Medicago Truncatula Gaertn Research Articles

Treatment of pea pods with Bruchin B results in up-regulation of a gene similar to MtN19

Differential display was used to examine changes in gene expression that resulted from treatment of pea ( Pisum sativum L.) with Bruchin B. This lipid-derived regulator, one of several closely related compounds collectively referred to as bruchins, is an insect elicitor that causes localized cell division and callus formation when applied to pods of pea and certain other legumes. A DNA fragment that was prominently displayed after bruchin treatment was cloned and sequenced, and a full-length cDNA was obtained. This cDNA exhibited a high degree of sequence similarity to a gene, MtN19, expressed in root nodules of Medicago truncatula Gaertn. MtN19 codes for a putative protein of unknown function that is similar in sequence to predicted proteins from a number of other plant species, both leguminous and non-leguminous. Quantitative real-time PCR showed that the expression of this MtN19-like gene was significantly up-regulated within 1.5 h of bruchin treatment, and increased more than 100-fold within 6 h of treatment. Given the rapid and strong up-regulation after exposure of pods to a bruchin, it is likely that this gene plays an important role in the response of pea to these insect elicitors.

Two genes encoding different truncated hemoglobins are regulated during root nodule and arbuscular mycorrhiza symbioses of Medicago truncatula

The MtTrHb1 and MtTrHb2 genes of the model legume Medicago truncatula Gaertn. encode proteins homologous to truncated hemoglobins (TrHb) from plants and a range of different microorganisms. Induction of MtTrHb1 in root nodules and expression of MtTrHb2 in root nodules, as well as in mycorrhizal roots, were shown by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The promoters of both genes were PCR-amplified and fused to the gusAint coding region. By analysing these gusAint-fusions in transgenic root tissues, we were able to localize their activity in root nodules and in roots colonized by arbuscular mycorrhizal (AM) fungi. Whereas the promoter of MtTrHb1 was activated in the infected cells of the nitrogen-fixing zone of root nodules, the MtTrHb2 promoter was predominantly active in the nodule vascular tissue. This expression pattern correlates with the presence of an 'organ-specific element' (OSE)-like sequence in the MtTrHb1 promoter, which is not present in the MtTrHb2 regulatory unit. Concerning the AM symbiosis, only the MtTrHb2 promoter mediated an expression in arbuscule-containing cells and in the root vascular tissue of mycorrhizal root segments colonized by the fungus Glomus intraradices.

CDNA arrays as a tool to identify mycorrhiza-regulated genes: identification of mycorrhiza-induced genes that encode or generate signaling molecules implicated in the control of root growth

Arbuscular mycorrhizas (AM) are symbiotic associations formed by fungi from the Glomeromycota and most angiosperms. Despite the widespread occurrence of the association, its ecological significance, and its potential importance in agriculture, relatively little is known at the molecular level about the development, functioning, and regulation of the symbiosis. We have selected Medicago truncatula Gaertn. 'Jemalong' and an AM fungus, Glomus versiforme (Karsten) Berch, for molecular genetic analyses of the AM symbiosis. Here we used macroarrays as a screening tool to enable the rapid identification of genes that show differential expression in mycorrhizal roots. Forty-three genes showing increased transcript levels and 18 genes showing decreased transcripts in mycorrhizal roots were identified. This set contained several genes predicted to encode regulatory proteins including an alpha-fucosidase implicated in the generation of signaling molecules that modulate plant growth and a gene encoding a putative peptide also implicated in the control of plant growth.Key words: legume, symbiosis, arbuscular mycorrhizal fungi.

Respective roles of the glutamine synthetase/glutamate synthase cycle and glutamate dehydrogenase in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula.

Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription-polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15NH4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine synthesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO3- assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis.

Common gene expression in Medicago truncatula roots in response to Pseudomonas fluorescens colonization, mycorrhiza development and nodulation.

