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Module 1: Plant Cell Bioreactors

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Genetic Transformation in Plant Cells

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So, Genetic variation it is necessary forcrop improvement in agriculture.So, what all different ways in which crop yields are improved or that traits are improve,hybridization.Now what is hybridization?The process of combining different varieties of the same plant; I will be talking aboutplant hybridization of the same plant such that then you select for or thehybrid lineswhich will have the combination of the good traits of the both the varieties.So, it although includes it again a random process so, this will include a selectionof the cell lines which will have all the positive traits which have come together.Then mutagenesis DNA may be modified either naturally or artificially by a number of physicaland chemical agents resulting in mutations and then again you do a screening and selection.Then polyploidy, you apply certain agents which can result in change in the ploidy inplants known so, which may lead to improvement in the traits desired.Now, plant cell cultivation in vitro generates a considerable amount.Now we know thatin under in vitro conditions, we have heard about somaclonal variation whichitself can be used to generate different cell lines of different genetic makeup becausethis can lead to polyploidy change in the carrier topic nature of the cell.So, somatic hybridization is what?It is a well known technique we have learnt about it.So, somatic hybridization in which somatic cells are fused together such that the cytoplasmas well as the nucleus will get fused and therefore, you will end up in a single cellwhich will have club of both the varieties of cells.So, generally it is observed that these hybrid cells which come out they will bemore vigorousin nature; for crop improvement you will see that hybridization of plants is very commonlyused.So, seedlessfruits like example, I do not know whether you have heard or seen seedlesspapaya orvery bigger size fruits.So, these are all because of hybridization.So, generally it is deliberately pollination is done; mixing is done such that the hybridsare generally observed to be more vigorous in nature in terms of their growth rates orin terms of yields.Now, the plants which are obtained through genetic engineering contain a gene usuallyfrom an unrelated.So, when we say transgene integration, it means that a gene which is not present inthe plant, but has been transformed in the plant chromosome.So, such genes are called transgenes and the plants containing the transgenes are calledtransgenic plants.Genetic transformation can be defined as the transfer of foreign genes isolated from plants,virus, bacteria into new genetic background.The first step in gene transfer is to select the cells that are capable of giving riseto whole transform cell.So, we will see what are the different ways in which the selection is done and what arethe different ways in which the transformation of plant cells is done.So, what is a gene construct?When we are transforming plant cell this will consists of this is common.So, gene construct for transgene integration will contain different assembly of DNA.Generally agrobacterium is used as a tool to transfer; this genetic material.There are other techniques also which we will be talking about.So, the genetic whole transaction which is transferred will have these many sectionsas numbered here.So, the construct will contain a promoter, we will contain an enhancer or silencer transcriptionalstart leader, sequence initiation, codon, exons, introns stop codon, untranslated regionsand poly a tail.Have you heard about these terms?You have heard.So, promoter is required for promoter is used for.. Be loud.. RNA polymerase is.. The transcription of the gene, then enhancerand silencer?. They regulate something.the winding of the . Right so, they regulate as he said the promoteractivity when it as a enhancing.So, it is enhancing the promoter.When it is a silencer, it will silence the promoter.This can have certain sequence or this can lead to whoselective induction of the promoteror by either exogenous addition of certain agents.So, this is how it may lead to regulation of the promoter activity.Then transcriptional, start leader sequence?These are the regions of MRNA for the regulation of translation part, then at the ribosomelevel because once the MRNA is formed it will attach to the ribosome for translation.So, their this leader sequence regulates that then initiation codon exons introns.So, what is responsible what where your desired gene is which part of the gene is responsiblefor expression introns or exons?Exons.Exons.So, there is RNA splicing which removes introns then, but regulatory role has been found ofthe intronic regions as well.Then stop codon which is the transcriptional stop, it is followed by untranslated regionsare there is 5 prime untranslated region 3 prime untranslated region.3 prime untranslated region will also have your poly a tail which is required for forstability of the MRNA for the processing.So, the promoter it provides the site for binding of RNA polymerase and is involvedin transcription initiation, then it is required for an efficient expression in plant cells.So, generally the promoter which are used are either can be inducible promoters or canbe constitutive promoters.What is inducible promoter, which means the promoter activities regulated in the presenceof certain chemical the promoter will be active.Then constitutive promoter irrespective of the condition, it is uniformly all the timeactive.So, CaMV 35S promoter have you