BIOTECHNOLOGY/GENETIC TRANSFORMATION/FUNCTIONAL GENOMICS

Regeneration and Agrobacterium-mediated genetic transformationof Terminalia bellerica Roxb.: a multipurpose tree species

Bhawna Dangi & Sumita Kachhwaha & S. L Kothari

Received: 17 August 2011 /Accepted: 9 March 2012 /Published online: 2 May 2012 / Editor: T. Klein# The Society for In Vitro Biology 2012

Abstract An efficient in vitro transformation and plant re-generation protocol was developed for Terminalia bellericausing cotyledonary node cultures. High-frequency shoot budproliferation was obtained on mediumwith 6-benzyladenine.Significant improvements in plant regeneration occurredusing elevated levels of CuSO4 and CoCl2. Rooting occurredon a half-strength Murashige and Skoog medium containingindole-3-butyric acid. The rooted plants were acclimatizedand transferred to field conditions. The genetic fidelity of theregenerated plants was confirmed using randomly amplifiedpolymorphic DNA analysis. An Agrobacterium-mediatedgenetic transformation protocol was developed for Termina-lia by varying several factors which influence T-DNA deliv-ery. Southern blot analysis of regenerated plants confirmedselectable marker gene integration in transgenic plants. Thistransformation protocol can be utilized for further geneticmanipulation of T. bellerica.

Keywords Cotyledonary node . CoCl2 . Inorganicmicronutrient . Micropropagation . Plant regeneration .

Clonal fidelity . Randomly amplified polymorphic DNA(RAPD) . Transgenic plants

Introduction

Terminalia bellerica Roxb. (Fam. Combretaceae) occursscattered in monsoon, mixed, or dry deciduous forests and isoften associated with teak (Tectona grandis). In the SouthAsian countries of Bangladesh, Bhutan, Cambodia, Indonesia,Laos, Nepal, India, Sri Lanka, Burma, Thailand, Malaysia,and China, T. bellerica grows up to 50 m tall, 3 m in diameterand has a rounded crown (Orwa et al. 2009). It is commonlyknown as myrobalan belleric and used for firewood, timber,tannins, and folk medicines against cough and gastrointestinaldiseases (Chowdhury and Koike 2010). It is commonly usedas one of the three constituents of “Triphala” and has antima-larial (Valsaraj et al. 1997) and antibacterial (Elizabeth 2005)activity. The fruits are used as purgative, astringent, and intreating hemorrhoids and dropsy (Kirtikar and Basu 1993).

Although T. bellerica is propagated conventionally throughseeds, this is inefficient because of the hard seed coat andheavy insect infestation of seedlings. Propagation throughstem cuttings also leads to low survival rates (Negi andTodaria 1997). Tissue culture and transgenic technology couldcontribute significantly to the improvement of T. bellerica andother forest trees through exploitation of existing andproduction of new commercially valuable genotypes.Transgenic technology provides rapid genetic modifica-tion by transferring only the gene of interest into a treegenotype as compared to the conventional breedingprograms (Merkle et al. 2007).

Efficient genetic transformation of medicinal plants may behelpful to understand the molecular basis and regulation ofsecondary metabolism in plants and to engineer them foroverproduction of specific metabolites (Pandey et al. 2010).In the only report ofAgrobacterium-mediated transformation ofTerminalia species, a C58 strain of Agrobacterium tumefaciens

In Vitro Cell.Dev.Biol.—Plant (2012) 48:304–312DOI 10.1007/s11627-012-9436-1

B. Dangi : S. Kachhwaha : S. L. KothariDepartment of Botany, University of Rajasthan,Jaipur 302 004, India

S. Kachhwahae-mail: kachhwahasumita@rediffmail.com

S. Kachhwaha : S. L. Kothari (*)Centre for Converging Technologies (CCT),University of Rajasthan,Jaipur 302 004, Indiae-mail: slkothari28@gmail.com

was used to produce transgenic plants of Terminalia chebula(Shyamkumar et al. 2007).

Development of an efficient in vitro regeneration protocolis essential for production of transgenic plants. Regenerationof T. bellerica through cotyledonary node culture (Ramesh etal. 2005) has been reported, but the number of shoots obtainedwas low. In the present study, shoot production was firstincreased by manipulating micronutrient levels in the basalculture medium. This improved shoot regeneration protocolwas then successfully used for genetic transformation of T.bellerica.

