Tissue-culture-Types-Hybridization. pptxpptx

Plant tissue Culture

1. 2. Enzymatic method Leaf s t er l i z at i on, epide r m i s r e m oval of M i xed en z y matic S eq u ential e n z y matic P ec tina s e + ce l l ul as e P ec tina s e is o lat e d c e l l s ce l l ul as e Proto p la s m Iso l at e d Prot o p la s m r e le a s e d R e le a se o f Proto p la s m r e le a s e d Plas m o l y s e d c e l l s Plas m o l y s e d c e l l s

2. ISO L A TION OF PR O T OP L A STS in bri e f • Disadvanta g e: 64 • Gene r ally us e d • Advantag e s: 1. La r ge qua n t i t y o f p r o t o p l asts o bta i ned 2. Cel l s not b r o k en 3. Osm o tic shrin k age is much less Unk n o w n e ff ects of en z ymes on p r o t op l ast • Used o c casi o n a l l y • Advantage: Unk n o w n e ff ects o f en z ymes o n p r o t o p l ast el i m i nated • Disadvanta g e: Cel l s m a y be b r o k en Enzymatic Mech a nical I s ola t ion, main l y f r om le a v es & pollen

3. P R O T OPL A ST C U L TU R E Isol a t e d pr o t opl a st are u s u a l ly c u l t ur e d i n e i t h er l i quid o r s e m is o l i d agar m e d ia p l a t e s . T h ey re q u i re so m a t i c pr o t e c t ion i n c u l t ure m e d i u m u n t i l t h ey g e n e rate stro n g cel l wal l . M e t h o d o l ogy o f P r o t o p l a st c u l tu re i s d e scri b ed b e l o w: Isola t ed P rotoplast is cleaned by centri f ugation and decanta t ion me t hod a The p r otopla s t s o lution ( 1 × 105 p r otopla s t / ml) is pou r ed on sterile and cooled mol t en nu t rient medium Mix t he t wo gently but quic k ly by r o ta t ing ea c h petridi s h Al l ow t he medium to s e t, seal petridi s hes with para f fin fi l m a nd in c ubate Protopla s ts c a pable of dividing und e r g o c e ll divisions from callus within 2 - 3 wee k s The c a llus is then s u b c ulture on fre s h medium 65 Embryogene s is begin from callus w h en it is tran s ferr e d to a pro p er nutrient medium and s ub s equen t ly w h ole plant de v elops

4. A p plica t ions of p r otoplast cultu r e T o deve l op Novel hybr i d plant t h ro u gh pro t opla s t fusion. I n single c e ll deri v ed c olon y , isola t i o n of m utan t s th r ou g h m utag e ns i s ea s ie r . Sing l e ce l l clon i ng can be ea s i l y pe r for m ed with pr o t o p l as t s . Regener a t i o n of entire plant or p l a nt i m prove m ent t h rough pro t opla s t cul t ur e . 1. 2. 3. 4. 5. Gene t ic ach i eve d . tr a nsfo r m at i on th r ough DNA uptake can be 6. Reproduci b le protoplast t o p l a n t s y st e m s for m any plan t s of ag r ono m ic valu e . are n ow avai l able 66

5. P r ot o plast fusion • Protop l ast fus i on • I t i s the technique i n w h i c h t w o o r m ore p r otopla s t are fused into a s i n g le cel l pr o to p l a st. • The p r oto p l a st fusion a l lows u s t o bring a n desi r a ble plant trait s i n co m bi n a t ion that are no t p o s s ible b y s e x u al m e a ns. • May occur betwe e n sa m e o r d i f ferent p l ant • Mut i n u cle a te p r o t o p lasm o b tained after f u si o n 67

6. Three m ain p h ases are t h ere f or f u s i o n : 1. Agg l u t inati o n : Plas m a m e m b ra n e of two o r more pr o t o p l ast are b r ough t int o cl o se pr o x i m i t y . (A and B) 2. Me m br a nes o f pr o toplasts agglutinated b y f u s og e n get the the f u s ed at the point of ad h esio n . This results in fo r mation pr o t o p l ast. of cytoplasmic brid g e be t ween 3. Rounding ex p a nsion off of the f u s ed protop l a st due to the of cy t oplas m ic br i dge f r o m i n g a s p he r ical he t r o k a r yon Binu l ce a te ( A - B) or h o m o karyons ( A -A or B - B ). nuclei he t e r o karyon s . The f u s ion of the results in a te t raplo i d hybrid c el l . Also c y b r id cell is 68 f ormed with a s e l e c t i v e ch r omos o m e loss .

