Immature Leaf Research Articles

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research.

Acquisition of callogenic capacity in date palm leaf tissues in response to 2,4-D treatment

While callogenesis in date palm can be initiated by culturing immature leaf segments on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), it is difficult to obtain callus from certain genotypes. To better understand the mechanisms of callogenesis in date palm, we studied the developmental events underlying this process at the cellular level. The callogenic capacity of leaf segments depends on the state of cell differentiation. The most callogenic segments were within the leaf elongation zone, required polar auxin transport to initiate callogenesis and contained the highest quantities of free endogenous indole-3-acetic acid. At the cellular level, callus induction involves two spatially and temporally separated events. The first event involves cells from the fascicular parenchyma in an equatorial plane perpendicular to the vascular axis that within 2 days of culture in the presence of 2,4-D, acquire structural and ultrastructural features typically observed in meristematic cells. The second event occurs 3 days later and is characterised by the modification of adjacent perivascular sheath cells. The latter cells become callogenic in that they reinitiated their cell cycles and undergo cell division leading to callus formation. These data provide evidence that callus initiation in leaf vascular tissue of date palm involves a sequential response of two distinct cell types to auxin and requires polar auxin transport.

Summer Dormancy and Drought Survival of Moroccan Ecotypes of Orchardgrass

Summer dormancy improves drought survival and autumn recovery of perennial forage species in Mediterranean areas. Morocco has served as a source of germplasm for orchardgrass (Dactylis glomerata L.) breeding, but little is known of the incidence and variability of the summer dormancy trait in Moroccan orchardgrass germplasm. This paper characterizes summer dormancy and summer drought survival in Moroccan ecotypes of orchardgrass. Ten Moroccan ecotypes and the cultivar Kasbah were studied in the field in Guich experimental INRA, Rabat, Morocco. Two trials were conducted in irrigated (to test summer dormancy) and in rainfed conditions (to test drought survival after summer). Under irrigation, Moroccan ecotypes of orchardgrass and Kasbah were summer dormant. According to a summer dormancy index and a combination of physiological responses, most ecotypes and Kasbah were found completely dormant. A single ecotype was considered incompletely dormant. After intense first year summer drought, plant survival was correlated (P < 0.001) with water soluble carbohydrate (WSC) content and osmotic potential in immature leaf bases (r = 0.67 and 0.62, respectively). Two Moroccan ecotypes exhibited higher post‐drought survival after summer drought (98%) than Kasbah (90%) (P < 0.01). After a second year with severe drought, an ecotype outyielded Kasbah for autumn recovery (P < 0.001). Moroccan germplasm exhibited good levels of dormancy and agronomic performance that would be desirable in breeding programs.

Callogenesis and rhizogenesis in date palm leaf segments: are there similarities between the two auxin-induced pathways?

In date palm (Phoenix dactylifera L. cv. Ahmar, Arecaceae), as for many monocotyledons, callogenesis is a prerequisite for the initiation of somatic embryogenesis, and requires the presence of auxin in the medium. Immature leaf explants were cultivated in medium supplemented with either 1 or 54 μM 1-naphtaleneacetic acid in order to induce either rhizogenesis or callogenesis. Histological studies performed throughout the culture period established that precocious cell reactivation is similar in both morphogenetic pathways. Early cytological modifications are associated with cell reactivation and are observed in the pluripotent cells of perivascular sheaths. Divergence between the callogenesis and rhizogenesis pathways is observed later, during the subsequent determination and morphological differentiation phases. We established that in date palm, the rhizogenesis and callogenesis pathways are initiated from the same cell type, the ultimate developmental fate depending upon auxin concentration.

Identification of the flavin-dependent monooxygenase-encoding YUCCA gene family in Populus trichocarpa and their expression in vegetative tissues and in response to hormone and environmental stresses

Auxin is a plant hormone that regulates many processes of plant growth such as apical dominance, cell growth, adventitious rooting, and fruit and seed development. Expression of a family of YUCCA genes important for auxin biosynthesis has been shown to be spatially and temporally regulated in Arabidopsis. In this study, we report on the identification of 12 YUCCA genes from the completely sequenced Populustrichocarpa genome database and characterized them by comparing them with ArabidopsisYUCCA genes. The PopulusYUCCA genes are distributed on eight chromosomes. All Populus and Arabidopsis YUCCA genes can be divided into two phylogenetic groups, one of which can be divided into two subgroups. Populus YUCCA genes are expressed in the shoot tip, immature and mature leaf, young root, stem, and bark tissues in an tissue-specific manner. Transcript accumulation of the Populus YUCCA genes is reduced by exogenous applications of various plant growth regulators except auxin. Steady-state mRNA levels of different Populus YUCCA genes vary considerably depending on the experimental treatments. These results suggest that, similar to the Arabidopsis and rice YUCCA genes, the Populus YUCCA genes may also have diversified roles in Populus growth and development, and their expression may be regulated in a complex manner by developmental and environmental cues.

