In vitro
regeneration of
Arachis hypogaea
L. and
Moringa oleifera
Lam.using extracellular phytohormones from
Aphanothece
sp. MBDU 515
Manickam Gayathri
,Azhagiya Manavalan Lakshmi Prabha
, Gangatharan Muralitharan
a,
a
Department of Microbiology, Centre of Excellence in Life Sciences, Bharathidasan University, Palkalaiperur, Tiruchirappalli 620 024, Tamilnadu, India
b
Department of Plant Sciences, Centre of Excellence in Life Sciences, Bharathidasan University, Palkalaiperur, Tiruchirappalli 620 024, Tamilnadu, India
a b s t r a c ta r t i c l e i n f o
Article history:
Received 9 July 2014Received in revised form 7 November 2014Accepted 17 December 2014Available online xxxx
Keywords: Aphanothece Arachis hypogaea
Cyanobacteria
Moringa oleifera
PhytohormonesPlant tissue culture
The present study aims to develop an ef
cient and simple protocol for
in vitro
plant regeneration usingcyanobacterial liquid medium. The ability of the unicellular cyanobacterium
Aphanothece
sp. MBDU 515 to pro-duce the phytohormone indole-3-acetic acid (IAA) in the medium was demonstrated and chemically analyzedbySalkowskiassay,Highperformanceliquidchromatography(HPLC)andGaschromatography
–
Massspectrom-etry (GC/MS). The effect of cyanobacterial extracellular product (CEP) for
in vitro
propagation was evaluated ontwo economically important plants
Arachis hypogaea
and
Moringa oleifera
. The effect of commercial phytohor-mones 6-Benzylaminopurine (BAP) and Indole-3-butyric acid (IBA) in shoot and root induction, respectivelywas compared with CEP in both the plants. In the present study, it was observed that 1 mL of CEP per 10 mL of MSbasalmediumshowedanincreasein%ofshootandrootresponses,shootandrootlengthsandaveragenum-ber of roots per shoot compared to the control (MS basal medium). These effects of CEP are comparable to thecommercial phytohormones
—
BAP and IBA, tested. The tested CEP markedly reduced the accumulation of phe-nolics in
A. hypogaea
explants. Our study suggests that CEP may be used for micropropagtion at industrial scaleinstead of commercially available phytohormones.© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Ef
cient
in vitro
regeneration of plants depends upon reliable tissueculturetechniques,whichcanbringaboutsuccessfulapplicationofbio-technologyincropimprovement[1].Currentlythistechnologyhasbeensuccessfullyappliedtoawiderangeofcrops,forestandfruit,withcom-mercial micropropagation laboratories worldwide. However, the tech-nology is capital, labor and energy intensive. Hence, it is necessary tohavelowcostoptionsfortissue-culturetechnology.Inlowcosttechnol-ogy,costreductionisachievedbyimprovingprocessef
ciencyandbet-ter utilization of naturally available resources in place of commercialchemicals[2].Twoclassesof phytohormones i.e.,cytokininsand auxinsare very important for
in vitro
regeneration of plants [3]. It is alreadywell evident that cyanobacteria are competent in both accumulatingand releasing a variety of phytohormones such as auxins, gibberellins,cytokinins, vitamins, polypeptides, and amino acids, which promoteplant growth and development [4,5].Cyanobacteriaareamongthemostgeographicallywidespreadgroupoforganismsknown,andevendominatesomeaquaticandterrestrialen-vironments [6]. They are the most proli
c source of wide variety of secondary metabolites [7]. There are many examples of cyanobacteriafrom different genera that produce both exogenous and endogenousIAAs[3,8
10].
Invitro
regenerationof
Oryzasativa
,
Triticumaestivum
,
So-lanumtuberosum
,
Brassicaoleracea
,
Nicotianatabacum
andtheornamen-tal plant
Lilium alexandrae
15].Prasannaet al. [14,16] screened the rhizosphere soil of twelve rice cultivars fromsix different regions of India and reported that
Anabaena
strains CW1andRP9exhibitedpromisingIAAproduction.Interestingly,the
Anabaena
strain CW1 was isolated from the rice
eld of Aduthurai, Tamilnadu,India. However, only few species of unicellular cyanobacteria havebeen tested so far for the production of phytohormones.Hence this study aims to determine the ef
ciency of unicellularcyanobacterial strain
Aphanothece
sp. MBDU 515 isolated from the rice
eld of Thanjavur district for its plant growth promoting potential in
Arachis hypogaea
and
Moringa oleifera
. Peanut (
Arachis hypogaea
) isone of the principle economic oilseed legumes and is largely cultivatedinmanytropicalandsubtropicalregionsoftheworld andtheyareveryrecalcitrantto tissuecultureregeneration.Therefore, manyefforts havebeen devoted to develop an ef
cient
in vitro
M. oleifera
described clonalpropagation through the use of nodal explants taken from non-speci
Moringa
species is thus of great concern from
⁎
Corresponding author.
