Proline Accumulates in Transgenic Red Banana ...abap.co.in/sites/default/files/Paper-9_18.pdf ·...

Current Trends in Biotechnology and PharmacyVol. 8 (4) 413-422 October 2014, ISSN 0973-8916 (Print), 2230-7303 (Online)

413

Proline Accumulates in Transgenic Red Banana (MusaacuminateColla) Transformed with P5CSF129A GeneSrinath Rao

1*, C.Nagappa

1, P.Surender Reddy

2, S.Anil Kumar

2, P. Hima Kumari

2 and

P. B. Kavi Kishor2

1Plant Tissue Culture and Genetic Engineering Laboratory, Department of P.G Studies and Research inBotany, Gulbarga University, Gulbarga 585106, India

2Department of Genetics, Osmania University, Hyderabad 500 007, India*For correspondence - [email protected]

AbstractAgrobacterium tumefaciens mediated

genetic transformation in red banana (MusaacuminataColla) is reported in the presentcommunication.Male flower buds and suckers ofred banana were transformed using A.tumefaciens strain LBA 4404 with the binaryvector pCAMBIA1301, having hygromycinresistance gene as a selectable marker,â-glucuronidase (GUS) as a reporter and a mutatedversion of the gene “1-pyrroline 5-carboxylatesynthetase (P5CSF129A) encoding a prolinebiosynthetic pathway enzyme P5CS (EC 2.7.2.11/1.2.1.41) isolated from Vignaaconitifolia. Maleflower buds proved to be better explants thansuckers for the regeneration and genetictransformation. Green shoots were recoveredfrom A. tumefaciens-transformed explants onselection medium consisting of Murashige andSkoog’s (MS) basal medium supplemented with5 mg/L 6-benzylaminopurine (BAP), 2 mg/Lindole-3-butyric acid (IBA), 250 mg/L cefotaximeand 25 mg/L hygromycin. Shoots were rooted onMS basal medium fortified with 3 mg/L IBA, 250mg/L cefotaxime and 25 mg/L hygromycin.Transient expression of the GUS was observedin transgenic plants. The integration of theP5CSF129A gene was confirmed by polymerasechain reaction (PCR) and Southern blottingtechniques. Transformation frequency was 11.3%and transgenic T0 plants accumulated 2 to 3-foldshigher proline when compared to untransformedcontrol plants. This work possibly paves the way

for exploiting other genes for engineering salt,drought and temperature stress tolerance in redbanana.

Key Words: Musa acuminata, P5CSF129A,proline accumulation, genetic transformation

IntroductionIrrigated lands are particularly prone to

salinization and salinity has profound effects oncrop production (1). Increasing the salt toleranceof crop plants is vital for sustained foodproduction. The response of plants to osmoticstress is complex, several physiological andbiochemical changes take place and arrays ofgenes are induced. Several of these genes andtheir products have been identified; some areinvolved in the biosynthesis of osmolytes suchas proline. Plants accumulate proline underosmotic stress (2, 3). The role of this amino acidduring abiotic stress tolerance in plants has beenwidely discussed (4). Also, transgenic plants thataccumulate high levels of proline are reported todisplay increased tolerance to salt and osmoticstress, as well as to cold (3, 5, 6, and 7). Variousstress protection mechanisms have beenproposed for proline. It is suggested to functionas an osmolyte and take part in osmoticadjustment (3), as a stabilizer of sub-cellularstructures, in protecting macro molecules (8), andas a free-radical scavenger (9). The synthesis ofproline is initiated from glutamate which isdehydrogenated to glutamyl ã-semialdehide andspontaneously converted to Ä1-pyrroline-5-

Transgenic Red Banana


414

carboxylic acid. Both steps are catalyzed by abifunctional enzyme pyrroline-5-carboxylatesynthetase (P5CS) which also behaves as alimiting step in proline biosynthesis (5). Moth bean(Vignaaconitifolia) cDNA clone encoding P5CSenzyme was first isolated by a functionalcomplementation technique using E. coli mutants(10). Transgenic plants over-expressing theP5CS gene from Vignaaconitifolia accumulatehigh proline levels and are more tolerant toosmotic stress (3, 11, 12).

