Kiran Makhani conducted experiments on in vitro germination of Loki seeds and tissue culture of Solanum Surattense. For Loki seeds, bleaching time and concentration affected contamination levels, with 20 minutes and 5% bleach showing the least contamination. For Solanum Surattense explants, apical and lateral meristems treated with 10% bleach for 20 minutes showed initial growth but became contaminated by day 4, while stamens remained uncontaminated and enlarged over time. The experiments assessed the impact of different bleaching parameters on contamination control for in vitro culture.

1. Kiran Makhani
PakistanCouncil for Scientific and
Industrial Research
Department of Plant Biotechnology
[INTERNSHIP REPORT]
- Effects of Bleaching Time and Bleach Concentrations on the Contamination Control of
In Vitro Germination on Loki seeds
-To Grow Solanum Surattense In Vitro and Observe the Effects of Bleach Concentration
and Bleaching Time

2. Effects of Bleaching Time and Bleach Concentrations on
the Contamination Control of In Vitro Seed Germination
Kiran A. Makhani
University of Karachi

3. Introduction
In natural environment,seedgerminationprobabilityisverylow,owingtoseveral bioticandabiotic
factors.It accounts fortwo phases:Phase one referstothe movementof seedfromparenttocertain
surface;Phase tworeferstothe vertical andhorizontal movementsof the seed.The distance andtype
of movement,seedmorphology,surface attributesall contributetothe viabilityandthe probabilityof
germinationof aseedinnatural environment.Incontrast,seedculture inacontrolledenvironmentwith
propermaintenance of asepticconditions andadequate nutrientavailabilitycanserve toincrease the
germinationprobabilityof the seedmanifold.Asaconsequence,plantseedculture hasgaineda
significantplace in variousapplicationsof botanical sciences.
Tissue culture isa methodof plantpropagationdone ina laboratory.There are manymethodsof plant
propagation,one of whichisbyusinga seed.
LOKI-The calabash,Lagenaria siceraria,alsoknownas opo squash,bottle gourd orlongmelon,isa
vine grownforits fruit,whichcaneitherbe harvestedyoungandusedasa vegetable,orharvested
mature,dried,andusedas a bottle,utensil,orpipe.The freshfruithasalightgreensmoothskinanda
white flesh.Roundervarietiesare called calabashgourds.Theycome ina varietyof shapes:theycanbe
huge and rounded,small andbottle shaped,orslimandserpentine,more thanametre long.
The calabash wasone of the firstcultivatedplantsinthe world,grownnotprimarilyforfood,butforuse
as a water container.The bottle gourdmayhave beencarriedfromAfricatoAsia,Europe and the
Americasinthe course of humanmigration,orbyseedsfloatingacrossthe oceansinside the gourd.It
has beenproventobe inthe NewWorldpriorto the arrival of Columbus. Itsharesits commonname
withthat of the calabash tree.
Methodology
2.1 Preparation of MS Zero Media- Sucrose andgellingagent-Phytagelwasaddedto Murashige and
Skoogzeromediumstocksolutionandwasallowedtodissolve onamagneticstirrer.
2.2 Autoclaving- Mediawas poured upto a centimeteranda half ineach of the twelve jarsandset aside
to be autoclaved.Tool setwaswrappedtobe autoclaved,whichincludedthreeforceps,one scalpel and
a pair of scissors.Three Petri disheswere wrappedseparately.Six jarsfilledathirdwithdistilledwater,
alongwiththe above mentionedmaterialswasautoclavedfor15 minutesat15 psi and 121 degrees
Centigrade.
2.3 Surface Sterilization- The surface of LaminarFlow hood wasthoroughly sterilizedwith70 percent
ethanol. The toolswere submergedintoajar havingethanol andthenflameduntil redhot. All the
bottlesandjars were wipedwithethanol before placingthemontothe hoodsurface.
2.4 Bleachingand Sonication- Bleachingsolutionswerepreparedatconcentrationsof 5,10 and 15
percent-fourjarsperconcentration. The dry,preservedLoki seeds were washed intapwaterto remove
preservativesand were addedthree eachinthe previouslypreparedjarsof bleachingsolution. Half of