• Beneficial rhizosphere microorganisms may share similar molecular steps during root colonization. To test this hypothesis, we compared Medicago truncatula Gaertn. gene expression in roots colonized, or not colonized, by Glomus mosseae BEG12, Pseudomonas fluorescens C7R12 or Sinorhizobium meliloti 2011. • Pseudomonas fluorescens C7R12 formed colonies on the surface of M. truncatula roots and colonized root tissues intercellularly and intracellularly in a way similar to that previously described for other plants. • Semiquantitative reverse transcriptase polymerase chain reaction of a set of 12 mycorrhiza upregulated M. truncatula genes revealed different expression profiles in roots 3weeks after inoculation with P. fluorescens or S. meliloti. Pseudomonas fluorescens colonization activated seven of the plant genes while nodulated root systems showed increased expression in only three genes and five appeared to be downregulated. • This first report of similar gene induction by a fluorescent pseudomonad and a mycorrhizal fungus in roots supports the hypothesis that some plant cell programmes may be shared during root colonization by these beneficial microorganisms. Less similarity existed in expression of the gene set with nodulation by S. meliloti.

Expression of the plastid-located glutamine synthetase of Medicago truncatula. Accumulation of the precursor in root nodules reveals an in vivo control at the level of protein import into plastids.

In this paper, we report the cloning and characterization of the plastid-located glutamine synthetase (GS) of Medicago truncatula Gaertn (MtGS2). A cDNA was isolated encoding a GS2 precursor polypeptide of 428 amino acids composing an N-terminal transit peptide of 49 amino acids. Expression analysis, by Westerns and by northern hybridization, revealed that MtGS2 is expressed in both photosynthetic and non-photosynthetic organs. Both transcripts and proteins of MtGS2 were detected in substantial amounts in root nodules, suggesting that the enzyme might be performing some important role in this organ. Surprisingly, about 40% of the plastid GS in nodules occurred in the non-processed precursor form (preGS2). This precursor was not detected in any other organ studied and moreover was not observed in non-fixing nodules. Cellular fractionation of nodule extracts revealed that preGS2 is associated with the plastids and that it is catalytically inactive. Immunogold electron microscopy revealed a frequent coincidence of GS with the plastid envelope. Taken together, these results suggest a nodule-specific accumulation of the GS2 precursor at the surface of the plastids in nitrogen-fixing nodules. These results may reflect a regulation of GS2 activity in relation to nitrogen fixation at the level of protein import into nodule plastids.

L-galactono-γ-lactone dehydrogenase is present in calcium oxalate crystal idioblasts of two plant species

Recent studies of calcium oxalate (Caox) crystal producing plant species have revealed that ascorbic acid is likely the primary precursor of the oxalic acid (OxA) used in crystal formation. Thus far, the evidence is based on pulse-chase labeling studies coupled with microautoradiography. In addition, some studies have used the same methods to investigate the biosynthetic pathway giving rise to the ascorbic acid being used to produce OxA. Strong evidence indicates that ascorbic acid is being produced via the pathway proposed by Wheeler and Smirnoff. Further evidence in support of this biosynthetic pathway is now being pursued in the form of identification and purification of key enzymes involved in the catalysis. The final enzyme in the synthesis, L-galactono-γ-lactone dehydrogenase (EC 1.3.2.3) has been isolated and purified from two plant species, cauliflower and sweet potato. Antibodies have been raised against the purified protein from sweet potato. In this study, we employed this antibody to gather further evidence that ascorbic acid is being produced in crystal idioblasts cells via the Wheeler-Smirnoff pathway by localization of this key enzyme. We used two Caox crystal producing species, Pistia stratiotes L. and Medicago truncatula Gaertn. as model systems.

Genomic organization of rDNA loci in natural populations of Medicago truncatula Gaertn.

