"

C H A P T E R
Protoplast Culture and Somatic
Hybridization
U.K. Tomar
1*
and P. K. Dantu
2
1
^rld Forest Research lnstltute, Hew Pall Road, Jodhur342005
2
Dets. ol Botany, Dayalbagh Educatlonal lnstltute, Dayalbagh, ^gra282 005
E-nall: uktonar_alrl.res.ln
*
Corresonding auIhor
In IanIs, vhere fairIy disIanI secies couId Le crossed, iI has aIvays noI Leen ossiLIe Io
oLIain fuII hyLrids LeIveen desired individuaIs Lecause of sexuaI inconaIiLiIiIy Larriers.
This has ofIen roved Io Le a serious handica in cro inrovenenI rograns Ihrough
hyLridizaIion. WiIh Ihe aLiIiIy of isoIaIing roIoIasIs fron IanI ceIIs using enzynes in Ihe
earIy 1960 Ly L. C. Cocking, Ihe inIeresI in geneIic nodificaIion of sonaIic ceIIs in higher IanIs
has deveIoed. WiIhin a decade, TakeLe c| a|. (1971) regeneraIed conIeIes IanIs fron Ieaf
roIoIasIs of IaLacco increased Ihe oIenIiaI of rIoIasI cuIIure Iechniques.
NoI onIy isoIaIed roIoIasIs LuI Iheir fusion roducI, a sonaIic hyLrid, can aIso Le
regeneraIed inIo vhoIe IanIs. IIanI roIoIasIs can aIso Iake u foreign DNA, Ihrough Iheir
naked Iasna nenLrane, under secific chenicaI and hysicaI IreaInenIs. IroIoIasIs aIso
rovide an exerinenIaI sysIen for a vide range of LiochenicaI and noIecuIar sIudies ranging
fron invesIigaIions inIo Ihe grovIh roerIies of individuaI ceIIs Io nenLrane IransorI.
IresenI chaIer descriLes Ihe neIhods of roIoIasI isoIaIion, urificaIion, cuIIure
Iechniques as aIso roIoIasI fusion Iechniques and sonaIic hyLrid seIecIion rocedures.
I. Fk0t0FLASt IS0LAtI0h
The Iern roIoIasI refers Io Ihe shericaI IasnoIysed conIenI of IanI ceII encIosed Ly
Iasna nenLranes or naked ceII viIhouI ceII vaII. Before going inIo Ihe deIaiIs aLouI
2nd Proof 31/5/8
Frotojiost Cuitur. orc Sorot:c H,or:c:;ot:or &%%
2nd Proof 31/5/8
roIoIasI cuIIure, iI is inorIanI Io isoIaIe as genIIy and as quickIy as ossiLIe viaLIe and
unin|ured roIoIasIs. The Ivo neIhods for isoIaIion of roIoIasIs are: nechanicaI and
enzynaIic neIhod.
Hecbaoica| Iso|atioo
KIercker (1892) vas Ihe firsI Io isoIaIe roIoIasIs fron IasnoIysed ceII of S|ra|ia|cs a|cidcs.
These sIudies vere IaIer exIended for roIoIasIs isoIaIion fron Iissues of onion LuILs. ScaIes
vere innersed in 1.0 M sucrose unIiI Ihe roIoIasI shrunk avay fron Iheir encIosing vaII
and Ihen Ihe IasnoIysed Iissue vas cuI viIh a shar knife aI such a Ihickness IhaI onIy Ihe ceII
vaIIs are cuI viIhouI danaging Ihe roIoIasIs in sIris. The roIoIasIs vere reIeased Ly
osnoIic sveIIing vhen Ihese sIris of Ihe Iissue are Iaced in Iov concenIraIion sucrose
soIuIion (Iig. 1).
I.2 ozmatic Iso|atioo
This neIhod invoIves Ihe use of enzynes Io dissoIve Ihe ceII vaII for reIeasing roIoIasIs.
IniIiaI sIudies vere carried ouI on roIoIasIs isoIaIion of yeasI ceII Ly digesIing Ihe ceII vaII
using gasIric |uices oLIained fron Ihe snaiI, Ha|ix pcnca|ia. Iovever, crediI of deveIoing high
yieId roIoIasI isoIaIion Iechnique fron higher IanI roIoIasIs goes Io Cocking (1960). Ie
enIoyed a crude ceIIuIase rearaIion fron Ihe fungus Mqrc|nccium tcrrucaria Io dissoIve ceII
vaII and reIease Ihe roIoIasIs fron IonaIo rooIs. LaIer Ihis neIhod viIh suiIaLIe
nodificaIions and using urified enzynes has Leen exIensiveIy used Ly oIher grou of
vorkers. The enzynaIic neIhod couId Le used as a one sIe neIhod (direcI neIhod), or as a
Ivo-sIe neIhod (sequenIiaI neIhod).
In Ihe one sIe neIhod, roIoIasIs are isoIaIed direcIIy fron Ihe Iissue Ly using Ivo
enzynes, ceIIuIase and ecIinase, sinuIIaneousIy. WhiIe, in Ihe Ivo-sIe neIhod, ceIIs are firsI
isoIaIed fron caIIus or Iissue Ly using ecIinase and Io Ihis ceII susension ceIIuIase is added Io
digesI Ihe ceII vaII and reIease roIoIasIs.
An enzynaIic soIuIion used for roIoIasI isoIaIion conIains Ihe enzynes and a sugar as
an osnoIicun Io revenI Ihe Iasna nenLrane fron ruIuring. Sone saIIs and nuIrienIs are
aIso used as osnoIicun. CeneraIIy 50 nM CaCI
2
is added Io increase Ihe sIaLiIiIy of reIeased
Fig. 1. When tissue is cut at the dotted lines (A) with a sharp razor blade, some cells release uncut complete
protoplast and rest of the cells produced broken dead protoplasts as shown in Figure 1B marked with stars (
*
).
Cut
A B
&%& C.iiuior orc B:oc|.r:coi Sc:.rc.s Boo
2nd Proof 31/5/8
roIoIasIs (Rose, 1980). The enzyne soIuIion is fiIIer sIeriIized Ihrough a 0.45 fiIIer
nenLrane and sIeriIized Iissue cuI inIo snaII ieces is added. IncuLaIion for sone Iine
reIeases roIoIasIs fron Ihe Iissue.