Material and Methods

Plant regeneration. Fruits of T. bellericawere collected fromplants growing in the Grassfarm nursery of the ForestryDepartment in Jaipur, India. The seeds extracted from maturefruits were thoroughly washed with 20 % (v/v) Extran®(Merck, Mumbai, India) for 5–7 min followed by surfacesterilization with a 0.1 % HgCl2 solution (w/v) for 10 minand then rinsing 3× with sterile distilled water. The sterilizedseeds were inoculated in culture tubes containing 25 ml 8 gl−1

water agar (Merck) for seed germination and kept in a culturechamber at 26±1°C under a 16-h photoperiod with25 μmol m–2 s−1 photosynthetic photon flux density providedby 40 W white fluorescent tubes (Philips, Mumbai, India).After 12–15 d, seedlings were used as the source of cotyle-donary node explants.

Murashige and Skoog (MS) (1962) basal medium supplementedwith 3 % (w/v) sucrose, solidified with 0.8 % agar (Merck), andadjusted to pH 5.8 before autoclaving at 1.06 kg cm−2 (121°C)for 20 min was used in all the experiments. Cultures weremaintained in the culture chamber at 26±1°C under a16-h photoperiod with 25 μmol m−2 s−1 photosynthetic pho-ton flux density. The medium was dispensed in 100 ml Erlen-meyer flasks (Borosil, Mumbai, India), each containing 40 mlmedium and sealed with non-absorbent cotton plugs.

The cotyledonary nodes were inoculated on medium con-taining the following concentrations of plant growth regulators(Sigma, Bangalore, India): 6-benzyladenine (BA; 2.2, 4.4, 8.8,13.2, and 22.2 μM), kinetin (Kn; 2.3, 4.6, 9.2, 13.9, and23.2 μM), and thidiazuron (TDZ; 0.45, 0.9, 2.3, 4.5, and9.1 μM). After 4 wk, shoot buds induced on MS mediumcontaining 8.9 μM BA were cut into pieces (approximatelyfour shoot buds in each piece) and subcultured on mediumcontaining 8.9 μM BA for proliferation.

Elongated shoots of 2–3 cm length were placed on 1/2-strength MS medium supplemented with different concentra-tions of 0, 1, 2.5, and 5 μM indole-3-acetic acid, indole-3-butyric acid (IBA), naphthaleneacetic acid, or phenylacetic acidfor rooting. Cultures were evaluated after 4 wk. Histological

preparations were made as previously described (Johansen1940).

Plantlets with well-developed roots were thoroughlywashed with running tap water to remove agar and transferredto screw-capped glass bottles containing sterile Soilrite mix(Keltech Energies Limited, Bangalore, India) irrigated with 1/2-strength MS solution and kept in a culture chamber at 26±1°C under a 16-h photoperiod with 25 μmol m−2 s–1 photo-synthetic photon flux density. Plantlets (5–6 cm long) weretransferred to pots containing sand/garden soil (1:1) and sand/garden soil/vermicompost (Agricultural Research Station,Durgapura, Jaipur, India) (1:1:1) and placed in a greenhousewith humidity at 70 % and temperature at 30±2°C for hard-ening. Successfully acclimatized plantlets were transferred tofield conditions.

MS medium supplemented with 8.9 μM BAwas modifiedwith different concentrations of CoCl2·6H2O (up to 50×),MnSO4·H2O (up to 5×), ZnSO4·7H2O (up to 5×), andCuSO4·5H2O (up to 50×), one nutrient at a time, to study theeffect of that particular nutrient on shoot bud induction fromcotyledonary nodes. After 4 wk of culture, all the shoot budswere transferred onto the medium with a modified concentra-tion of a particular nutrient as used in the induction medium.

Randomly amplified polymorphic DNA (RAPD) markerswere used to evaluate the genetic fidelity of micropropagatedplants of T. bellerica over 20 passages in culture for 1 yr. Theplantlets used to assess clonal fidelity were derived from asingle seedling which served as the mother plant (Mp). Fiftyplantlets were obtained from a single cotyledonary node (after20 subcultures). DNA was extracted from the leaves of 14randomly selected regenerated plants and from the Mp. Eachsample was powdered in liquid nitrogen and stored at −20°Cuntil used for DNA extraction by cetyltrimethylammoniumbromide method (Doyle and Doyle 1990). Ten RAPD primerswere taken to assess the clonal fidelity of the regeneratedplants. PCR amplification conditions were an initial denatur-ation at 94°C for 5 min followed by 35 cycles of 94°C for 30 s,50°C for 45 s, and 72°C for 1 min and a final extension at 72°Cfor 5 min.

