35 Int.J.Curr.Biotechnol. Volume 2; Issue 12; December, 2014

International Journal of CurrentBiotechnology

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Subiramani Sivakumar, Ganesan Premkumar, Govindarajan Siva, Mogilicherla Kanakachari, Manickam Vigneswaran, Sadhasivam Vinoth, ThiruppathiSenthil Kumar and Narayanasamy Jayabalan, Optimization of factors influencing microinjection method for Agrobacterium - Mediated transformation ofEmbryonic Shoot Apical Meristem in Cotton (Gossypium hirsutum L. cv.SVPR-2), Int.J.Curr.Biotechnol., 2014, 2(12):35-40.

Optimization of factors influencing microinjection method for Agrobacterium - Mediatedtransformation of Embryonic Shoot Apical Meristem in Cotton (Gossypium hirsutum L. cv.SVPR-2)

Subiramani Sivakumar¹*, Ganesan Premkumar¹, Govindarajan Siva¹, Mogilicherla Kanakachari1,Manickam Vigneswaran¹, Sadhasivam Vinoth¹, Thiruppathi Senthil Kumar 2 and Narayanasamy Jayabalan¹

1Department of Plant Science, Bharathidasan University, Tiruchirappalli - 620 024, Tamil Nadu, India.

2Department of Industry University Collaboration, Bharathidasan University, Tiruchirappalli, Tamil Nadu - 620024, India.

A R T I C L E I N F O A B S T R A C T

Article History:Received 05 December 2014Received in revised form 19 December 2014Accepted 25 December 2014Available online 28 December 2014

Key words:Gossypium hirsutum, ESAM,Hygromycin-B, GUS, Microinjection,Agrobacterium, uidA gene and hptII gene.

A simple and rapid protocol was developed for Agrobacterium-mediated genetictransformation of pCAMBIA1304 vector harbouring uidA and hptII genes into theEmbryonic Shoot Apical Meristem (ESAM) of germinated cotton seeds through mi-croinjection method. Various parameters influencing transformation were standard-ized for getting high frequency transformation. ESAM of the cotton seeds was micro-injected with bacterial inoculum for direct gene delivery to get high frequency oftransformation. After co-cultivation, seeds were transferred to selection medium con-taining Cefotaxime and Hygromycin-B. Resistant plants were grown inside plantgrowth chamber for acclimatization. GUS analysis was performed to check the expres-sion of uidA gene in resistant cotton plants. The method was so simple and rapid bywhich more transgenic plants were developed within a short period of time with 20%transformation frequency.

*Corresponding author.Email address: shivaplant1385@gmail.com

IntroductionCotton is the most important fiber yielding crop plantbeing cultivated worldwide with leading production byChina (6840 metric tons) followed by India (5321 metrictons) and US (3598 metric tons). Although Gossypiumincludes more than 50 species only four major speciesare cultivated namely G.hirsutum, G.arboreum,G.herbaceum, G.barbadense. Among these four species,G.hirsutum is cultivated over 90% of the agricultural fieldsfor commercial production (TianZi et al., 2010). Thuscotton plant occupies an inevitable place in worldeconomy. Production of good quality cotton fiber is aprerequisite for profitable marketing in cotton industry.There are several factors that can affect the growth andyield of the cotton plant. Among them biotic and abioticstress greatly result in yield reduction and poor qualityof cotton fiber which subsequently leads to economicloss. Hence it is desirable to generate cotton plants thatcan tolerate the adverse environmental conditions.Creating hybrid cotton plants with desirable characters

ISSN: 2321 - 8371

through cross breeding is time-consuming and laborious.Genetic engineering offers promising result forsuccessful production of transgenic cotton plants withdesirable characters within stipulated duration.Agrobacterium mediated transformation of cotton plantsusing various regeneration methods have been wellreported by researchers from different parts of the world.Hence it is necessary to generate transgenic cotton plantsfrom cotton cultivars confined to different parts of theworld. Cotton plants are naturally recalcitrant to in vitromanipulation and their regeneration is highly genotype-dependent. Developing specific regeneration protocolis important required for successful establishment oftransgenic cotton plants of local cotton cultivars. Cold,salinity, drought and heavy metal stress are consideredas major factors in limiting plant development andproductivity. Today there is increasing use of saline waterin agriculture in arid and semiarid regions, where theavailability of fresh water is scarce. Water-deficit stress,cold stress and salinity stress are major causes of plantgrowth inhibition due to reduction in water availability,sodium ion accumulation and mineral imbalances andthese are leading to cellular and molecular damage.

