Technicsl report
I
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EVALUATION OF SPECIFIC PARAMETERS IN THE
ISOLATION OF PROTOPLASTSFROM MESOPHYLL CELLS
OF THREE MULBERRY CULTIVARS
u. PAVAN, A. V. RAO, V. YASHODHARA, N. RAMA SWAMY & A. SADANANDAM*
PlantBiotechnologyResearchUnit, Departmentof Botany,Kakatiya University,
Warangal- 506 009, India.
Significanceof type of mulberry (Morus indica L.) cultivar, enzymetreatmentduration and level
of the solute during protoplast isolationfrom mesophyllcells of mulbeny has been investigated.For
this purpose,axenicshoot cttlturesof three ntulberrycultivars i.e. Kz, Sn and 536were raisedvia
culture ofnodal explants.From the results, it may be concludedthat protoplast yield in ntulbeny is
cultivar dependent.I 2- I 3 hours prolonged incubation of cv. Szoresulted in higher protoplast yield
compared to cvs. K2 and 53. However, t hours of short duration incubation is pre.ferable to get
ntaximuntprotoplastyield.from cvs.Kz and Sn. 0.5 M concentrationof mannitolas osntoticuntwas
.found to be more appropriatefor protoplast isolation.
Keywords: Morus indica,cvs. K2, St: and 536,protoplastisolation,cultivar dependent,cellulase
onozukaR-10,macerozyme
R-10.
INTRODUCTION
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Mulberry (Morus indica L.) plantsplay a significantrole in the silk industryandforestry(Hossain
et al.,1992;Zamanet al.,1993,1996;Kathiravanet a|.,1995;Tewaryet al.,1996).Selectionof novel
genotypesshowinghigh yield, suitabilityto silkworm rearingandresistance
to differentabiotic stress
factorsi.e.,toleranceto waterstress,alkalinity and salinityis alwaysdesirable.Protoplastcultureand
fusion can facilitate the combinationof such useful traits by producing somatichybrids. Somatic
hybridizationprovidesan alternativemethodto conventionalhybridisationfor improving mulberry.
Theseuniquepropertiesofprotoplasthaveopenedup an entirelynew areaoffundamentalandapplied
researchin experimentalbiology and somaticcell genetics.However,for the successfulapplication
ofthese techniquesthe availabilityofan efficientprocedurefor protoplastisolationis a prerequisite.
In mulberry not much work hasbeendone in this regardexcepta few reportson isolation(Ohyama
and Oko, 1975;Ohnishiand Kiyama,1987 Katagiri,1988;1989;Tewaryand Lakshmisita,1992;
Tewaryetal,,1995:Chandetal.1996;Tohjimaetal.,1996).
The experimentdescribedin this paperwas carriedout to improvethe yield of protoplastsfrom
mesophyllcells by analysingthreespecificparametersi.e.,the type of mulberrycultivar usedduring
protoplastisolation(Kz, Sr: and S:o),the effectof durationof enzymetreatment(prolongedand short
durationincubationperiod)and the effect of osmoticumlevel (0.4M,0.5M and 0.6M).
* Correspondingauthor.
2000 - Sericologia40(3) 469-474
469
ISOLATIONOF PROTOPLASTS
OF THREEMLJLBERRY
CULTIYARS
MATERIALSAND METHODS
Axenic shootculturesof threemulberrycultivarsof Morus indica namelyK2, St 3 and S3owere
usedfor the isolationof mesophyllprotoplasts.Leavesfrom in vitro grownplantswere alwaysfound
to be more promisingfor protoplastisolationthan thoseof in vivo plants(Tewary and Lakihmisita,
1992).To initiate axenic cultures,the nodal explantswere found to be the best sourcefor rapid
micropropagationof mulberry throughin vitro cloning (Zamanet al., 1996).
Young fully expandedleavesof 2-3 cm in lengthand I - I .5 cm in width were excisedfrom all
the partsof the shoots(3-4 cm) approximately10-15daysfrom the beginningof the subculture.The
leaveswere cut into piecessmallerthan I mm and incubatedin filter sterilisedenzymesolution.The
enzymesolution consistedof 2Yo cellulase"Onozuka" R- I 0 and lYo macerozymeR- I 0 preparedin
MS (Murashigeand Skoog, 1962)saltsat pH 5.5 with mannitol as osmoticum.Different l-evelsof
osmoticum (0.4 M, 0.5 M and 0.6 M) were tried to optimisethe level of osmoticumto get better
protoplastyield.
