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Module 1: Plant Development

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Organogenesis and Regeneration

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So, nowbefore we begin the specific requirements of different types of cultures, let us seeinorder to redifferentiate and dedifferentiate what happens in the plant cells and what isneeded.So,ah whenever you bring an explant now explant is a tissue or an organ which have you havebrought.So, it is a mixture of many different kinds of cells is not it?So,if you want that cell to come back to now it is a differentiate itself performing aspecific function . Now, if you want to bring back the cell intosomeother form of tissue or organ, the first that cell has to be reprogrammed.Although all the genetic information in the plant cells as we say they are totipotentin nature, which means that they have have the capacity to redifferentiate into someother form.So, which means in turn all the genetic information available for carrying out those metabolicactivities is available.It is just that at a particular particular time and there is only specific sets of geneswhich are expressed . So, under in vitro conditions when you bring an explant, first thing thecells within that explants have to be reprogrammed.Now, that reprogramming should be involving two things one is cytodifferentiation andthe other is organogenesis in order to do regeneration from that explant into some otherform.So,what are the kind of cells in an organ or a tissue which you bring out from the nature?These are non dividing differentiating quiescent cells now they are matured.So, now, they are performing a specific function.So, they are non dividing generally these cells of the explant.So, when grown on a nutrient medium, they first undergo changes to achieve the meristematicstate.So,these programmed cells or matured cells have to be reprogrammed to come back to theiroriginal meristematic state.Now, the phenomena of mature cells reverting to this meristematic state and forming anundifferentiated callus tissue is what is called as dedifferentiation.So, if you know we were saying that callus is a dedifferentiated tissue.So, the process of reverting back a matured cell into a meristematic cell, well is calledas dedifferentiation that is what forms that callus blogNow, since the multicellular explant comprises cells of diverse type, the callus is therefore,heterogeneous in nature.Now heterogeneity arises due to two factors one is inherent variation in the parent plantmaterial because there are so, many different kinds of cells the second is cytological andgenetical differences.Now all these cells are performing different functions which means that some amount ofgenetic information is expressed and some genetic information is not expressed in thesedifferent types of cells.Now, the ability of the component cells of the callus to differentiate into the wholeplant is what we know as redifferentiation or the totipotency is the capability.So, as to dedifferentiate and redifferentiate.It is not necessary that the explant has to go to a callus stage before regeneration.So, now the cells in a callus we know they are parenchymatious in nature which meansgenerally all these cells are parenchyma cells.Now the differentiation of these cells to a variety of other forms of cells needs thecells to be redifferentiated.Now this redifferentiation stage 1 is called cytodifferentiation.So, In-vivo or In-vitro in plant cytodifferentiation main emphasis is forming these vascular tissue.So, which is for example, as I said xylogenesis forming of phloem and xylem elements throughtheir connections.Now various factors for example, various chemical and physical factors are responsible for thiskind of differentiationfor example, you can take auxins, cytokinins, carbon source whichis sucrose or gibberellins.All genes as I said earlier all genes which are responsible for this process now the cellsbeing totipotent all genes are present, but due to the environmental conditions or thestimuli present in vitro, some of these cells the genes will get expressed to for the reprogrammingto happen and the cells to redifferntiate into some other forms of cells and therebyforming another type of tissue.Now for development of an adult organism from single isolated cells from these differentiatedtissues, which are generally non dividing and quiescent cells the differentiated cellfirst undergoes dedifferentiation and redifferentiation .Now, when I say cytodifferentiation, one is that formation of vascular bundles.Now then and it has to in order to regenerate into a whole plant what is needed?There has to be a shoot bud formation or primordium which is called as.Now once this is done shoot bud primordium would form directly then it is direct regenerationhappening or somatic embryos are formed through somatic cells.If somatic embryos are formed then subsequent development of bipolar structures which isshoot primordium and root primordium will form.So, organic differentiation can be of two forms direct formation of shoot bud or rootbud or formation of somatic embryos, thereby then forming bipolar structures one leadingto shoots and the other leading to root.Now, regeneration how is it useful in comparison to whole plant reproduction through whichprogenies are produced, there are three ways in which it is useful.It is more efficient process you can do clonal propagation of the species, which is whichmeans less time taken and what else?The potential for the production of much higher number of plants.So, the number of plants the rate of production of the progenesis is also enhanced.So, this is how under in vitro conditions if plant biotechnology tools are used