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SiNAC transcription factor gene isolated from Setariaitalica transcribes characterized protein which containsa NAM (Number of Apical Meristem), ATAF(Arabidopsis thaliana Transcription Factor) and CUC(Cup Shaped Cotyledon) transcription factor with transactivation activity. Several reports describe and supportthe hypothesis that the endogenous functions of thesetranscription factors regulate their target gene expressionthrough binding to the cognate cis-elements in thepromoters of the stress-related gene (Puranik et al., 2011;Hu et al., 2006).

The present study was carried out with the aim ofdeveloping a simple and efficient transformation methodusing germinated cotton seeds as explants. pCAMBIA1304 vector harboring SiNAC gene construct with uidAand hptII genes was used to transform the cotton plant.Various factors influencing transformation efficiency ofthe plant were optimized. Hm-B resistant plants weresuccessfully hardened in growth chamber.

Materials and MethodsPlant materialSeeds of cotton cultivar SVPR-2 were procured fromCotton Research Station, Tamil Nadu AgriculturalUniversity, Srivilliputtur, Tamil Nadu, India. Seeds weredelinted with conc.H2SO4 and washed under running tapwater for complete removal of the acid. Few drops ofTeepol solution was added and stirred for 5 minutes toremove the dust that reside on the seed surface. Theseeds were then kept under running tap water for 1 hour.The seeds were then washed with 70% ethanol for 50seconds followed by three washes with sterile distilledwater. 0.1% mercuric chloride was then added and rinsedslowly for 4 minutes followed by three washes with steriledistilled water. Surface sterilized seeds were theninoculated on petriplates containing sterile cotton wetwith 1/2 strength MS medium. Plates were kept underdark conditions overnight.

Bacterial strain and vectorThe SiNAC gene construct was kindly provided by Dr.Manoj Prasad, Scientist, National Institute of PlantGenome Research, New Delhi, India. Binary vectorpCAMBIA 1304 was used for the transformation study.The gene construct contains the following parts in itsbackbone: GUS reporter, CaMV 35S promoter, SiNACgene, kanamycin and hygromycin resistant genes, NOSterminator, Ori and EcoRI restriction site.

A. tumefaciens strain GV3101 was used for carryingpCAMBIA 1304 vector. Bacterial culture was initiated bystreaking 50µl stock culture on solid YEM medium. Thesingle colony obtained on solid medium was inoculatedinto 50ml YEM medium. Kanamycin (50mg/l) andRifampicin (10mg/l) were added to the medium prior toinoculation.

Optimization of minimal inhibitory concentrationThe germinated seeds were cultured on 1/2 strength MSmedium supplemented with Cefotaxime (200mg/l) andHygromycin (0-20mg/l) in order to find out the minimalinhibitory concentration of the antibiotics in which onlytransformed seedlings can grow and non-transformedseedlings will die.

TransformationBacterial cultures at different OD values were taken formicroinjection of cotton seeds. Overnight grown culturewas centrifuged at 5000 rpm for 15 minutes at 4°C. Thebacterial pellet was dissolved in 1/2 strength MS medium.

Acetosyringone was added to the culture at differentconcentrations and kept at 120 rpm for 1 hour. Seed coatswere removed prior to infection. 100µL of the culture wastaken in an insulin syringe and microinjected into theembryonic shoot apical meristem of germinated cottonseeds. The culture was microinjected (0.5-1.0 mm depth)one to five times to check the effect of number ofmicroinjection in and around the embryonic shoot apicalmeristem. Excess bacterial culture was removed byblotting the infected seeds on sterile filter paper(Whatman No.1). The seeds were co-cultivated on 1/2strength MS medium for two days in dark condition. Afterco-cultivation the seedlings were washed with Cefotaxime(200 mg/L) and transferred to selection medium containingCefotaxime and Hygromycin-B antibiotics. Cotton plantsthat survived on selection medium after two subculturewere transferred to paper cups containing soil andvermicompost and grown inside artificial plant growthchamber for acclimatization.