The sliced leaf pieceswere incubatedin l0 ml of enzyme solution at 27" C and shakenat
40-50 rpm for 5 hours.After being left stationarilyover a periodof I - I 0 hours,the protoplastswere
then purified by 60 pm steelmesh.The filtrate was collected in screw cap centrifuge tube and
centrifugedat 50 g for 5 minutes.The supernatant
was discardedandthe pellet containingprotoplasts
was floatedon20%osucrosesolutionfor purificationand centrifugedat l00g for l0 minutestoget a
distinctprotoplastband.The bandwas takenin a screwcap centrifugetube and washedwith S ml of
0.5M mannitol by centrifugingat 50g for 5-7 minutes.The pellet was suspendedin culturemedium
containingMS + 0.5M mannitol + 2,4-D and BAP and the protoplastyield was estimatedby using
hemocytometer.Upto 15 hours from the start of incubation,protopl;st in 500 pl aliquoteswere
removedand centrifugedevery hour and usedfor microscopicinvestigations.
RESULTSAND DISCUSSION
Effect of duration of enzymetreatment on protoplast isolation:
In the caseof two mulberry cultivarsKz & St:, prolongedincubationperiodsi.e., 12-13hours
were observedto be unfavourablefor a good protoplastyield. Longer durationofincubation caused
shrinkageof protoplastsin thesecultivars.Slicedexplantsof cvs.Kz and St: immersedin the enzyme
mixture shakenat 40-50rpm in dark for 5 hoursandbeing left stationaryfor 4 hours,resultedin better
yield of protoplasts.The numberof protoplastsincreasedwith treatmenttime and it reacheda peak
at t hoursofincubation(5 hoursshakenand4 hoursstationary)in dark.Beyondt hoursofincubition
the protoplastyield graduallydecreased
and furlher resultedin shrinkageofprotoplastsat l2-13 hrs
of incubation.From theseresults,it is estimatedthatthe adequatetime for enzymetreatmentto isolate
maximum numberof protoplastsfrom mesophyllcellsof cvs.Kz (Fig. 1a)and Sr: (Fig. lb) is 9 hours
(5 hoursshakenand 4 hoursstationaryincubation)in dark.
Severalauthorshavereportedthat cell digestionand protoplastyield in caseqfcvs. Kz and SI:
were fairly good during l2- I 3 hoursincubationin the dark (Tewaryand Lakshrr{isita,1992,Tewary
et al., 1995.Chandet al., 1996)in contrastto our observations.
However,our findingsagreewith the
result of Tohjima et al., (1996) who have examineda similar effect of enzymetreatmenttime for
shorterduration(4 hours)on protoplastisolationfrom seedlingcotyledonsof mulberry.
Contradictoryto the abovefindings,a prolongedincubationin dark for 12-13hoursresultedin
much increasein protoplastyield from mesophyllcells of cv. S:o (Fig. 2). The numberof mesophyll
protoplastsisolatedfor every hour, afterbeing incubatedin the enzymesolutionfor increasingtime
(up to l5 hours)is shownin Fig. 3. In the caseof cvs.K2 and S13,the numberof protoplastsincreased
with treatmenttime and reachedpeak at t hours(6 x l0o and 5.6 t l0o respectively).tn caseof cv.
536,the number ofprotoplastsincreasedduring longer incubationperiod i.e., l2-13 hours (5 hours
470
2000- Sericologia
40(3)469-474
U. PAVAN...
shakenand 7-8 hours stationaryincubation)and reacheda maximum of 6.2 x 106.Tohjima et al.,
(1996) have opined that cell walls are not completelydigestedin shortertreatmenttimes,but some
protoplastsget releasedduring longertreatmenttimes.The abovestatementwas found to be correct
in accordancewith our findingsthatthe cell walls of cvs.Kz and Sr: get completelydigestedin shorter
enzymetreatmenttime, but protoplastsof cv. S:o get releasedduring longertreatmenttimes.
The result of the presentinvestigationhas revealedthe importanceof incubation time on
protoplastyield which in tum is cultivar dependent.Earlier studieson plant induction from winter
buds(Oka, 1985),axillarybuds(Jainet al.,1990),leafexplants(Wenet a\.,1990)andnodalexplants
(Tewary et al., 1996) of mulbeny genotypesshowed that the in vitro regenerationis genotype
dependent. Even if the in vitro regeneration is genotype dependentas th-eprotoplast y-ield,ihe
mechanism involved in the two physiological behavioursis probably completely different and
dependingon different factors.
