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Module 1: Plant Development

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Nutritional Requirements of Plant Cells

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We all know that any kind of living cells would be needing a carbon source mineral nutrients.Now,whole plants they they take their mineral nutrients and water from the soil, but howis it different in plant cell and tissue cultures that generally in whole plants vitamins andyour plant growth regulators they are synthesized at the meristematic regions or in young plants.Probably the reason is because that those are the sides where they are most needed andthen after the production then they can be transported for various otherdevelopmentalreasons to matured plants,parts of the plant.So,vitamins and plant growth regulators as I said are synthesized in the meristematicregion.Now, apart from mineral nutrients and carbon plant growth regulators they are two kinds,yesterday also I mentioned.One are endogenous which means the cells themselves produced these plant growth regulators.So, there they are called as plant hormones, now one if they are synthetically producedor they are exogenously being present as signaling molecules for example, which are theninducingthe endogenous plant hormones, then they are also called as plant growth regulators .So, initial attempts which were made in media formulations and plant cell and tissue culture,they included application of knop solution.Now, knop solution is a very old media which is majorly composed of major salts.Have you heard about, what what kind of salts come under major class of salts?There are have you heard of micro nutrients?Yes.And then major nutrients.So, any examples for major nutrients?Potassium.Nitrogen, carbon, phosphorous, sulfur, magnesium as you said sodium, calcium and micro nutrientsfor example, boron and . Manganese.Manganese Manganese.Cobalt Iron .Copper.Copper.So, knop solution was initially it was only composed of major salt solutions.So, initial preparations of plant tissue culture media, when they were being discovered ormade it then knop solution used to be used or or whole plant extracts or like for example,coconut water we were mentioning.So, coconut milk, coconut milk was being used or extracts or juices from the plants or anyother biological origin.So, thenmajor formulations of these synthetic media; so, initially it started with non-definedmedia and now we are at a stage where media is completely defined.Why do you think there is a need to come?Anyways you see that it grows so well in complex media, living organisms isn't it; same wasthe case here coconut milk was being used very nicely.Juices and extracts were found to be working well, but in 1930s to 50s there was a shiftand now we are at a stage where you can find readymade media, but they are completely definedmedia.Why do you think there was a need to shift from a complex to a well defined media?Batch to batch variation can be there, what else?Any other logical reason?Depending on how much impurities you get, but what other logical reason do you thinkpeople might have?Do you think downstream processing that time would have been the reason?What did I say in the earlier classes that even though these complex media found to beworking well, but everything is in turned dependent on nothing is full proof, is dependenton so many different factors.The species, the type of explant, the genetic makeup.So, oh up depending on what culture do you want to induce also the media compositionhas to be varied.So, oh depending on what was the objective of the study there could be a better formulationof the media.So, until and unless you have the clarity of the medium composition and how are theseeach factors affecting your objective, it will always be a hit and trail isn't it.So, gradually depending on the purpose of the study these different media formulationscame into the picture.So, starting from knops, then you got Miller's media.Then what else?Murashige and Skoog B 5, but there has been a difference.For example, in Murashige and Skoog and between Gamborg's medium which is also called B 5medium there is one major difference.The ratio between the ammonia to nitrate, in B 5 the ratio of ammonia to nitrate isvery less.Now, why this difference is important for example, as I said it is species specificand the kind of culture you want to induce.For example, when we were working with as a directent where woody plant species likeneem, neem is a wood woody plant.And, we were finding it very difficult to induce in vitro cultures and MS media is generallyused when you want to do shoot but formation, shoot multiplication on in vitro culturesso,but it was not working so, but B 5 started working.So, when we started doing and that also I will come back to when we will talk aboutmedia optimization.When we did statistical optimization which has the advantage of of doing studying theinteractive effects and even studying the single effects, we could find that if youremove ammonia from the medium um and have only nitrate as nitrogen source it