Hello everyone.Welcome to the practical demonstration course of Plant Developmental Biology.Today I have Mister Tushar Garg with me, a senior PhD student in my lab.So, Tushar is going to practically demonstrate you some of the practical aspects of plantdevelopmental biology.So, if you can recall our previous classes; so, two very important component or aspectwhich we consider while studying plant developmental biology is analyzing the expression patterntemporal and spatial expression pattern of the genes.So, Tushar will show some of the process associated with it.And second important component is you have to genetically modify the plant to understandthe developmental biology or the aspect of plant development.So, Tushar is also going to show you or demonstrate you how to make transgenic plant and how todo some genetic manipulation in it.Hello everyone.So, you have gone through the lectures and you must be knowing that to study plant developmentbiology; the genetic modification of plant is a very essential process.So, for genetic modification, the first thing that we need is an explant.So, explant is any plant material which can be used to make or regenerate callus and thosecalluses are then transformed using two methods.The first is the biological method and the second is the physical method.So, biological method is the one where we use bacteria and this bacteria contains ourconstruct or the gene which we want to modify and this is then transferred into the callus.The second one is, where the gene gun like instrument is used for the transformationwhere the gold particles are used and the gene that we have to transform is coated onthe gold particle.And, then this machine sends this gold particle along with the gene into the cells and thecells which are transformed they regenerate and make the new plant.So, first we will be going through the biological method of transformation, where in our labwe use rice as an explant.So, here you can see this is the dehusked dehusked rice and here the embryo; the embryogives the callus which is under the influence of hormones.So, when you give a certain hormone component then the embryo generates dedifferentiatedcallus and this callus is further used for our further process.Now, we will see how we proceed.So, the first step is where we sterilize the seeds.Now, for sterilization it is called as Surface sterilization.So, in surface sterilization, certain chemicals are used which kills the contamination likebacteria, fungus.So, the most common surface sterilization which is done is using 70 percent ethanol.Now, this is done for 90 seconds not more than 90 seconds.So, once 90 second is complete then we use Rin Ala.So, this is Rin Ala which contains sodium hypochlorite.Some of the others chemical agent which is used for surface sterilization is HgCl 2.In Rin Ala it is done for 15 minutes.So, once it is done so, these seeds are taken on the blotting sheet and then using forcepsit is placed on a media.Now, this media has auxin.So, auxin is the hormone which causes the embryo to make dedifferentiated tissue orthe callus.This plate is placed under dark condition for about 30 days.And, once the 30 days is complete, it creates a callus which you can see you over here.So, these are the calli.Now the cream-colored embryogenic calli, they are selected and placed on the subculturemedia.Basically, it is placed for 2 to 3 days for having a proper amount of growth.So, that it is ready for Agrobacterium infection.Now, once my callus is ready for Agrobacterium infection so, I use the Agrobacterium whichI will show over here.So, this is the overnight grown culture of Agrobacterium.This Agrobacterium has my gene of interest.So, this is pelleted down on centrifuge.And, then a 5 percent sucrose solution is added to it.Now, this is vortexed to suspend in the solution.Now, we take the suspended Agrobacterium in the flask, and put our callus in it.In this way we take all the calli and put acetosyringone in it.Acetosyringone is basically a signaling molecule, which helps Agrobacterium in its growth.Now, we keep this for 15 minutes on a shaker and after that, we take out we throw the media,and we take the callus and put it on a co-cultivation plate.So, co-cultivation means, here we have bacteria as well as the callus.Now this co-cultivation plate is placed under dark condition for 2 to 3 days and after thatthese callus; so, you can see here there is little amount of growth of the bacteria.Now, this callus is taken again in the flask and washed using water.So, enough amount of washing is done.So, that there are no Agrobacterium left, there are no threads coming out of the Agrobacteriumand then it is placed on a selection media.Selection media is the media which contains our selection marker; over here suppose mygene of interest which I have included in the Agrobacterium and it has been if my callushas been transformed.So, along with the my gene, there is selection marker such as hygromycin.So, when I grow this callus on hygromycin plates, so this, callus which are transformedthey will grow.But, those which are not transformed they will die due to the hydromycin which is anantibiotic.So, once placed on selection media, here you can see some of the calluses are dying whereas,some are not dying.So, those which are not dying they give out new cells and which proliferate and to forma regenerated calli which once it is ready and enough growth has taken place we placeit on a shooting media.So, shooting media is a media which has high cytokinin to auxin concentration, under theinfluence of high cytokinin the callus forms shoot apical meristem and regeneration takesplace.Now, here you can see with time this bottle has been placed under light condition andwith time you can see that there are green colored tissue coming out.These are basically shoot apical meristem where new organs are being formed.Now, once this greening is enough and there are enough organogenesis occurred.So, this is taken on a rooting media.So, this rooting media has high auxin concentration, auxin helps in the initiation of root formation.So, here you can see this greening plant has enough shoot and roots are also coming outonce this plant is about this the length of the bottle.So, from here it is transferred into the soilrite for hardening, which we will see in the greenhouse.Now, we are done with the biological method of transformation then, now we will see thephysical method of transformation.So, I will just show you the machine over here which is called as gene gun.Gene gun is used for the physical method of plant transformation.Here the principle for gene gun is that, the gold particles or the tungsten particle theyare coated with the gene of interest.The gene or the construct that we were transforming into the bacteria, here those construct arecoated on the gold particles.And, this gold particle is forced on to the callus; it means that it is projected ontothe callus with lots of pressure or a force.And, then these gold particle insert inside the plant callus where it goes and integratewith the genome of the plant or the callus.Now, we will take this callus and it will go through