E-mail address:
drgm@bdu.ac.in (G. Muralitharan).
1
Equally contributed.http://dx.doi.org/10.1016/j.algal.2014.12.0092211-9264/© 2014 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Algal Research
journal homepage: www.elsevier.com/locate/algal
2. Materials and methods
2.1. Cyanobacterial strain and growth conditions
Thefreeliving
Aphanothece
sp.MBDU515wasisolatedfromtherice
elds of Thanjavur district, Tamilnadu and was maintained in axenicform in the germplasm of Department of Microbiology, BharathidasanUniversity, Tiruchirappalli. Theorganism wasgrownina250mLErlen-meyer
ask containing 100 mL BG 11 medium and incubated at 25 °Cunder a light-
ux density of 50
μ
mol/m
2
2.2. Preparation of cyanobacterial extracellular product (CEP)
TheCEPwaspreparedbyharvesting15daysoldcultureofthetestedcyanobacterium at 10,000 rpm for 15 min at 4 °C and supernatant wascollected. The supernatant was sterilized by
ltration before beingused at various concentrations along with basal Murashige and Skoog(MS) medium [12].
2.3. Extraction and identi
cation of IAA
The cyanobacterial culture broth was centrifuged at 10,000 rpm for15minat4°Candthesupernatantwascollected.The40mLofsuperna-tantwasextractedwith80mLofethylacetateinseparatingfunnel.Ethylacetate fraction was recovered and evaporated. The residue was dis-solved in methanol and used for further analysis. HPLC equipped witha constant
ow pump (Shimadzu, Japan) was used. Separation was ac-complished under reversed phase isocratic conditions with (Shim-PackCLC-Octadecyl silane) ODS-C
18
column (4.6 mm ID × 25 cm) and guardcolumn (Shim-Pack G ODS) (4 mm ID × 1 cm) and mobile phases of 100% methanol. The
ow rate was adjusted to 1 mL/min for analysisand a UV absorbance at 254 nm was used as a detector. Prior to GC/MSanalysis,sampleswereresuspendedinanappropriatevolumeofmetha-nol.Samplesof1
μ
LinmethanolwereinjectedintotheGC/MSequippedwith HP5 fused silica capillary column (30 m × 0.25 mm × 0.25
μ
m).Heliumwasusedasacarriergasata
owrateof1mL/minandoperatedin a splitless mode.Injector anddetector temperatures were bothset at220°C.Theoventemperaturewasheldat80°Cfor2min,thenrampedat a rate of 20 °C min
−
1
and then
nally programmed from 220 °C to250 °C at a rate of 10 °C min
−
1
for 10 min. MS was performed with anAGILENT Gas chromatograph coupled to a GCmate
™
II Joel (Japan)ion trap mass spectrometer using electron ionization (EI) in apositive mode with ionization energy of 70 eV. Spectra wererecorded from
m
/
z
100 to 2500 and with a scan rate of 0.5 s scan
−
1
.
2.4. Plant material and culture media
Healthyandnodalexplantsof
Arachishypogaea
and
Moringa oleifera
werecollectedfrom45daysoldplantsgrownundergreenhousecondi-tion at Bharathidasan University, Tiruchirappalli, Tamilnadu, India.Shoot tip, internode and nodal explants were washed thoroughly inrunning tap water for 20 min, before being dipped in 70% ethanol for60 s and rinsed with sterile distilled water and surface sterilized using0.1%mercuricchloridesolutionthenagainwashedwellwithsteriledis-tilled water (3 to 5 min each). Apical and nodal regions were used forthe following experimental setup.