is veryuseful or 100 percent efficiency for callus induction in directent.So, it is sometimes explants specific, species specific.So, B 5 was then used rather than MS media because, it has lower ammonia to nitrate ratio.Then the initial discovery alsowhich was remarkable and which was very successful was the discoveryof auxins.The first auxin which was discovered was IAA, IAA in plant is synthesized by through tryptophanbio synthesis pathway.So, then synthetically also IAA subsequently was produced.Then the discovery came with cytokinins, now again in cytokinins also there are syntheticcytokinins and which are endogenously present, naturally present cytokinins.Now, addition also please note that add exogenous additions of these plant hormones, even ifthey are present endogenously or not present endogenously e[ffect]- they also effect thesequestration, the production and theconjugation of these endogenously present hormones orthe production within the cell.So, it has to be optimized, then came the discovery of totipotency and regulation ofmorphogenesis.Now, first came the callus induction, then there was a need to find a media which couldinduce root, then there was a need to find the objective became regeneration or shootmultiplication micro propagation.So, gradually with thedifferent objectives in mind different media formulations cameinto the picture, but I must say that Murashige and Skoog medium which came in 1962 till dateis one of the most successful media which formulations, which areable to give resultsin most of the in general almost all the species.But, still that does not meet it will always work, one has to optimize the compositionof the media then as well as the growth hormones.So, rest of the factors have to be optimized even the environmental conditions play a role.So, these are some of the examples of different media which are now available in the marketGamborg as I said is B 5, it contains B vitamins.So, then that was another discovery, whenvitamin addition in the formulated media was alsodiscovered where at 3 major vitamins pyridoxine, thiamine in were added and along with yourvitamin B 5.So, the nutrient mediais composed of these different things, one is inorganic nutrients,then carbon sources, vitamins, growth regulators and organic supplements.Organic supplements here any idea?Complex nitrogen sources or like for example, organic nitrogen sources, any organic nitrogensource which is well known well used in microbial fermentations . What else?Amino acids are also added; yeast extract, peptone, malt extract.So, coming on to the inorganic nutrients, now inorganic nutrients as I said apart fromcarbon, hydrogen, oxygen there are 12 other elements.Now, what are those elements?The major elements include nitrogen, phosphorous, sulfur, calcium, potassium and magnesium;the micro nutrients generally added include iron, manganese, copper, zinc, boron and molybdenum.So now, let us talk about the nitrogen which is supplied.So, in plants, in whole plants in nature nitrogen is assimilated in plant in the form of nitrateor ammonium ions.Now, there are ammonia transporters so,nitrate there are many nitrate transporters.So, inorganic salts of nitrates they are assimilated by the plant mixed in the xylem and the otherthrough minerals, they come to the plant either in the root or in the shoot itself.It is then converted into nitrate and then from nitrate it is converted into ammoniumand assimilated in the form of amino acids and other nitrogen compounds.So, which pathway have you heard of any pathway which is known in plants for assimilationof nitrates, where nitrate reductase nitrate reductase is involved?What is that pathway called?Gogat pathway, have not heard ok; so, please go back and read.It is about how nitrogen is fixed for the by the plant, nitrogen in the form of nitratemajorly.So, it is an energy driving process, it requires energy.So, active transport of nitrate happens; sulfur, sulfur is supplied as sulphates.Then it is utilized where all?In protein synthesis, inorganic sulfur maybe supplied by organically bound sulfur; organicallybound sulfur is present in the form of cysteine.Cysteine.So, phosphorous is a major nutrient ok.So, then calcium, magnesium, potassium they play a role in cell metabolism.Let us see for example, magnesium; magnesium plays a role in translation, it acts as acofactor.Then calcium, calcium it acts as an activator and inhibitor in various enzyme activitieswith in glycolysis.Then it is required in cell wall formation as calcium pectate.So, calcium is a important nutrient, then calcium pumps and calcium binding proteinswhich are present they demonstrate that it has a significant role as a in the cell wallformation as well.Now, calcium is also used as a secondary messenger.So, primary messengers are the signal molecules outside the cells and secondary messengersin response to the primary messengers, there is a signal cascade inside the cell whichthen regulates the metabolism of the cell.So, calcium is one of the secondary messengers in the cells, then micro nutrients for example,your boron, manganese or copper or iron