forregeneration, then these are the advantages in comparison to natural plant production.Now, organize development is successfully achieved through proper selection.So, what things have to be kept in mind under in vitro conditions for regeneration to happen,you need to have a good starting material which means the inoculum.Then proper choice of the medium, the media composition matters then a balanced compositionof growth hormones which means what kind of the effect ofauxins cytokinins gibberellinsas I said ah.So, these have to be optimized and moreover it is controlled physical environment.Now, what happens in organogenesis?The cells are forced to undergo changes which can lead to the production of unipolar structuresas I said generally shoot.So, once you will see that when shoot multiplication is done for regeneration, it is shoot budprimordium which comes out you get multiple shoots and then later you put it into a rootinducing medium such that now these shoots will have roots and before they are transferredto the fields.So, in organogenesis shoot bud primordium is the objective when you are trying to regenerateinto new plantlets.Now root and shoot differentiation is a function of quantitative interaction between auxinsand cytokinins whether auxin.So,last class also as I said that depending on what species are you working whether dicot,monocot, herbs or tree species, the effect of the same auxin may vary.Now high in general what has been found that higher ratio of auxin to cytokinin would leadto a production of root and lesser ratio will induce shoot formationNow, usually high cytokinin concentration favors shoot formation as I said and 2, 4D is generally used for cell proliferation, but it suppresses cellular and organ.So, these things you can note down although this is specie specific , but in general howit helps that when you are working inwith a species for which no information is availablethis can be the starting point.Now, 2,4 D promotes cell proliferation and suppresses cellular and organ differentiationin dicot plants.I hope you know what are monocots and dicots monocotyledonous dicotyledonous dicotyledonsthat much you people know.Now abscisic acid and gibberellins now what as abscisic acid and gibberellins?These are plant growth regulators plant hormones.Now, they are also sometimes use to be promoting organogenesis in the medium.Now sometimes nitrogen complex nitrogen sources like casein hydrolysate it andamino acidsare also known to affect shoot bud formations.Now light qualityintensity period which meansphotoperiod, then temperaturecan also affect organogenesisin in vitro cultures.So, how does the organ development begin?Differentiation begins with the changes in the individual cells in response to the externalstimuli.So, generally what happens that if suppose there is a cell which is competent enoughfor dedifferentiation to happen.So, there are competent cells which are ready for differentiation dedifferentiation whichmeans meristematic which have the ability to quickly go through meristematic state.So, it begins from there around that cells there develops many meristematic centers .So, from these meristematic centers the development of organogenesis takes place.So, the there is a cell which is determined to undergo differentiation and read dedifferentiationaround that cells after multiplications, there are some more meristematic centers which getformed and these meristematic centers together will lead to formation of your shoot bud orroot bud primordium . Now, the morphogenetic signals setup the conditionswhich allow competent cells.So, these are called as competent cells.So, not all cells and sometimes it is very species specific sometimes you may try outmany hormonal combinations, but you will see that you are not getting successful organogenesishappening.So, which means that the explant it depends on the nature of the explant the age of theexplant the season in which you have collected the explant it may not end up the cells inthat explant may not be competent for coming back to a meristematic state and then redifferentiatiating.It also depends on the environmental conditions which you have given.So, there are multiple factors which are responsible for successful regeneration to happen andeven the genotype and the type of explant which you have used will be playing a crucialrole in this.So, why did I say?Because, there has to be cells which should be competent enough to undergo these reprogramming.So, morphogenetic signals set up the conditions which allow competent cells to undergo internallythis controlled program of differentiation to happen.Let us talk about somatic embryogenesis.The process of embryo developmentfrom somatic cells is called somatic embryogenesis.Now when we say somatic cells somatic cells are non germ cells the cells do not participatein the sexual reproduction of the plants.Now, somatic embryos can be formed from callus, cell cultures, protoplast or any find kindof organized structures from the plant.Now plant regenerations via somatic embryogenesis can be divided into two phases one is inductionsof cell with embryogenic competence.now this happens when when you have very high concentrations of auxins in the medium generallyand the development of these cells subsequently into embryos once vascularization, which meanscytodifferentiation and organogenesis both have taken place.Now, once organogenesis also has taken place which means coming up of the shoot and theroot primordium in these.So, for subsequent growth you need a combination of cytokinins and auxins.Now 2, 4 D is the most commonly used auxin in somatic embryogenesis sometimes thidiazuronor zeatin these are other kinds of cytokinins which may also help in subsequent growth ofdevelopment