Agrobacterium strain and kanamycin sensitivity. The A.tumefaciens strain EHA105 with plasmid p35SGUSINT(Vancanneyt et al. 1990) was used with the nptII gene asselectable marker and the uidA (gus) reporter gene undercontrol of the CaMV 35S promoter (Fig. 1). Sensitivity ofthe cotyledonary nodes for kanamycin was determined byculturing these on shoot bud induction medium (MS+8.9 μM BA) supplemented with different concentrations ofkanamycin (0, 20, 30, 50, 75, and 100 mg l−1).

Genetic transformation, selection, and regeneration of trans-formants. A. tumefaciens (EHA105) was grown in yeastextract mannitol medium (Vincent 1970) with 50 mg l−1

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kanamycin and 20 mg l−1 rifampicin at 28°C, with continuousshaking (200 rpm), to an optical density of 0.5, 0.8, 1.0, or 1.5at 600 nm. Bacterial cells were centrifuged at 4,000 rpm for5 min and resuspended in liquid MS medium to 0.8 OD.Cotyledonary nodes pre-cultured for 3, 5, and 7 d on MSmedium supplemented with 8.9 μM BAwere pricked with aneedle and immersed in an A. tumefaciens suspension for 20,30, 45, and 60 min with gentle shaking. After infection,explants were blotted dry on sterile filter paper. The effect of100 μM acetosyringone was also tested on co-cultivationmedium. Explants were then transferred to antibiotic free co-cultivation medium (MS+8.9 μM BA) supplemented with100 μM acetosyringone and cultured in the dark at 26±1°Cfor 2–7 d. The co-cultivated nodal segments were transferredto regeneration medium (MS+8.9 μM BA) with or withoutelevated CuSO4 (5×) and containing 300 mg l−1 cefotaximeand 50 mg l−1 kanamycin for selection of transformed shoots.The cultures were subcultured on the same medium at 20-dintervals. After three cycles of selection, elongated shootswere transferred for rooting.

β-Glucuronidase assay. Agrobacterium-infected explantswere tested for transient β-glucuronidase (GUS) expressionafter 7 d of co-cultivation (Jefferson 1987), and stable GUSexpression was tested in regenerated shoots. GUS was assayedby placing plant parts in a microfuge tube containing GUSbuffer (1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide(X-gluc)), 100 mM sodium phosphate buffer pH 7.0, 0.5 mMpotassium ferricyanide, 0.5 mM potassium ferrocyanide, and0.1 % Triton X-100). Tissues were incubated in GUS assaymix overnight at 37°C, washed once with sterile distilled water,and dipped in 70 % ethanol overnight to extract any chloro-phyll present in the tissue. Percent GUS expression wascalculated as 100 (number of explants expressing the GUS/number of explants infected).

PCR analysis. DNAwas isolated from putative transformantsand control shoots of T. bellerica using a Qiagen mini DNA kit(Genetix Biotech, Bangalore, India). Desalted nptII specificprimers: forward (5′-CAA TCG GCT GCT CTG ATG CCGCGG-3′) and reverse (5′-AGG CGA TAG AAG GCG ATGCGC TGC-3′) were obtained from Integrated DNA Technolo-gies (IDT, Coralville, IA). Genomic DNA (approximately40 ng) along with 1.5 mM MgCl2 and 50 ng of nptII forward

and reverse primers in 25 μl were subjected to PCR amplifica-tion (DNA engine, Bio-Rad, Hertfordshire, UK). The PCRprogram had an initial denaturation for 3 min at 95°C, denatur-ation for 1 min at 94°C, annealing at 64°C for 30 s, primerextension at 72°C for 1 min for 30 cycles, followed by a finalextension of 7 min. To check Agrobacterium contamination,DNAwas tested using PCR and vir C gene specific forward (5′-ATC ATT TGT AGC GAC T-3′) and reverse (5′-AGC TCAAAC CTG CTT C-3′) primers under the following PCR con-ditions: denaturation at 94°C for 45 s, annealing at 50°C for 30 s,extension at 72°C for 45 s, and final extension for 5 min. Theamplified products were separated on 1.2 % agarose (Himedia,Mumbai, India) gel through electrophoresis and photographedusing Gel Documentation System (Bio-Rad).