GUS assayThe seedlings derived from microinjected cotton seedswere subjected to GUS assay to check the expression ofuidA gene after co-cultivation and antibiotic selection.GUS analysis was performed according to the protocoldescribed by Jefferson et al., (1987). For analysis of theputative transformants, seedlings that were tested andfound free of residual Agrobacterium were used. Theseedlings were incubated overnight at 37°C in a solutioncontaining 0.1 M phosphate buffer, pH 7.0, 2 mM X-Gluc,5mM each of potassium ferricyanide and ferrocyanideand 0.1% Triton X-100. The seedlings were later soakedwith 75% ethanol to clear chlorophyll. The gene transferefficiency was calculated as the average number of ESAMshowing GUS staining. Also the efficiency was expressedas the number of Hm-resistant and GUS positive SAM/initial number of ESAM inoculated × 100%.

Results and DiscussionOptimization of Hygromycin B and MicroinjectionIn this study the ESAM of cotton seed was used as anexplant for gene delivery. Successful gene delivery wasassessed by GUS analysis and growth in the presence ofinhibitory concentrations of Hm-B. Differentconcentrations of Hm-B (0, 5, 10, 15, 20 mg/l) andCefotaxime (200 mg/l) was tested to find the optimalconditions, which would allow the efficient selection oftissue that was microinjected. Among differentconcentrations tested, Hm-B at 20 mg/l was found to besuitable for efficient selection of transformed plants.Germination of cotton seeds reduced or totally stoppedabove 20 mg/l concentration. High concentrations of Hm-B (> 20 mg/l) reduced the survival rate of the seedlings.Thus 20 mg/l Hm-B was taken as minimal inhibitoryconcentration for further selection of transgenic plants(Fig.1).

Optimization of factors influencing Microinjection ofcotton seedsEmbryonic shoot apical meristem of cotton seeds(cv.SVPR-2) was used as an explant for gene deliveryusing microinjection of A. tumefaciens (Fig 1). 100µlculture of A. tumefaciens at different OD600 values (0.2,0.4, 0.6, 0.8, and 1.0) was microinjected 5 times to evaluatethe optimal bacterial density for efficient gene transfer.After co-cultivation, the explants were transferred toselection media supplemented with 1/2 strength MS + B5Vitamins + 1% sucrose + Hygromycin-B (20 mg/L) (Table1). Five days old and 15 days old seedlings weresubjected to GUS assay (Fig.2, 3). In the present studymicroinjection of cotton seed ESAM was performed with

37 Int.J.Curr.Biotechnol. Volume 2; Issue 12; December, 2014

Conc. of Agrobacterium (OD600)

Conc. of Acetosyringone (mM)

Number of co-cultured Embryonic Shoot Apical Meristem yielding Hm-B resistant (percent)

GUS positive (percent)

0.2 0 0/100 (0%) 0/100 (0%) 0.2 50 0/100 (0%) 0/100 (0%) 0.2 100 2/100 (2%) 0/100 (0%) 0.2 150 1/100 (1%) 0/100 (0%) 0.2 200 0/100 (0%) 0/100 (0%) 0.4 0 0/100 (0%) 0/100 (0%) 0.4 50 3/100 (3%) 0/100 (0%) 0.4 100 8/100 (8%) 2/100 (2%) 0.4 150 4/100 (4%) 0/100 (0%) 0.4 200 1/100 (1%) 0/100 (0%) 0.6 0 6/100 (6%) 1/100 (1%) 0.6 50 12/100 (12%) 3/100 (3%) 0.6 100 20/100 (20%) 8/100 (8%) 0.6 150 15/100 (15%) 4/100 (4%) 0.6 200 8/100 (8%) 2/100 (2%) 0.8 0 2/100 (2%) 0/100 (0%) 0.8 50 6/100 (6%) 1/100 (1%) 0.8 100 12/100 (12%) 3/100 (3%) 0.8 150 8/100 (8%) 2/100 (2%) 0.8 200 3/100 (3%) 0/100 (0%) 1.0 0 0/100 (0%) 0/100 (0%) 1.0 50 0/100 (0%) 0/100 (0%) 1.0 100 0/100 (0%) 0/100 (0%) 1.0 150 0/100 (0%) 0/100 (0%) 1.0 200 0/100 (0%) 0/100 (0%)

Table – 1: Transformation efficiency in five times microinjected embryonic shoot apical meristem of cotton seeds(cv.SVPR-2).

Figure – 1: Effect of hygromycin on seed germination. Seeds were germinated and grown on ½ strength MS basalmedium containing 0, 5, 10, 15 and 20 mg/L hygromycin for 10 days.

