Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l -1 NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l -1 NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l -1 benzyladenine (BA) and 0.1 mg l -1 NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l -1 BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0-1 mg l -1 ) of ABA while no significant differences were observed at higher concentrations (2-5 mg l -1 ) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.

An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine (BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1.0 mg/ l indole-3-butyric acid and 40 mg/l kanamycin. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective transformation and direct regeneration of E. tereticornis Sm.

The present work investigated the effect of NaCl and seawater salinity (SWS) on accumulation of proline and glycinebetaine (betaine) and expression of two key enzymes of their biosynthetic pathway, betainealdehyde dehydrogenase (BADH) and Δ 1 -pyrroline-5-carboxylate synthetase (P5CS) in two aquatic macrophytes, salt-tolerant Najas gramenia and moderately-salt-tolerant Najas indica. N. gramenia did not show any accumulation of betaine or expression of BADH in response to NaCl or SWS treatments. The content of proline increased significantly in response to the salt treatments. High expression of P5CS was also observed, however, the transcript abundance did not correlate with the amount of proline. N. indica, exhibited significantly greater accumulation of proline in response to SWS, which contained Ca 2+ in addition to NaCl, than in response to NaCl alone.Additional key words: betaine aldehyde dehydrogenase, NaCl, Najas gramenia, Najas indica, Δ 1 -pyrroline-5-carboxylate synthetase, seawater salinity.

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and 12 superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay. The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA) n and its complementary nucleotide (GT) n repeat motifs amplified relatively higher number of bands with an average of 6.0 ± 3.5 and 6.3 ± 1.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5¢ or 3¢ end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C. equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT) 4 distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.

Eucalyptus is planted worldwide for raw material in paper and rayon industry. It is a potential out-crosser and the natural populations are highly heterogeneous displaying strong inbreeding depression. Eucalyptus hybrids have been intensively utilized for their vigor, higher wood quality and resistance to diseases. Identification of species for hybridization is predominantly based on morphological characters and is not always reliable. Hence, DNA marker based species identification and hybrid validation is an important and efficient tool in breeding programs. In the present study, attempts were made to identify species - diagnostic markers for six eucalypt species (E. camaldulensis Dehnh, E. citriodora Hook, E. grandis W. Hill ex Maiden, E. pellita F. Muell, E. tereticornis Sm and E. urophylla S.T. Blake) using ISSR-PCR fingerprints. PCR amplification using seven ISSR primers resulted in significant polymorphism among the population from different species. E. citriodora and E. tereticornis showed monomorphic frequency of maximum 37.5% and minimum 14.3% respectively. Twenty species-diagnostic markers were identified for E. camaldulensis, E. citriodora, E. grandis and E. urophylla while no marker was detected for E. pellita and E. tereticornis. A maximum of eleven and a minimum of one species-diagnostic marker were recorded for E. citriodora and E. camaldulensis respectively. Among the twenty markers, nine were present in all the individuals of a particular species.