Fk0t0FLASt F0kIfI6AtI0h
The successfuI cuIIure of roIoIasIs requires a ure ouIaIion of inIacI and viaLIe
roIoIasIs aI a high yieId. So Ihe roIoIasIs require Io Le urified Ly renoving Ihe
undigesIed naIeriaI (deLris), LursI roIoIasIs and enzynes.
kemova| o 0ebris aod ozmes
DeLris can Le renoved fron Ihe roIoIasI susension Ly fiIIering Ihe rearaIion Ihrough a
sIeeI or nyIone nesh of 100 ore size. Lnzyne is renoved Ly cenIrifuging Ihe roIoIasI
susension aI 600 rn for 5 ninuIes. The roIoIasIs seIIIe Io Ihe LoIIon of Ihe cenIrifuge IuLe
vhiIe Ihe suernaIanI is renoved viIh Ihe heI of a ieIIe. The roIoIasIs are Ihen re-
susended in a vashing nediun conIaining an osnoIicun onIy or osnoIicun viIh nuIrienI
nediun or hydraIed caIciun chIoride. The susension is cenIrifuged again Io seIIIe Ihe
roIoIasIs and Ihe vashing nediun is decanIed. Traces of enzyne are renoved Ly vashing
Ihe roIoIasIs Ivice or Ihrice viIh Ihe nediun.
kemova| o 8rokeo Frotop|asts
InIacI roIoIasIs are searaIed fron Ihe Lroken deLries Ly susending Ihe roIoIasI
rearaIion in 20-40% sucrose soIuIion and cenIrifugaIion aI 350 rn for Ihree ninuIes. InIacI
roIoIasIs coIIecI aI Ihe Io of Ihe sucrose soIuIion and are carefuIIy renoved viIh a ieIIe
(Cregory and Cocking, 1965, Iover and Cocking, 1970, Lvans c| a|., 1972).
Schenk and IiIderLrandI (1971) used ficoII soIuIion vhiIe Larkin (1976) used densiIy
Luffer for urificaIion of roIoIasI. In Ihis neIhod 0.5-3.0 voIunes of crude roIoIasI
rearaIion afIer fiIIeraIion Ihrough sIeriIe nusIin cIoIh is Iayered on Iynhore in Ihe
cenIrifuge IuLe and sun aI 50-200 g for aLouI 10 ninuIes. The roIoIasIs coIIecI as a ring
LeIveen Ihe enzyne soIuIion and Iynhore and deLris seIIIe Io Ihe LoIIon (Iig. 2A). The
roIoIasIs are renoved fron Ihe inIerhase viIh a asIure ieIIe (Iig. 2B).
Frotop|ast Viabi|it aod 6e|| Wa|| formatioo tests
To a snaII voIune of Ihe roIoIasI susension add equaI voIune of 0.1% caIcofIour soIuIion,
incuLaIe for 5 ninuIes and Ihen oLserve under fIuorescenI nicroscoe. The ceII vaII viII
fIuoresce and roIoIasIs renain dark.
Frotop|ast Viabi|it
IIuorescein diaceIaIe (IDA) soIuIion in aceIone (5 ng/I) is added Io roIoIasIs susension Io
give a finaI concenIraIion of 0.01%. AfIer 5 ninuIes aI roon IeneraIure Ihe roIoIasIs are
exanined using fIuorescenI nicroscoe. nIy viaLIe roIoIasIs can Le seen.
Frotojiost Cuitur. orc Sorot:c H,or:c:;ot:or &%'
2nd Proof 31/5/8
fA6t0kS Aff6tIh6 th Fk0t0FLAStS IS0LAtI0h Ah0 ItS VIA8ILItY
To achieve a high yieId of viaLIe roIoIasIs, iI is necessary Io sIandardize Ihe Iye and
concenIraIion of osnoIicun (e.g., sucrose, nanniIoI, sorLiIoI, gIucose and oIher sugars).
ManniIoI is Ihe nosI connonIy used osnoIicun. The oIher condiIion Io Le sIandardized is
IhaI of enzynes (ceIIuIase, ecIinase, heniceIIuIase, neiceIIase, ecIinoI, or dresiIase) vheIher
Ihey are Io Le used singIy, in conLinaIion or sequenIiaIIy viIh Ihe osnoIicun.As a IhunL ruIe,
Iov enzyne concenIraIion aI Iov IeneraIure and high I (5-8) for shorI incuLaIion eriod
roved Io Le LeIIer Ihan Ionger incuLaIion eriods viIh high enzyne concenIraIion, high
IeneraIure and Iov I vaIue.Though Ihe ionic saIIs vhen used viIh osnoIicun degrade Ihe
enzynes LuI increase Ihe sIaLiIiIy of roIoIasIs. IroIoIasIs isoIaIed in resence of Ca
++
or
Mg
++
ions shoved a greaIer caaciIy for ceII vaII regeneraIion as conared Io roIoIasIs
isoIaIed in Ihe aLsence of Ihese ions (Rose, 1980).
Fk0t0FLASt 60Lt0k
AfIer viaLiIiIy IesI Ihe roIoIasIs are cuIIured aI a knovn densiIy. DifferenI neIhods have
Leen used for cuIIuring Ihe isoIaIed roIoIasIs.
Suspeosioo 6u|ture
In Ihis neIhod roIoIasIs are susended in a Iiquid nediun viIh a suiIaLIe concenIraIion of
osnoIicun. IroIoIasIs aI a densiIy of 10
5
/nI is generaIIy IaIed on 25-50 nI of nediun in an
LrIenneyer fIask. The cuIIures are shaken sIovIy Io rovide sufficienI aeraIion for grovIh.
SoneIines shaking can cause LursIing of roIoIasIs, so rn of shaking required for a given
secies shouId Le sIandardized. Lvans and Cocking (1975) suggesIed IhaI 2 nI susension of
roIoIasIs couId Le cuIIured in 25 nI fIasks Io faciIiIaIe aeraIion. VasiI (1976) suggesIed Ihe
addiIion of ficoII Io Ihe nediun Io kee roIoIasIs fIoaIing and IhereLy aIIoving LeIIer
grovIh.
Fig. 2B. Purified protoplast population
B
Fig. 2A. Purified Protoplast ring formation and debris
get settled at the bottom
Enzyme solution
Purified proplasts
Lymphprep
Debris
A
&& C.iiuior orc B:oc|.r:coi Sc:.rc.s Boo
2nd Proof 31/5/8
haogiog 0rop Hetbod
Kao and his grou deveIoed Ihe hanging dro neIhod in 1970 vhich vas suLsequenIIy used
Ly oIhers (Bava and Torrey, 1971). In Ihis neIhod, a susension of roIoIasIs aI a densiIy of
10
4
/nI Io 10
5
/nI is Iaced as 50 I dros in IasIic IeIri IaIes, seaIed viIh arafiIn and
incuLaIed in an inverIed osiIion aI 25-30C under Iv IighI inIensiIy (100-500 Iux) or even in
dark. AfIer ceII vaII regeneraIion and Ihe iniIiaIion of ceII division, fresh nediun is added Io
nake ceII susension. The snaII size of dro heIs in roviding enough aeraIion Io roIoIasIs
(VasiI, 1976).