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Figure – 2: Microinjection of ESAM of cottona). One day old germinated seeds, b). Agrobacterium culture (SiNAC), c). Hygromycin-B resistant plants, d). GUSexpression in root and meristematic tissue, e). GUS expression in cotyledon, f). Control.

Agrobacterium which favored the penetration of bacteriainto cotton shoot meristems. Agrobacterium aftermicroinjection can transfer its T-DNA to the cells of apicalmeristems Baskaran et al. (2011). Various densities ofAgrobacterium and Acetosyringone concentrations wereevaluated for gene transfer efficiency. The efficiencyvaried at different concentrations of Agrobacterium &acetosyringone in co-cultivation media. The efficiencyof 20% was achieved with a density of Agrobacterium0.6 at OD600 and containing 100mM acetosyringone. Table- 1 represents the optimal concentration ofAgrobacterium for efficient gene transfer. Increasing ordecreasing concentration of Agrobacterium (OD600 > 0.8< 0.4) reduced the efficiency of transformation. Bothtissue culture-dependent tissue culture-independent inplanta transformation procedures have been developedfor economically important crop plants such as Hibiscus(Kojima et al., 2004), Rice (Supartana et al., 2005, Sekharet al., 2014) and wheat (Supartana et al., 2006), Brinjal(Subramanyam et al., 2013), Bell pepper (Kumar et al.,2009), Pigeon pea (Rao et al., 2008), Sunhemp (Jyosthnaet al., 2012), Sugarcane (Subramanian et al., 2013), Peanut(Rohini et al., 2000), Soybean (Chee et al., 1989),

Arabidopsis thaliana (Feldman et al., 1987). Use ofmicroinjection method was first reported by Baskaran etal., in 2011 for Agrobacterium-mediated transformationby targeting the Embryonic Shoot Apical Meristem ofthe one day old germinated rice seeds. Microinjection ofESAM has also been achieved in tomato by Vinoth et al.,in 2013 to get transgenic plants within short period. Thesame technique was adopted for cotton (SVPR 2) genetictransformation. Huang et al. (1999) described a protocolfor introduction of exogenous DNA into cotton via thepollen-tube pathway with GFP as a reporter gene. TianZiet al. (2010) developed transgenic cotton plants byinoculating pistil drip into a solution of Agrobacteriumand got transformation efficiency of 0.46-0.93%.Keshamma et al. (2008) developed an in plantatransformation using apical meristem of the differentiatedembryo of the germinating seedling. Transient expressionof GUS in the absence and presence of acetosyringone(0 - 200 mM) was evaluated 4 days post co-cultivationand after antibiotic selection on MS medium.

39 Int.J.Curr.Biotechnol. Volume 2; Issue 12; December, 2014

GUS assayGUS assay was performed to confirm the expression ofthe GUS reporter gene from putatively transformed plantssurviving on Hygromycin selection medium. Leavesshowing blue colour formation after incubating with X-Gluc substrate were considered to be GUS-positive(Table.1). Transformation efficiency was calculated bynumber of primary transformants showing blue colorregions.

ConclusionThe present work is the first attempt to perform genetictransformation of cotton through microinjection methodby using one day old germinated seeds. Previously thistechnique was used to transform foreign genes into theESAM of rice and tomato plants. As in vitro regenerationof cotton is genotype dependent generating transgeniccotton plants through callus culture and directregeneration consume more time. Hence it is necessaryto apply an alternate way to overcome this problem. Inplanta transformation is the best way of getting morenumber of transgenic plants that are recalcitrant within ashort period of time. The present study also emphasized

the importance of in planta transformation in cotton,which avoids unnecessary time consumption for in vitroregeneration. The transformation frequency was higherwith microinjection when compared to othertransformation protocols. However optimization ofseveral factors is a pre-requisite for any transformationprotocol to get high transformation frequency. Thepresent work a simple protocol used to generatetransgenic cotton plants in a genotype independentmanner within a short period of time, approximately 45days. This method can be applied to create transgeniccotton cultivars that are recalcitrant for in vitromanipulation.

AcknowledgementsThe authors are grateful to the Department ofBiotechnology (DBT), Government of India, for thefinancial support to carry out the present work. Authorsare thankful to Dr. Manoj Prasad, Scientist, NationalInstitute of Plant Genome Research, New Delhi, India forproviding SiNAC gene constructs to carry outtransformation work.

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