2 Manipulating plants for the benefit of mankind PLANT BIOTECHNOLOGYManipulating plants for the benefit of mankindA process to produce a genetically modified plant by removing genetic information from an organism, manipulating it in the laboratory and then transferring it into a plant to change certain of its characteristicsImproved food cropsHigher yieldsImproved nutritionEnvironmental tolerancesImproved production of valuable moleculesProduction of novel molecules
3 It chiefly involves the introduction of foreign genes into economically important plant species, resulting in crop improvement and the production of novel products in plants.Today, biotechnology is being usedas a tool to give plants new traits that benefit agricultural production, the environment, and human nutrition and health.
4 The goal of plant breeding is to combine desirable traits from different varieties of plants to produce plants of superior quality.This approach to improving crop production has been very successful over the years.Plant Biotechnology is a rapidly expanding field within Biotechnology.Today plant biotechnology encompasses two major areas, plant tissues culture and plant genetic engineering (Transformation).
5 Transgenic Plants:A transgenic crop plant contains a gene or genes which have been artificially inserted instead of the plant acquiring them through pollination.The inserted gene sequence (known as the transgene) may come from another unrelated plant, or from a completely different species
6 Tissue cultureTissue culture is the term used for “the process of growing cells artificially in the laboratory”.It involves both plant and animal cells.Tissue culture produces clones from the explant. (In clones all the produced plants have the same genotype).Tissue culture of every living cell of every plant is possible.
8 Exogenous genes (non-plant genes) pathogen-derived genesbacterial genesany other organismExogenous genes (non-plant genes)Applications:transgenicPathogen resistanceHerbicide resistancebioreactorsDelivery systems
9 Historical Background Gottlieb Haberlandt (1902) - Who grew palisade cells from leaves of various plants. (Father of Tissue Culture) White , Developed tissue culture medium for in vitro culturing of meristematic cells of tomato [medium containing salts, yeast extract and sucrose and 3 vit B (pyridoxine, thiamine, nicotinic acid)] Miller and Skoog, proposed that root-shoot differentiation is regulated by auxin – cytokinin ratio.1959 Braun Regenrated first plant from plant cells.1962 Murashige and Skoog developed M&S medium.Guha & Maheshwari Developed embryoids from anther culture, produced androgenic haploid plants by pollen culture of Datura stramonium.
10 • 60’s and 70’s Murashige cloned plants in vitro – raised haploid plants from pollen grains. – used protoplast fusion to hybridize 2 species of tobacco into one plant contained 4N • 70’s and 80’S beginning of genetic engineering. A more recent advance is the use of plant and animal tissue culture along with genetic modification using viral and bacterial vectors and gene guns to create genetically engineered organisms.
11 IMPORTANT TERM & DEFINITIONS Totipotency:It is the ability of a single plant cell, (with protoplasm) to divide and to produce all the differentiated cells in an organism.Medium:The basic nutritive material on which explant is placed in laboratories for callus or organ formation is called medium . It can be in liquid or solid.
12 Explant:Cell, tissue or organ of a plant that is used to start in vitro cultures.( in vitro = in + glass)Any part of the plant can be used as explants for micro propagation, but axillary buds and meristems are most commonly used.
13 Callus: A. Is a natural response of the plant tissue to wounding. B. A unorganised mass of actively dividing undifferentiated cells produced by plant tissue explant.Usually a explant with morphologically uniform cells produces a uniform type of callus and explant with variety of cell types produces a mixed callus.Callus cultures are transferred to fresh medium at 4 to 6 weeks interval.Failure of transfer to fresh medium leads to death of callus due to exhaustion of nutrient.
14 Embryogenesis: (embryo + genesis-birth) Organoganesis: ( organ + genesis- birth)The process of initiation and development of root, stem & leaf (but not embryo) in plant tissue culture. Starts due to presence of chemicals in the medium especially growth hormones.Embryogenesis: (embryo + genesis-birth)The process of initiation and development of embryo or embryo like structure from somatic cells in tissue culture.( somatic embryogenesis).