Agar F|atiog Hetbod
The neIhod is aInosI siniIar Io Ihe one deveIoed Ly Bergnann (1960) Io grov caIIus ceIIs of
IoLacco and Leans and vas firsI enIoyed for Ihe roIoIasI cuIIure Ly NagaIa and TakaLe
(1971). The na|or advanIages of Ihis Iechnique are: (i) a Iarge nunLer of roIocoIs can Le
handIed sinuIIaneousIy, and (ii) IaIing efficiency can Le deIernined easiIy. AI Ihe sane Iine
Ihe na|or dravLack of Ihis Iechnique is IhaI afIer regeneraIion of ceII vaII and inducIion of ceII
division, osnoIic oIenIiaI of Ihe nediun can noI Le aIIered Ly addiIion of fresh nediun
Iacking osnoIic sIaLiIizer, since Ihe iniIiaI nediun is seni-soIid. Iovever, Ihis roLIen can Le
soIved Ly Iransferring LIocks of agar (in vhich roIoIasIs are cuIIured iniIiaIIy) Io a fresh
nediun viIh Iesser concenIraIion of osnoIicun or aIIogeIher in iIs aLsence. This Iechnique has
Leen nodified in differenI vays viz., as feeder Iechnique Io suorI division of ceIIs IaIed aI
Iov densiIy (Raveh eI aI., 1973), as nicro vesseI (BuIIon, 1978), and as nuIIiIe dro arrays
(IoIrykus eI aI., 1979).
Hicro 6u|ture tecboique
This Iechnique deveIoed Ly Jones eI aI. (1960) for cuIIuring isoIaIed ceIIs vas used Ly VasiI
and IiIderLrandI (1965) Io raise IoLacco IanIs fron isoIaIed ceIIs. Micro cuIIure Iechnique has
Leen successfuIIy used for cuIIuring roIoIasIs of IoLacco and Pc|unia (Durand eI aI., 1973). A
dro of cuIIure nediun conIaining one or nore roIoIasIs is uI on a nicroscoic sIide. n
eiIher sides of Ihis nicroscoic sIide are keI Ivo cover sIis. A Ihird one is uI over Ihese Ivo
cover sIis Io shieId Ihe roIoIasIs susensions.
Hu|tidrop Arra (H0A) tecboique
This is a furIher inrovenenI of hanging dro neIhod and aIIovs screening of Iarge nunLer
of hornonaI and nuIriIionaI facIors (u Io 4900) using snaII anounI of IanI naIeriaI
(IoIrykus eI aI., 1979)
fA6t0kS Aff6tIh6 Fk0t0FLAStS 60Lt0k
SuccessfuI grovIh and regeneraIion of roIoIasIs is deendenI on a nunLer of facIors ranging
fron Ihe sIaIus of Ihe donor IanI Io Ihe cuIIure condiIions.
Frotojiost Cuitur. orc Sorot:c H,or:c:;ot:or &&
2nd Proof 31/5/8
F|aot Species aod Varieties
II is a veII docunenIed facI IhaI even very snaII geneIic difference Ieads Io varying roIoIasI
resonses Io cuIIure condiIions.
F|aot Age aod 0rgao
A second inorIanI facIor for Ihe successfuI cuIIure is Ihe age of Ihe donor IanI and
deveIonenIaI sIage of Ihe donor arI used for isoIaIion of Ihe roIoIasIs. The sIages viIh
resecI Io Iheir resonses are gerninaIing enLryos, foIIoved Ly seedIing of one veek,
IanIIeIs, Ieaves, |uveniIe Iissue fron naIure IanI and naIure Iissue. Though having Ihe sane
geneIic infornaIion roIoIasIs isoIaIed fron Ihese arIs Lehave differenIIy in cuIIure
condiIions. IIanI regeneraIion has Leen nosI successfuI fron Ieaf roIoIasIs of herLaceous
secies.
Fre 6u|ture 6ooditioos
Besides Ihe facIors nenIioned aLove Ihe Lehaviour of roIoIasIs in cuIIure is highIy
infIuenced Ly cIinaIic facIors and re cuIIure condiIions. DifferenI cuIIure condiIions of
seedIing yieIds roIoIasIs having differenI resonses vhen cuIIured.
Fretreatmeot to tbe tissue, 8eore Iso|atioo o Frotop|asts
IreIreaInenIs such as coId IreaInenI, IasnoIysis, and hornone IreaInenI Io Ihe Iissue
increases Ihe chances of recovery of viaLIe roIoIasIs and Iheir IaIing efficiency.
hutritiooa| kequiremeot or Frotop|asts
NuIriIionaI requirenenI for Ihe grovIh of roIoIasIs differ fron Ihe nuIriIionaI requirenenI
for Iissues and ceII cuIIure. IroIoIasIs Ieak in cuIIures as Ihey are devoid of ceII vaIIs, and
noreover, Ihey have a greaIer surface area for Ihe diffusion of neIaLoIiIes Ihan Ihe Iissues. So
Ihe concenIraIion of differenI neIaLoIiIes in roIoIasIs is Iess Ihan IhaI in Ihe Iissues and ceIIs.
To conensaIe for Ihese Ioses fron roIoIasIs Ihe nunLer of neIaLoIiIes rovided in Iheir
cuIIure nedia is nore (Kao and MeichyaIuk 1975).
Frotop|asts 0eosit
IroIoIasIs densiIy is very cruciaI as iI infIuences Ihe IaIing efficiency and LeIIer surviving of
roIoIasIs. When IaIed aI higher densiIy roIoIasIs coneIe viIh one anoIher vhiIe aI
Iover densiIy Iosses of neIaLoIiIes fron roIoIasIs is nore. The IaIer siIuaIion of roIoIasI
Ieakiness can Le circunvenIed Ly addiIion of required neIaLoIiIes Io nake nediun isoIonic.
AIIernaIiveIy, one can use nurse caIIus or feeder Iayer Iechnique Io rovide essenIiaI organic
noIecuIes Io roIoIasI for survivaI and Iheir suLsequenI grovIh (MenczeI eI aI., 1978).
Fk0t0FLASt f0SI0h 0k S0HAtI6 hY8kI0ILAtI0h
As IanI ceIIs have an inhiLiIing ceII vaII iI is very difficuII Io fuse Ihen. BuI isoIaIed
roIoIasIs vere oLserved Io fuse sonIaneousIy Lecause nov Ihe onIy Larrier LeIveen Ihe
&& C.iiuior orc B:oc|.r:coi Sc:.rc.s Boo
2nd Proof 31/5/8
cyIoIasn of Ivo ceIIs is Ihe Iasna nenLrane. AfIer IoI of refinenenIs Ihe Iechniques for
roIoIasI fusion Lecane inorIanI Io roduce hyLrids fron sexuaIIy inconaIiLIe secies.
IroIoIasI fusion is ossiLIe even LeIveen a IanI ceII and an aninaI ceII and Ihus can have
vider aIicaIion. The rocess of sonaIic hyLridizaIion invoIves: (i) roIoIasI isoIaIion,
(ii) roIoIasI fusion, (iii) seIecIion of sonaIic hyLrids, and (iv) cuIIure of sonaIic hyLrids Io
regeneraIe conIeIe IanIs. DifferenI neIhods of roIoIasI fusion are descriLed Ly Bengochea
and Dodds, (1986). Three inorIanI neIhods are generaIIy used for roIoIasI fusion: (a) high
Ca
++
and high I, (L) oIyeIhyIene gIycoI (ILC), and (c) eIecIric fieId. LiIeraIure survey
indicaIes IhaI Ihe second and Ihird neIhods are nosI connonIy used due Io LeIIer resuIIs. The
Ihree neIhods are discussed LeIov.SexuaI hyLridizaIion occurs vhen haIoid ceIIs generaIed
in a revious neiosis fuse. The fusion of sonaIic diIoid ceIIs shouId generaIe a IeIraIoid
fusion roducI rovided IhaI Ihe nucIei fuse, Ioo. If Ihis is Ihe case, iI is soken of a synkaryon.
