Unit 1: Plant Tissue Culture

Un-1)((plant Tissue culture)) Introduction Pront tissue culture is a method of growing lounge numbere of Isolaten cells on tissues under sterile and controlled condiflon. The idea of tissue culture technique was conceived by Chottlieb Haberlandt of Bersin in 1902. He reported culture of lovlated single parisade con From leaves in knop's satt solution enriched with sucrose. He is known as Father of tissue culture. The term plant tissue cultive is commonly used home in a very wider sense to include in vitrom cultivation of plant ceus in an enorganised massed the callus cultures. The terem tissue culture was coined by American pathologist montrose Thomas PourULD WS. Delination Tissue culture at is a mothed in which Fragments froma plantor animae are trans frimed to an artificial mediom ore environment in whien they can continue to Sutuvive grow and function ho the cultured tissue orbea Singee cell, a population of cells on a whole on pant of an ongan. plant Tissue culture - It is grow pant cells, tissues on nutrient culture medium a collection of technique used to maintain on organs stenite conditions on a of known composition. 'Ranjan Kumare Sahoo YT channel" ✓ Plant tissue is defined as R-culturing plant Seed, ongans, explants, OR protoplast on a Hissues, cens on nutrient media Chemically defined synthette enter sterile and controlled U comation of right, temperature and hiroldidy. Aseptic I Tissue culture Techniques - Aseptic tissue etttture culture moans cultu means Suchas viruses, bacteria diseres. and a a septie of the absence the absence of genms, organisme that can cause other microrganisme a technique Aseptic tissue culture technique is designed to provide a bannior betoveen the mices organisms in the environment and the stenile cell culture, und a set of depends upon a Procedures to reduce the Condeurmination from these Sarred. probability & Thus technique primantly solves two basic problems tre (a) to keep the plant disque on cells or explants free from infection of microbes, viruses cind fungi and (5) providing Suitable nutrient media and other en environmental condition for efficient culturing of cells on tissue. The general technique on procedure on steps of plant tissue culture in a aspe aseptic conditim is described below: (1) Explant serection - Sterilization - Tissue culture, laboratory. (vi) Regeneratim (vii) Handenling/Arctimatization (1) Preparation of nutrient on culture mealium (vil) plant trong Fer. (v) Lonoculation callus colture & Suspension culture (۷) E Pallis Fesmaltion & its culture. Sub culture (1) Εχρίαπί δεκαετίνη - Εχprant if the plant post used For: Hasue culture. Explant irsen hew thy and young pant of the plant is creed, parenchyma foom stem, nhixumemes, tubers, noot is easily accessible and respond quickly to culture condition, which have meristematie activity. ii) Tissue culture labonatrry - Fore plant tissue culture, a wel furnished Spacious and planned laboratory with at least one media coom and one culture noom and requined. It should have the Following facilities: Carwashing, drying, and storage off of culture verseis, tubes, conical Flasks and othere gialy wounes preparation of nutrient matte Sterilization Cleaning and Striage of meolia and Othon items. Condition of the A septic condition Culture Room. 5. controlled environmentes conditions for maintaining culture. 6. onservation and evaluation of the cultres The colture room should have controlled temps usually of 85° fac which is main teured bin air conditiorlens and room heaters. The culture racks a be fitted with light (1000 lux on lower generated by tor ate fluorescent tubes and automatic • A shaken timert may be used may be used for agitation liquid cultures. A generator set must be there to Provide pouce in case of electrity Fallure. The tissu culture laboratory Share distillation appanaty, autoclave, dries, physical and chernicou boucances, centrifuge, laminan am Frow machine, agitater, stereoccopic microscope, burners, water bath, oven, refrigerator, PA meter, different glasswanes etc. Borosilicate orce pyrex glas, wines and toltire vessers should be used. The witure tobes on Flask emtaining Sterilized nutrient medium and plant material one plugged cuith non adsorbent Setsi stelsive condition cotton on metal caps Sterilization Sternization means exemination of all living wing anganism (in) (perticularly miento