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Module 1: Plant Development

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Somaclonal Variation and Micropropagation

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So, today we are going to talk about somaclonal variation, and then the applications how itis useful, and what are the demerits of somaclonal variation in plant tissue culture.And then how we do micropropagation?Now, first let us talk about the somaclonal variation.So, the growth of the plant cell and the regeneration is an asexual process which involved mitoticdivision of cells.So, we know now, the ability of the plant cells to regeneration because they are totipotentin nature, are is because of this success continuous dev division of plant cells, sowhich is because of the mitotic process Now,whenever the cell has to or the callushas to form or the callus has to regenerate into a plant or any organ, the cell has tocontinuously keep on dividing.Every cell, single cells undergoes hundreds of division cycles, finally, to lead to differentiationand the regeneration process because of this rapid multiplication there is instabilitywhich is in many different forms.You can see it in ultimately in phenotype, there will be in terms of chromosomal instability,genetic insatiability, when I say genetic instability it would involve in terms of size,numberof chromosomes.Then it can alsoshow up as change in biochemical instability, which means suddenly you willsee some new compounds getting formed or the yield of the existing compounds changing.Then,I was talking about phenotypic variability.Phenotypic variability you can see if the regenerants have come out ofthis somaclonal,after this somaclonal variations you will find that they are not clonal progenies.There will be variation in terms of which is visible in terms of may be flower color,maybe in terms of leaf size, height of the plant, branching patterns.So, these are different kinds of variations which can happen because of this somaclonalvariation.So, if the objective is to do clonal propagations of the plant then somaclonal variation ishas a demerit.So, the occurrence of an uncontrolled variation during the callus regeneration is termed assomaclonal variation.Now, this leads to what?This leads to variation found in the plants regenerants when you are using cells or callusin in vitro cultures.And because we are using somatic cells generally in in vitro cultures, so therefore, the termsomat somaclonal variation.So, cell and tissue cultures, they undergo frequent genetic changes and these get expressedin the form of biochemical and at molecular level changes.Now, what causes somaclonal variations?Now, we know that the plants are totipotent and as I said every cell needs to divide anumber of times before that redifferentiation and dedifferentiation, and then again redifferentiationhappens.So, because of this sometimes there is a ploidy change.Sometimes you will find the karyotypic alterations.When I say karyotypic alterations which means you will find that they are recombinationshappening or there may beactivation of transpose on elements.There can be single gene mutations which might happen.Or, when I say activation of transpose on elements and different recombination eventsmight happen which means that these are those gene elements which can change their relativeposition in the plant chromosome.When I am saying plant chromosome because I am talking with respect to plant cellSo, the when these get activated.Now, why this is happening?We were talking.This may happen because of many reasons.For example, it depends on the genetic makeup of the explant which has been used, the ploidylevel of the explants which has been used.Then it also depends on the culture conditions, sometimes the if there is very high concentrationof phytohormones that can also lead to this.If there are stress conditions,those who work in plant tissue cultures it is recommendedthat the time of subculture, the uniformity of the inoculum which you use which meansthe quality of inoculum has to be taken care of.The reason, to avoid the somaclonal variations.If the objective whether it is to produce a secondary metabolite, the product qualityand the product quantity has to be kept consistent.If you need to have reproducible results then therefore, these have to be taken care thatthe inoculum which you use for subculture has to be same in quality and age, thentheculture conditions have to be kept same, sudden changes in conditions can also lead to epigeneticvariations.So, when we say somaclonal variations some of the epigenetic variationslike for example,DNA methylation can happen, so which is also a part of an example of which can cause somaclonalvariation.Then,I said some even addition of chemicals like colchicines.Now, this may bring about a change in the chromosome number and the ploidy levels.So, that can also cause then somaclonal variations.But, generally you would not use mutagenic agents in the medium.So, when you are doing plant tissue culture, it may be sudden changes in the culture conditionsor if you delay the subculture cycle for very long periods.So, it happens when there is a very long term, it takes time, it does notimmediately happen,but if there are stress conditions continuously provided for a number of subculture