Banana (Musa sp.) is developing world’sfourth most important field crop after rice, wheatand maize, in terms of gross production. The cropis grown in many countries throughout the tropicsand sub-tropics, with an annual world productionof around 95.6 million tons on an average areaof 4.84 million hectares. India accounts for 27.4%of world’s banana production with a yield of 26.2million tons during 2010-11(13). It is grown in arange of environments and production systemsand provides nutritionally staple food and asignificant source of revenue all around the year.In India, bananas are grown in the states ofAndhra Pradesh, Karnataka, Kerala,Maharashtra, Tamil Nadu and to some extent inwestern and central India. However, thedevelopment of new banana varieties throughconventional breeding programs remains difficultbecause of sterility and polyploidy of most ediblecultivars. Genetic transformation offers a viablemeans for introduction of agronomically importantgenes into popular high yielding lines. Therefore,an efficient regeneration and transformationprotocol is crucial in selected varieties of bananafor its improvement, particularly for drought andsalt tolerance. Genetic transformation using A.tumefaciens is reported earlier (14, 15, 16, 17)and also by particle bombardment (18, 19). Thesereports include studies on transient expressionof transformed genes, stable transformation andregeneration of transgenics and heritability oftransgenes in banana. Since there are no reportsof transformation in red banana thus far, presentstudy aims at standardizing parameters forgenetic transformation using Agrobacterium.

Materials and MethodsPlant material: The terminal male flower budsand suckers were collected from the field grownplants and sterilized with 70% (v/v) absolutealcohol under sterile laminar airflow for oneminute. The outer enveloping bracts wereremoved and inner parts (2-3 cm in length)containing male flower primordia were isolatedand used as explants. These explants were usedfor genetic transformation after sterilizing with0.1% HgCl

2 (w/v) for 5 minutes and washing with

sterile distilled water 3-4 times to remove tracesof HgCl

2.

Agrobacterium strain, plasmid vector, culture,infection and co-cultivation : Transformationwas performed using A. tumefaciensstrainLBA4404 harboring the binary vectorpCAMBIA1301. This construct contains hptIIgenefor resistance to hygromycinsulphate, GUSreporter gene encoding â-glucuronidase (GUS)intron and the mutated version of VignaaconitifoliaP5CSF129A cDNA under the control of CaMV35Spromoter. To prepare the inoculum, a singlecolony of Agrobacterium was grown in 50 mL ofliquid YEB medium containing 50 mg/Lkanamycin for 24 h in dark at 28 0C, on a gyratoryshaker (Orbiteck, India) at 200 rpm. Bacteria werethen pelleted by centrifugation at 4,000 rpm for 5min followed by resuspension in Murashige andSkoog’s (MS) liquid medium20 supplemented with100 mM acetosyringone and the pH was adjustedto 5.7. The culture was allowed to grow till itattained an optical density (OD) of 0.5 atA600nm(5x109 cells/mL). One hour prior to co-cultivation of explants, 50-100 µM acetosyringonewas added. A total of 300 explants were used fortransformation. Explants were gently pricked fewtimes to make wounds using a sterile needle.Excised male flower buds were immersed for 30min in Agrobacterium. Explants were blotted dryusing sterile Whatman Number 1 filter paper andinoculated (one explants/culture tube) ontoregeneration medium consisting of MS basal saltssupplemented with BAP (5 mg/L) and IBA (2 mg/L). Co-cultivation was performed for 0-5 days withan interval of 1 day, under a 16 h photoperiod

Srinath Rao et al


415

with a light intensity of 40 µmol m-2 s-1 and keptat 25 ± 2 0C.

Selection of transformants : Differentconcentrations (0-25 mg/L) of hygromycin wereused in the present study to determine the lethallevel at which wild type explants do not exhibitany growth. On medium supplemented with 25mg/L, necrosis and death of explants wasnoticed, therefore, this concentration ofhygromycin was used for selection of putativetransgenics. After co-cultivation, the male flowerbuds were rinsed thoroughly with sterilizeddistilled water containing filter sterilizedcefotaxime (250 mg/L), dried on sterile WhatmanNumber one filter paper and transferred to MSmedium supplemented with 5 mg/L BAP + 2 mg/L IBA + 250 mg/L cefotaxime and 25 mg/Lhygromycin. The explants with emerging shootbuds were subcultured on the same medium withthe same composition for 4 weeks. Regeneratedshoots (4-5 cm long) were transferred to MSmedium supplemented with 3 mg/L IBA for rootinduction. Finally, the plantlets were transferredto plastic pots containing garden soil mixed withvermiculite and sand (2:1:1) for hardening andthen subsequently transferred to green house.