7. Stages of P r otoplast f usion a n d Hybrid i zation Pro t opl a s t A Pro t opl a s t B A+B 2A 2B A - A Ho m okar y on B- B Ho m okar y on A - B Hetro k a r yon C y brid (Cytopl a s mic hyb r id ) A - B H y brid 69

8. Me t ho d s of P r otoplast f usion T e c h n i q ue s /Meth o ds of Proto p la s t F u sion (Fusi o n of pr o to p l a sts of t w o d i f fer e nt g e n o mes) 1 . Spo n t a neous F u s i o n 2 . I n d u ce d F u s i o n In t raspecific In t ergeneric Che m ical Mechanical Ele c trical

9. T echniques/ M e t hods of Protoplast Fusion S pontaneous Fusion Protopl a st f u se spon t an e o u s ly du r ing i s olation proc e ss mainly physi c al c o ntact am o ng similar pa r ental p r otopla s t. due to  Protopl a st f rom their adjoini n g c e lls fu s e th r ou g h the i r  pla s mode s ma t a t o form a mult i nu c leate p r otopla s t. T wo t y pes of s p ontaneo u s fusion • Intraspe c if i c p r odu c e homo k a r yon e s • Intergeneric have no importan c e Indu c ed Fu s ion Fre e ly is o la t e d p r o t oplast f rom di f f e r ent sou r ces a re help of fusion indu c ing age n ts.  f u s e d with t he  Fu s ion n e e d s an ind u c i ng ag e nt t h at a c t ually r e duces ele c t r o  neg a ti v ity and allo w s the is o lated p r otopla s t t o fus e . The v ario u s fus o ge n s u s e d f o r ind u c e d f u sion a re various cl a ss as b e lo w : cl a s sified u nde r  ◦ ◦ ◦ a) C h emic a l fusion b) Me c hani c al fusi o n c ) Electri c al fusion

10. a) Chem i c a l Fusion Ca 2+ T y p es of Polyvin y l n ega t i v e f u s o gens a re u s ed l i k e PEG, N a No 3 , ion s , a l c o hol (P V A ) etc. T h e cell m e mbra ne p os s es c h a rg e a nd a f t er tr e a t ment w i th s u c h c h e m i c a l a gen t s w he n cell m e mb r an e a re b ro ught i n to c lo s e phys i cal contact they fuse. Mech a n ical Fus i on I t i s n ot dep en d e nt upo n t h e p r e se nce o f f usi o n i ndu c i n g b) a gen t . P h y sical f usi o n of p r o t o plasts is d one un der micro s c op e micr o p i pe t t e . by usi n g micr o ma ni p ulat o r and per fu s i o n c) Electr i cal Fusion Pro t op l ast a r e pla c e d i n t o a s m a ll cu l t u re c e ll c o n t ain in g m - 1 ) elect r od es a nd a p ot en tial d i f f e ren ce ( 1 kv i s a ppl ie d th e n t he pr ot o plast w i l l l i n e u p ( f o rm ch ain) b et we en t h e e l ectrod e s . Fu s ion o f pro to plas t s o f cha i n is in d u c e d by t h e f o r m - 1 ) a p p l i c at i on o f hi g h str e n gth e lec t r i c fie l d ( 100 kv

11. Somatic hybrid i z a tion Defina t io n : The t e c h nique o f hyb r id p r oduct i on t hro u gh the fus i on of i s olat e d somat i c p r ot o plast un d e r i n - v i t r o condit i o n s a nd subse q u e nt de v e l op m ent of their product to a hybrid plant is called so m a t ic h y bridiza t io n . 73

12. V arious sta g es of s o matic h y bridization are as fol l o w s 2. Fusion of the protoplas t s of desired species/ v arieties 5. Regeneration of h y br i d p l ants 4. Culture of the h y brid cel l s 3. Identification and Se l ec t ion of somatic h y brid cel l s 1. Isolation of pro t opla s t