Southern blot hybridization. Approximately 8–10 μg ge-nomic DNA of transformed plants was digested with BamH1or EcoR1 at 37°C. Digested plasmid p35SGUSINT served asa positive control, and DNA from a nontransformed plant was

Figure 1. Linear (partial) restriction map of the p35SGUSINT T-DNA cassette. LB/RB left/right border sequences, NOS-P/NOS-pA nopalinesynthase promoter/terminator, CaMV 35S-P/CaMV-pA cauliflower mosaic virus 35S promoter/terminator (Vancanneyt et al. 1990).

Table 1. Effect of cytokinins on shoot bud proliferation from cotyle-donary nodes of T. bellerica cultured on MS medium

Cytokinin Concentration (μM) % Response Shoot budsMean±SD

BAP 2.22 100 1±0e

4.40 100 5.3±0.2b

8.90 100 7±0.8a

13.30 90 3.5±0.3c

22.20 90 1±0e

Kinetin 2.32 50 1±0e

4.60 75 1.6±0.5de

9.30 100 2±0.6de

14.90 100 3.3±0.5c

23.20 100 5.1±0.9b

TDZ 0.45 100 2.3±1.0d

0.90 100 2.5±0.7d

2.30 100 2.3±0.6d

4.50 100 2±0de

9.10 100 2.1±0.1d

Values are the mean of 45 replicates. Means in a column followed bydifferent letters are significantly different at P00.05 from each other

SD standard deviation

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used as a negative control. DNAs were electrophoresed on0.8% agarose gel and blotted on Immobilon-NY+membranes(Millipore, Bangalore, India) as described by Sambrook andRussell (2001). PCR amplified nptII gene, labeled with DIG-dUTP, was used as the probe. Blotted membranes werehybridized with the probe using the standard protocol providedin DIG High prime DNA labeling and detection starter kit(Roche, Mumbai, India) for color detection with nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl phosphate.

Statistical analysis. Each experiment of axillary shoot pro-duction consisted of 15 replicates, and all experiments wererepeated 3×. Suitable controls were maintained for eachexperiment. Data for explant establishment, shoot prolifera-tion, and rooting of micro-shoots were collected after 4 wk.In transformation experiments, each treatment was repeated3×, and each experiment consisted of at least 50 explants.Data were subjected to one-way analysis of variance byFischer’s least significant difference (P00.05) (Gomez andGomez 1984).

Results and Discussion

Regeneration from cotyledonary node explant. Sixty percentof T. bellerica seeds germinated within 7 d on water agarmedium. Cotyledonary nodes excised from 15-d-old seedlingswere used as explants, and their morphogenic response tovarious cytokinins (Table 1) was documented. On mediumcontaining cytokinin, axillary buds formed within 10 d andappeared as small green protuberances on the cotyledonarynodes that elongated into shoots after 20 d of culture. Thefrequency of shoot proliferation was lower on Kn containingmedium than on BA, and TDZwas the least effective of all thecytokinins tested. The shoots induced on TDZ failed to elon-gate and were fasciated, as reported for several otherspecies such as Dalbergia sissoo (Pradhan et al. 1998),Oroxylum indicum (Dalal and Rai 2004), and Balanitesaegyptica (Anis et al. 2010). The optimum shoot inductionand growth was observed on MS medium containing8.9 μM BA alone, which produced up to eight shoots after30 d culture (Fig. 2a). BA has been used extensively for

Figure 2. (a–g) (a)Proliferation of axillary buds atthe cotyledonary node onMS+8.9 μM BA after 4 wk ofculture. (b) Enhanced shootbud proliferation fromcotyledonary nodes on MSmedium with 3.3 μM CoCl2and 8.9 μM BA. (c, d) Shootbuds in section with leafprimordia (bar0100 μM). (e)Rooting of regenerated shoot on1/2-strength MS medium with2.5 μM IBA. (f) Regeneratedplant transferred in pot. (g)RAPD profile of regeneratedplants (Primer OPF-07) (lanes:M molecular size marker, Mpmother plant, 1–14 regeneratedplants).