Histochemical GUS staining, polymerasechain reaction (PCR) and Southern blotanalysis : All putative transformants (T0) wereanalyzed for GUSexpression following themethod described by Jefferson (21). Thetransformed shoot tissues were immersed in 1mM X-Gluc solution (Sigma-Aldrich, USA), 100mM NaH2PO4 (pH 7.0), 0.5 mM potassiumferricyanide, and 0.1% triton X-100 and incubatedin the dark overnight at 37 0C. After staining, thematerials were treated with 70% ethanol for threedays to remove chlorophyll before observation.Blue spots were recorded using a stereoscopicmicroscope. Genomic DNA from leaves of controland T0 progeny of five transformants wasextracted according to Doyle and Doyle (22).PCR reaction was performed with purifiedgenomic DNA (30 ng) using two convergentprimers that are complementary to theP5CSF129A DNA.

F: ACC ATA TGT GCT CTA AAG GCT ATT GCR: GCG TCG ACG AAT TCC CGA TCT ATG AAAmplification reactions consisted of one cycle of4 min at 94 °C, followed by 25 cycles(60s, 94 °C, 45s 58 °C, 2 min 72 °C) and finallyan extension cycle of 10 min at 72 °C23. The finalvolume of PCR reaction was 25 µl (F primer 0.5µl, R primer 0.5 µl, dNTP 1 µl, Taq polymerase 1µl, PCR buffer 5 µl, DNA sample 5 µl and distilledwater 12 µl). PCR products were separated on1% agarose gel and stained with ethidiumbromide. Integration of the P5CSF129A gene intransgenic banana plants (T0) was confirmed bySouthern blot analysis. Twenty µg of genomicDNA was isolated from leaves of each PCR-positive transgenic plants along withuntransformed controls and was digested withEcoRI. The samples were resolved on 0.8%agarose gel and blotted on a Hybond N+ nylonmembrane (Bioscience, Little Chalfont, Bucks,UK) membrane, following the method describedby Sambrook and Russell (23).

Proline measurement in T0 plants: Prolinecontent was determined according to the methodof Bates et al. (24). One gram leaf tissue fromtransgenics (T0) and untransformed controlplants was quickly frozen and ground in liquidnitrogen, the tissue was extracted with 3%sulfosalicylic (m/v) coloring reaction containingninhidrin solution and glacial acetic acid. Themixture was boiled until the colour developed.Colour was extracted with toluene, and thechromophore was measured with aspectrophotometer at ë 520.

Results and DiscussionRegeneration of shoots and roots fromexplants of red banana : Male inflorescenceand the bunch of red banana fruits are shown inFigure 1a and 1b respectively. From the fieldgrown plants, suckers and male flower buds wereused as explants for initial shoot regenerationon MS basal medium supplemented with 5 mg/LBAP plus 2 mg/L IBA. Shoots were rooted onMS medium containing 3 mg/L IBA. Plantregeneration frequency was found better from



416

male flower buds compared to suckers (data notshown). Thus, shoot regeneration wasstandardized before genetic transformationexperiments were taken up.

Co-cultivation with Agrobacterium andsensitivity of explants to hygromycin:Recombinant vector was constructed withP5CSF129A gene driven by CaMV35S promoterand the map is shown in Figure 2. To optimizegenetic transformation, experiments wereperformed with the choice of explant, pre-cultureand co-cultivation period, effective density ofAgrobacterium, acetosyringone concentration,and the effect of hygromycin on regeneration.Explant type and co-cultivation period influencedthe transformation efficiency (Table 1). Male flowerbuds used for co-cultivation were found better fortransformation compared to suckers and areshown in Figure 3a. Of different periods of co-

cultivation (0-5 days), 3-day co-cultivation wasfound ideal. A 30-minute immersion in bacterialculture followed by a co-cultivation period of 3-days was optimal which resulted in significantlyhigher rate of GUS expression. Co-cultivationbeyond 3-days led to bacterial overgrowth andresulted in the death of explants (Table 1). In thepresent study, a 5-day pre-culture of explants inthe shoot induction medium was found critical toachieve high frequency transformation. After oneweek of culture, shoots were produced from maleflower buds and suckers but 98% of them wereescapes probably due to pre-emergence of shootbuds. When explants were co-cultivated withoutpre-culture, 90% of the explants were unable towithstand the infection and eventually died. Hence,in subsequent experiments, explants pre-culturedfor 5-days on shoot induction medium were used.The period of agro infection and co-cultivation ofexplants with Agrobacterium is an important factor

Fig. 1.(a) Male inflorescence (b) Bunch of red banana fruits

Fig. 2. Recombinant vector map of pCAMBIA1301-P5CSF129A gene construct.