of somatic embryogenesis say embryos because even in the development of somaticembryos there are different stages involved we will take up that later once we come tosomatic embryos.They are first globular structures, then torpedo heart shaped structures though this is a gradualdevelopment.So, for this gradual development also you need variation in the nutrient.So, abscisic acid is used in combination with cytokinins for maturation of the somatic embryos.Nowsometimes in some species even activated charcoals can impact the induction of somaticembryos.So, it is the stimuli which can induce either the determined cells for this maturation tohappen or some of the cells will be induced to undergo embryogenic changes.So, a high nitrate to low ammonium nitrogen also has been found to favor somatic embryogenesis.Reduced nitrogen is required during embryo development.So, induction is one thing and subsequent development is another.So, it is not necessary what has worked for inducing will work for subsequent development.If you see that once callus has induced or somatic embryo has induced you will you mayfind that keeping in the same medium does not lead to further growth even if it haslead to successful induction.So, which means that and it needs revision of the medium ok.Somatic embryogenesis of single or multicelled origin is known to occur.Now they can occur even from single cells or multiple cells together.Somatic embryos of a single cell origin they are produced from synchronous cultures synchronouscultures means all the cells have the similar metabolic activity.Now, somatic embryogenesis can be induced directly from the explant or can be indirectlyfrom the callus phase.So, in case when the callus phase is involved once you bring the explant then it developsinto callus then you do a variation and it develops into somatic embryos that is calledas indirect somatic embryogenesis and when directly an explant which means a leaf givingrise to directly somatic embryos then it is called somatic embryogenesis direct.It is presumed that the cells of the explants have the competence and determined to producesomatic embryos.Now, there are two kinds of cells what is the hypothesis the explant tissue containscells that are already determined two embryogenic development.So, these are called as preembryogenic determined cells these group of cells are already determinedthat if given the chance, they will form somatic embryos others are called as redeterminationthrough a period of culture which means induced embryogenic determined cells which means theyhave they can be brought into somatic embryogenic stage, but after reprogramming which meanskeeping them under theright stimuli so, as to reprogram them into induce them into somaticembryogenesis which means that it will take much longer time.Now, embryogenic cells they produce embryos more readily than the differentiated explantsor or inducedembryogenic competent cells.So, it is presumed that highly differentiated cells more epigenetic changes.So,when we change the environmental parameters or we give stimuli then what is happening?The cells are undergoing these genetic changes genetic changes in the sense the genetic machinerywhich was not expressed then starts getting expressed which means which was scryptic isnow expressed thereby leading to some DNA replication and translation, thereby leadingto biochemical changes, histological changes and cytological changes which then you cansee the morphogenetic events like somatic embryos .Now, it is presumed that highly differentiated cells require more epigenetic changes.Now what do you understand by epigenetics?Right which can bring about a change in the gene expression now in some species it isnot possible to induce such epigenetic changes and it depends on on as I said in the natureof the explant and also depends on the conditions which you give in vitro.If those conditions are not adequate enough to bring them into induce embryogenic stageor to bring about such epigenetic changes then you will not get successful results .Now, what are the factors which can affect regeneration?It is the type of the explant, sometimes the size of the explant, season of collectionage of the explant.So, younger plants and younger explants they can easily get into and can be manipulatedand can be they are moreconducive for epigenetic changes.Reason because they are still dividing cells they will the majority of the cells will beat a meristematic stage because they are younger they have still not completely differentiatedand become organized to carry out a matured function now they can be easily manipulated.The explants can give rise to organs and embryos directly or indirectly via callus.So, as I said what happens that, there is a single cell there may be a single cell whichis determined to undergo or cytodifferentiation and organogenesis as it divides there aremultiple meristematic centers around that region these meristematic centers under growreprogramming and lead to your shoot or root primordium formation .So, what kind of nutrient media helps in regeneration?In organic micro and macro nutrients major difference as we said yesterday we were talkingthat although define media can be used, but there is a difference in these.So, if it is high ammonium to nitrate.So, generally what induces somatic embryogenesis?Then the it is a very high nitrate to ammonium ratio or suddenly you give very high concentrationsof sucrose.So, or if you can increase the concentration of auxin.So, although dependent on species, but in general it may be noted if no informationis available, these things can be tried and rest of the environmental factors anywayscan play a role in generating these organized structures that is it I will stop here today.