A fusion roducI vhere Ihe nucIei sIay searaIe is caIIed a heIerokaryon.
higb 6a
++
aod higb ph Ioduced Frotop|ast fusioo
IhysicaI conIacI of Ivo roIoIasIs is essenIiaI for Iheir fusion. Iovever, roIoIasIs do noI
fuse easiIy due Io Ivo nain reasons: (i) Ihey have a neI negaIive charge on Iheir nenLrane
surfaces and force of reuIsion vorks LeIveen roIoIasIs, and (ii) iI is difficuII Io renove
vaIer fron hydrohiIic surfaces of roIoIasIs vhich aIso creaIe a reuIsive force LeIveen Ivo
roIoIasIs. II vas oLserved IhaI osiIiveIy charged ions reduce Ihe neI negaIive charges of
nenLranes reducing Ihe reuIsive force consideraLIy. KeIIer and MeIcher (1973) found
caIciun ions vere suiIaLIe for such uroses and deveIoed Ihis neIhod of roIoIasI fusion
using high Ca
++
ions in a high I soIuIion. LaIer Ihe neIhod vas inroved Ly MeIcher and
LaLiL (1974). The Iechnique invoIves Ihe foIIoving sIes: IreshIy isoIaIed roIoIasIs of
seIecIed arenIs are nixed in a raIio of 1:1 viIh a finaI densiIy of 2.5x10
5
roIoIasIs er nI. The
roIoIasIs are coIIecIed as a eIIeI Ly cenIrifuging aI 50 g for 3 Io 5 ninuIes. The suernaIanI is
renoved and 2 nI of fusion nixIure conIaining 50 nM CaCI
2
.2I
2
, 50 nM CIycine-NaI
Luffer and 400 nM ManniIoI is added. I of Ihe nixIure is ad|usIed Io 10.5. IroIoIasIs are re-
susended in soIuIion Ly genIIy shaking Ihe cenIrifuge IuLes.The roIoIasIs are coIIecIed Ly
cenIrifuging aI 50 g for 3 Io 5 ninuIes. The cenIrifuge IuLes viIh roIoIasIs are incuLaIed in a
vaIer LaIh aI 37C for 10 Io 30 ninuIes. The fusion nixIure is reIaced Ly vashing nediun
(600 nM ManniIoI and 50 nM CaCI
2
.2I
2
) and roIoIasIs are IefI for 30 ninuIes.The
roIoIasIs are vashed Ivice viIh Ihe vashing nediun.The roIoIasIs are re-susended in
cuIIure nediun. IroIoIasI fusion roducIs can Le oLserved under nicroscoe. These
roIoIasIs can Le cuIIured on seIecIion nediun, vhich aIIovs onIy sonaIic hyLrids Io grov.
F6 Ioduced fusioo
IoIyeIhyIene gIycoI (ILC) induced roIoIasI fusion vas deveIoed Ly Kao and MichayIuk
(1974). This Iechnique is reIaIiveIy nore efficienI Ihan Ihe revious one. IoIyeIhyIene gIycoI
noIecuIes have oIariIy Iike nenLrane hoshohoIiid noIecuIes and geI aIIached viIh
nenLrane roIeins. When Ihe aIIached ILC LeIveen Ivo roIoIasIs is renoved iI resuIIs in
Lreakdovn of nenLranes aI Ihe conIacI oinIs causing roIoIasIs Io fuse viIh suLsequenI
re|oining of IasnanenLarnes of Ihe ad|acenI roIoIasIs. The foIIoving sIes are invoIved
for roIoIasI fusion Ihrough oIyeIhyIene gIycoI. Mix Ihe freshIy isoIaIed roIoIasIs (vhiIe
Frotojiost Cuitur. orc Sorot:c H,or:c:;ot:or &&!
2nd Proof 31/5/8
sIiII in enzyne soIuIion) of seIecIed arenIs in a raIio of 1:1. Iass Ihe susension Ihrough a 62
n ore size fiIIer and coIIecI Ihe fiIIraIe in a cenIrifuge IuLe. SeaI Ihe nouIh of Ihe IuLe viIh
screv ca. CenIrifuge Ihe fiIIraIe aI 50 g for 60 ninuIes Io sedinenI Ihe roIoIasIs. Renove Ihe
suernaIanI viIh asIure ieIIe. Wash Ihe roIoIasIs viIh 10 nI of soIuIion I (soIuIion I: 500
nM CIucose, 0.7 nM KI
2
I
4
.I
2
and 3.5 nM CaCI
2
.2I
2
and I 5.5). Renove and vash
Ihe roIoIasIs in soIuIion I and nake a susension viIh 4-5% v/v roIoIasIs er nI. IuI a 2-
3 nI dro of siIicon 200 fIuid (100 cs) in a 60 x 15 nn sIeriIe IeIri dishes.IIace a 22x22 nn cover
sIi on Ihe dro. IieIIe 150 I of roIoIasI susension onIo Ihe cover sIi viIh a asIure
ieIIe. AIIov aLouI 5 ninuIes for Ihe roIoIasIs Io seIIIe on Ihe cover sIi forning Ihin Iayer.
Add dro-Ly-dro 450 I of ILC soIuIion (50% ILC-1540, 10.5 nM CaCI
2
.2I
2
, 0.7 nM
KI
2
I
4
.I
2
) Io Ihe roIoIasI susension and oLserve Ihe roIoIasI adhesion under
inverIed nicroscoe. InocuIaIe Ihe roIoIasI in ILC soIuIion and kee aI roon IeneraIure
(24C) for 10 Io 20 ninuIes. CenIIy add 0.5 nI aIiquoIs of soIuIion II (50 nM CIycine, 50 nM
CaCI
2
.2I
2
, 300 nM CIucose, I 9-10.5) aI 10 ninuIes inIervaIs. AfIer anoIher 10 ninuIes add
1 nI of roIoIasIs cuIIure nediun. Wash Ihe roIoIasIs five Iines aI five ninuIes inIervaIs
viIh 10 nI of Ihe fresh roIoIasIs cuIIure nediun. AI Ihe end of each vashing do noI renove
enIire nediun fron Ihe cover sIi LuI Ieave Lehind a Ihin Iayer of Ihe oId nediun adding fresh
nediun Io iI. If arenI roIoIasIs are disIinguishaLIe visuaIIy, iI nay Le ossiLIe Io assess Ihe
frequency of heIerokaryon fornaIion aI Ihis sIage. CuIIure Ihe fused roIoIasIs IogeIher viIh
Ihe unfused roIoIasIs on Ihe sane cover sIi in a Ihin Iayer of 500 I of cuIIure nediun. IuI
addiIionaI 500-1000 I nediun in Ihe fronI of droIeIs around Ihe cover sIi Io nainIain
hunidiIy inside Ihe IeIri IaIes.
|ectric fie|d Ioduced fusioo
In Ihis neIhod, deveIoed Ly I. Zinnernan, roIoIasIs are Iaced in an eIecIric fieId and are
exosed Io high inIensiIy eIecIric uIse for a shorI duraIion (nano Io nicro second). This
exosure Io eIecIric fieId reversiLIy increases erneaLiIiIy of ceII nenLrane. LocaI eIecIricaI
charge Lreak dovn occurs in Ihe Iasna nenLranes resuIIing in fusion of ad|acenI roIoIasIs.