cyclesthen this can lead to somaclonal variations in the culture.Now, what are the methods of assessment?How will you come to know that there has been processively a somaclonal variation in theculture?So, there can be three methods, one isyou will see a change in phenotypic parametersyou can find out from that.Now, in phenotypic parameters they can be two ways quantitative, qualitative.Now, quantitative people measure plant height.The clonal propagation is happening.So, actually you will see the same you should see similar phenotypic parameters, but ifyou see a change in the leaf size or a plant height or in the internodal regions, the length.So, then that is an indication.Now, qualitative.For example, in flowering patterns, the color change happening.Then physiological parameters,physiological parameters means functional.Now, the minute there is a change in functional characteristics, you can see a change in theprotein characteristics, so which means the differential expression of proteins or eventhe total protein content, so which you can see on the gel electrophoresis.Now,then again your secondary metabolite.Like for example, we sawin viola odorata, I was mentioning in the earlier classes duringthe introductory that we were working on class of plant peptides called cyclotides from amedicinal plant called viola odorata.Now, what we observed was when the plant wasbrought in vitro and we could generate the in vitroplantlets and we did a study onthe array of cyclotides being produced from the same plantmaterial which we got from the horticulture which was be maintained at IIT, Madras.We saw some novel cyclotides coming out.So, suddenly, so which means that some of these variations which might have happenedbecause of the in vitro conditions which may be stress to the culture or which may havecaused these variations ultimately then leading to variations in its biochemicals in synthesis.It is not that the genes were not present, but may be these genes were not expressed,but because of the conditions which were provided inside the lab could lead to expression ofthe genes of other cyclotypes.Now, what are the factors which can affect somaclonal variation?The source of the explants I said, which means the genotype of the explants or the ploidylevels.In plants it is well known, so then medium composition, medium composition which meansas I said if there is higher amounts of phytohormones or plant growth regulators then it can actas a stress.For example, BAP and 2, 4, D, then they can bring about karyotypic alterations.So, please remember when I say genetic variability which can happen is in terms of chromosomenumber, in terms of chromosome shape, size, these can be the changes.What other factors?Culture period, generally long term cultures.You keep them for very long under a conditions then you will see these changes happening.The subculture period can be a factor, then the subculture frequency, so it is recommendedkeep things consistent whatever conditions are being kept.Now, what is the molecular basis of this variation?Now, this can arise due to single gene mutations which can happen in the cultured cells whichcan be because of the conditions which you provide.Then changes in the cytoplasmic genome might happen which can be also a part of somaclonalvariation.Now, when I say changes in the cytoplasmic genome which means what?Where are these changes happening?. Or the chloroplast.So, sometimes it is also observed some plasmids they get intig extra chromosomal materialgetting integrated into mitochondrial genome and bringing about a change.Then as I said trans, activation of transposable elements.What are transposable elements?Transposable elements, these aresmall DNA sequences which can change their relativeposition in the chromosome.So, when their recombination happens because of this jumping, these are called jumpinggenes.So, then there can be a genetic variability.Now, somaclonal variation, it can also happen because of the mitotic crossing over.Generally, crossing over it isah natural phenomena to bring about to increase genetic variationin the progenies which we know it is a well-known part of meiosis.But when it happens in mitosis then this can also cause because somaclonal variations thecell division is happening because of mitosis, the cells are continuously dividing becauseof the process of mitosis.If during this continuous division some crossing over happens which means the gene transferin the homologous pairs of the chromosomes then this can cause a genetic variabilitywhich means chromosomal instability.Now, changes in organelle, DNA, then also because of there is protein variation as wehad spoken about, so there can be ultimately a change in enzymes.You can have same enzymes same sequence, but their functionality might change or differin the amino acid sequence, but they catalyze the same reactions.Now, what therefore, if you see all these things, now I hope you can make out that howcan be exploit somaclonal variations.Now, because there is a chance by inducing epigenetic variations by changing cultureconditions you can bring about a change in its phenotypic parameters, their physiologicalparameters therefore, it also gives you a chance to make a desirable change in the cellline.So, this can be therefore used for choosing a disease resistant cell line