PLANT BIOTECHNOLOGY6th week of Biotech. Lectureby :Lanny Hartanti, M.SiMICROPROPAGATION

Plant Tissue CulturesThe culture of parts/organs of plants (protoplasts, cells, tissues, organs, embryos, or seeds) in vitro. In vitro means : in glass/test tube/bottle a controlled environmentIndependent from geographic and climate conditionFree from microorganism, insect, other plants pathogen-free, virus-freeSterile inoculation aseptically No nutrition competitionNo interfering metabolic products from others living cells

Contamination of plant tissue culture

Applications of Plant Tissue CulturesAgro-biotechnologyThe growth and development control of plants (vegetative, generative & reproduction/propagation)The protection of plants towards biotic & non biotic stresses (improvement of plants traits)

Industrial Plant Cell BiotechnologySee 7th week lectures

Classification of Plant Tissue CulturesBased on its cultivation methods :HeterotrophicRequiring organic substances as carbon sourcePhotoautotrophicAble to synthesis carbon source needed by photosynthesis processDoesnt need carbon source addition into its mediumMixotrophicCombination of culture type 1 and 2

Classification of Plant Tissue CulturesBased on types of culture :Culture of intact plants used a seed that is sown in vitro from which a seedling, and finally a plant developsEmbryo culture an isolated embryo is grown after removal of the seed coatOrgan culture an isolated organ is grown in vitro types : meristem culture, shoot-tip culture, root culture, anther culture, etc. also known as explant culture.

Classification of Plant Tissue CulturesBased on types of culture :Callus culture if a differentiated tissue is isolated, allowed to de-differentiate in vitro and a so-called callus tissue produced, the process is termed callus culture.NOTES : callus is actively dividing non-organized tissues of undifferentiated and differentiated cells often developing from injury (wounding) on tissue cultureSingle cell culture The growing of individual cells which have been obtained from a tissue, callus or suspension culture, with the aid of enzymes or mechanically.Protoplasts culture The culture of protoplasts obtained from cells by enzymatic digestion of the cell wall.

Classification of Plant Tissue CulturesBased on types of culture :Cell suspension cultures The culture in which (single) cells and/or clumps of cells grow and multiply while suspended in a liquid medium placed on rotary shaker (+ 100-150 rpm) to aerate the culture or to reduce the size of clumps cell.Immobilized plant cells cultureHaploid cultureThe culture that is obtained from repeatedly monoploid spore division (from a microspore or unripe pollen), which is induced from anther or pollen to yield haploid tissues/plant. Haploid plant/cell is plant/cell with half the number of chromosomes (denoted by n) due to reduction division of the diploid (=2n)To produce certain plant seed.

Initiation of callus and suspension cultures

Summary of the different cultural manipulations possible with plant cells, tissues and organs

Hairy roots cultureThe culture which is obtained from transformation of plant cells by Ri-plasmid (from Agrobacterium rhizogenes)The culture, which has smooth hairy like roots, is able to produce endogenous auxins and able to grow in medium without addition of any plant growth hormones. Fast growth, able to produce certain secondary metabolite (ex :alkaloid) which is stabile enough in a long period.

Classification of Plant Tissue Cultures

Diagram of the control of differentiation exerted by interaction of auxin and citokinin

MICROPROPAGATIONVegetative propagation of plants in vitroThe cell theory of Schwann and Schleiden (1838-39) CELLULAR TOTIPOTENCY : Potential of cells or tissues to form all cell types and/or to regenerate a complete plant (unless lacking a nucleus or enclosed by a rigid, secondary wall).Methods :Propagation from axillary budsThe development of adventitious shoots/ somatic embryos direct & indirect morphogenesis

Some of the pathways by which micropro-pagation can be achieved

MICROPROPAGATIONThe applications give benefits in several ways:Yield identical plant population geneticallyFast propagation in an enormous amountYield the best traits of plant stocksCould be used in plant conservationSaving in space (greenhouse), time and energyThe external factors is more controllable

The in vitro growth and development of plant is determined by a number of complex factors :The genetic make-up of the plant Nutrients : water, macro- and micro-elements, and sugarsPhysical growth factors : light, temperature, pH, O2 & CO2 concentrationSome organic substances : regulators, vitamins, etc.

Composition and Preparation of Murashige & Skoog (MS) Medium

Plant Growth RegulatorsAuxins (IAA, IBA, NAA, 2,4-D)induce cell elongation, or in some cases cell division; often inducing adventitious roots and inhibiting adventitious buds (shoots)IAANAA2,4-D

Plant Growth RegulatorsCytokinins (BA, 2iP, BAP, Zeatin)induce cell division and often adventitious buds (shoots) in most cases inhibit adventitious root formationdecrease apical dominance

Plant Growth RegulatorsGibberellins (GA3)Induce cell elongation and cell division, not generally usedInhibit adventitious root and shoot formationEthylene natural hormonesAbsicic acid has a negative influence

Scheme to show propagation of plantlets by tissue culture from callus

The Stages in MicropropagationStage 0 : selection & preparation of host plant :Healthy disease-free, virus-free, pathogen-free (from glasshouse); having best traits that we wantsDipped into ethanol 70% for a few seconds, followed by bleach/sodium hypochlorite 10% before cutting into explant (Ca(ClO)2 & HgCl2 could also be used).The addition of Tween 20 or 80 to the sterilizing fluid is also necessary to give better surface contact.

Stage 1 : isolation and inoculation aseptically in Plant Tissue Culture System

The Stages in MicropropagationStage 2 : Propagation by repeated subculturing avoid callus formationStage 3 : Rooting preparation for plant growth in its natural environment (soil)direct roots already grown in stage 2indirect induced by cutting and wounding the plantlets, followed by dipping in rooting powder contain auxinsStage 4 : Transfer of plants to its natural environment critical stage :change of relative humiditythe cuticula layer is thin Need adaptation aclimatization in certain room with environment control and regulation (temperature, light, humidity)

Transferring plantlets to its natural environment

Strategies to improve the plants traitsGenetic manipulation, which can be done in several ways :Conventional ways Cell fusion somatic hybridization of protoplasts of 2 different speciesTransformation by Agrobacterium tumefaciens tumors inducing (crown-galls)Transformation by Agrobacterium rhizogenes hairy roots diseaseDNA transformation method, 4 ways :As pure DNA biolistic method / Gene GunAs calcium phosphate-DNA co-precipitateAs DNA encapsulated in liposomesBy direct micro-injection of DNA

The end of our session

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