The originaI roerIies of nenLrane are resIored viIhin nicro second Io ninuIes deending
on Ihe exerinenIaI condiIions and nenLrane roerIies (Zinnernan and Scheurich, 1981,
Zinnernann and Vienken, 1982). An insIrunenI designed Ly I. Zinnernan is used for
roIoIasI fusion and for geneIic IransfornaIion Ihrough eIecIrooraIion. The rocess of
eIecIric fieId induced roIoIasI fusion can Le exIained in Ihe foIIoving five sIes:
DieIecIrohoresis
MuIuaI dieIecIrohorasis
MenLrane conIacI
LIecIric Lreak dovn of nenLranes, and
IroIoIasI Iusion
(i) Dic|cc|rcpncrcsis: CIose nenLrane conIacI of Ivo roIoIasIs is one of Ihe rerequisiIe
for roIoIasI fusion. This sIe is achieved Ly dieIecIrohoresis. DieIecIrohoresis is concerned
viIh noIion of neuIraI arIicIes in a non uniforn eIecIric fieId. NeuIraI arIicIes Lecone
oIarized in Ihe resence of eIecIric fieId viIh negaIive side Iovards anode and osiIive charge
of Ihe sane nagniIude on Ihe oosiIe side, i.e. Iovards caIhode.In uniforn eIecIric fieId Ihe
fieId sIrengIh is equaI on LoIh Ihe sides. And in such a fieId Ihese induced LioIar arIicIes viII
&&" C.iiuior orc B:oc|.r:coi Sc:.rc.s Boo
2nd Proof 31/5/8
noI nove Io eiIher side due Io equaI eIecIric force on LoIh sides (Iig. 3A). Iovever, under non
uniforn fieId (fieId sIrengIh on Ihe Ivo sides of arIicIes is unequaI) a neI force acIing uon Ihe
arIicIe resuIIs in Iinear noIion Iovards Ihe region of highesI fieId inIensiIy (Iig. 3B). This is
Ierned as dieIecIrohoresis.
(ii) Mu|ua| Dic|cc|rcpncrcsis: ne roIoIasI aroaching anoIher oIarized roIoIasI
during Ihe novenenI Iovards Ihe region of highesI fieId inIensiIy, viII encounIer an
enhancenenI of IocaI fieId divergence and viII Iend Io nove Iovards IhaI roIoIasI since Ihe
fieId sIrengIh is higher near IhaI ceII. As a resuII roIoIasIs in non uniforn fieId forn chain
Iike aggregaIes (so caIIed earI chain) viIh oinI Io oinI nenLrane conIacI (Iig. 3C). This
effecI is Ierned as nuIuaI dieIecIrohoresis.
The aIIracIion forces arising fron dioIe generaIion viIhin roIoIasIs overcone LoIh Ihe
eIecIrosIaIic reuIsive forces LeIveen nenLrane surfaces of neighLoring roIoIasIs Learing
neI negaIive charges and reuIsive hydraIion forces. ReuIsive hydraIion force is assuned Io
Le a consequence of vork required Io renove vaIer fron hydrohiIic surface as Ihe roIoIasIs
aroach one anoIher. DieIecIrohoresis and earI chain fornaIion usuaIIy have Io Le
erforned in virIuaIIy non conducIive soIuIion (conducIiviIy Iess Ihan 10
-4
cn
-1
).
(iii) ||cc|ric 8rca| Dcun: ReversiLIe eIecIric Lreakdovn in Ihe zone of nenLrane conIacI is
Ihe rinary rocess resonsiLIe for Ihe iniIiaIion of fusion (Iig. 4). IhoshoIiids are arranged
in a Ianer Li-Iayer inIo vhich eriheraI or inIegraI sIrucIuraI carrier roIeins are enLedded
in nosaic Iike fashion. The IaIeraI fIuidiIy of hoshoIiids is very high, vhiIe Ihe novenenI
verIicaI Io Ihe nenLrane surface is severeIy IiniIed. A fIi-fIo novenenI of Iiids in Ihis
direcIion is, Iherefore, highIy difficuII.
In an equivaIenI eIecIricaI circuiI Ihe nenLranes can Le reresenIed Ly a IaIe caaciIor of
secific caaciIance (Cn) and resisIance (Rn) connecIed in araIIeI (Iig. 4A). The aqueous
exIernaI soIuIion and oIar heads of Iiquids reresenI Ihe IaIe of caaciIor, vhiIe nenLrane
inIerior forns a dieIecIric viIh reIaIive dieIecIric consIanI of 2 Io 3. The resisIance of aqueous
exIernaI soIuIion R
L
is in series viIh Ihe nenLrane. The secific resisIance of ceII nenLrane is
in Ihe order of 10
2
Io 10
4
Wcn
-2
, vhiIe IhaI of secific nenLrane LiIayer is Ihree Io four orders
of nagniIude higher. The secific caaciIances of arIificiaI and LioIogicaI nenLranes on Ihe
oIher hand are conaraLIe of having vaIues of 0.3 Io 0.7 mIcn
-2
and 1 mIcn
-2
,

resecIiveIy.
Fig. 3. Movement of protoplast in non uniform electric field towards higher strength electric field and forms
protoplast chain.
A. UniIorm Iield developed equal Iorce on both side oI charged protoplasts B. Unequal electrical Iield developed net Iorce
towards higher Iield strengthC. Non uniIorm electric Iield thus Iorms pearl chain oI protoplasts
Frotojiost Cuitur. orc Sorot:c H,or:c:;ot:or &&#
2nd Proof 31/5/8
II is knovn IhaI caaciIors can onIy Le charged Io a cerIain naxinun voIIage. ALove Ihis
voIIage IeveI (vhich is deendenI on searaIion of IaIes, connon area LeIveen IaIes and
dieIecIric naIeriaI of IaIes) eIecIricaI (dieIecIric) Lreakdovn is oLserved in Ihe caaciIor.
LIecIricaI Lreakdovn is associaIed viIh an exIrene increase in Ihe eIecIricaI conducIiviIy of Ihe
caaciIor, vhich is usuaIIy irreversiLIe i.e. Ihe caaciIor is desIroyed. In Ihe seIf-regeneraIing
caaciIors on Ihe oIher hand Ihe originaI resisIance and caaciIance is resIored under cerIain
exerinenIaI condiIions. BioIogicaI nenLranes and arIificiaI Iiid LiIayer Lehave in an
anaIogous vay Io Ihe eIecIric Lreakdovn of Ihe seIf-regeneraIing caaciIors descriLed aLove.