bes). The tissue culture is canning ou Completely aseptic condition for which there is a need of to propency sterilize the glan wane, culture media, instromuments explant ete. These sources of can be sterilized by one of the σε methods- Following ways weight Head- Glass were, culture vessels, and instaments ane Sterilized by day heat in an oven at 160°-180°c The instrument glass bees may include scalpes, Forrests, needies etr ent may bead sterilizer (300°c) for shoung.J Recen Used for Sterninization lizaeronty ty of these instruments. different (b) flame sterilization: The instruments may be fiathe sterilized by dippines them in 95% alcohol followed by firming ] Generatiy mouth of culture vessels arre Flame Sterilized prior to inocularlin on cub culture. (Autociaving culture well vessels, media, fat oultures, plastic wanese etc Contaminate glass waros terilized by inan autoclaven to 121°C at 15 P.S-i (pounds per squand heating inar -Tech, 1.06xg/cm²) For Is minutes. (80-saree mealim) to 40minutes (8.0 lit medion). After autoclaving, it should be opened only when the presure gauge indicates Zeno premure. (4) Filter Genlition : Heat rablime compounds like growth regulators (gibbermell! cauid on Ging, abscisic acid, zealine), certain vitamins and enzyme ane Fritene Sterilized by passing their soutine through HEPA (High efficiency Partioulate air Filtern which flitens cut all particies longer than 0.3 µm ] The die blows at a speed of los km/h which keeps the enclosed woske ing working anon aseptic. (e) wiping with 70% Ethanol The surface of the equipments which can't be Sterilized by Athen technique ane sterilized for example, the piad from of the laminate flow with 701. ethanol, and ause hands of cabinet the operators etc are sterilixed by this process.. (4) Sunfare sterilization of is the sterilization of sunfare of the plant material de explant. The explants are sunfare sterinized by repeated washing in stenite water using disinfectants such as miercuri Chloride, hychogen peroxide, Sodiom or calcium hypoclorite ele. This process depends mainly on the sousres and lymf type of tissue of the explant and its leveing tol ercame to the verifi sterilizing agente. (iv) preponatim & composition of nutrient mella -The culture medium is preparcetin aseptic condition. The nutsient media Provide nutrition to the growing cells, tilsine on explants. Generally all Only nutrientcncdicain Cosynthetic consisting of chemically definal compounds. Fow media contain complex oreganic Substances potato textracte. For different types of culture different type of nutrient medla anes are used because a single medium does not suit for an types of culture experiments. The different types of culture imedia are me medium 6. C munashige and skoog, 1962), (ii) white's mediom (PR white 1963) 60) FR medium (E-R. Falksson, 1965). (v) BS mediom (Ommborg et al., 1964) The most commonly used media ane me and BS media. The basic constituent of any culture medicm ane A Inorganic nutrients: Inorganic nutrients include mamanutrients, Salt of nitrogen and sulphur and phosphonous, pottoussium micro nutrient calcium like buran, molybdenum Chionine, cay zinc, Jaon and chloride. magnesium copper, A source of comboni - suctrote is used as the lastre of carbon. A Growth hormones and vitamins- Severial Growth houromes the like 2,4D, cytokinins-benzylamino pusine kinetin, myolnositol, TAA, NAA are used and vitamins like Inositol, thiamine, Pyridoxine vitamin B1, B2, B6, niacin ay vitaminc etc ane used in or added in a nutrient mediem. - Aftere the addition of all the ingredients in appropriate proportion, agar. agare is added to prepanse a solid melium, in some types of culture (like rool culture) liquid medium is. Use). is used (no-agen-agar is (V) Inoculation & Inoculation is the process of the treanfers of engunt to suitable nutrient mention contained in cololtre vealed. This I's done in sterile condition either in an inaculation chamber on under laminan ain Flow. After the inocuation the cultore vessels ane main tamen in controlled temps and light. The suitable temps for tissue cultore franges botueen 11-25%. vi) calus Formation and its culture: ( A callus isan Cells phous mass of 100 Saly pannanged thin wewind porrent developing from proliferating cells of porrent fitsue the nutrient medios Supplemented toith auxin's Padure cel division and soon the upper surface of expant is covered