Fig l. Freshlyisolatedmesophyll
p r o t o p l a s t sa f t C r t h o u r s o f
incubationin enzymesolution
from (a) Kz cultivar (b) Srr
cultivar.
Fig. 1, Protoplqstesdu misophylle
fratchement isolAsdu cultivar K2
(a) et du cultivar Srs ft) apris 9
heures d'incubation dans une
solutionenzymatique.
2000- Sericologia
40(3)469-474
471
ISOLATIONOF PROTOPLASTS
OF THREEMULBERRYCULTIYARS
Fig. 2. Freshly isolated mesophyll
protoplastsfrom Srocultivar after 13
hours of incubation in enzvme
solution.
Fig, 2. Protoplastes du misophylle
Jratchement isolts du cultivar Sj6
apris 13 heures d'incubation dans une
solu ti ott en 4ym ati qu e.
3'hF
6 hF (5'+1")
t hre (5'+4")
l2 hF (5'+7-)
15 hF (5'+10")
Duration of €Dyme treatrent
;4r'
Fig. 3. Time schedulefor the releaseof mesophyll protoplasts isolated from three mulberry cultivars
(K2, S13 and 536).
Leaves
wereexcised
afterI 0-I 5 daysfromsubculture.
Protoplasts
wereprepared
with0.5M mannitol.
Thebarshows
standard
error.
Fig. 3. Cinitique de Ia libiration
desprotoplastes isolds du misophylle sur trois cultivars de mfirier (K2,
S1-3et 536).
Les.feuilles
sontprdleviesde la sous-culture
aprdsI 0 d I 5jours. Lesprotoplastes
sontprdpardsavec0,5M de mannitol.
La barrentontrel'6cart g,pe.
(* = shaken/ agltt : ** = stationary
/ stationnait.e)
472
2000- Sericologia
40(3)469-474
U. PAYAN...
,
Effect of osmoticum levels
on protoplast isolation:
Threeconcent
so,u,ion(d;;;:ffi
Xt:;':t*#i-f
n"i::ffi1:t[,tJff
:ifiT:itri,ff
fi:fl
Jilib?:iif;
:
concentrations
tried'0'5M was rounoiJu" the
mostappropriate
osmoticum
andwas.reporred
rever
forkeeping
to 6eil,e.igl.,t
-"i;;';.;toprast
investigated'
Thisisrontia-dic;""
,l'"itirrl
i"ilii;;ii';J
iTr=curtivars
rrz, sr: andS:o)
r"portr
l" *rri"r-,
wasconsidered
o.eM concentrati-on
to be-ot" fuuoutubr.'ror'r"ropr',yl
"u.ti"r p.topiuri'iJorution
oimunnitor
(TewaryanaLatstrrnisita,
iff"';"l:ffi';lfi:;
G;;,;i;, oqM
it^;:^::^t:;;"fi?;i#ifi*#i:"i?$,
",i,"
However'
o'o
v to"tln,tu,i"#.i*i*ar;
;TtJlli,l"
ot,ro,u."o
proroprasrs
""r".g""ri*
andcaused
In conclusion'it may be said
that optimisationof protocols
forprotoplastisolationfrom
cultivars
needs
a otiotuna"tstunoin!"firr
murberry
."gqg
r" ,ri",r"r*'"rlulriu* ur"ain the
confirmtheimportance
process.
our dara
iryyu"w?'rti""rin*,
tn protoplast
time
and
isolation
rever
of osmoticum
andits yitro' v*" research
"i;6;"##ent
,ro"ro6r-r"'anted to find the
conditions
pe*nitting
better
favourabre
cuttivarl"a"p""ru"io.,oor"Jivt"ri
ilJ,nr"ropt y1 cersof murberry.
ACKNOWLEDGEMENTS
Theauthors
arethankfur
ro Dr R. K. Da*a,Director,
csRTI,yr::.: for providing
murberry
assrsrance.
A.V.Ris grateful
theawariorn"seuictarro"iii.rr,io..,rs'w,a,
,Jb.sr[, N"r.bJrr,iro.
:li:ln::ii,o]:T,*'-;"-irii*"n"o,ogy,NewDe,hia
itt'tr',il6i;";Tf#ffi
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2000- Sericologia
40(3)469-474