REGENERATION AND AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION 307

multiple shoot induction in Capparis decidua (Tyagi andKothari 1997), D. sissoo (Pradhan et al. 1998), Sesbania ros-trata (Jha et al. 2004), and O. indicum (Dalal and Rai 2004).

Following subculture of the shoots induced on MS mediumcontaining 8.9 μM BA into pieces (three to four shoot budsper piece) on the same medium, a continuous mass ofmultiplying shoots was obtained which did not decline evenafter 1 yr of culture. After the third subculture, elongatedshoots (2–3 cm) were transferred to rooting medium.

At first, the composition and ratio of plant growth regu-lators were manipulated to optimize the quality and numberof shoots initiated. However, many species and varieties donot respond to this classical approach and require additionaloptimization (Ramage and Williams 2002). The effect ofdifferent micronutrients on morphogenesis and proliferationhas been studied in many plants such as Jatropha curcas(Khurana-Kaul et al. 2010), Capsicum annum (Joshi andKothari 2007), Kodo and finger millet (Kothari-Chajer et al.2008), barley (Chauhan and Kothari 2004), chili pepper(Kintzios et al. 2001), and Holostemma ada-kodien (Martin2002).

Four micronutrients (CuSO4, ZnSO4, MnSO4, andCoCl2) were selected to study their effect on shoot budinduction and proliferation. Reduction in the regenerationcapacity of cotyledonary nodal explants was observed in

response to increasing concentration of Zn and Mn, whilean increase in concentration of Cu and Co in MS mediumimproved regeneration response up to a threshold beyondwhich there was decline (Table 2).

When the concentration of CuSO4 was increased(0.55 μM), a maximum of 16 shoot buds were formed. Shootsformed on the medium devoid of copper were pale green.Copper is an essential component of several enzymes involvedin electron transport, protein, and carbohydrate synthesis(Kothari-Chajer et al. 2008). A maximum of 22 shoots formedon medium with 3.3 μM CoCl2 (Fig. 2b) compared to 11shoots on medium with the basal concentration of cobalt.Increased CoCl2 has been shown to greatly improve regener-ation of other plant species (Lai et al. 2000; Sharma et al.2008). Co2+ inhibits ethylene production by blocking the con-version of l-aminocyclopropane 1-carboxylic acid to

Table 2. Effect of inorganicmicronutrients on plantregeneration from culturedcotyledonary nodes of T.bellerica on MS mediumsupplemented with8.9 μM BA

Values are the mean of 45replicates. Means in a columnfollowed by different letters aresignificantly different at P00.05from each other

SD standard deviation

Inorganicmicronutrient

Concentration ininduction medium(μM)

Shoot buds ininduction medium

Concentration inproliferationmedium (μM)

Shoots in proliferationmedium

Mean±SD Mean±SD

CuSO4 0.1 7.3±0.8 0.1 11±0.8b

0.5 7.4±0.8 0.5 16±0.8a

1.0 6±1.1 1.0 9.8±0.3c

2.0 6.4±0.8 2.0 8.6±0.7d

3.0 4.4±0.5 3.0 6.8±0.7e

CoCl2 0.1 6.8±1.2 0.1 12.1±0.7a

0.5 8.3±0.7 0.5 13.3±0.5ad

1.1 8.3±0.5 1.1 13.5±1.0ad

2.2 8.4±0.7 2.2 17.1±0.7bd

3.3 10.6±1.1 3.3 21.1±0.7b

5.5 5.5±0.7 5.5 8.1±0.7c

ZnSO4 29.9 7.1±0.7 29.9 12.5±0.5a

59.8 5.5±0.5 59.8 10.8±0.9b

89.7 4.7±0.8 89.7 9±0.6bc

119.6 4.6±0.7 119.6 8.3±0.5c

149.5 3.6±0.7 149.5 7.3±0.5c

MnSO4 100.0 7.1±0.9 100.0 11.1±0.9a

200.0 5.6±0.5 200.0 8.8±0.4a

300.0 4.1±0.6 300.0 6.8±0.7bc

400.0 3.5±0.5 400.0 5.6±0.5bc

500.0 3.5±0.7 500 5.5±1.0c

Figure 3. (a–h) (a) Effect of kanamycin on shoot regeneration fromcotyledonary node explants of T. bellerica. (b–g) The effect of differentparameters on % transient GUS expression. (b) Preculturing of cotyle-donary node (d). (c) Bacterial density (OD600). (d) Infection time(min). (e) Acetosyringone concentration. (f) Co-cultivation period (d).(g) Co-cultivation temperature. (h) Effect of CuSO4 on shoot budinduction on control (0.1 μM CuSO4) and modified (0.5 μM CuSO4)regeneration medium following infection with Agrobacterium. TransientGUS expression0(Number of explants expressing the GUS/Number ofexplants infected)×100. Values are the mean of three experiments con-sisting of 50 explants each. Data represent the mean±SD.