Medicago truncatula Gaertn. is an annual self-pollinating species characterized by a diploid complement 2n = 16 and low DNA content. It responds very well to transformation methods so it is used as a model species for Leguminosae. In contrast with the advanced studies in molecular biology, cytogenetic research has remained limited even though it is an extremely valuable approach to the analysis of the genome structure. In the present study we examined the chromosomal distribution of rDNA sequences in five natural populations of M. truncatula, explored the genomic diversity of this species and found markers for chromosome identification. FISH experiments revealed three distribution patterns of rDNA sequences, distinguished by one, two and three loci of 5S genes; 18S-5.8S-25S genes were always localized at a single locus. The results add information to the genome structure of M. truncatula, revealing a pattern of distribution of rDNA genes unobserved previously, which consists of 5S genes clustered at a single locus. The physical mapping of rDNA sequences is a first contribution towards the construction of a detailed molecular karyotype of M. truncatula.

Proteome analysis and identification of symbiosis-related proteins from Medicago truncatula Gaertn. by two-dimensional electrophoresis and mass spectrometry.

Time-course analysis of root protein profiles was studied by two-dimensional gel electrophoresis and silver staining in the model plant Medicago truncatula, inoculated either with the arbuscular mycorrhizal fungus Glomus mosseae or with the nitrogen fixing bacterium Sinorhizobium meliloti. Protein modifications in relation to the development of both symbioses included down- and upregulations, as well as newly induced polypeptides. Matrix assisted laser desorption/ionization-time of flight-mass spectrometry after trypsin digestion clearly identified one polypeptide induced in nodulated roots as a M. truncatula leghemoglobin. Internal sequencing with a quadrupole time-of-flight mass spectrometer and database searches confirmed the induction of proteins previously described in root symbioses, and revealed the implication of other proteins. In nodulated roots, one polypeptide was identified as an elongation factor Tu from S. meliloti, while another one could not be assigned a function. In mycorrhizal roots, analyzed proteins also included a protein of unknown function, as well as a glutathione-S-transferase, a fucosidase, a myosin-like protein, a serine hydroxymethyltransferase and a cytochrome-c-oxidase. These results emphasize the usefulness of proteome analysis in identifying molecular events occurring in plant root symbioses.

Fate of annual pasture legumes seeds on a two-way thermo-gradient plate

Effects of diurnally alternating temperatures (5/5–45/45�C) were examined on a two-way thermogradient plate for non-dormant seeds of 14 annual pasture legumes. Seed fates (germination, temperature induced non-viability and temperature induced dormancy) were determined from daily seed counts over a 14-day period on-plate and a further 14 days after removal from the plate. These data clearly demonstrate the existence and extent of seed fates other than germination. Maximum dormancy occurred over a broad range of temperatures for seeds of Trifolium subterraneum L. var. subterraneum (Katzn. et Morley) Zohary and Heller cv. Woogenellup (40/35–30/25�C), Astragalus hamosus L. cv. Ioman (40/25–30/5�C) and Trifolium hirtum All. cv. Hykon (35/30–30/5�C). In contrast, maximum dormancy occurred over a narrow temperature range for Medicago truncatula Gaertn. var. truncatula cv. Paraggio (35/30–30/25�C), M. aculeata Willd. var. inermis (Aschers.) Heyn (30/25 and 30/20�C), and M. minima L. (35/20–35/5�C) seeds. Generally, non-viability was highest in all legumes when temperatures were greater than 35/30�C and not significantly different from the maximum value (100%) at temperatures greater than 40/30�C. At temperatures less than 35/30�C non-viability was less than the maximum (P<0.05) for seeds of all legumes, except M. truncatula cvs. Jemalong and Sephi, M. aculeata, and Vicia villosa ssp. dasycarpa (Ten.) Cav. cv. Namoi. Compared with the other subterranean clover cultivars germination levels T. subterraneum var. brachycalycinum (Katzn. et Morley) Zohary and Heller cv. Clare seeds were not significantly different from the maximum value at higher temperatures. In all annual medics, except M. aculeata germination was not significantly different to the maximum at temperatures greater than 25/20�C, with high germination (P<0.05) occurring at 30/25�C in Jemalong and M. scutellata (L.) Mill cv. Sava and at 30/20�C in Paraggio, Sephi, M. minima and M. polymorpha L. Maximum germination in Ioman and Hykon seeds occurred over a broader temperature range (35/20–15/10�C and 30/20–15/5�C, respectively) than in Namoi (30/15�C and 25/20–25/5�C). Probable implications of these data for field emergence of non-dormant seeds and soil seed reserves are discussed.