13. 1. Isola t ion of P r otoplast Sa m e as dis c ussed be f ore Fusion of i sola t ed P r otop l ast Sa m e as dis c ussed be f ore Identi f ica t ion and Selec t ion of somat i c hy b rid ce l ls 2. 3. The proto p lasts of two parents m ay be labe l led by d i f fe r en t fluore s c e n t co m pounds, which will then ena b le for t h e iden t ifi c a t i o n and s e le c tion of hybr i d s . Ident i fic a t i on is based on di f ference b etween t h e pa r e n tal c e lls and hybr i d c ell w i th re s pec t t o P ig m enta t ion, Cyto p las m ic m ar k er s , Presen c e of c h lo r o p las t , Nuclear st a in i ng et c . I n t h e m ix t u re of both fused and unfused protop l ast S e lecti o n of hybrid i s d one on the bas i s of se v eral p roced u res like Ge n et i c co m ple m en t ation, a n ti bi o te c - r esis t ant c ell li nes, isoenz y m e autotr o ph y , an a lys i s by an a lys i s, Phyt o tox i n s , Spe c ific a m i n o a c id, A uxin Auxotrophic and m et a b o l ic m utant s , Chro m os o m al hybi r d ce l l, Herb i cid e s et c . 75

14. 4. C u l t u r e of the hy b rid cel l s Hybrid c e l l s are cul t ur e d on su i tab l e m edium prov i ded with the approp r ia t e cul t ure condi t ions. 5. Rege n erat i on of hy b rid plants Plan t s are induc e d to re g ene r ate from hybr i d ce l ls. These hybr i d plan t s m ust be at least pa r t i ally fert i le, in addi t ion to having so m e sche m es. useful prop e r t y , to be of a ny use i n br e eding 76

15. Adv a n t a g es of so m a t ic hybr i d i zati o n Novel ap p ro a ch for i n t r od uc i n g or inc r easing ge n et i c  varia b il i ty a t nu c lear or extra nu c le a r o r gan e lle ge n o m e level, i n higher plan t s Production of novel int e rspeci f i c and int e r gen i c hybrid  ◦ e.g. Po m ato (Hyb r id of pota t o and to m ato) S o m atic hybr i d iz a t i on i s signi f ica n t i n i m prov e m ent of p l a nts such as banana, pota t o, suga r cane, and ya m .  Produc t ion of f e r ti le d i pl o ids and polyp o ids f rom sexually  st e r i le haplo i ds, t r ip l o i ds and aneup l oids T ra n sfer g e ne for dise a se resistan c e, a b i o tic stress resi s tanc e , he r bic i de re s is t ance and m any other qual i ty cha r ac t er s . Produc t ion of he t e r o zygous l i n es i n the s in g le species w h ich cannot be prop a gat e d by vege t at i ve m eans Stud i es on the fate of pla s m a genes Produc t ion of unique hybr i ds of nu c l e us and cytop l asm    

16. Limitations of S o matic hybridization  Poor re g ene r ation of hybr i d plan t s  No n - viab i l i ty of fused produ c ts  Not succ e ssful i n all plan t s .  Produc t ion of unfavorab l e hybr i ds  Lack of an e f ficient m ethod for se l e c t i on of hybr i ds  No con f i r m ation of e x press i on of part i cu l ar tra i t in so m at i c hybr i d s .  Eli m ina t ion of ch r o m os o m es from the hybr i d ce l l

17. PARENT PLANT A PARENT PLANT B TR E A T M E NT WI T H FU SI ON C H E MI C A L I SOLATED PRO T OPLASTS B I SOLATED PRO T OPLASTS A AG G REGAT I ON PRO T OPLAST F U S I ON 79 PRO T O P L AST F U S IO N FROM 2 DIF F E R ENT P L A N T S P E C IES & LA T ER P L A T I N G & SE L ECT I O N O F HYBR I D C O L ON IES & REGE N ERAT I O N O F “ SO MA T IC HYBR I D”

18. PLA TI NG OF F U SED PROTOPLAST SELECT I ON OF H Y BRID COLONIES TRAN S FER OF C ALL U S TO D I FFERENT I AT I O N M E DI U M SELECT I ON OF S O M AT I C HY BRID PLA N TS 80