The seeds of Litchi (Litchi chinensis Sonn.) cv. “Bedana” were obtained from Bihar Agricultural University, Sabour, Bhagalpur, Bihar, India. The Agrobacterium tumefaciens strain LBA4404 containing binary plasmid pBI121 with SAMDC gene from Datura stramonium under the control of cauliflower mosaic virus CaMV35S promoter and nopaline synthase terminator, and hygromycin phosphotransferase (hpt) as plant selection marker was used for litchi transformation [22] (Figure 1). An empty pBI121 vector not carrying SAMDC gene was also transferred into Agrobacterium tumefaciens strain LBA4404 as positive control.

Litchi immature seed explants, collected from about 6 - 7 weeks, were used for transformation. The Agrobacterium culture grown to an O.D. (A600) of 0.1 - 0.2 was used for infection (10 min) on 2 days old precultured explants (zygotic embryos), grown on calli regeneration medium (CRM). Without antioxidants presoaked zygotic embryos were carefully removed and transferred onto induction medium (MS1), which consisted of MS salts and B5 vitamin with 2 mg∙l⁻¹ 2, 4-D (2, 4-dichlorophenoxyacetic acid), 50 g∙l⁻¹ sucrose and 8 g∙l⁻¹ agar (pH 5.8). With antioxidants pre-soaked zygotic embryos were transferred onto induction medium (MS1) containing the tested concentrations of anti-browning agents (combination of ascorbic acid and citric acid) were 160 mg∙l⁻¹. MS1 medium was sterilized by autoclaving at 120˚C and 1.1 kg∙cm⁻² for 15 min. The Cultures were maintained in darkness at 26˚C ± 2˚C. The embryogenic cultures were pale yellow and friable appeared after 6 - 8 weeks [23] . The embryos were sub cultured in every 2 weeks interval on fresh MS1 or MS2 (MS1 plus STS 29.4 μM)

with addition of antioxidants. The STS stock solution was prepared as follows: 0.1 M sodium thiosulphate and 0.1 M silver nitrate were prepared separately in distilled water; 0.02 M STS (Sodium thosulphate) stock solution was prepared by slowly pouring 20ml of 0.1 M silver nitrate to 80ml of 0.1 M of sodium thiosulphate. The STS solution was filter sterilized and added to autoclaved MS1to form the final concentration of 29.4 μM [24] . According to a number of workers [25] , culturing on medium supplemented with 2, 4-D followed by growth of the callus onto medium devoid of 2,4-D, gives rise to somatic embryos and eventually to plantlets. For germination of naked white to yellowish, opaque, cotyledonary-stage somatic embryos were cultured on MS agar medium containing B5 macrosalts, MS microsalts, iron EDTA (Ethylene diamine tetra acetic acid), organics with 3% sucrose and 0.7% agar supplemented with 2.9 μM GA₃ (Gibberellic acid) with addition of higher concentrations (0.04, 0.2 and 0.4 μM) of ABA (Abscisic acid). Each experimental treatment was repeated five times.

The putative transgenic plants were analyzed by PCR for the integration of the transgene. DNA was isolated from the leaf explants by CTAB (Cetyl trimethyl ammonium bromide) method. About 100 ng of DNA from untransformed plants as well as putative transgenic lines was taken and mixed with 100 mM of primer pair, 7.5 μl of PCR buffer, 100 mmol∙l⁻¹ dNTP mix and 0.5 U of Taq DNA polymerase for 50 μl reaction mixture (MBI, Fermentas). The PCR program included denaturation at 94 ˚C for 5 min followed by 30 cycles of denaturation at 94˚C for 30 s, annealing at 56˚C for 45 s and synthesis at 72˚C for 45 s and finally 1 cycle of 7 min at 72˚C. The primer pairs specific for the amplification of a 1.9-kb fragment of SAMDC gene 5’-GTATACCCGTGGGAG CTTCA-3’and 5’-TCTCCCTGTCCAAACCAGTC-3’.

Litchi genomic DNA (10 mg) was restricted with EcoRI to detect the copy number of the transgene. Southern blots were prepared by standard procedure [26] using Hybond-N Nylon membrane (Pharmacia). The hpt gene probes were prepared using ³²P-labeled dCTP by nick translation as the manufactures guidelines (Gibco-BRL). Hybridization was carried out for 18 - 22 h at 65˚C. Signals were detected by exposure of storage phosphor screens, which were scanned in a Typhoon TM 9400 (GE Healthcare).

Total RNA was isolated using TRIzol^® (Invitrogen) as a template and the cDNA was made using SMARTTM PCR cDNA Synthesis Kit according to the manufacture (Clontech). The 20 μl of reaction mixture included 1 × PCR reaction buffer, 400 mM of dNTPs, 1.2 mmol∙l⁻¹ of each primer, 2 U Taq Polymerase and 1.2 μL cDNA. The reaction mixture was heated to 95˚C for 10 min, followed by 30 cycles of 30 s denaturation at 94˚C, annealing at 53˚C for 30 s, extension at 72˚C for 1 min and final extension for 10 min. The PCR products were analyzed on 1% agarose gel. Twenty micrograms of total RNA were electrophoresed through a 1.2% (w/v) denaturing formaldehyde/agarose gel, blotted to a Nitran filter by the capillary blot method. Prehybridization and hybridization were done in 50% formamide buffer using an [³²P]-labeled SAMDC cDNA probe at 42˚C. Yeast specific SAMDC cDNA 1 fragment was labeled using Random Priming Labeling Kit (Promega, Shanghai, China) with α-[³²P] dCTP. Washing of the filter was carried out first with 2 × SSC, 0.5% SDS for 7 min and then 1 × SSC, 0.5% SDS at 55˚C for 4 min.