by coulus. The callus has the bon biological potention to debesop Normal root, shoots ang Ultimatery forming a plant. - callus is formed through I and cen division three duelopmental differentiation by the Courre of exp stages: Paduction, fiation callus Formalin plant, nutrition is govereneg composition of the medidn and enviromental Factors. During the induction pening the metabolic rate of cen is stimulated owing to intrended melabolic nate cells enten to ren division stage. I on the thlind phayse, Cellular differeren tiation and expression of certain metaboric path ways stants leading to serdedany producte. when calivs one grown on a evention to Onutrient mestion Fora long time it becomes Sobraltire it in fren enelia due to nutrient depretion and medion creying. There are tud types of coltre invitore growth culture fuch as (), callus contre - Tissue and cons coltired on an agar gelled mellom Form an unonganised mass of rella cale callus coalus culture need to be subculture 25 weeks in view of medion drying and ank cell growth every numbent depetlay and There fore canuses are every to main thin (b) meoliom produce of low to mast whilary to used a The tiesure and ceus cultures in a liquid Suspension of Singte and clumps of mang cells these ane kallal as sasprincion cultures. generally by a try lagitated, liquid cultures must be constantey gyratory shaker at 100-200 kpm. (b) Suspension *Sue culture ?- Aflere a period of time of colturing cells on fistve in a nutñent mestim, it become thees necessary, chiefly due to nutrient depretim melioom dru drying, to tranfere origan and tissues to Fresh inoculated into new colture tubes er media that means Flask This is known as Sub cu Hurle adhiring sob cultured every In general, callus culture are 4-6 weeks while suspansion cultures need to be subcultured every 2-14 days. vil Regane maution - It refers to the development of Onganized ( Struckres like root, shoot, buds, flowers, somatic embryar etc... Room cultured cens on tissues on explants. This may be also known as ongano genesis lamber ke aisin and cytokinein Growth regulatores iške Nay an important role in groot and root negeneratim. Pudure root Formation, regeneration. Generenity highes auxin/ cyfekir onmation, higher cytokinin/auch induces shoots shast Equadration of auxin & cytokinin ytokinin hduces louth root and shoot regeneration Howevert, variations may occur in responses of different pampe in to growth regulation during ongan regeneration. Alfalfa, application of auxin and and lowers kinetin during Canus initiatim results shoot regeneration in subsequent and on other hand, higher kinetih and lowes auxin concentration indures noot regeneration ina Similan treat ment Regenenation leads to formation a plant-lets. (vii) Hardening at is a proces where plantlets are gradually exposed For acciimatization to the environmental conditions. Accii mali zatim means the adaptation of plantlets to a new environ menfur condition. Handering is a method in which the live culture plants developed in artificial media are habituated to grow in natural environment. Firstly these plants are taken out ferm som nutrient mestia and washed thoroughly cwith anter. Then these plant are grown in nested plastic Pots Freed with liquid mediums and kept in green house fire 6-8 This is called Primary handening Afterwards the plants are transformad to poly bage plued with Potting mixture and grown under shad al house for 6 weeke. This is called secenotony handening ane sulitable Alt carmen condry handen ing in farmen's For growing in Fields the plants (x) Plant Trans Fen when plantiete are hardened, there ane transfes ed to suitable pare like initially to green house and then to Fierd. Serection of plant part (Explant) cells on tiseve Sterilization of Expants 4 Inaccusation of Exoplant on the surface off sorid fieq an liquid nutrient medium ↓ Incubation Chenormally at 85° ± 20 in 508-60% retative humidity ( and 16 has photoperiod in tissue culture noom to modure canus on noots and ↓shoots) Regeneration (Formation of plantlets) Handening/Acclimatizatim Plant Trans Fern (To green house and then to rnd) (Basic Steps Forn, regeneration of complete plant by tissue culture Technique) to promote con division (perticularly in dicots), regeneratim of shoots, ef SE (Somatic Embryo) induction and to growth of auxillary beide enhance prosliferation and Abscisic acid (ABA) promotes