Srinath Rao et al


417

for efficient transformation as revealed in thepresent investigation. An immersion period of 30min was optimal for agro infection, while 15 minwas not sufficient and immersion for 40 minresulted in the overgrowth of bacteria. Aninoculum density of 0.5 OD (A600 nm) showed highfrequency of GUS activity. It was noticed that ODgreater than 1 resulted in bacterial overgrowth.Transformation efficiency was 80% at 0.5 OD anddensities lower and higher than this resulted indecreased transformation efficiency (data notshown). Addition of acetosyringone at differentconcentrations (0-120 µM) was found as animportant factor for increasing the frequency oftransformation. Among different concentrationsof acetosyringone tested, 100 µM was found thebest beyond which frequency of transformationand number of shoots formed per culture declined(Table 2).

Selection of transformants and theirmolecular confirmation: In order tofind outtheappropriate concentration of the selection agentin red banana, different (0-25 mg/L) concentra-tions of hygromycin were employed during shootbud production (Table 3). On medium without andwith lower concentrations of hygromycin, luxuriantgrowth of shoots was observed. With an increase

in the concentration of hygromycin to more than5 mg/L, shoot bud induction was reduced from100 to 60%, and at 25 mg/L, all the explantsdisplayed necrosis and none of them survivedafter 8 days (Table 3). Three hundred explantsinoculated on shoot induction mediumsupplemented with 25 mg/L hygromycin resultedin a transformation frequency of 18.66%.Transient expression of GUS activity was noticedin the transformants but not in untransformedcontrols (Fig. 3b). Shoots from male flower budswere regenerated (Fig. 3c) on MS mediumfortified with 5 mg/L BAP + 2 mg/L IBA + 3%sucrose, 250 mg/L cefotaxime and 25 mg/Lhygromycin (shoot induction medium). Well-developed shoots were excised and rooted onMS basal medium supplemented with 3 mg/L IBAand 25 mg/L hygromycin (Fig. 3d). Regeneratedplants were first transferred to polycups and laterinto pots containing soil, sand and vermiculite(Fig. 3e). A total of 10 plants were transferred topots for hardening, out of which 4 plants survived(data not shown). PCR analysis of putativelytransformed (T0) plants resulted in the detectionof an amplification product of a 2.8 kb with genespecific primers corresponding to the codingregion of P5CSF129A gene but the same was

Fig. 3. Different stages of Agrobacterium - mediated transformation of red banana: (a) Excised male flowerbuds used for co-cultivation (b) Transgenic leaf tissue showing transient GUS expression (c) Shoots regeneratedon MS medium containing 5 mg/L BAP + 2 mg/L IBA + 250 mg/L cefotaxime and 25 mg/L hygromycin (d) Rootdifferentiation from transgenic shoots (e) Hardened transgenic plants growing in green house.



418

not noticed in untransformed controls (Fig. 4).Amplification of hptII gene in transgenics usinggene specific primers resulted in a band size of750 bp (Fig. 5a). Southern blot analysis wasperformed for the two PCR positive lines, usinggenomic DNA isolated from leaf of T0 anduntransformed control plants. Genomic DNA wasdigested with Eco RI, followed by hybridizationwith the P5CSF129A probe, and as expectedthe whole cassette measuring 3.8 kb wasobserved in the two transgenic lines but nohybridization signal was detected in theuntransformed control plants (Fig. 5b). Insupport of this, proline content was analyzed inuntransformed controls as well as in T0 plants,after one month of transfer of plants to thegreenhouse. Two to three-fold increases in prolinecontent were noticed without imposing any stressin the transgenic lines compared to controls(Fig. 6).