PAs were estimated in seedlings of the wild-type and transgenic lines according to the protocol of [27] . Litchi leaves (0.4 g) from 5 seedlings were pooled and powdered with nitrogen, then extracted with 10 volumes of 4% perchloric acid (PCA) and centrifuged at 20,000 g for 30 min at 4˚C. Aliquots (0.2 ml) of the supernatant containing free polyamines were dansylated, extracted in toluene and analysed by HPLC on a reverse phase C18 column using a programmed acetonitrile: water step gradient, respectively, with 1 ml∙min^?1 flow rate. Eluted peaks were detected with a fluorescence spectrophotometer at excitation and emission wavelengths of 360 and 506 nm and their areas were recorded and integrated relative to those of standard PAs (Sigma, USA). Three extractions of polyamines were made from each sample and each extraction was quantified by HPLC in duplicate.

Many concentrations and combinations of ascorbic acid (AA) and citric acid (CA) were used to define an efficient antioxidant to check browning as well as promote calli formation for litchi regeneration from explants. Calli formation was observed only in zygotic embryos cultured on MS1 medium added with antioxidant. Embryos failed to respond in the absence of antioxidant. Antioxidants lowered down the polyphenol oxidase (PPO) activity, at the time of incision of zygote. PPO activity was found maximum but it gradually reduced the PPO activity under the control of different antioxidant in different ratio with time interval (Table 1). The combination of AA and CA increased the percentage of somatic embryogenesis and the development of the somatic embryos. While, in MS1 medium, without antioxidant we found that percentage of somatic embryogenesis and the development of the somatic embryos from explants were very low. Combination of AA and CA initiated somatic embryos formation. Optimum values for calli induction from zygotic embryos were obtained in MS2 medium (MS1 plus STS 29.4 μM) with addition of equal conc. of AA and CA (160 mgl⁻¹). One hundred percent of explants cultured on this medium turned light pale color and we observed the proliferation of callus (Figure 2). Primary somatic embryos were generated from zygotic embryos when cultured on MS1 medium, they were subcultured on either NN basal or NN medium supplemented with IBA and within 4 - 6 weeks embryogenic callus was formed (Figure 3(a)), which differentiated into many globular to cotyledonary stage secondary embryos. The globular stage somatic embryos reached the cotyledonary stage in 4 weeks (Figure 3(b)). The cotyledons (closed) of most of the somatic embryos became opaque and milky-white to yellowish in colour (Figure 3(c)) to light-green or green after transfer to light. Exogenous abscisic acid (ABA) treatment prevented precocious germination of immature somatic embryos in several species [28] [29] . The visible shoots emerged in the co-cultivated explants after 2 - 3 weeks in shooting culture medium. The growth of shoots derived from leaf explants was slow and the shoots rooted in rooting medium after more than 3 - 4 weeks of culture. However, the plantlets grew well when transferred to pots for 20 - 30 days (Figure 4).

The presence of SAMDC in putative transgenic plants was confirmed by PCR analysis. The expected amplified product of 1.9 kb, specific to SAMDC gene was obtained (Figure 5). The PCR positive plants were analyzed by Southern hybridization to identify the integration and copy number of the transgene. The transgenics showed one or two copy insertions of the transgene in tested plants (Figure 6). The transgenic lines showed high trans-

gene expression at the transcript level by RT-PCR using SAMDC gene-specific primers, while not in wild type plants (Figure 7). The inserted SAMDC was further confirmed by Northern blot analysis, transgenic plants showed high transgene expression at the transcript level (Figure 8). The expression levels were, however, variable among the transgenic lines.

The transgenic lines showed significant increase in Spd and Spm levels when compared with wild-type plants and pBI121 plants. In wild-type plants, the Spd and Spm contents were 25.2% and 38% respectively in normal condition. However, in the transgenic lines, Spd contents were increased by 94% - 135% while Spm contents increased by 51.7% - 93.9% and, when compared with wild-type plants. The levels of accumulated polyamines were much higher in all transgenic litchi plants than in wild-type ones. On average, Spd and Spm in transgenic lines were increased by 114% and 69.7% respectively under normal condition compared with wild-type plants. Different transgenic lines showed variations in the increased levels of PAs. Transgenic lines showed significantly different in putrescin (Put) level grown under control condition. Put content increased from 14.4% - 30.3% compared with the wild-type plants (Table 2). On the contrary, Put level in the non-transformed plants and pBI121 plants remained unchanged.

Values (nmol/g fresh weight) represent average of data from three independent experiments and are shown as means ± S.E. Different letters in column indicate significant differences (P < 0.05) between means of different transgenic lines and wild-type with Duncan’s multiple range test.