SE. and shoot bud megeneration in many Species and mank eduy improves SE maturation. Of the over 200 gibberelling, known, GA₃ is almost exclusively used; it promotes Shaol generation and SE germination The common range of oncentrations used ane as follows: auxin, 01-3 mg/1; cytokinin 0.1-3 mgle; Arot a lang le ang GAs 0.1-1 mg18. complex organic Additives so earlier studives, compex additives like Yeast extract, coconut milk, casein hydrolysesate, corn milk, malt extract and tomato juice were used to support planit tissue growth. But Such additives are no more preferred. In many cases, their effeus could be reproduced by a single acid z'e L-asparagine or miglutamine. Such additives should be used only when "Synthetic Solidifying Agents Agan in tissue culture media Foom ured most commonly preporabition & a ned age, Gelidian amango. isa as solidi fyingagent pe polycaccianide ccharide cotalme PH The pit of the medium maintatied at 5.0 to 60 fer maximum growth and development of cultured wens on tiesues. Organogenesis In pant tissue culture, enganogenesis isa process of differentiation by which plant ongons like resis Foremed Fram the shoot, buts exc. an'e Unusual points of enigin of an organixeg explants whenel a preformed meristem is sacking. onopnogenesis starts with stireviatial caused by the chemical of mediom Substances)contri ed avere from the exiginal explants and enitegenous ty compound for pistetuced by the culture. Onganogenesis can be indue caux'in and eytokinin. Skoog and induced with the appiloution vanying proportion of miller demonstrated theet a high a high ratio of auxin Cytokinin (3:0.02) stimulated noot formation in to barro Callus but a low ratio of some (8.0.2) induced sheet Fermation, - plant production through onganogenesis achieved by twis modes! Can be achieve ) Emergence of adventitious organ directly from the explant ) Angorogenesis through carus fromation reactivation of genes concerned (d Dineed Adevintl the Formation Every cell. Adventitious organ formation X genome. The te through Frir ma fie mito tic divisim's adventitious with of plant is derived from containing the complete buds depends on the embryonic phase of development The coleti in suitable medium supplemented with growth hormones the formatie tissues of higher plants and capable of regenerating adventitious bas bud/shoot. These bude ane Fesmed dinecdiy & Farm a Plant onyon or any piere of fistue with out forming any Caling structure. Composition of Nutrient medium In generas - Ho, segueyter Sequestnene 335 Fe The nutrient media contains the following components in Inorganic minerals: gonerar! Macronutrients like micronutrient ilke fe, ma, zn, tu, Na, B, in the nirment medium in the fami N, Pk, ca, s, mg etc. and mo, ci ffed and used form of salt in different concentratim as pere requirement. Areino Acid The most widery used amins auld ane L-asporragine, L-glutamine, L-arginine, glycine, etc. ③ vikumins: fac quimum callus growth, the following vitamins are required inosite), thiamine, Pyridoxine and nicotinic acid of which thiamine is essential and the rest ane promatrry pants thenic acid is also known to be promotory, but is not incuded in most of the recipes. Thus the vitaming used are mainly vitamin A, Be, niacin, vilmamine. Carbon Source Sucrose (20-95091) is the most commonly sed Couriern Source for all cultured plant matériaus including even green 2001- Autoclaving hytoxyber Sucrases, which enhances its availability to pront celo In some m System że monocuts, glocose maybe Superior to Sucrose.. plant tissues can utilize othen sugaas like maltose, gavautose lactose, mannose and even sitarich, but these are ranely used. ⑤ Growth Regulates Auxin die Inn (indole-3- acetic acid) IBA (Indole-3-Butyric acid), NAA (napthaiene acetic acid) NOA (Napthoxy acetic acid), 2-4-D(2,4-dichlorophenoxy acetiz ald) ete are growth regulates which are commonly used to Supprit ces division and callus growth (especiany 2,4-D), Somatic embryo incluction, rooting etc. Cytokinins like kinetin (6-FunFunylamino Purine), PAP (Ben amino purine), Zeadin, zip (kopentye adenine), TDZ (thi dia zunan, a compound having Cytokinin activity) ane employeshad In plant tissue culture technique, an explant is taken this to Humreal on nutrient mealiom under contain emdition & finary we obtain a whole new plant. This will happers due to the inherent capacity of plant cell that arre differentiation