they are inducers of enzyme synthesis.Now, boron is used for membrane function and it also affects thesequestration of otherelements as well.So therefore, this is also one of the crucial elements which might have come into the picturewhile formulating media.Now, carbon sources, it is again very species specific; as I said generally sucrose is used.Now coming to the question, why do you think generally sucrose is used?Ultimately, it has to be broken down to the most easily metabolisable sugar which is glucose.So, sucrose one is it is less costly, there are sucrose transporters present in the cellwall and also the invertase enzyme.So, it is it is not that all the sucrose in the media at one time initially itself getsbroken down, this is a simultaneous phenomena.Sucrose is been taken in and it is also being divided into or broken down into glucose andfructose.And, separately glucose and fructose are also being taken in in and it is then divert towardsglycolysis and sucrose also has sucrose metabolism is present in the cell.So, sucrose is also taken up by the cells.So, as she said if there is only glucose present, the growth can be faster because it is aneasily metabolisable sugar readily available to the cell can be taken up.But, once is the cost and the second is the not all glucose is needed at one given time.So, slow assimilation is also one of the reasons.Now, vitamins; vitamins for example, thiamine is essential vitamin, then vitamin B 3, pyridoxine.So, as I saidyour vitamins which are included in the media they include pyridoxine, nicotinicacid, niacin in and your thiamine which is present in these media which has called asB vitamins to induce the growth.Other vitamins which might be added depending on the species or the objective are vitaminC, D and E. So, amino acids and organic supplements they serve as reduced nitrogen, complex organicsupplements like casein hydrolysate, then yeast extract coconut milk they are till datebeing used as complex nitrogen sources to reduce; the why are they still being preferreddespite the fact we were saying that it can lead to batch to batch variation this andthat?. To see if you can control the batch to batchvariation, these things can bring down the cost of the production media because, manyof these are obtained as?Waste by products.Waste by products, by products which are in the waste of the industries.So, now let us talk about plant growth regulators, organic compounds which affect the morphologicalstructure and physiological process in plants in very low concentrations; they form theplant growth regulators.So, as I said these plant growth regulators can be native, can be synthetic which meansexogenously added which can modify the plant growth.When they are native or endogenous they are called as plant hormones and rest are alsocalled as growth regulators.Now, as plant growth regulators some of these signaling molecules they are also termed asplant growth regulators.Now, endogenous which is one of the hormones which is volatile agent is your, any hormoneswhich you know is a volatile agent and forms a part of plant signaling?Ethylene.Ethylene, very nice even these jasmonates when they are salts of jasmonate they areexogenously added like methyl jasmonate, otherwise jasmonic acid is a part of the signal cascadeinside the cell as well.So, auxins, ethylene, abscisic acid, cytokinins and gibberellins these are major class ofplant growth regulators or hormones.Thenbecause, these are naturally present, then auxins and cytokinins they interact andthey are important for regulating growth and development of the plant.Now, ethylene abscisic acid and gibberellins they generally form the regulatory role inplant metabolism or in the in vitro cultures.Now, synthetic compounds which are not natural plant hormones as I said they are called asplant growth regulators.Although, it is mentioned here jasmonates polyamines and salicylic acid, now salicylicacid is also produced as a secondary metabolitan many plants.But, when exogenously added it forms a signaling part of the signal cascade agent and alsojasmonate salts they then are termed as plant growth regulators because they are being exogenouslyadded.Otherwise jasmonic acid is a part of endogenous signal cascade mechanism in secondary metabolism.So, the response to plant growth regulators on growth and development in culture varieswith culture conditions, type of explants and genotype.Now, exogenously applied plant growth regulators they affect cellular mechanism and as I saidalso the sequestration, activation, transport and sensitivity even towards the endogenousgrowth substance of the same kind or even of the different kind.So, auxins they strongly influence processes like cell expansion, cell wall acidification.Now, it is said that auxins they they are useful in the growth or extension of the plantroots shoots length.So, by the mechanism which is cell elongation and cell wall acidification.So, now how does it happen?Auxins they not only help inexpression or controlling the expression of certain proteinsin the cell wall which are called as extensions; so, which loosen the cell