heard about CaMV which is a very widely used cauliflowermosaic virus promoter for genetic transformation in plant cells.Now is this a constitutive promoter or inducible promoter?This particular promoter is responsible for the entire gene transcription of mosaic virus.It is a constitute promoter and very high activity that is why and it is well knownto be infecting the plant cells.So, this particular promoter being a constitutive promoter or strong promoter is used for genetictransformations in plant cells.Among all the promoters known CamV35S promoter is very commonly employed in transformationexperiments.The promoter of the 35S RNA is a very strong constitutive promoter that leads to the transcriptionof the whole CaMV35S.So, it is well known for its use in plant transformation.A suitable enhancer sequence is also required to control the activity of the promoter.Now either; so, why do you think thisenhancer sequence how is it useful?If suppose you want to express it at a particular developmental stage or you want an expressionin a particular tissue so, then you need to regulate the activity of the promoter.So, that can be done using your enhancer or your what else silencer sequence.The enhancer is defined as the DNA sequence which enhances the activity of the promotersand the DNA sequence suppressing the activity of the promoter is known as silencer.So, how do we recover transgene cell lines; suppose we have done transformation usingthis construct so, how do you find out that which particular cell line is transformedor not how do you select?You would be needing a marker system.Now, what is the market system antibiotics?So, generally used is antibiotic resistance.So, it is a selective market system.Now in which what does it mean, only when it is expressed in the because it is presentin the transgene which is integrated into the plant chromosome; it will express in theplant chromosome, then only it will be able to show antibiotic resistance.Now, even an antibiotic resistance generally the antibiotic, you would like to choose anantibiotic resistance against an antibiotic to which the plant cells are highly sensitive.So, what kind of antibiotics?Generally hygromycin is used because plant cells canamycin we have heard in bacterialtransformations, but plant cells are found to be highly sensitive to hygromycin becauseof their uptake and their reach availability to plant cells and effect them.So, hygromycin system is used as a reporter system or as a marker system.So, selective agents differ in the toxicity to different plant species.The different developmental stages of plant cells or tissues give different response tothe selectable marker.It is necessary to use the correct concentration of the selective agent, then herbicides aregenerally more toxic to plant cells than antibiotics.Now, callus and cell cultures are better than organised explants to achieve transformationsand for the selection of transformed cells.Now, reporters system , how is it different from your marker system? Generallyyou areGUSyou are GFP GFP; you must have heard isn't it why is it used.Spectro photometric So, try to connect it with saying the wordreporter gene.You want to know what levels of protein are being expressed.So, they can correlated with.It can be quantification quantified as well as qualitative in nature.So, if it is qualitative in nature, it will tell you what?. Expressed or not.So, generally these systems in plant cells you will heregus as says very frequently usedwhich is glucoronidase luciferase.So, in which blue fluoresce or blue colouredproduct comes so on the cells which are once theyare exposed to X-Gluc substrate.So, this particular enzyme if it is express properly in the transgenic cell lines thenwe will be able to break down the substrate X-Gluc into product which is blue colour andyou can visualise that and then this becomes an indication of successful transgene integration.Now, the genes need to be tagged with another genes which are called as reported.Now I was saying that you can confirm whether it is a successful transformation or not usingreporter systems.Now the other thing how it can what else can be the use of reporter system?It can also be used to check promoter activity or the activity is in the promoter is activeor not.So, then if you use the same promoter which you have used for your desired transgene integrationto for this particular GUS for luciferase and if that gets expressed then there is ahigh chance that the promoter is in tagged; the promoter activity is in tagged is ableto show good activity in this particular plant species.So, a reporter gene is a coding unit whose product is easily assayed like GUS whose productcan be easily detectable.So, it is called a histochemical assay.So, this gene may be connected to any promoter of interest.So, that the expression of the gene can be used to assay promoter function that is itdescribes the transfer and activity of the promoter.Two genes that are widely used is beta glucuronidase and the other is luciferase, the coding regionsof these non plant genes.So, these are transgenes non plant genes have been fused to plant promoters and polyadenylationsequences which means you need a 5 prime utr and 3 prime utr.So, 5 prime utr you are using the same promoter which you are using for your desired geneand 3 prime utr for the entire construct to happen your GUS or beta glucuronidase or yourluciferase enzyme for expression.So, plasmid mediated gene transfer.How many types of plasmids are used for gene transfer?These are conjugative plasmid on transmissible plasmids, the others are non conjugative plasmidswhich are also called as non transmissible plasmids.So, what are plasmids?Self replicating extra extrachromosomal