and cellular to tipatendy. The potential of a plant cell to grow and develop into a whole new multicesnular plant is called totipotency. OR The property of a single cell to differentiate into many other cell types leading to the formation of a complete plant is defined as totipotency. The tenso "totipotony" war coined in 1901 by T.Il mongan. During tissue culture practice, an expians is taken forma Mature tissue. It means the cell in diviăng and quiescent in nature. ma differentiated, explants ane generally non- To show totipatemy which neven Petron into a meristematic state Such mature, non-dividing codengues a changes This phenomenon is Phenomenon of reventing back of makere cens to dividing state is Cassed as de differentiation. have the ability to from a whole plant of plant organ is known as Now there re- differentiation. dediffement atmed -Thil phenomenon Usually the defft defferentiation of the explant leads to the Formation of callers. How ever the emp embryonic explants, sometimes result in differention of cost and shoot without an interme Slate. Callus state. diary The prrepenty of totipotency of plant ces indicate that even up unditherms Hated cells of a callus carry the cabentiae genetic information required for regeneration of a whole plant. It is also clear that an the genes Orepensibue dedifferentiation and πε differen Hation are present within the individual sens and they become active Firexpresion enden adequate to culture condition. It is to be noted here that only thing fand plant cons have the ability to negenerate a complete plant and are totipotent where af dead cen's cubich lack cytoplasm and nuciens (trach eids, vesel elment etc.) arve nos totipotent at all Totipotency is not similar in all pran pants of the plantaivas Application off Totipotency - Favowing Follow ane various application of tipotency: It has potential appilcation in the corcap plant improvement. var micro-propagation of commercially important plants. 3. production of artificial of Synthetic seeds. It helps in conservation of germ plam. 9. This ability is utilized for haprold produckim. 6. It is applied in production of n Somatic buy bride & Cybrides 7. It is helps to study cytological and histological differentiation. 8. 9. For high scale and efficient production of secondonry meta hesites. very minute and diffiwit to germinate. yt also helps in cultivation of those plants whose seeds are This type of onganogenesis & mastry Fund in herbaceous plants. 11) Grupnogenesis enough canus Formatim: ( (ie Sometimes plant regeneration form Cultured explant Rotyledons, hypocoty, stem, leaf, sheet apere, shoot, root, young Pafiorescence, embryos, Callus then etc) in voives the initiation of shoot bud differentiation. basa For differen Species different kinds of explants may be necessary forts Successful plant regeneration. Through the expants with mitotically Shoot tips, axinary, ane used su alfie cell te mel meristems , buds, immature leaf, hay immature embryes fon sucress for, to callus initiation and cuso can the oned success fully to tatt initiate plantlets through Ongathogenesis. mbryogenesis (Somatic and zygotic embryogenesis) - The process as emany genformation and desnesopent of an embryo is caured Embayo geneti's Jenesis stants Froom a single embryogenic cell, that can be a Zygote (the product of the Fusion of an egg and sperm during fentillization) on an undifferentiated casus (er). Embriyos are of too to Following type! Kygatte ombeyas Non-Zygatic embryos . Zygetic embryos one Formed by the zygote on the Fertilized egg. Non-zygotic ombay os can be further be segregatool into Samoulic Einbeyes These are fromeel by the sporophytic cell inan in-vitro scenario. These somatic embryos directly amenging from orthon, organs on ane Reffered aquentive embryos. Embryos Andriogenetic embryos & Formed patthieno capipic Embryos: Un Fentil by the male gametiphytes I formed by the (Zygatie Ermbeya gene The procese of Formation of embryos and its development firm Zygote is called as zygotic embryogenesit. The embryos that are foram from Zygote is known as Zygotic embryos are often simply embriyes. Zygotes, when cultured in a nutrient medlim devido and developed into giotulan stage, they one heart shape ori tempedo- shop Shuped, cotyledomany stage and It can Fineury they germinate and produce plantlet. excute naturally of atch Ficiary (by tissue culture tech)