wall.So, and prepare the cell wall material extra cell wall material, nowcell wall acidificationis another mechanism where auxins are involved in which they induce H ion concentration inthecell wall region.Because, of this what happens now H ion concentration has increased, the water gushes inside andcausing a turgor pressure.The vacuole gets extended, forces itself against the cell wall.The cell wall has been loosen by the combined effect of via auxin by having these expressionof extensions which then loosen the cell wall and therefore, the cell wall is now extendedand expanded.So, this is how the cell increases in size, in length elongate and cell wall materialis formed and the length is of the plant or that tissue is extended.Now, inside the cells the free auxins are synthesized and stored as conjugates, nowthere is a purpose why auxins are stored as conjugates.So, that they can be use later otherwise free auxins can get broken down.So, in order to prevent their break down they are stored as conjugates with amino acidsor alcohols or sugars thereby, protecting them from the oxidative break down.And, whenever needed enzy[me]- it is an enzymatic process through which the enzy[me]- theseauxins are set free in the cytoplasm or in the cell.Now, IAA as I said is a naturally occurring auxin which is synthesized from tryptophanbio synthesizes pathway.Now, cytokinin; cytokinin induces cell division by accelerating or inducing mitosis.So, it regulates the activity of a protein which then activates mitosis in the cellsso, thereby enhancing cell division . So therefore, if you think go back and think why a callusinduction is an event which happens, because of the combination of cytokinin and auxinit looks logical.Because, there is cell elongation, cell expansion and cell division which is happening and itis also in in vitro plant tissue culture.Higher concentrations of cytokinins can even lead to release of lateral or lateral buddormancies which means branching can happen then .So, cytokinins exert control over events leading to mitosis and they can also in some speciesthey have been found that at they can even promote chlorophyll biosynthesis.So, most commonly used cytokinins are zeatin, kinetin, benzyl adenine.So, you will find majorly benzyl adenine being used, evenyour thidiazuron is also used.So, cell differentiation and organogenesis in tissue culture is controlled by interactionbetween cytokinins and auxins.So, now some general guidelines, but let me tell you these general guidelines are generalbecause for most of the species they have found to work.But, still it does not mean depending on what species are you are working; you can takeit as a guideline, but it is not necessary that the same concentration or the same hormonewould work in that particular species.So, optimization is still essential.So, what are these general guidelines?Plant growth regulators a combination is required for sustaining the growth of in vitro cultures.Now, when you need cell proliferation which means cell multiplication to happen 2, 4 dichlorophenoxy acetic acid which we know as 2, 4 D or one napthelene acetic acid which is NAAor a cytokinin.So, these are auxins then cytokinins like kinetin, benzyl adenosine and I said thidiazuron.Now, for regeneration which needs organogenesis to happen.So, for regeneration what is in general found to work is you reduce the concentration ofauxins and increase the concentrations of cytokinins.So, generally it is seen that once you reduce the concentration of auxin to cytokinin whichis a ratio of auxin and cytokinin, then it leads to organogenetic events.2, 4 D in dicots induces cell proliferation, but suppresses differentiation while, in monocotsin herbaceous species it has been found that high concentrations of 2, 4 D can lead tomorphogenesis or organogenesis thereby, leading to even somatic embryo formationsSo, how is the media prepared?The media is prepared in the form of generally these are the recommended guidelines.You use10X to 50X stock solutions of your major salts, micro salts are autoclaved andseparately and including your iron it is in the form of stock autoclaved separately.And, then they are mixed at diluted concentrations depending on your media formulation, thenvitamins because some of these vitamins are found to be heat sensitive, heat labile.So, vitamins depending on what vitamins you are using care should be taken whether youcan afford to add them before autoclaving or they should be added and filter sterilizeafter autoclaving the major and micro nutrients.Now, in general theplants grow well in acidic pH which is nearly acidic in the sense slightlyacidic pH which is around 5.8 to 5.8 around so or 6, 5.8 to 6.So, the pH isthe initial pH of the media is then maintained after autoclaving because,during autoclaving the pH can get disturbed.So, after autoclaving the pH should be ideally maintained to 5.8 to 6 and then gelling agentsare added, if you want to work with solidified medium; acidic pH would prevent gelling.So, the concentrations in general preferred are 0.8 to 1 percent weight by volume of agaror any gelatinous substance.