containing autoclaved nutrient medium. This is called inoculation.Culture to be incubated in a tissue culture room where temperature is maintained at 56° ± 5° for 3 – 4 weeks in low light (relative humidity 60%). Calli will be developed.Transfer the callus into a liquid medium.Regeneration of plants from cell suspension culture.Regenerated plants are transferred to pots and kept in glass house for acclimatization.This is called hardening effect in natural conditions.
9 Nutrient MediaCulture media used for in vitro cultivation of plants cells are composed of three basic components:1. Essential elements, or mineral ions, supplied as a complex mixture of salts2. An organic supplement supplying vitamins and / or amino acids3. A source of fixed carbon; usually supplied as the sugar sucrose
11 Plant growth regulators AuxinPromotes cell division and growthPromotes root formationCytokininsPromote cell division (purine derivatives)Promotes shoot formationGibberelinsRegulate cell elongationImportant in determining plant height and fruit-setAbscisic acidInhibits cell divisionUsed in tissue culture to promote distinct developmental pathways such as somatic embryogenesisEthyleneMost commonly associated with fruit ripening
12 Ratio of auxin and cytokinin determines the type of regeneration Intermediate ratioCallus formationLow auxin to cytokinin ratioShoot formationHigh auxin to cytokinin ratioRoot formation
13 Applications of cell and tissue culture Production of somaclonal variationsVariation seen in plants that have been produced by plant tissue culture.Chromosomal rearrangements are an important source of this variation.Results areAneulpoidsSterile plantsMorphological variantsAll resulting in crop improvement
14 EmbryogenesisSomatic embryogenesis is an asexual form of plant propagation in nature that mimics many of the events of sexual reproduction.Plants may be produced artificially by the manipulation of tissues and cells in vitro.In vitro somatic embryogenesis is an important prerequisite for the use of many biotechnological tools forgenetic improvementmass propagationProduction of haploidsProduction of triploids
16 What is a transgenic? Concept Based on the Term Transgene Transgene – the genetically engineered gene added to a speciesTransgenic – an organism containing a transgene introduced by technological (not breeding) methods
17 Transgenics are a Biotechnology Product Transgenic plants possess a gene or genes that have been transferred from a different species.The term "transgenic plants" refers to plants created in a laboratory using recombinant DNA technology.The aim is to design plants with specific characteristics by artificial insertion of genes from other species or sometimes entirely different kingdoms.
18 Transgenic plants with beneficial traits Stress toleranceWithstand biotic and abiotic stressBiotic stressAttacks by insect pests, viruses, bacteria, fungi etc.Abiotic stressCaused by non-living environmental factorsDroughtExtreme temperaturesSoil conditionsHigh winds
19 Why are transgenics important? We can develop organisms that express a “novel” trait not normally found in the speciesExtended shelf-life tomato (Flavr-Savr)Herbicide resistant soybean (Roundup Ready)