The reversiLIe eIecIricaI Lreakdovn of roIoIasI nenLranes Ieads Io erIurLaIion of
nenLrane sIrucIure, vhich erniIs an exchange of naIeriaIs LeIveen Ihe roIoIasI and iIs
environnenIs. In figure 4B iI is shovn IhaI Ihe eIecIric fieId effecIs Ihe roIoIasI adhering Io
each oIher in Ihe orienIaIion Io Ihe fieId direcIion onIy. In Ihis case, eIecIricaI Lreakdovn occurs
aI Ihe oIes of Ihe ceIIs and zone of conIacI LeIveen Ivo roIoIasIs.
Herits o |ectrica|| Ioduced Frotop|ast fusioo
1. The fusion rocess LeIveen any Ivo roIoIasIs of differenI secies in a nixed
ouIaIion can Le foIIoved under nicroscoe. This vouId Le of arIicuIar inIeresI
vhen roducing hyLridona ceIIs.
2. Iusion rocess is synchronous and exIends over a shorI duraIion. So Ihe hyLrids do noI
Iose Ihe viaLiIiIy.
3. ViaLiIiIy can aIso Le affecIed Ly fusogenic conounds, vhich are Ihus aLIe Io inIeracI
viIh IoIaI nenLrane surface in an unconIroIIed nanner. In Ihis neIhod Ihere is no use
of such fusogenic conounds.
4. YieId of sonaIic hyLrids is very high.
5. During Ihe fusion, Ioss of inIerceIIuIar suLsIances is generaIIy very Iov.
I. SL6tI0h 0f S0HAtI6 hY8kI0S
SuccessfuI sonaIic hyLridizaIion requires Ihe inducIion of roIoIasI fusion resuIIing in Ihe
fornaIion of heIerokaryocyIes and sonaIic hyLrid, Iheir survivaI, seIecIion and recovery of
Fig. 4. (A) Diagrammatic representation of Charge pulse technique, (B) Electric break down occurs at the poles of
the cells and the zone of contact between two protoplasts.
&&$ C.iiuior orc B:oc|.r:coi Sc:.rc.s Boo
2nd Proof 31/5/8
hyLrids, suLsequenI division of fusion roducIs and norhogenesis giving rise Io hyLrid
IanIs.
DifferenI neIhods have Leen deveIoed for Ihe seIecIion of sonaIic hyLrids. These
neIhods can LroadIy Le divided as foIIovs:
Crou 1 SeIecIion of sonaIic hyLrids Ly cuIIuring Ihen on such a nediun on vhich onIy
sonaIic hyLrid roIoIasI can grov (Iover eI aI., 1977).
Crou 2 ConIenenIary seIecIion of sonaIic hyLrids on secific cuIIure nediun.
Crou 3 MechanicaI isoIaIion Ly visuaI neans and knovIedge of idenIificaIion of sonaIic
hyLrids.
Crou 4 MorhoIogy of Ihe IanI afIer regeneraIion.
Grcup 1 - Sc|cc|icn Mcdium: SniIh eI aI. (1976) used a nodeI sysIen invoIving Ihe knovIedge
fron sexuaI inIersecific Nicc|iana hyLrids IhaI can Le cuIIured and seIecIed on nedia IhaI faiIs
Io suorI arenIaI secies. Chueau eI aI. (1978) vere Ihe firsI Io denonsIraIe Ihe successfuI
use of Ihis Iechnique for seIecIing sonaIic hyLrids. Iusion of roIoIasIs fron Nicc|iana
|cngsdcrfii and N. g|uca vere induced using ILC. IarasexuaI hyLrid coIonies vere seIecIed for
Iheir aLiIiIy Io grov viIhouI grovIh suLsIances (Iig. 5).
SiniIarIy roIoIasIs of sexuaI hyLrid IanIs of Pc|unia parcdii and P. nq|rida can grov and
regeneraIe conIeIe IanIs on secific nediun conIaining AcIinonycin D, vhere as
roIoIasIs of arenIs can grov and forn coIonies on Ihis nediun LuI can noI regeneraIe
conIeIe IanIs. Iover eI aI. (1977) successfuIIy used Ihis nediun for seIecIing sonaIic
hyLrids.
Grcup 2 - Ccmp|cmcn|arq sc|cc|icn: This neIhod deveIoed Ly MeIcher and LaLiL (1974)
uIiIizes LiochenicaI narkers such as aILino and chIorohyII deficienI nuIanIs (fig. 6). SonaIic
hyLrids can Le seIecIed on Ihe Lasis of conIenenIaIion of characIers exressed Ly Ihe
hyLrids, vhich are noI resenI in any of Ihe arenIs. SoneIines, hyLrid is seIecIed on Ihe Lasis
of characIers of arenI roIoIasIs resenI in sonaIic hyLrid.
MeIcher and LaLiL (1974) Iook Ivo nuIanIs of Nicc|iana |a|acum, one of vhich vas
suLIeIhaI, IhaI did noI grov nore Ihan fev inches (2-3 inches) in Iov IighI inIensiIy (800 Iux),
LuI aI higher IighI inIensiIies (10,000 Iux) vas aLIe Io grov inIo conIeIe IanIs. The oIher
Fig. 5. Flow chart showing selection of hybrids using selection medium on which only hybrid can grow.
Frotojiost Cuitur. orc Sorot:c H,or:c:;ot:or &&%
2nd Proof 31/5/8
nuIanI vas chIorohyII deficienI IhaI couId noI grov nore Ihan fev inches. When roIoIasIs
of Ihese Ivo nuIanIs vere fused Ihe hyLrid roduced green coIonies in cuIIure and IaIer on
regeneraIed inIo fuII IanIs in Iov IighI inIensiIy (800 Iux).
CIeLa eI aI. (1975) deveIoed a siniIar conIenenIaIion seIecIion rocedure IhaI invoIves
conIenenIaIion LeIveen IasIone and genone chIorohyII nuIanI and does noI uIiIize IighI
sensiIiviIy as a seIecIion narker. Cocking eI aI. (1977) used aILino roIoIasIs of P. nq|rida and
green Ieaf nesohyII roIoIasIs of P. parcdii (Iig. 6). These sonaIic hyLrids incIuded sIricI
anhidiIoids.
Grcup 3 - Mccnanica| |sc|a|icn: In Ihis neIhod heIerokaryons are seIecIed and isoIaIed visuaIIy
under nicroscoe. This neIhod is arIicuIarIy usefuI for Ihose inIersecies fusions vhere Ihe
osI zygoIic inconaIiLiIiIy revenIs sexuaI hyLridizaIion. In such cases of inIersecies fusion
chronosone eIininaIion nay resuII in faiIure Io seIecI Ihe sonaIic hyLrids, vhiIe oIher
inIersecies fusion nay resuII in sonaIic hyLrid roIoIasIs viIh no evidence of secific
chronosone eIininaIion. In Ihese insIances Ihe sIaLIe condiIions nay faiI Io Le nainIained on
organogenesis. Through Ihis seIecIion aroach such Larriers Io vhoIe sonaIic hyLrid IanI
fornaIion can Le idenIified and erhas overcone.
AuIonaIic searaIor of sonaIic hyLrids fron nixed ouIaIion of roIoIasIs afIer fusion
rocess has Leen denonsIraIed. In Ihis neIhod arenI roIoIasIs are sIained viIh Ivo
differenI coIour dyes and sonaIic hyLrids exhiLiIing nixed coIour are sorIed.