the process similar to that ofthe biological transformation.So, it will go through the process of selection, then regeneration, then rooting and furtherhardening.So, the process of hardening is carried out in the greenhouse where we provide the optimallight, temperature and humidity condition.So, the plant that we saw in the bottle, that is taken in a pot which has soilrite in it.So, initially the plant is covered with plastic so, that there is 100 percent humidity condition.So, that the plant does not come under stress and after a few week this polythene is cut,as you can see over here and once the plant is healthy and has good strength then it istransferred into the clay pot.So, here you can see this is a transgenic line which is about 4 months old and thenthe seeds mature and here the seeds dry.The dried seed is then collected and used for selection.So, we take the seeds and grow them on the half MS plate containing hygromycin.So, hygromycin acts as a selection marker and only those plant will grow or the onlythose seeds will germinate which has hygromycin in it.Now, we will see how transformation is done in a model dicot plant which is Arabidopsis.So, similar to the procedure in rice we also surface sterilize the seeds of Arabidopsisand these sterilize seeds are placed on the half MS plate.So, this is the half MS plate where the seeds were put and after about 2 weeks the plantsare of this length.Now, once my plant is having enough root and healthy shoot this is transferred on a soilrite.Now, once on soilrite this plant grows of this height in around 1 month and this ismy fully grown plant which you can see here it has silliques, flowers.For the transformation what we do in a younger stage when the flowers are not even openedthat is it is just a floral bud.The Agrobacterium which is having my gene of interest is used for a transformation.So, for that what we do first we pellet the Agrobacterial culture as we did similarlyfor rice.Now, this pelleted culture is then suspended in sucrose solution along with silvett-77,silvett-77 is a chemical which allows the Agrobacterium to stick to the Arabidopsisplant.Now, that the suspended culture is then applied on the Arabidopsis using the pipette.So, I will put the culture on the buds.Previously people use to completely dip the plant in the culture and, but now that methodis not used anymore.Now, this plant is placed under 100 percent humidity condition and once the plant formsthe new seeds, it can have both the kinds of seed.The first one can be the non transformed one and the second one will be the transformedone.These seeds are collected and grown on half MS plate with hygromycin.So, hygromycin will be my selection marker over here.Once, the seeds grow only those plants will generate which will have my gene or my selectionmarker.So, here this is a transformed Arabidopsis.Here you can see that the plants which I have taken, it is the transformed one and theyare growing healthy.So, these are the transformed Arabidopsis.Here we have placed DR5 promoter and downstream of DR5 promoter we have placed GFP.So, wherever there will be auxin maxima DR5 will get activated and hence GFP protein willbe made and a green signal will be observed.So, we will take this and see under the microscope how and where the auxin maxima is formed.So, we will take this Arabidopsis and place it on a slide.And, see it under the microscope.So, here at the tip you can see that the signal for GFP is high which means that an auxinmaxima is present over here.So, in this fashion we can use this method to temporarily or spatially understand thegene expression pattern also.So, in the lecture you have seen how the temporal and spatial expression for gene is checked,the method is called as in situ hybridization.So, I will show you how we prepare the sections or the tissue for in situ hybridization.This is a wax block which has a stem base of the rice embedded in it.So, we have taken the stem base of the rice, gone through the steps of dehydration thenthrough xylene series and finally into the wax.These wax blocks, are then used for sectioning.So, this is a Microtome.Microtome is the machine which makes thin sections, here we will be making 8 micrometersections of the stem base and then those sections will be taken on the slides and fixed to theslides.We make a 8 micrometer section using this microtome, here you can see the ribbons areformed.Now, these ribbons will be taken on the slide.So, the slides used here are poly-L-lysine coated slides.So, poly-L-lysine basically creates a positive charge on the slide and it helps the tissueto stick to it.Because, you have seen that through the steps of in situ hybridization, the slides goesthrough various amounts of steps and there is a chances of that tissue will fall off.Therefore, the positive slides are used and the tissue does not fall off.Now, we will use nucleus free water.So, that our RNA is not degraded.And we will pour few drops over here, now this ribbon will be taken and placed on theslide.Now, we will place the slide on the hot plate which is at around 40 degree celsius.So, as the water will heat the wax will, the ribbon will starts spreading.The excess water is removed using the tissue paper.So, here you can see that my tissue is spread evenly on the slide.Now, this slide is kept overnight on the hot plate so, that the tissue sticks to the slidecompletely.The next day you will have the tissue completely stuck to the slide as you can see over hereand now this slide is ready to go for the process of in situ hybridization.So, once the slide is dried overnight as I have shown you.The slides are now dewaxed using xylene and then goes through a series of step as youhave seen in the lecture.And, then at the end once the slide is probed and the signal is detected, it is fixed usingentellan and a cover slip is placed on it.So, the slides look like this.If you can notice there are some blue spots over here which is basically the signal, thisis the color reaction.So, therefore, a color has developed and it is seen in particular tissue only.So, now here you on the screen you can see.So, this is the rice crown tissue and this is a primordia which is at a younger stagewhereas, this is the primordia which is at older stage.So, you can see how differential expression of gene is taking place; in the younger tissueyou can see the intensity, the color signal is higher which means, that the gene is highlyexpressed in the younger stage whereas, when the primordia is old the expression of thegene goes down and therefore, the color signal is lower.Therefore, in situ helps us to understand the differential expression pattern of thegene in the tissue, that is why called as in situ in its own place.So, these are the few basic techniques that we use in the study of plant developmentalbiology.And, I hope that this session will help you in understanding the topic much better.So, this practical demonstration is going to be the last class of the plant developmentalbiology.So, we hope that you have learnt something in this field and we wish you all the verybest for your examination and future career.Thank you very much.