Annual Medic and Berseem Clover Dry Matter and Nitrogen Production in Rotation with Corn

Annual medic (Medicago truncatula Gaertn.) and berseem clover (Trifolium alexandrinum L.) have potential to fix atmospheric N2 for use by subsequent crops. Our objectives were to determine the dry matter and N production of annual barrel medic and berseem clover, and their effect on grain yield of a subsequent corn (Zea mays L.) crop. First‐year treatments included spring‐seeded annual medic–oat (Avena sativa L.) and berseem clover–oat intercrops; summer‐seeded annual medic following spring‐seeded oat; and a spring‐seeded oat monoculture. The second‐year corn crop was fertilized with four N rates. In three of four environments, berseem clover had greater average fall dry matter (4392 kg ha−1) and N yield (125 kg ha−1) than spring‐ or summer‐seeded barrel medic. Spring‐ and summer‐seeded medics had similar fall dry matter (1309 kg ha−1 avg.) and N yields (38 kg ha−1 avg.) in two environments, but spring‐seeded medics had greater dry matter and N yield than summer‐seeded medic in two environments (avg. dry matter yield of 5304 and 2428 kg ha−1, respectively, and N yield of 138 and 70 kg ha−1, respectively). Berseem clover and medic did not consistently differ in their effects on soil NO3–N or corn grain yield when no N fertilizer was applied. Legume treatments increased second‐year corn grain yields from 9% for the silt loam soil to 82% for the loamy sand soil compared with the no‐legume treatment when no fertilizer N was applied; however, this effect decreased as N fertilizer rate increased.

Spatial effects and rare outcrossing events in Medicago truncatula (Fabaceae).

In order to elucidate the mechanisms underlying the large amount of RAPD polymorphism found in 1990 in a population of the selfing annual Medicago truncatula GAERTN. (Fabaceae), we have analysed most of the individuals (n = 363) from the same population 6 years later using microsatellite loci. We confirm the result of the earlier study, namely that this population is very polymorphic and highly subdivided, with approximately 37% of the variance distributed among subpopulations, only 50 m apart one from another. We use standard F-statistics analyses, linkage disequilibria, minimum spanning network, multilocus assignment tests and spatial autocorrelation analyses to test the hypotheses that spatial structure and outcrossing events are involved in maintaining the large amount of genetic diversity at the level of each subpopulation. Interestingly, fine-scale spatial structure could be observed in only one subpopulation suggesting that other mechanisms are acting elsewhere. To the best of our knowledge, this is the first study of fine spatial genetic structure in a predominantly selfing species.

Use of Cover Crop Mulches in a No-till Furrow-irrigated Processing Tomato Production System

No-till processing tomato (Lycopersicum esculentum Mill.) production in four winter cover crop-derived mulches was evaluated in 1997 and 1998 in Five Points, Calif. The effectiveness of two medics, `Sava' snail medic (Medicago scutellata Mill.) (sava), and `Sephi' barrel medic (Medicago truncatula Gaertn.) (sephi), and two cereal/legume cover crop mixtures, triticale/`Lana' woolypod vetch (X Triticosecale Wittm./Vicia dasycarpa Ten.) (triticale/vetch) and rye/`Lana' woolypod vetch (Secale cereale L./V. dasycarpa) (rye/vetch), was compared with two conventionally tilled fallow controls (with and without herbicide) (fallow+h and fallow-h) in suppressing weeds and maintaining yields with reduced fertilizer inputs. The comparison was conducted as a split plot, with three N fertilization rates (0, 100, and 200 lb/acre; 0, 112, and 224 kg·ha-1) as main plots and cover crops and fallow controls as subplots. Tomato seedlings were transplanted 3 weeks after the cover crops had been mowed and sprayed with herbicide. There were no significant differences in weed cover in the no-till cover crop treatments relative to the fallow controls in 1997. Early season weed suppression in rye/vetch and triticale/vetch plots was similar to herbicide-treated fallow (fallow+h) in 1998, however, later in the 1998 season weed suppression was best in the fallow+h. Tissue N was highest in the fallow treatments in both 1997 and 1998. Yields were highest in the triticale/vetch and fallow and lowest in sephi treatments in 1997, but there were no differences among treatments in 1998. These results demonstrate the feasibility of no-till mulch production of furrow irrigated processing tomatoes and identify opportunities for further optimization of the system.