Efficient transformation and subsequentregeneration of plants, using A. tumefaciensmediated transformation are dependent onseveral factors such as type of explants,genotype, hormonal composition, duration of co-cultivation, duration of selection, type andconcentration of antibiotic marker used forselection (25). In the present study, both suckersand male flower buds from red banana were usedas explants for transformation. Different explantssuch as embryogenic cell suspensions (14, 26),male flower buds (15) and intercalary meristems(27) were used for A. tumefaciens-mediatedtransformation in banana. The superiority of maleflower buds for transformation studies over thatof suckers indicates that the explant type isimportant. Transgenic banana plants werereported earlier using A. tumefaciens mediatedtransformation (14, 17). The frequency oftransformation was 18.66% in the present studycompared to 35.1% as reported by Matsumotoet al. (28). This could be due to the differences inthe genotypes used. In the present study, aninoculum density of 0.5 OD (A600 nm) was foundideal for co-cultivation of explants, OD greaterthan 1 resulted in bacterial overgrowth. Inoculumdensity corresponding to OD value varies widely

Fig. 4. PCR amplification of P5CS F129A gene usinggene specific primers (T0 transformed plants): LaneM = 1 kb molecular weight marker; Lane +C =amplification of vector DNA; Lane C = untransformedcontrol plants; Lanes 1 and 2 = putative transgenicT0 lines.

Fig. 6. Accumulation of proline in leaves ofuntransformed controls (C) and T0 transgenic lines(1 and 2) of red banana without any stress.

Fig. 5a and 5b. PCR amplification ofhpt II gene (750bp) not observed in untransformed control but noticedin T0 transgenics (1 and 2): Fig. 5a-Lane M =Molecular weight marker (1 Kb ladder); Lane +C =Vector DNA, Lane C = untransformed control, Lanes1 to 2 = T0 transgenics. Fig. 5b-Southern blot analysisof transgenic banana: Lane +C =vector control, LaneC = Untransformed control plant without insert, Lanes1, 2 = Independent transgenics (1 and 2) showingP5CSF129A gene insert measuring 3.8 kb.

Srinath Rao et al


419

Table 1. Effect of co-cultivation period and explant type on transformation efficiency of red banana

Explant type Co-cultivation period and transformation frequency*

2 Days 3 Days 4 Days 5 Days

Sucker 34.0 ± 0.98c 50.4 ± 0.45a 40.3 ± 0.35b 30.0 ± 0.00d

Male flower bud 45. 0 ± 0.00c 70.0 ± 1.50a 65.4 ± 0.50b 40.2 ± 0.33d

*MS medium supplement with 5 mg/L BAP + 2 mg/L IBA was used for shoot regeneration. Data representaverage of three replicates; each replicate consists of 25 cultures. Mean ± standard error, Mean followed bythe same superscript in a column is not significantly different at P = 0.05 levels.

Table 2. Effect of acetosyringone concentration on transformation frequency

Concentration of Explantsacetosyringone* Male flower bud Sucker

(µM/L) Frequency No. of Frequency (%) No. of(%) shoots/culture shoots/culture

00 00 0.0 ± 0.00 00 0.00 ± 0.0020 30 6.0 ± 0.50

e20 3.0 ± 0.00

e

40 30 9.4 ± 0.45d

20 7.0 ± 0.00d

60 70 11.0 ± 0.86c

50 9.4 ± 0.00c

80 90 14.0 ± 0.56b

70 11.8 ± 0.00b

100 100 20.7 ± 0.34a

90 15.3 ± 0.00a

120 80 12.0 ± 0.50c

70 10.1 ± 0.00c

*MS medium supplement with 5 mg/L BAP + 2 mg/L IBA was used for shoot differentiation. Data representaverage of three replicates; each replicate consists of 25 cultures. Mean ± Standard error, Mean followed bythe same superscript in a column is not significantly different at P = 0.05 levels.

Table 3. Frequency of shoot regeneration, rooting and survival of plants after gene transferthroughAgrobacterium

Concentration of Explantsacetosyringone* Male flower bud Sucker

(µM/L) Frequency No. of Frequency (%) No. of(%) shoots/culture shoots/culture

0 100 30.6 ± 0.34a 100 14.8 ± 0.21a

05 80 20.7 ± 0.25b 60 11.4 ± 0.25b

10 60 14.0 ± 0.33c 40 8.0 ± 0.00c

15 50 7.8 ± 0.50d 10 6.4 ± 0.25d

20 30 4.0 ± 0.00e 00 1.0 ± 0.00e

25 0 0.0 ± 00 0 0.0 ± 00

*Data represent average of three replicates; each replicate consists of 25 cultures. Mean ± standard error,Mean followed by the same superscript in a column is not significantly different at P = 0.05 levels.