4. the jars of each concentrationwere placed inaSonicatorfor10 minutesandthe otherhalf for20
minutes.
2.5 Washingwith DistilledWater- The seedsfrom5% bleachingsolutionjarsthatwere sonicatedfor
tenminutesandwere immersedinajarcontainingdistilledwater.The jarwasshakenforten minutes.
The procedure wasrepeatedforthe otherbleachingconcentrationsaswell.
2.6 Inoculation- In a laminarflowcabinet, the seeds were removed fromeachjarwiththe helpof
forcepsand were placedontothe agar inagar-containingjarsinduplicates(threeineach).Itwasmade
sure that the forcepswere heat-sterilizedaftereveryuse.
Observation
S.No Seed Sonication
Time
Bleach
Concentration
Replicates Contamination
Day
1
Day
2
Day
3
Day
4
Day
5
Day
6
Day
7
Day
8
01. LOKI 10 min 5% Jar 1 No Yes
(3B)
Yes
(3B)
Yes
(3B)
Yes
(3B)
Yes
(3B)
Yes
(3B)
Yes
(3B)
02. Jar 2 No No No No Yes
(1B)
Yes
(3B)
Yes
(3B)
Yes
(3B)
03. 10% Jar 1 No No Yes
(1B)
Yes
(1B)
Yes
(3F)
Yes
(3F)
Yes
(3F)
Yes
(3F)
04. Jar 2 No No Yes
(1B)
Yes
(1B)
Yes
(2B)
Yes
(2B)
Yes
(2B)
Yes
(3B)
05 15% Jar 1 No No No No No No No Yes
(1B)
06. Jar 2 No No No No No Yes
(3B)
Yes
(3B)
Yes
(3B)
07. 5% Jar 1 No No No No No No No No
08. Jar 2 No No No No No No No No
09. 10% Jar 1 No No Yes
(1B)
Yes
(1B)
Yes
(3F)
Yes
(3F)
Yes
(3F)
Yes
(3F)
10. Jar 2 No Yes
(1B)
Yes
(1B)
Yes
(3F)
Yes
(3F)
Yes
(3F)
Yes
(3F)
Yes
(3F)
11. 15% Jar 1 No No No No No No No No
12. Jar 2 No No Yes
(3B)
Yes
(3B)
Yes
(3B)
Yes
(3B)
Yes
(3B)
Yes
(3B)

5. Contaminated Loki seeds
Non-contaminated Loki seeds

6. Result
Three out of twelve jarsdevelopedcontaminationinaperiodof three weeks.
Discussion
Higherbleachconcentrationswere expectedtogive little ornocontamination.This seemsthe case for
the 15% bleachingsolutionssonicatedfortenminutes,inwhichcontaminationwasobservedlater
duringthe observationperiod. Onthe contrary,5% bleachingconcentrationswere more successful than
10% and15% in twentyminutessonicationperiod.Thissuggeststhatthere mightbe some handling
error duringthe procedure.
On the otherhand,time factor hassomewhatappearedtofollow the expectedresult.Three outof six
jars sonicatedfortwentyminutesturnedouttobe contamination-free.Therefore,itmaybe concluded
that twentyminutesisthe minimumtime forbleachingandsonication.

7. To Grow Solanum Surattense In Vitro and to Observe the
Effects of Bleach Concentration and Bleaching Time
Kiran Makhani
University of Karachi

8. Introduction
Plantsgrowinginnatural environmentencounterseveral stresses:physical,chemical,biological etc.The
harsh sunlightandweatherextreme bringaboutadecline inthe qualityof the plantanditsfruits.
Nutritional deficiencies,too,hinderpropergrowth.Moreover,the microorganismthatattackplant,not
onlylimititsgrowthbuthave a long-termeffectonitsfruit,flower,andmostimportantly,itsprogeny.
Tissue culture of explantobtainedfromahealthyplantcantherefore give rise toawhole fieldof its
progenyaftersubsequent multiplicationinaninvitro,controlledenvironment.Inthisexperiment,tissue
culture hasbeenusedto propagate the plant SolanumSurattense.
SolanumSurattensehasastrong medicinal valueandrecentresearcheshave shownittoscavenge
oxidizingradicals(Sridevi Muruhan,SenthilSelvaraj,andPugalendi KodukkurViswanathan,2013).
Hence,itcan have a strongprospectas an anti-agingtherapy.
Methodology
2.1 Preparation of MS Zero Media-Sucrose andgellingagent-Phytagel wasaddedto Murashige and
Skoogzeromediumstocksolutionandwasallowedtodissolve onamagneticstirrer.
2.2 Autoclaving-Mediawaspouredupto a centimeterandahalf in eachof the twelve jarsandset aside
to be autoclaved.Tool setwaswrappedtobe autoclaved,whichincludedthreeforceps,one scalpel and
a pair of scissors.Three Petri disheswere wrappedseparately.Six jarsfilledathirdwithdistilledwater,
alongwiththe above mentionedmaterialswasautoclavedfor15 minutesat15 psi and 121 degrees
Centigrade.
2.3 Pickingthe DesiredPlant- SolanumSurattensewaspickedfromthe field andimmersedinwater
until the nextstep.Itwasmade sure that several apical andlateral shootsandbuds are picked.
2.4 Bleachingand Sonication-Bleachingsolutionswere preparedatconcentrationsof 10 and 15
percent-twojarsperconcentration.The dry,preservedLoki seedswere washedintapwaterto remove
preservativesandwere addedthree eachinthe previouslypreparedjarsof bleachingsolution. Half of
the jars of each concentrationwere placedinaSonicatorfor10 minutesandthe otherhalf for20
minutes.
2.5 Surface Sterilization-The surface of LaminarFlow hoodwasthoroughlysterilizedwith70 percent
ethanol. The toolswere submergedintoajar havingethanol andthenflameduntil redhot.All the
bottlesandjarswere wipedwithethanol before placingthemontothe hoodsurface.
2.6 Washingwith DistilledWater- Inside the laminarflow cabinet,bleachingsolutionsfromthe jars
havingthe plantcuttingswasdrainedandthe cuttingswere re-immersedindistilledwater. The jarwas
shookfor tenminutes.The cuttingsfromthe firstjarwere removed andimmersedintoanotherjar
havingdistilled water.Again, the jarwas shakenfortenminutes.Finally, the cuttings were re-immersed
inthe thirdjar havingdistilledwaterandshakenforanothertenminutes.