Arbuscular mycorrhiza development regulates the mRNA abundance of Mtaqp1 encoding a mercury-insensitive aquaporin of Medicago truncatula.

The genome of the model legume Medicago truncatula Gaertn. was screened for the presence of genes encoding tonoplast intrinsic proteins, and a gene family was identified. The cDNA fragments of two members of the multigene family were cloned from roots inoculated with an arbuscular mycorrhizal fungus. Transcript accumulation in roots could be detected for both cDNA fragments, but only one gene was induced in the symbiosis when compared to non-mycorrhizal control roots. A full-length cDNA clone was obtained from the arbuscular-mycorrhiza-regulated gene, and injection of in-vitro-transcribed RNA into Xenopus oocytes revealed that the encoded protein MtAQP1 specifically facilitates water transport. The possible role of MtAQP1 in buffering osmotic fluctations in the highly compartmented vacuole of arbuscule cells is discussed.

Allocation of carbon to shoots, roots, soil and rhizosphere respiration by barrel medic (Medicago truncatula) before and after defoliation

The allocation of carbon to shoots, roots, soil and rhizosphere respiration in barrel medic (Medicago truncatulaGaertn.) before and after defoliation was determined by growing plants in pots in a labelled atmosphere in a growth cabinet. Plants were grown in a 14CO2-labelled atmosphere for 30 days, defoliated and then grown in a 13CO2-labelled atmosphere for 19 days. Allocation of 14C-labelled C to shoots, roots, soil and rhizosphere respiration was determined before defoliation and the allocation of 14C and 13C was determined for the period after defoliation. Before defoliation, 38.4% of assimilated C was allocated below ground, whereas after defoliation it was 19.9%. Over the entire length of the experiment, the proportion of net assimilated carbon allocated below ground was 30.3%. Of this, 46% was found in the roots, 22% in the soil and 32% was recovered as rhizosphere respiration. There was no net translocation of assimilate from roots to new shoot tissue after defoliation, indicating that all new shoot growth arose from above-ground stores and newly assimilated carbon. The rate of rhizosphere respiration decreased immediately after defoliation, but after 8 days, was at comparable levels to those before defoliation. It was not until 14 days after defoliation that the amount of respiration from newly assimilated C (13C) exceeded that of C assimilated before defoliation (14C).

Hydrogen peroxide accumulation in Medicago truncatula roots colonized by the arbuscular mycorrhiza-forming fungus Glomus intraradices

The diaminobenzidine (DAB) staining technique was used to examine the accumulation of H2O2 in parts of roots of Medicago truncatula Gaertn. colonized by the arbuscular mycorrhiza (AM)-forming fungus Glomus intraradices Schenk and Smith. At the cellular level, the combination of bright-field and fluorescence microscopy revealed that a brownish stain, indicative of H2O2 accumulation was present within cortical root cells in the space occupied by arbuscules. Accumulation of H2O2 was especially pronounced in cells containing arbuscules that were clumped and less branched. Moreover, H2O2 accumulated around hyphal tips attempting to penetrate a host cell. In contrast, no H2O2 accumulation was observed in hyphal tips growing along the middle lamella, or in appressoria or vesicles. On the basis of these findings we suggest that a locally restricted oxidative burst is involved in the temporal and spatial control of the intracellular colonization of M. truncatula cells by the AM-forming fungus G. intraradices.

Comparison of quantitative genetic parameters between two natural populations of a selfing plant species, Medicago truncatula Gaertn.