420

in banana. While Matsumoto et al. (28) reportedan OD value of 0.4, Maziah et al.(27) noticed thatan inoculum density of 0.7 OD as optimum fortransformation in banana cv. Rasthali (AABgenome). Likewise, Huang et al. (26) and Tripathiet al. (29) reported 0.8 OD for efficienttransformation in the cv Mass (AA) and EastAfrican highland banana respectively. The aboveresults clearly show that inoculum density ofbacterial cultures vary from cultivar to cultivar. Co-cultivation duration for banana transformation hasbeen reported to vary between 2-5 days. In thisstudy, it was found that a co-cultivation period of3-days was suitable for red bananatransformation. Earlier, Ganapathiet et al. (14),Tripathi et al. (16) and Sreeramanan et al. (30)also reported a 3-day co-cultivation period for highfrequency transformation in banana. In thepresent study, 25 mg/L hygromycin was foundsuitable for selecting the putative transformants.Our results are in agreement with the results ofTripathiet et al. (29). Plant specific phenoliccompounds that induce the expression ofvirgenes are important for the gene transfer (31).Acetosyringone has been reported to enhancethe frequency of transformation both in dicots (12)and monocots (32). Addition of acetosyringoneto the co-cultivation medium has been reportedto induce vir genes, which extends the host rangeof some Agrobacterium strains and found mostessential for banana transformation (27). Ourobservations are in confirmation with the findingsof Tripathiet et al. (16) who reported that inclusionof 100 µM acetosyringone was inevitable fortransformation. However, Matsumoto et al. (28)reported that 200 mM acetosyringone wasrequired for Nanicao and CNPMFF varieties ofbanana for high frequency transformation. Thisdifference in the requirement of differentconcentrations of acetosyringone may be due tovarietal differences. Transformed To plantsdisplayed GUS expression in leaf tissues but thesame was not observed in the untransformedcontrols. These findings are in agreement withthe reports of others (14, 29, 26). These resultsestablish that A. tumefaciens can be employedfor stable genetic transformation in red banana.

Transgenics showed elevated levels ofproline (2 to 3-folds) under non-stressedconditions compared to the untransformed plants.This clearly confirmed the functional expressionof P5CSF129A gene in the banana genome. Thefirst plant to be transformed with P5CS gene wasthat of tobacco, which showed elevated levels ofproline (5). Similar to our results, the primaryP5CSF129A – transgenic wheat plants displayedmore than ten times proline content than their wildtype counterparts without any stress (33).Recently, Yamchiet et al. (11) observed that therewas a 26-times more proline production intobacco plants transformed with P5CSF129Agene as compared to the untransformed controls.Kumar et al. (34) also reported high prolinecontent in rice transformed with P5CSF129Agene. Introduction of P5CSF129A gene is shownto confirm abiotic stress tolerance due to elevatedlevels of proline in several plants like rice (34),wheat (33), carrot (7), chickpea (12) andpigeonpea (35). However; abiotic stress tolerancehas not been carried out in the present study inred banana. Successful insertion of P5CSF129Agene has been achieved in the T0 generation ofred banana as revealed by PCR and Southernanalysis. The gene was functionally expressedas indicated by elevated levels of prolineaccumulation in transgenics as compared to theuntransformed controls. It is concluded that maleflower bud is the ideal explant for genetictransformation of red banana. Thus, this workpaves the way for developing transgenic redbanana with improved agronomic traits.

AcknowledgementsThe authors are grateful to the UGC, New

Delhi for financial assistance under the Non-SAPprogram to the Department of P.G Studies andResearch in Botany, Gulbarga University. PBK isthankful to the CSIR, New Delhi for sanctioningCSIR-Emeritus fellowship.

References1. Rengasamy, P. (2006). World salinization

with emphasis on Australia. J. Exp. Bot., 57:1017-1023.

Srinath Rao et al


421

2. Greenway, H and Munns, R. (1980). Mecha-nisms of salt tolerance in non-halophytes.Annu Rev Plant Physiol.,31: 149-190.