2.5. Phytostimulation experimental setup
To assess the effectof CEP on shoot regeneration,the explants weresubculturedintofreshculturetubescontainingMSbasalmedium(MS),MS+BAP(1.0
–
2.0mg/L)andMS+CEP(0.5
–
1.0mLofCEPin10mLof MS basal medium, (hereafter 0.5 mL/10 mL and 1.0 mL/10 mL,respectively)andallowedtogrowfor15days.Theculturetubecontain-ing MS medium without the addition of plant growth regulators wasserved as control. After 15 days, the explants from each experimentmentionedaboveweretransferredtonewculturetubescontainingcon-trol,MS+IBA(1
–
2.0mg/L)andMS+CEP(0.5
–
1.0mL/10mL)forrootregeneration.Foreachtreatmenttenreplicateswereused.Allthetreat-ments were repeated at least three independent times. The treated ex-plants were maintained in a growth chamber at 25 ± 2 °C and16 hour photoperiod and 8 hour dark period with PPFD of 50
μ
mol m
2
/s provided by cool white
uorescent lamps for 15 to30 days to obtain shoot and root development [22]. The number of ex-plants responded to shoot development (% of shoot response) was cal-culated for each treatment. Also, the length of shoot responded wasmeasured, averaged and compared between control and treatments.Similar measurements were carried out to calculate the percentage of root response, the number of roots per explant and the length of roots.
2.6. Salkowski assay
The screening for the presence of IAA-like compounds was carriedout as described earlier [23]. The Salkowski reagent (1 mL of 0.5 MFeCl
3
, 50 mL of 35% HClO
4
) [24] was added to the culture supernatantin a ratio of 1:1 (v/v). The concentration of IAA in the culture mediumwas determined by comparison with the standard curve. Auxin-likesubstances were estimated by OD at 535 nm after 30 min in the darkatroom temperature. Salkowski assay was used for the determinationsof released, Salkowski-positive indolic compounds, particularly auxins.The presence of IAA in the CEP was further con
rmed using HPLC andGC/MS analyses.
2.7. Statistical analysis
All experiments were conducted with ten replicates per treatmentand the mean values were compared using Duncan's Multiple RangeTest (P
b
0.05) using SPSS v.20 for windows 7 Basic.
3. Results and discussion
In this study, MS medium was complemented with two differentconcentrations(1and2mg/L)ofBAPandIBA,forshootandrootinduc-tionrespectivelyandwerecomparedwiththatofCEPactivity.McKentlyet al. [25] reported that the MS medium supplemented with BAPshowed maximum shoot regeneration from the explants taken fromtheseed of peanut. In
Arachis hypogaea
, theMS mediumsupplementedwith0.5mLand1mL/10mLofCEPshowedatwofoldincreaseinthe%of shoot response compared to the control. However, % of shoot re-sponse between the MS medium supplemented with BAP and MS sup-plemented with CEP at both the concentrations tested was statisticallysimilar. In addition, the shoot length was increased two fold in the MSmedium supplemented with CEP (1 mL/10 mL) when compared totheMSmediumsupplementedwithbothconcentrationsofBAP,where-astheshootlengthintheMSmediumsupplementedwithCEP(0.5mL/mL)wasfoundtobestatisticallysimilar(Table1aandFig.1a).Although
there is an increase in % of shoot response in BAP treated explants, theshoot length is decreased when compared to control (MS basal medi-um)inbothcases(Table1aandFig.1a).Thisisduetotheaccumulation
ofphenolics(Fig.2b)duringshootdevelopment.Theabsenceofpheno-lics might be attributed to the two fold increase in shoot length in CEPtreated explants (Table 1a).For root induction in
Arachis hypogaea
, % of root response, averagenumber of roots/shoot and the root length were measured for each of the treatments (Table 1b). The MS medium supplemented with CEP(0.5mLand1mL/10mL)showedsimilar%ofrootresponseandaveragenumberofrootsproducedpershootcomparedtoIBAatbothconcentra-tions(1and2mg/L),whereas,theMSmediumsupplementedwithCEPat 1 mL/10 mL and 0.5 mL/10 mL concentrations showed a three-fold
101
M. Gayathri et al. / Algal Research 7 (2015) 100
–
105