9. 2.7 Preparing SmallerCuttings- The cuttingswere laidontothe Petri platesand,withthe helpof blade
and forceps,the plantpieceswere furthercutintosmallerbits.The bleachedtipswere cutoff andthe
apical as well aslateral shootswere cutoff verynear to theirnodes.
The buds obtained,were gentlyopenedwiththe helpof twoforcepsandthe stamenswere setfree
ontothe agar-containingjars.
2.8 Inoculation-Ina LaminarFlowcabinet, the capof an agar-containingjarwasheat-sterilizedand
opened.The mouthof the jar wassterilizednext. The plantcuttings were removed fromthe Petri plates
withthe helpof forcepsandwere placedontothe agar inagar-containingjars(fourineach).Itwas
made sure that the forcepswere heat-sterilizedaftereveryuse.

10. Contaminated explant cutting
Stamen culture

11. Observation
Plant
Name
Explant
Part
Sonication
Time
Bleach
Concentration
Day 1 Day 2 Day 3 Day 4
Solanum
Surattense
Apical and
lateral
meristems
20
minutes
10%
Contamination
was observed
inone out of
fourcuttings
Contamination
was observed
inone out of
fourcuttings;
shoot
sproutingwas
observedat
the node and
an increase in
apical shoot
was also
observed
Contamination
was observed
inone out of
fourcuttings;
further
increase in
apical and
lateral shoots
were observed
The whole
agar surface
got
contaminated
withbacterial
lawn;
although
there wasa
veryslight
increase in
shootsize
Stamens
No
contamination
was observed
The stamens
were observed
to swell
The stamens
were observed
to swell
further
Most of the
stamens
remained
swollen,
however,
some opened
up
Apical and
lateral
meristems
15%
Contamination
was observed
intwo out of
fourcuttings
Contamination
was observed
intwo out of
fourcuttings;
Leaflets
sproutingwas
observedat
the apex and
the shootsize
increased
Contamination
was observed
intwo out of
fourcuttings;
shootsize
increased
considerably
and lateral
shootarose
The whole
agar surface
got
contaminated;
no growth
was observed.
One of the
cuttings
wilted
Stamens
No
contamination
was observed
The stamens
were observed
to swell
The stamens
were observed
to swell
further
Most of the
stamens
remained
swollen,
however,
some opened
up

12. Result
Both the jars withplantcuttingsdevelopedcontamination onDay4 of observationwhile boththe jars
containingstamensremainedintact.
Discussion
Neither10%nor 20% of bleachconcentrationsprovedtobe effective topreventcontaminationinthe
plantcuttings.The thornyand roughsurface of the plantstemmay account forthe observedresultas
such surfacesharborbacteria.Probably,ahigherconcentrationof bleachmayprove effective.
The two jars havingstamenswere free fromcontaminationasthe budsprovide asterile conditiontothe
stamens.These stamenswere neitherexposedtothe bleachtreatmentnorto the Petri plate andwere
directlyreleasedfromthe budintothe jars.
Reference
In vitro antioxidantactivitiesof Solanumsurattenseleafextract
Sridevi Muruhan,SenthilSelvaraj,andPugalendi KodukkurViswanathan,2013
Acknowledgements
I am grateful toDr.BeenaNaqvi andthe staff at the Departmentof PlantBiotechnology,PakistanCouncil
for ScientificandIndustrial Researchfortheirimmensesupportand the learningexperience providedby
them.