3. Kavi Kishor, P.B., Sangam, S., Amrutha,R.N., Sri Laxmi, Naidu, K.R., Srinath Rao,Reddy, K.J., Theriappan P. andSreenivasulu N. (2005). Regulation ofproline biosynthesis, degradation, uptakeand transport in higher plants: itsimplications in plant growth and abioticstress tolerance. Curr Sci., 88: 422-443.

4. Hare, P. and Cress W. (1997). Metabolicimplications of stress induced prolineaccumulation in plants. Plant Growth Reg.,21: 79-102.

5. Kavi Kishor, P.B., Hong, Z., Miao, G., Hu,C.A.A. and Verma, D.P.S. (1995). Over-expression of Ä1-pyrroline-5-carboxy-latesynthetase increases proline productionand confers osmotolerance in transgenicplants.Plant Physiol., 108: 1387-1394.

6. Zhu, B., Su, J., Chang M., Verma, D.P.S.,Fan, Y.L. and Wu, R. (1998). Over-expression of a Ä1-pyrroline-5-carboxylatesynthetase gene and analysis of toleranceto water and salt-stress in transgenic rice.Plant Sci.,139: 41-48.

7. Han, K.H., and Hwang, C.H. (2003). Salttolerance enhanced by transformationP5CS gene in carrot.J Plant Biotechnol., 5:149-153.

8. Schobert, B. and Tschesche, H. (1978).Unusual solution properties of proline andits interaction with proteins. BiochimBiophysActa., 541: 270-277.

9. Smirnoff, N. and Cumbes, Q.J. (1989).Hydroxyl radical scavenging activity ofcompatible solutes. Phytochem.,28: 1057-1060.

10. Hu, C.A.A., Delauney, A.J. and Verma,D.P.S. (1992). A bifunctional enzyme (Ä1-pyrroline-5-carboxylate synthetase)catalyzes the first two steps in prolinebiosynthesis in plants. Proc Natl Acad Sci,USA., 89: 9354-9358.

11. Yamchi, A, Jazii F.R, Mousav A, KarkhaneA. and Renu, A. (2007). Prolineaccumulation in transgenic tobacco as aresult of expression of Arabidopsis Ä1-pyrroline-5-carboxylate synthetase (P5CS)during osmotic stress.J Plant BiochemBiotechnol., 16: 9-15.

12. Ghanti, S.K., Sujata, K.G., Vijay Kumar,B.M., Nataraja Kabba, N., JanardhanReddy, K., SrinathRao, M. and Kavi Kishor,P.B. (2011). Heterologous expression ofP5CS gene in chickpea enhances salttolerancewithout affecting yield. BiologiaPlant., 55: 634-640.

13. Singh, H.P, Uma, S, Selvarajan, R. andKarihaloo, J.L. (2011).Micropropagation forproduction of quality banana plantingmaterial in Asia-Pacific. Asia-PacificConsortium on Agricultural Biotechnology(APCoAB), New Delhi.

14. Ganapathi, T.R., Higgs, N.S, Balint-Kurti,P.J. and Van-Eck J. (2001).Agrobacterium-mediated transformation of embryogeniccell suspensions of banana cultivar Rasthali(AAB). Plant Cell Rep., 20: 157-162.

15. Khanna, H., Becker D., Kleidon J. and Dale,J. (2004). Centrifugation assistedAgrobacterium tumefaciens-mediatedtransformation (CAAT) of embryogenic cellsuspensions of banana (Musa spp.Cavendish AAA and Lady Finger AAB).MolBreed., 14: 239-252.

16. Tripathi, L, Tripathi, J.N. and Hughes, J.D.A.(2005). Agrobacterium mediatedtransformation of plantain (Musasps)CvAghagba. Afr J Biotech., 4: 1378-1383.

17. Sreeramanan, S., Mazia, M., Abdullah,M.B.and Saria Xanier, R. (2006). Transientexpression of GUS and gfp gene inAgrobacterium-mediated banana trans-formation using single tiny meristematicbud. Asian J Plant Sci., 1: 1-13.

18. Sagi, L., Panis, B., Remy, S., Schoofs, H.,Smet, K.D., Swennen, R. and Cammue,B.P.A. (1995). Genetic transformation of



422

banana and plantain (Musa spp.) via particlebombardment.Biotechnology., 13: 481-485.