or salt resistant,drought resistant plants, then environmental stress tolerance.can be because of salt or drought any kind of stress can be used.Now, talking about micropropagation.Now, what is micropropagation?Micropropagation is multiplication of plants through plant tissue culture.Now, in plants even highly mature and differentiated cells they retained we know the ability toregress to meristematic state.So, the vegetative method of propagating plants is termed as micropropagation or cloning tissueculture.Now,the what are the different applications?For example, in medicinal plant if you need to preserve the medicinal plants then thiscan be of use.Under in vitro conditions you can generate large amount of plantlets of the same geneticmakeup and then those can be planted.But this is again a very slow process because once you are generating these, they need specific.This is all being done under controlled conditions.So, very gradually then and hardening of these plantlet us is required, so as to get themacclimatized to environmental outside conditions and then they are transferred.So, what is required?The differentiated cell, undergoes dedifferentiation, redifferentiation, and then a new plant isobtained.Now, let us see what are the different kinds of micropropagation methods.So, development of shoots from these preexisting meristems or nodal regions ensures geneticstability of the regenerants and is termed as axillary budding.Now, similarly this is also a method to develop virus free plants, as we were talking aboutin the last class because these are meristem regions rapidly dividing and so there is lesschance that the virus can get propagated from the infected areas of the plant to these regionsat the same rate as the cells are dividing.And moreover on top of it the vascular bundle is still not formed, so the chances that thevirus can propagate to these regions is missing or is less.Now, the principle method for propagation in industry is axillary budding.Now, what is adventitious budding?Adventitious budding is from directly the explants, from the organs you see new de novomeristems in induction which means that at they are not from the existing meristems region,but new meristematic regions get formed and directly from the explants you see organogenesishappening.So, it is de-novo formation of adventitious buds, adventitious buds which means not fromthe pre-existing meristems may occur directly from the tissues of the explants.Now, they also form indirectly, so there is always a chance where a callus visit, it isnot that if you are not it depends on the species.Sometimes it may not happen, the same conditions you are trying it depends on the genetic makeupthe species on which you are working.So, if direct is not possible then generally it becomes more conducive if an indirect phasewhich means the callus phases involved.Even in somatic embryo induction you will find that because it is so much dependenton the species response and depending on the objective then it is preferred to use a callusphase and then regenerate into organ cultures.Now, as I said axillary bund budding, then adventitious budding, and you can even domicropropagation using somatic embryos.Now, somatic embryos which we know as synthetic seeds.This can also be used to generate large amount of plantlets in smaller amount of time andspace.Now, what is a demerit included here?Although, you can get large amount of plantlets, large amount of plant material in less timeand space.Now, because it is through somatic embryos and if you are using an intermediate callusphase which means indirect somatic embryogenesis then there will be genetic variability inthese plantlets.So, if the purpose is it depends on the objective, if the purpose is not clonal propagation iftheclonal or the genetic stability is not a requirement, it is suppose it is being usedfor fuel if you just need to do forestry or if you need to propagate a weedjust for asake of biomass to be used for biofuels generating biomass, so then clonal propagation mightnot be a need.So, then if you can compromise then synthetic seeds is a good way of multiplication of thoseplants.So, somatic embryos can be used for pro production of synthetic seeds.So, if you can see in the picture these are gel beads.So, it is nothing but, like you do plants cell immobilization similarly somatic embryosare immobilized.So, what is critical here?The viability of the somatic embryos in the gel.So, what are the advantages?Small space requirement in case of micropropagation, then plantlets produced are free from insectsand microbes because they are been under controlled conditions, then virus elimination is possible.Why?You know now the reason because we are using making use of the meristems to bring aboutthe micropropagation.Then reproducible system as the conditions are being maintained, under controlled environmenteverything is being done, so it is reproducible then production is unaffected by the seasonalvariations as which might happen in the natural conditions of propagating a plant.So, these are the stages which will be involved.Selection and establishment of an aseptic culture, then multiplication of the Propagule,then plantlet regeneration and preparation and transfer to the field.The step of preparation and transfer to the field is a long drawn.It takes time.