15 Stem cells: undifferentiated cells that are able to differentiate into more than one cell type. Stem cells can divide to produce differentiated type of cells and also retain the ability to divide to maintain the stem cell population. Pluripotent: The most versatile stem cells that have the ability to give rise to all tissues of the body are also called pluripotent stem cells.
16 Micropropagation: (Murishige 1974) Also known as clonal propagation in vitro culture It is nothing but multiplication of genetically identical copies of plant by asexual or vegetative method. As minute sized propagules are used in culture so is named as micropropagation. It is beneficial as it produces large number of plants in short period.
17 Basic Requirements for Invitro Culture Tissue culture laboratory:requires all the nutrient and physicochemical factor mention in all laboratory.Good laboratory must have:Nutrient medium preparation, sterilization, cleaning and storage of supplyAseptic condition for working the living materialA control environmental condition for the growth and development of cultureObservation and evaluation of the culture as hope.Recording the observation during the experiment.But the basic facility that an individual needa. Washing and storage facilityb. Media preparation roomc. Transfer area (LAF)
18 2. Nutrient Media Composition and Preparation Vital activity of a cell as the absorption of nutrient through cell membrane and rapid proliferation into innumerable cell.Nutrient medium contains inorganic salts, sugar, vitamins, growth hormones and a few amino acids.a. INORGANIC:It included N, P, K, Ca, Mg, S.Microelements:B, Mo, Cu, Zn, Mn, Fe, Clb. Growth Hormones:It will stimulate the biological activities in culture materials.c. Organic Constituents:serve as a source of carbon and energy.Sucrose and de-glucose are commonly use but glycerol and myoinosital are also the principal source of carbon.Other organic compound: peptone, yeast extract, coconut water, tomato juice etc.d. Vitamins:The are required in trace amount.The catalyze the various enzymatic reaction in the cell.e.g. vitamin B1 (Thimine) is the most commonly use vitamin for plant tissue culture.Others, niacine (nicotinic acid), vitamen B2( Ribofliven) B6 (pyrodxin) C (ascorbic asid) vitamen H (Biotene) B12 (cyanocoblamin).
19 2. Nutrient Media Composition and Preparation e. Amino acid:Serve as a source of nitrogen. Most commonly use aminoacid are L-aspartic acid, L-Aspragin, L-glutanic acid, L-glutamine, L-argine.f. Solidifying agent:Most commonly ager (0btain from seaweed) i.e. red alge, Glidium amansii.It do not react with constituent of media and not digested by enzyme. Generally from 0.5 to 1%.g. pH Effect:Affect the uptake of ions, optimum 5-6 is required for development of culture tissue. pH should be mention before sterilization of media.
20 3. Maintenance of Aseptic Environment a. Sterilization of glass waves ( C, 2-4 hours)b. Sterilization of Instruments:The metallic instruments will be claim sterilize (dipping them in 75% ethanol followed by flaming and cooling. It is called incineration. Sterilization of culture room and transfer area.c. Sterilization of Nutrient media (15 psi) 121C for 20 minutes.Vitamins, plant extract, amino acid and hormone are denatured by auto calving, they should be sterilized by 0.2 um filter paper.d. Sterilization of Plant Material:disinfectant (hypo chloride, hydrogen per oxide, mercuric chloride or ethanol).Then 6-8 time washing with sterile distil water.
21 Basic Steps in Tissue Culturing The basic step required for regeneration of a whole plant from plant cell tissue or organ culture:Preparation suitable nutrient medium (per objective of the culture and transferred into a suitable container and then sterilized)Selection of ex-plant: Always young and healthy part of the plant are selected as an ex-plant.Sterilization of ex-plantInoculation (under aseptic condition)Incubation:Growth chamber/tissue culture room at 25± 2c, relative humidity and 16 hours of photo period.Regeneration:A portion of Callus transferred into another medium (induction medium, shoot and root)Hardening:hardening is the gradual exposure of plant let for acclimatization to the environmental condition.Plant Let Transfer:After hardening plant let are transferred to green condition.