19. Becker, D.K., Dugdale, B., Smith, M.K.,Harding, R.M. and Dale, J.L. (2000).Genetic transformation of Cavendishbanana (Musa spp. AAA group) cv GrandNeyne via microprojectile bombardment.Plant Cell Rep., 19: 229-234.

20. Murashige, T. and Skoog, F.A. (1962). Arevised medium for rapid growth and bio-assays with tobacco tissue cultures. PhysiolPlant., 15: 473-497.

21. Jefferson, R.A., Kavanagh, T.A. and Bevan,M.W. (1987).GUS-fusions: â-glucuronidaseas a sensitive and versatile gene fusionmarker in higher plants. EMBO J.,6: 3901-3907.

22. Doyle, J. and Doyle, J. (1990). Isolation ofplant DNA from fresh tissue. Focus.,12: 13-15.

23. Sambrook, J. and Russell, R.W. (2001).Molecular cloning: A laboratory manual.Cold SpringHarbour Laboratory Press, ColdSpring Harbour, USA.

24. Bates, L.S, Waldren R.P. and Teare, I.D.(1973). Rapid determination of free prolinefor water stress studies. Plant Soil., 39: 295-208.

25. Indurker, S., Misra H.S. and Eapen E.(2010). Agrobacterium-mediated trans-formation in chickpea (Cicerarietinum L.)with an insecticidal protein gene:optimization of different factors. PhysiolMolBiolPlants., 16: 273-284.

26. Huang, X, Huan L X., Xiow W., Dai X M.,Chen Y.F. and Li, X.J. (2007). Highly efficientAgrobacterium mediated transformation ofembryogenic cell suspension of Musaacuminatacv Mas (AA) via a liquid co-cultivation system.Plant Cell Rep., 26: 1755-1762.

27. Maziah, M., Sreeramanan, S.P., Uad A. andSariah, I. (2007). Production of transgenicbanana cultivar Rasthali (AAA) via

Agrobacterium mediated transformationwith rice chitinase gene. J Plant Sci., 2: 501-517.

28. Matsumoto, K., Cabral G.B., Batista-Teixeira, J. and Castro Monte, D. (2009).Agrobacterium-mediated transientexpression system in banana immaturefruits.Afr J Biotech.,8: 4039-4044.

29. Tripathi, L., Jaindranath T. and Fateera W.T.(2008). Rapid and efficient production oftransgenic east African island banana(Musa. sps) using intercalary meristematictissue. Afr J Biotech., 7: 1438-1445.

30. Sreeramanan, S., Maziah, M. and Xavier,R. (2009). A protocol for Agrobacterium-mediated transformation of banana with arice chitinase gene. Emir J Food Agric., 21:18-33.

31. Stachel, S.E., Messens M., Van-Montagu,P. and Zambryski, P. (1985). Identificationof the signal molecules produced bywounded plant cells which activates the T-DNA transfer process in Agrobacteriumtumefaciens. Nature, 318: 624-629.

32. Rashid, H., Chaudhry, Z. and Khan, M.H.(2011). Effect of explant plant source andacetosyringone concentration ontransformation efficiency of wheat cultivars.Afr J Biotech., 10: 8737-8740.

33. Sawahel, W.A. and Hassan, A.H. (2002).Generation of transgenic wheat plantsproducing high levels of the osmo-protectantproline. Biotech Lett., 24: 721-72.

34. Kumar, V., VarshaShriram, KaviKishor P.B.,Jawali, N. and Shitole, M.G. (2009).Enhanced proline accumulation and saltstress tolerance of transgenicindica rice byover-expressing P5CSF129A gene. PlantBiotech Rep., 4: 37-48.

35. Surekha, C., Nirmala Kumari, K., Aruna,L.V., Suneetha, G., Arundhati, A. and KaviKishor, P.B. (2014).Expression of theVignaaconitifolia P5CSF129A gene intransgenic pigeonpea enhances prolineaccumulation and salt tolerance. Plant CellTiss Organ Cult., 116: 27-36.

Srinath Rao et al

Proline Accumulates in Transgenic Red Banana ...abap.co.in/sites/default/files/Paper-9_18.pdf ·...

Documents

Transcript of Proline Accumulates in Transgenic Red Banana ...abap.co.in/sites/default/files/Paper-9_18.pdf ·...