In this paper we compare mean values, heritability estimates, coefficient of genetic variation, and genetic correlations among several fitness components of two natural populations of a selfing plant species, Medicago truncatula L. It is shown that the population that had been found most polymorphic for molecular markers in a previous study was also the most variable for quantitative characters. Depending on the traits, the larger heritabilities in this population were due to either larger coefficients of genetic variances or smaller coefficients of environmental variances. Whereas genetic and phenotypic correlation matrices were very similar within each population, they were quite different between populations. In particular, although a positive correlation between age and size at maturity was found in both populations, the correlation between age at maturity and reproductive success was negative in the more variable population (late flowering plant, with a larger size at flowering, produced fewer pods), whereas no correlation was observed in the less variable population. We suggest that while in the less variable population all individuals have a high reproductive effort, several strategies coexist in the more variable population, with some early-flowering genotypes showing a high reproductive effort and other late-flowering genotypes showing a larger competitive ability through increased vegetative growth.

High level of polymorphism and spatial structure in a selfing plant species, Medicago truncatula (Leguminosae), shown using RAPD markers

Using RAPD markers and one morphological marker, we studied the among‐ and within‐population structure in a selfing annual plant species, Medicago truncatula GAERTN. About 200 individuals, sampled from four populations subdivided into three subpopulations each, were scored for 22 markers. It was found that the within‐population variance component accounted for 55% of the total variance, while the among‐population variance component accounted for 45%. Eighteen percent of the total variance was due to within‐population structure (i.e., among subpopulations). Thus, 37% of the total variance was within subpopulations. Using a multilocus approach, it was found that no multilocus genotype was common to two populations. Two of the four studied populations were composed of few (≤6) multilocus genotypes, whereas the other two had many (≥15) multilocus genotypes. In the most polymorphic population (37 genotypes), only one genotype was found to be common to two subpopulations. Resampling experiments show that, depending on the population, three to 16 polymorphic loci were necessary and sufficient to score all multilocus genotypes in the population. When these data are compared to published results, it appears that on some occasions, the number of genotypes per population of selfing species might be larger than would be expected from the sole consideration of effective population size. The large within‐subpopulation genetic variance observed in some populations could be explained by either small neighborhood sizes within subpopulations, or by outcrossing following migration through seed and/or pollen.

Transformation of barrel medic (Medicago truncatula Gaertn.) by (Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12

Fertile and stable transgenic plants of the model legume Medicago truncatula Gaertn. were obtained through transformation of leaf tissue with the disarmed Agrobacterium tumefaciens strain LB4404 and in vitro regeneration via somatic embryogenesis. An optimised transformation/regeneration protocol has been established for two genotypes of the cultivar Jemalong, including a previously described highly embryogenic line (Nolan et al. 1989, Plant Cell Rep. 8: 278-281). Using this protocol, transgenic plantlets were obtained within 4-10 months following cocultivation with Agrobacterium. We have introduced into M. truncatula a chimeric fusion between the early nodulin MtENOD12 promoter and the gus (β-glucuronidase) reporter gene, and shown that symbiosis-specific gene expression can be elicited in the roots of such transgenic plants following the addition of purified Rhizobium nodulation factors.

Agrobacterium-Mediated Transformation and Expression of Foreign Genes in Medicago truncatula

Medicago truncatula Gaertn. (barrel medic) is a pasture legume and a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Using the highly regenerable seed line Jemalong 2HA and Agrobacterium tumefaciens (LBA4404), Medicago truncatula can be reliably transformed. The binary vector utilised was pBI121 containing the nptII and uidA genes under the control of the NOS and CaMV 35S promoters, respectively. Kanamycin-resistant embryogenic calli were produced from 17% of the initial leaf explants and on average each one of these embryogenic calli produced a single regenerated plantlet. Regenerated plants showed varying gene copy number and gene location. GUS activity was detected in roots, stems and leaves. The progeny of a regenerated plant containing a single gene copy segregated in a Mendelian fashion. This study confirms the value of Jemalong 2HA for producing transgenic plants for biological and agricultural studies.