Grcup 4 - Mcrpnc|cgq cf |nc p|an| af|cr rcgcncra|icn: In Jhis neIhod, Ihe sonaIic hyLrids are
regeneraIed inIo vhoIe IanIs. The norhoIogicaI characIers of Ihe hyLrid IanIs are
conared viIh Ihose of Ihe Ivo arenIaI Iines Io esIaLIish Ihe siniIariIies. This neIhod is Iine
consuning and is ossiLIe vhere regeneraIion of IanIs and hyLrids is veII esIaLIished.
60h6L0SI0hS
IroIoIasI IechnoIogy has various aIicaIions oIher Ihan regeneraIion of conIeIe IanIs and
roducIion of hyLrids of sexuaIIy inconaIiLIe secies. In facI, iI has noI roduced any
Fig. 6. Flow Chart showing complementary selection of somatic hybrids on specific culture medium.
&&& C.iiuior orc B:oc|.r:coi Sc:.rc.s Boo
2nd Proof 31/5/8
vonderfuI research resuIIs so far as iI has ronised in earIy 1960s. There is noI even a singIe
successfuI roducIion of agrononicaIIy usefuI hyLrid IanI Ihrough Ihis IechnoIogy.
NeverIheIess, Ihese Iechniques have Leen insIrunenIaI in generaIing Lasic scienIific
infornaIion on ceII LioIogy, IanI inconaIiLiIiIy, nenLrane funcIions, ceII organeIIe sIudies
and ceII vaII regeneraIion. IIIrasIrucIure and noIecuIar archiIecIure of IanI ceIIs. These
Iechniques are nov Leing used for Iransfer of cyIoIasnic naIe sIeriIiIy. IroIoIasI can Iake u
nacronoIecuIes (nucIeic acids and roIeins), viruses, ceII cononenIs Iike chronosones and
chIoroIasIs Ly hagocyIosis.
kfkh6S
Bava, S.B. and Torrey, J.C. (1971) Budding and NucIear Division in CuIIured IroIoIasIs of Corn,
ConvoIvuIus, and nion. Tnc 8c|. Gaz. 132: 240-245.
Bengochea, T. and Dodds, J.I. (1986). IIanI IroIoIasIs: A LioIechnoIogicaI TooI for Cro InrovenenI.
Bergnann, L. (1960) CrovIh and division of singIe ceIIs of higher IanIs in viIro. ]curna| cf Gcncra|
Pnqsic|cgq 43: 841-851.
BuIIon, J. (1978) The effecI of sone corLohydraIe on grovIh and organizaIion of CiIrus ovuIar caIIus. Z.
Pf|anzcnpnqsic|. 88: 61-68.
CarIson, I. S., SniIh, I. I. and Dearing, R. D. (1972) IarasexuaI inIersecific IanI hyLridizaIion. Prcc.
Na|. cad. Sci. IS 69: 2292-2294.
Chueau, Y., Missonier, C., IonneI, M-C, Cou|aud, J. (1978): SonaIic hyLrids of IanIs Ly fusion of
roIoIasIs. Mc|ccu|ar and Gcncra| Gcnc|ics 105 (3): 239-45.
Cocking L.C. (1960) A neIhod for Ihe isoIaIion of IanI roIoIasIs and vacuoIes. Na|urc 187: 962-963.
Cocking, L. C , Ceorge, D., Irice-Jones, J. J., Iover, J. B. (1977) SeIecIion rocedures for Ihe roducIion of
inIer-secies sonaIic hyLrids of Pc|unia nq|rida and P. parcdii. II AILino conIenenIaIion seIecIion.
P|an| Scicncc |c||crs 10:7-12.
Durand, J. IoIrykaus, I. and Donn C. (1973) IIanIes isuues roIoIasIes de IeIunia. Z. Pf|anzcnpnqsic|. 69:
26-34.
Lvans I. K. and Cocking L. C. (1975) The Iechniques of IanI ceII cuIIure and sonaIic ceII hyLridisaIion..
In: Ncu Tccnniucs in 8icpnqsics and Cc|| 8ic|cgq (R. I. Iain and B. J. SniIh, Lds.), 127-158, John
WiIey and Sons LId.
Lvans, I. K., KeaIes, A. C. and Cocking, L. C. (1972) IsoIaIion of roIoIasIs fron cereaI Ieaves. P|an|a 104:
178-191.
CIeLa, Y. Y. (1979) NonchronosonaI InheriIance in Iigher IIanIs as SIudied Ly SonaIic IyLridisaIion.
In: Prccccdings |cur|n nnua| Cc||cgc cf 8ic|cgica| Scicnccs Cc||cuium, P|an| Cc|| and Tissuc Cu||urc-
Princip|cs and pp|ica|icns, I.775-788. hio SIaIe Iniv. Iress, CoIunLus.
Cregory, D. W., and Cocking, L. C. (1965) The Iarge-scaIe isoIaIion of roIoIasIs fron innaIure IonaIo
fruiIs. ]. Cc|| 8ic|. 24:143-146.
Jones, L. IiIdeLrandI, A. Riker, A. Wu, J. (1960) CrovIh of sonaIic IoLacco ceIIs in nicrocuIIure. m. ].
8c|. 47: 468-475.
Kao, K. N. (1976) A neIhod for fusion of IanI roIoIasIs viIh oIyeIhyIene gIycoI. In: Cc|| Gcnc|ics in
Higncr P|an|s (D. DuddiIs eI aI. Lds.). .233-237.Akad. Kiado, BudaesI.
Kao, K.N. KeIIer, W.A. and MiIIer, R.A. (1970). CeII divisions in nevIy forned ceIIs fron roIoIasIs of
soyLean. |xp. Cc|| Rcs. 62: 338-340.
Kao, K. N. and MichayIuk, M. R. 1974) A neIhod for high-frequency inIergeneric fusion of IanI
roIoIasIs. P|an|a 115:355-67.
Frotojiost Cuitur. orc Sorot:c H,or:c:;ot:or &&'
2nd Proof 31/5/8
Kao, K.N., ConsIaLeI, I., MichayIuk, M.K. and CanLorg, .L. (1974) IIanI roIoIasI fusion and grovIh
of inIergeneric hyLrid ceIIs. P|an|a 120:215-27, 1974.
KeIIer, W.A. and MeIchers, C. (1973) The effecI of high I and caIciun on IoLacco Ieaf roIoIasI fusion.
Z. Na|urfcrscn. 28, 737-741.
KIercker, J. von (1892) Line MeIhode zur isoIier IeLender roIoIasIen. Oftcrs Vc|cns| |ad |crnand| 49:
463-474.
Larkin, I.J. (1976). IurificaIion and viaLiIiIy deIerninaIions of IanI roIoIasIs. P|an|a 128, 213-216.
MenczeI, L., Lazar, C. and MaIiga, I. (1978) IsoIaIion of sonaIic hyLrids Ly cIoning Nicc|iana
heIerokaryons in nurse cuIIures. P|an|a 143: 29-32.