and two-fold increase in root length compared to theMS medium sup-plemented with IBA (1 mg/L), respectively (Table 1b and Fig. 1b). This
clearlydemonstratedthesimilareffectsofCEPandIBAinrootinductionof
Arachis hypogaea
. Our results agree with those obtained byextracellular products of cyanobacterium
Phormidium subincrustatum
on the regeneration of
Wedelia trilobata
L [26].Browning and blackening due to excessive accumulation of pheno-lics are the major drawbacks of
in vitro
culture of many economicallyimportantplants.Interestingly,wefoundtheabsenceofphenolicsecre-tion in shoot and root induction during CEP treatment in
Arachishypogaea
(Fig.2dande).Whencellsaredamaged,thecontentsofcyto-plasmandvacuolesaremixedandphenoliccompoundscanreadilybe-come oxidized by air, which may inhibit enzyme activity and result inthe darkening of the culture medium and subsequent lethal browningof explants or causes rooting de
ciencies. The oxidized phenols (poly-phenoloxidase)arenegativelycorrelatedthatproducehighlytoxicqui-nones and polymerized material causing discoloration of the mediumand death of the cultured explants [27]. Notably, the CEP used in ourstudy also inhibited the accumulation of phenolics compared to thatof control treatment (Fig. 2a and b).Another economically important plant
Moringa oleifera
was alsotested for root and shoot development with CEP. In
Moringa oleifera
,the MS medium supplemented with 0.5 mL and 1 mL/10 mL of CEPshowed a similar level of % of shoot response as that of control and MSmedium supplemented with BAP in 1 mg/L and 2 mg/L concentrations,whereas the length of the shoot in medium supplemented with bothBAP(Fig.4b) and CEP (Fig. 4d) at both concentrationswassigni
cantlyincreasedthreefoldwhencomparedtocontrolshoots(Table2a,Figs.3a
and4a).OurresultsindicatethatalthoughCEPdoesnotplayanyrolein% of shoot response, it has a prominent function in the regeneration of shoot in
Moringa oleifera
. Similar effects were observed in % of root
Table 1
Effect of different concentrations of CEP on shoot response in
Arachis hypogaea
L. com-pared to the commercial phytohormones BAP and IBA. (a) Shoot response and shootlength in
Arachis hypogaea
L. (b) Root response, average number of roots per shoot androot length in
Arachis hypogaea
L.aTreatments % of shoot response Shoot length (cm)MS basal (Control) 40
d
4
b
MS + BAP (1 mg/L) 70
c
3
b
MS + BAP (2 mg/L) 80
b
2.5
b
MS + CEP (0.5 mL/10 mL) 80
b
4
b
MS + CEP (1 mL/10 mL) 90
a
6
a
MS
—
Murashige and Skoog; CEP
—
cyanobacterial extracellular product;BAP
—
6-Benzylaminopurine; values in the table represents mean (
n
= 10).Mean having the same letters in column does not differ signi
cantly at P
b
0.05(Duncan's Multiple Range Test).bTreatments % of rootresponseAverage no. of roots/shootRoot length(cm)MS basal (Control) 0 0 0MS + IBA (1 mg/L) 75
d
1
c
3
c
MS + IBA (2 mg/L) 90
b
3
a
7
b
MS + CEP (0.5 mL/L) 85
c
2
b
6
b
MS+ CEP (1 mL/10mL) 92
a
3
a
9
a
MS
—
Murashige and Skoog; CEP
—
cyanobacterial extracellular product; IBA
—
Indole-3-bu-tyric acid; values in the table represents mean (
n
= 10). Mean having the same lettersin column does not differ signi
cantly at P
b
0.05 (Duncan's Multiple Range Test).
Fig.1.
EffectofdifferentconcentrationsofCEPon
Arachishypogaea
L.comparedtothecom-mercialphytohormonesBAPandIBA.(a)Shootresponsein
Arachishypogaea
L.(b)Rootre-sponse in
Arachis hypogaea
L. (Values indicated the root length and average no. of rootresponse).Meanshavingthesamelettersdonotdifferentsigni
cantlyatP
b
0.05(Duncan'sMultiple Range Test).
Fig.2.
Thephotographsshowingtheorganogenesisof
Arachishypogaea
L.(a)MSmedium(control), (b) shoot developmentintheMS medium+ BAP (2.0mg/L), (c)root develop-mentintheMSmedium+IBA(2mg/L),(d)shootdevelopmentinMSCEP(1mL/10mL),and (e) root development in MS + CEP (1 mL/10 mL).102
M. Gayathri et al. / Algal Research 7 (2015) 100
–
105
Ihre Neugier belohnen
Alles, was Sie lesen wollen.
Jederzeit. Überall. Auf jedem Gerät.
Keine Verpflichtung. Jederzeit kündbar.