DNA maintained as independent molecules; generallythey were they are found in bacterial.So, plasmid DNA can be circular or linear in nature the size of the plasmids variesis from 1.15 kb to 1500 kb.So, conjugative plasmids what are these they can mediate their own transfer between bacteriaby the process of conjugation.So, there is a appendages, yeah which are formed and then the two cells will come togetherso as to facilitate the transfer of the genetic material.So, then conjugative plasmids can mediate their own transfer between bacteria by theprocess of conjugation, then non conjugative plasmids are not self transmissible.So, generally when we are using non conjugative plasmids their transfer is done with the helpof conjugative plasmids.Now, plasmids can be classified also in terms of lower and high copy numbers; low copy numberswhen it is nearly 1 to 4 copies high copy numbers greater than 100.So, conjugative plasmids are large with stringent control of DNA replication and are presentin low copy numbers.Non conjugative plasmids are small high copy number and have relaxed control of DNA replicationX.So, if suppose you are working for cloning multiplication, what kind of plasmid you willprefer?. Non conjugative plasmids; large number ofcopies are produced because of the relax control of DNA replication.High copy number plasmids are useful for expression of cloned genes to higher yields.So, when I say binary vector these plasmids can get expressed in more than one differenttypes of organisms.Like when in plant transformation binary vectors can PCaMVS, they can get expressed in e colias well as multiply in e coli as well as in agrobacterium.When such plasmids are used as vectors, they completely utilise the host metabolism.So, what is the demerit?They will utilise the host metabolism if they are expressed in a organism.Each plasmid has an origin of replication can you make out that why do we say it isa metabolic burden, then any kind of transgene integration.Each plasmids has an origin of replication plasmid DNA in bacteria may be 0.1 to 5 percentof the total DNA.Now, gene transfer methods; one is indirect gene transfer and the other is direct genetransfer.We will take up the direct gene transfer later.The indirect gene transfer methods are the most frequently used in transgene integrationsin to plant cells indirect gene transfer.This is vector mediated gene transfer vectors are DNA carriers into which foreign DNA orgenes of interest are inserted to make a recombinant DNA.Now, what are the different kinds cloning vectors and expression vectors?Cloning vectors which are required for if you want to multiply want large number ofcopies, expression vectors these expression vectors are used when you want to expressthe transgene into the plant used for expression of the clone gene to produce the product.Now most vectors carry what we have already studied this a reporter gene which allowsrecognition example antibiotic resistance.So, plasmid mediated gene transfer.Plasmids are most commonly used vectors in gene cloning.What are the different types of plasmids which are used?It is a different types of plasmid which are used, we you will come across Ti plasmidsor Ti plasmids.Ti plasmid is tumor inducing plasmids or root inducing plasmids.Now some of these once disarmed.Now I will come tocertain diseases in nature or because of bacteria which is agrobacteriumwhich contain these plasmids.Now, these plasmids will have set number of genes.Now why do you think a bacteria infects the plant should be a purpose behind this.So, it contains certain genes which cannot be expressed when the plasmid is present inthe bacteria, but as it gets integrated into the plant they will express these genes andthese genes lead to opine production these opines are carbon and nitrogen sources forthe bacteria.So, now in this course the portion which gets integrated will have certain oncogenic genes,now plant is allowing this infection to happen.So, it has certain auxin and cytokinin gene as well which get integrated which will causesome biochemical changes to happened leading to neoplastic growth at the point of infections.Now morphogenetic events can either it can either lead to rapid multiplication of cellsleading to crown gall formation or organogenic or adventitious root form for which are calledas hairootz.Now, this itself therefore, these agrobacterium and the plasmid containing it and as thenbeen modified or disumed; disumed removing of these oncogenic genes which means you areauxins and cytokinins genes and putting an MCS their multiple cloning site with differentrestriction sites.So, that the desired gene can be integrated now this whole casset is included in the T-DNAregion that portion of the construct which gets integrated into the plant chromosome,this is called as T-DNA.Now, this portions contains a right border and a left border.These are direct repeat sequences twenty base pairs around long right border and left borderthen the entire outside this T-DNA also there are different regions of the Ti plasmid orthe Ri plasmid the genes which are responsible for the breaking and the multiplication ofthis T-DNA processing of this T-DNA, then transfer of this T-DNA into the plant cellprotecting their till it reaches the chromosome of the plant cells and gets integrated.So, there are virulence genes then there are other genes which will which are requiredin the entire packaging of the T-DNA.So, it is not alone T-DNA, but it is T-DNA with other protein which are involved in theentire process of movement till the plant chromosome even with its interaction withthe plant with its interaction with the plant protein thereby facilitating the integrationin the plant chromosome .