Iover, J. B. and Cocking, L. C. (1971) Iusion of IanI roIoIasIs. Scicncc Prcgrcss (Oxfcrd) 59. 181-98.
Iover, J. B., Berry, S. I., Irearson, L. M. and Cocking, L. C. (1977) SeIecIion rocedures for Ihe roducIion
of inIer-secies sonaIic hyLrids of Pc|unia nq|rida and P. parcdii. I NuIrienI nedia and drug
sensiIiviIyconIenenIaIion seIecIion. P|an| Scicncc |c||crs 10: 1-6.
Raveh, D., IuLernan, L. and CaIun, L. (1973) |n ti|rc cuIIure of IoLacco roIoIasIs. Ise of feeder
Iechniques Io suorI division of ceIIs IaIed aI Iov densiIies. |n Vi|rc 9: 216-22.
MeIchers, C. and LaLia, C. (1975) SonaIic hyLridizaIion of IanIs Ly fusion of roIoIasIs. SeIecIion of
IighI resisIanI hyLrids of haIoid IighI sensiIive varieIies of IoLacco. MoIec. Cen. CeneI. 135:
277-294.
NagaIa, T. and TakeLe, I. (1971) IIaIing of isoIaIed IaLacco nesohyII roIoIasIs on agar. IIanIa VoI. 99:
12-20.
IoIrykus, I. Iarns, C.T. and Lorz, I. (1979) MuIIiIe-dro-array (MDA) Iechnique for Ihe Iarge scaIe
IesIing of cuIIure nedia variaIions in hanging nicrodro cuIIures of singIe ceII sysIens. I. The
Iechnique. IIanI Sci. LeII. VoI. 14, 231-235.
Raveh, D. IuLernan, L. and CaIun, L. (1973) |n ti|rc cuIIure of IoLacco roIoIasIs. Ise of feeder
Iechniques Io suorI divison of ceIIs IaIed aI Iov densiIies. In ViIro 9: 216-22.
Rose, R. J. (1980). IacIors IhaI infIuence Ihe yieId, sIaLiIiIy in cuIIure and ceII vaII regeneraIion of. sinach
nesohyII roIoIasIs. us|. ]. P|an| Pnqsic|. 7: 71-725.
Schenk, R. I., and IiIdeLrandI, A. C. (1969) IroducIion of roIoIasIs fron IanI ceIIs in Iiquid cuIIure
using urified connerciaI ceIIuIases. Crcp. Sci. 9:629-631.
SniIh, I.I. Kao, K.N. and ConLaIIi, N.C. (1976) InIersecific IyLridizaIion Ly roIoIasI Iusion in
Nicc|iana. The J. IerediIy 67: 123-128. 1976.
TakeLe, I., LaLiL, C. and MeIchers, C. (1971) RegeneraIion of vhoIe IanIs fron isoIaIed nesohyII
roIoIasIs of IoLacco. Na|uruisscnscnaf|cn 58: 318-320.
VasiI, I. K. (1976) The rogress, roLIen, and rosecIs of IanI roIoIasI research. dt. grcn., 28: 119-
160.
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MicrocuIIures. Science VoI. 150: 889-892.
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8icpnqsica c|a 694: 227-277.
Zinnernan, I. and Scheurich, I. (1981) Iigh frequency fusion of IanI roIoIasIs Ly eIecIric fieIds.
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Zinnernann, I. and Vienken, J. (1982) LIecIric fieId-induced ceII-Io-ceII fusion. ] Mcm|r 8ic| 67: 165-182.
Suggested keadiogs
1. Bengochea, T. and Dodds, J.I. (1986). IIanI IroIoIasIs: A LioIechnoIogicaI TooI for Cro
InrovenenI. Chanan & IaII.
&' C.iiuior orc B:oc|.r:coi Sc:.rc.s Boo
2nd Proof 31/5/8
2. Dodds, D.J. and Bengochea, T. (1987) IIanI IroIoIasIs: A BioIechnoIogicaI TooI for IIanI
InrovenenI, Chanan & IaII.
3. Davey, M.R. and Kunar, A. (1983) Iigher IanI roIoIasIs: reIrosecI and rosecI. In: P|an|
Prc|cp|as|s. |n|crna|icna| Rcticu cf Cq|c|cgq, (CiIes, K.L. Ld.) Su. 16, 219-263. Acadenic Iress,
Nev York.
kVIW q0StI0hS
Loog questioos
1. WriIe a noIe on five inorIanI aIicaIions of roIoIasI Iechniques.
2. WriIe shorI noIe on roIoIasI isoIaIion Iechniques and vhich one is nore efficienI?
3. WriIe a noIe on ceII vaII and roIoIasI viaLiIiIy IesI.
4. WhaI are Ihe neIhods of roIoIasI fusion? DescriLe any one neIhod in deIaiI.
5. DescriLe Ihree neIhods of sonaIic hyLrid seIecIion.
Hu|tip|e 6boice questioos
1. Who discovered enzynaIic neIhod of roIoIasI isoIaIion?
(a) KIercker (L) L.C. Cocking
(c) I. K. VasiI (d) I. Zinnernann
2. Which enzyne is used Io dissoIve niddIe IaneIIa in IanI ceIIs?
(a) CeIIuIase (L) IacIinase
(c) BoIh of aLove (d) Non of aLove
3. Crude ceIIuIase enzyne vas isoIaIed fron vhich organisn?
a) BacIeria (L) SnaiI
(c) Iungus (d) AIgae
4 Which chenicaI is used in ceII vaII IesI?
(a) Saffranin (L) CaIcofIour
(c) IIuorescence diaceIaIe (d) Non of Ihe aLove
5 Which chenicaI is used in roIoIasI viaLiIiIy IesI?
(a) AnaIine LIue (L) CaIcofIour
(c) IIuorescence diaceIaIe (d) Saffranin
6 Iigh Ca
+
ions and high I is used for roIoIasI fusion Io
(a) Increase osiIive charge (L) Increase nenLrane fIuidiIy
(c) Make roIoIasI neuIraI (d) AII of aLove
7. WhaI effecI on roIoIasI can Le oLserved under an eIecIric fieId
(a) BursIing (L) Shrinking
(c) DioIe fornaIion (d) Non of Ihe aLove
8. RegeneraIion of conIeIed IanIs Ihrough roIoIasIs vas firsI reorIed in
(a) DaIura (L) IeIunia
(c) Niccciana |a|cum (d) Daccus carrc|a
Frotojiost Cuitur. orc Sorot:c H,or:c:;ot:or &'
2nd Proof 31/5/8
9. IirsI successfuI regeneraIion of conIeIe IanIs fron sonaIic hyLrid have Leen
reorIed Ly
(a) TakeLe, I. eI aI (L) L.C. Cocking,
(c) I.K. VasiI (d) J.B. Iover
10. Who deveIoed eIecIric induced roIoIasI fusion Iechnique?
(a) I. S. CarIson (L) L.C. Cocking
(c) I. K. VasiI (d) I. Zinnernann
Aoswers
1. (L) 2. (L) 3. (c) 4. (L) 5. (c) 6. (d)
7. (c) 8. (c) 9. (a) 10. (d)