b

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ethylene (Yang and Hoffman 1984). Thus, the enhancementof shoot regeneration with increased Co2+ is hypothesized tobe due to a decrease in the level of ethylene.

Histological studies revealed a distinct meristematic zoneof densely stained cells in the axil of each leaf present over adifferentiated zone after 10 d of culture (Fig. 2c). In the

a) b)

c) d)

e) f)

g) h)

REGENERATION AND AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION 309

cultured nodes, development of multiple shoots was observedafter 2 wk of culture (Fig. 2d).

Auxin supplements were important for root induction.Of the four auxins used individually, the best rootingresponse was observed with 2.5 μM IBA (Fig. 2e), interms of percent rooting and root number. Optimumrooting response using IBA has been reported in severaltree species such as Semacarpus anacardium (Panda andHazara 2010), B. aegyptica (Anis et al. 2010), and Aeglemarmelos (Nayak et al. 2007).

A low survival frequency was observed when rootedplants were directly transferred from agar medium tosoil. Yu et al. (2000) suggested that aeration is animportant factor in the formation of adventitious roots.Therefore, plantlets were transferred from agar mediumto sterile Soilrite (Keltech Energies Limited) fortifiedwith 1/2-strength MS nutrients in culture bottles keptin a culture chamber for 4 wk. A similar approach wasreported for T. chebula (Shyamkumar et al. 2003/2004)and Crataeva adansonii (Sharma et al. 2003). After4 wk of initial hardening, plants were transferred tothe greenhouse in pots containing different substrata.Vermicompost increased the survival of plants in thegreenhouse; thereafter, primary hardened plants weretransferred to a mixture of sand, soil, and vermicompostin the green house (Fig. 2f).

Genetic fidelity was checked with ten random primers outof which seven generated distinct, reproducible products. A

total of 180 amplification products were detected. Fingerprint-ing profile of regenerants was monomorphic, and there was novariation among mother- and tissue-culture-raised plants(Fig. 2g).

Optimization of transformation parameters. Different con-centrations of kanamycin (0–100mg l−1) were used to estimatethe tolerance level of cotyledonary node explants (Fig. 3a).Increasing doses of kanamycin reduced shoot regeneration. At50 mg l−1 kanamycin, the percentage of regenerating explantsdecreased to 16.6 %, and the shoots that still formed werealbino. Therefore, 50 mg l−1 kanamycin was used in all furtherexperiments.

Explants directly infected with Agrobacterium showed low-er GUS expression than precultured explants. Preculturing ofexplants for 5 d on regeneration medium increased transientGUS expression to 60.5 % (Fig. 3b). Explants with activemorphogenetic activity have a higher probability of Agrobacte-rium infection (Sangwan et al. 1992). Preculturing explants hasbeen shown to increase transformation efficiency in Eucalyptustereticornis (Aggarwal et al. 2011), Sesbania drummondii(Padmanabhan and Sahi 2009), and Leucaena leucocepahala(Jube and Borthakur 2009).

The bacterial suspension density used in infection and theduration of infection also influenced transient GUS activity.At an optical density of ≥1.0, Agrobacterium overgrowth wasseen and resulted in tissue necrosis. Maximum transient GUS

Figure 4. (a–f) Transformation of Terminalia using cotyledonarynodes: (a) regeneration of green and albino plants on modified regen-eration medium supplemented with kanamycin (50 mg/l) after 4 wk ofculture; (b) elongated shoots on kanamycin containing selection media;(c) GUS staining of transgenic shoots (right) and nontransformedcontrol (left). PCR analysis for (d) detection of 650 bp of the nptII

gene; (e) detection of virC gene (lanes: M molecular size marker(100 bp ladder), P positive control (plasmid DNA), N negative control(nontransgenic plant), 1–6 putative transgenic plants). (f) Southern blothybridization of putative transformants of T. bellerica. P positivecontrol, N negative control (non transgenic), lanes 1–5 DNA fromtransformants (digested with restriction enzyme BamHI).