22 Basis for Plant Tissue Culture Two Hormones Affect Plant Differentiation:Auxin: Stimulates Root DevelopmentCytokinin: Stimulates Shoot DevelopmentGenerally, the ratio of these two hormones can determine plant development: Auxin ↓Cytokinin = Root Development Cytokinin ↓Auxin = Shoot DevelopmentAuxin = Cytokinin = Callus Development
23 Factors Affecting Plant Tissue Culture Growth MediaMinerals, Growth factors, Carbon sourceEnvironmental FactorsLight, Temperature, PhotoperiodExplant SourceTypesUsually, the younger, less differentiated the explant, the better for tissue cultureGenetics1. Different species show differences in amenability to tissue culture2. In many cases, different genotypes within a species will have variableresponses to tissue culture; response to somatic embryogenesis hasbeen transferred between melon cultivars through sexualhybridizationEmphasize the implications for genetic involvement: Could there be undesirable genes linked to genes influencing tissue culture response?
24 Choice of explant Shoot tips Axillary buds Seeds Hypocotyl (from germinated seed)LeavesDesirable properties of an explantEasily sterilisableJuvenileResponsive to culture
25 Medium Constituents Inorganic salt formulations Source of carbohydrate VitaminsWaterPlant hormones - auxins, cytokinins, GA’sSolidifying agentsUndefined supplements
26 CarbohydratesPlants in culture usually cannot meet their needs for fixed carbon. Usually added as sucrose at 2-3% w/v.Glucose or a mixture of glucose and fructose is occasionally used.For large scale cultures, cheaper sources of sugars (corn syrup) may be used.
27 Inorganic Salt Formulations Contain a wide range of Macro-elements (>mg/l) and microelements (<mg/l).A wide range of media are readily available as spray-dried powders.Murashige and Skoog Medium (1965) is the most popular for shoot cultures.Gamborgs B5 medium is widely used for cell suspension cultures (no ammonium).
28 Vitamins A wide range of vitamins are available and may be used. Generally, the smaller the explant, the more exacting the vitamin requirement.A vitamin cocktail is often used (Nicotinic acid, glycine, Thiamine, pyridoxine).Inositol usually has to be supplied at much higher concentration (100mg/l)
30 Auxins Absolutely essential. Only one compound, Indole-3-acetic acid. Many synthetic analogues (NAA, IBA, 2,4-D, 2,4,5-T, Pichloram) - cheaper & more stableGenerally growth stimulatory. Promote rooting.Produced in meristems, especially shoot meristem and transported through the plant in special cells in vascular bundles.
31 Cytokinins Absolutely essential. Single natural compound, Zeatin. Synthetic analogues Benyzladenine (BA), Kinetin.Stimulate cell division (with auxins).Promotes formation of adventitious shoots.Produced in the root meristem and transported throughout the plant as the Zeatin-riboside in the phloem.
32 Gibberellins (GA’s)A family of over 70 related compounds, all forms of Gibberellic acid.Commercially, GA3 and GA4+9 available.Stimulate etiolation of stems.Help break bud and seed dormancy.Produced in young leaves.
33 Abscisic Acid (ABA) Promotes leaf abscission and seed dormancy. Plays a dominant role in closing stomata in response to water stress.Has an important role in embryogenesis in preparing embryos for desiccation.Helps ensure ‘normal’ embryos.
34 ‘Plant Growth Regulator-like substances’ Polyamines - have a vital role in embryo development.Jasmonic acid - involved in plant wound responses.Salicylic acid.Not universally acclaimed as plant hormones since they are usually needed at high concentrations.
35 Undefined Supplements Sources of hormones, vitamins and polyamines.e.g. Coconut water, sweetcorn extractsNot reproducible