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activity (68.5 %) was observed at OD60000.8 (Fig. 3c). Anincrease in infection time to more than 30 min resulted inbrowning of the target tissue (Fig. 3d). Our observations are inagreement with similar previous studies (Sharma et al. 2011)reported in Eleusine coracana. Explants were wounded byneedle prior to infection.

Addition of 100 μM acetosyringone to the co-cultivationmedium increased transient GUS activity from 52 % (onmedium lacking acetosyringone) to 67 % (Fig. 3e). Acetosyr-ingone is used to induce vir genes and enhance T-DNAtransfer (Stachel et al. 1985).

The influence of a co-cultivation period onAgrobacterium-mediated transformation has been reported for many plantspecies (e.g., Mohan and Krishnamurthy 2003; Uranbey etal. 2005). A co-cultivation period of 5 d in the dark resulted inoptimum GUS expression (70.6 %) (Fig. 3f). A co-cultivationperiod of more than 5 d resulted in bacterial overgrowth andtissue necrosis.

Cotyledonary nodes co-cultivated at 24°C showed thehighest transient GUS expression (66.6 %, Fig. 3g) and sur-vival. Salas et al. (2001) stated that co-cultivation at 25°Cappears beneficial for plant cell susceptibility to infection andfor stable T-DNA insertion into the plant chromosomes. How-ever, 19°Cmay be the best temperature for the Agrobacteriumtransfer machinery.

Agrobacterium-infected cotyledonary nodes cultured onmodified regeneration medium (5× CuSO4) showed bettershoot recovery as compared to normal regeneration medium(MS level of CuSO4) (Fig. 3h). Recently, Sharma et al. (2011)also suggested that an elevated level of CuSO4 (10×) in themaintenance and plant regeneration medium improves callusgrowth and recovery of plantlets for E. coracana.

Selection and regeneration of transformants. Following theoptimized conditions, 150 cotyledonary nodal explants wereco-cultivated and then cultured on modified regenerationmedium containing 50 mg l−1 kanamycin and 300 mg l−1

cefotaxime for shoot regeneration. Putative transgenic shootclusters were selected (Fig. 4a) and transferred onto freshmedium containing the same concentrations of antibiotics for2 wk, until the shoots attained a height of 2–3 cm (Fig. 4b).After three cycles of selection, putative transgenic green shootswere transferred to rooting medium containing 1/2-strengthMS salts, 2.5 μM IBA, and 3 % sucrose. GUS expressionwas examined in leaves of putative kanamycin-resistant trans-genics to confirm the transformation (Fig. 4c). The putativetransformed plantlets were established in soil.

Molecular analysis. The six putative transformants were ana-lyzed for the presence of nptII gene through PCR, and all gavethe expected 650-bp PCR product for the nptII gene (Fig. 4d).The positive plants were checked for Agrobacterium contami-nation using 729 bp vir C gene-specific primers. Amplification

of the vir C gene was not observed in any of the samples(Fig. 4e).

Initially, a transformation frequency ([number of PCR-positive plants obtained from individual explant/number ofexplants infected]×100) of 4 % was obtained. This result isanalogous to earlier studies in Withania somnifera (Pandey etal. 2010) and Bixa orellana (Parimalan et al. 2011).

Southern blot analysis of PCR-positive plants indicated thatthe T-DNA region was inserted in the T. bellerica genome andthat the insertion site was different between the transformedplants (Fig. 4f; lanes 1, 2, 4, and 5). No signal was detected inthe negative control (Fig. 4f). Absence of a hybridization signalin one of the PCR-positive plants (Fig. 4f; lane 3 sample) mayhave resulted from the production of a chimeric plant from thetransformation of few sectors of the meristematic region in thecotyledonary node explants.

The in vitro regeneration protocol described here for T.bellerica using cotyledonary nodes from seedling is efficientenough to be useful for genetic transformation. This is the firstreport, to the best of our knowledge, of successful genetictransformation of T. bellerica. The information may be usefulfor work involving the in vitro propagation and/or geneticmodification of this important tree species.

Acknowledgments Senior Research Fellowship to Bhawna Dangiwas awarded by the Council of Scientific and Industrial Research(CSIR), New Delhi, India. This is gratefully acknowledged.

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