Molecular cloning and characterization of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (hmgr1) gene from rubber tree (Hevea brasiliensis Muell. Arg.): A key gene involved in isoprenoid biosynthesis

Natural rubber (cis-1,4-polyisoprene) is a secondary metabolite produced in the laticiferous tissue of Hevea tree. Mevalonate synthesis, which is the first step in isoprenoid biosynthesis, is catalyzed by the enzyme 3-hydroxy-3-methylglutarylcoenzyme A reductase 1 (hmgr1). We have cloned and characterized a full-length cDNA as well as genomic DNA for hmgr1 gene from an elite Indian rubber clone (RRII 105). The nucleotide sequence of the genomic clone comprises 4 exons and 3 introns, giving a total length of 2440 bp. The sequences of 42 bp 5' UTR and 69 bp of the 3' UTR were also determined. The hmgr1 cDNA contained an open reading frame of 1838 bp coding for 575 amino acid protein with a theoretical pI value of 6.6 and the calculated protein M W was 61.6 kDa. The deduced amino acid sequence showed high identity with other plant hmgr1 sequences. The amino acid sequence of the Hevea hmgr1 revealed several motifs which are highly conserved and common to the other plant species. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural and/or catalytic properties of the enzyme. Southern blot analysis of genomic DNA from Hevea probed with a genomic fragment indicated that there were at least three isoforms of hmgr in Hevea. This result reveals that hmgr1 is one of the members of a small gene family. (Northern blot analysis showed that hmgr1 mRNA transcripts were noticed in all tissues - latex, leaf, immature leaf, and seedlings), however, the abundance of transcript level was higher in latex cells. As one step towards a better understanding of the role that this enzyme plays in coordinating isoprenoid biosynthesis in plants, hmgr1 cDNA was over expressed in transgenic Arabidopsis plants. Transgenic plants were morphologically distinguishable from control wild-type plants and an increased expression level of hmgr1 mRNA was detected. These data suggest that hmgr1 gene expression is playing an important role in isoprenoid biosynthesis.

Essential Oil-Bearing Plants from Nigeria: Studies on Vernonia perrottettii (Leaf and Stem Bark), Young Leaves from Eucalyptus decaisneana and Immature Leaves of Hyptis suaveolens

The essential oils from four medicinal plants from Nigeria were obtained by hydrodistillation and analyzed by means of GC and GC/MS. The main constituents of the leaf and stem oils of Vernonia perrottettii Sch. Bip. (Asteraceae) were sesquiterpene hydrocarbons (92.1%) with abundance of β-caryophyllene (28.1% & 39.8%) and germacrene D (34.5% & 16.0%) in the leaf and stem oils, respectively. On the other hand, the leaf oil of Eucalyptus decaisneana Sm. (Myrtaceae) was dominated by the monoterpene hydrocarbons (90.6%) with α-phellandrene (31.1%), p-cymene (20.8%) and γ-terpinene (11.7%) being present in large quantities. The oil composition of immature leaf of Hyptis suaveolens L. (Lamiaceae) revealed the abundance of β-caryophyllene (22.3%), α-phellandrene (10.6%) and caryophyllene oxide (10.3%). The compositional pattern may probably lead to the delineation of a new chemotype of this species.

Construction and evaluation of a maize (Zea mays) chimaeric promoter with activity in kernel endosperm and embryo

Chimaeric promoters contain DNA sequences from different promoters. Chimaeric promoters are developed to increase the level of recombinant protein expression, to precisely control transgene activity or to combat homology-based gene silencing. Sets of chimaeric promoters, each containing different lengths of DNA from maize (Zea mays) 27zn (27 kDa gamma-zein) endosperm-specific promoter and the Glb1 (Globulin-1) embryo-specific promoter were created and tested in a transient expression assay of GFP (green fluorescent protein). Promoter fragments with the highest activity were combined to create the chimaeric promoter A27znGlb1. In the context of the chimaeric promoter, the selected Glb1 promoter fragment was necessary and sufficient to activate expression in embryo tissue and was functionally equivalent to the native Glb1 promoter. Similarly, the selected 27zn promoter fragment in the chimaeric promoter was necessary and sufficient to activate expression in endosperm tissue and was functionally equivalent to the native 27zn promoter. Maize transgenic plants containing the A27znGlb1 chimaeric promoter fused to GFP were produced to characterize this promoter in vivo. Quantitative reverse-transcriptase PCR was used to determine that the promoter was active in the embryo, endosperm, pericarp and immature leaf tissues. GFP activity in plants containing the chimaeric promoter was not significantly different in endosperm than the activity of GFP fused to the full-length 27zn promoter, nor was it different in embryo from the activity of GFP fused to the full-length Glb1 promoter. Transgene copy numbers were shown to be between 4 and 12 copies in different events.

Seasonal changes in optically assessed epidermal phenolic compounds and chlorophyll contents in leaves of sessile oak (Quercus petraea): towards signatures of phenological stage.

Seasonal patterns of dry mass invested in chlorophyll and epidermal phenolic compounds (EPhen) were investigated in vivo using optical methods, in leaves of 2-year-old oaks (Quercus petraea Matt. (Liebl.)) grown under semi-controlled conditions. The plasticity of the seasonal pattern was investigated by applying stem girdling treatment. In control young expanding leaves, leaf dry mass per area, dry mass investment in chlorophyll and abaxial EPhen content increased. In late May, at leaf maturity, these variables reached a plateau, and adaxial and abaxial EPhen contents became similar. Thereafter, as leaves aged, dry mass investment in chlorophyll gradually decreased, whereas it remained steady for EPhen. Girdling treatment impacted this seasonal pattern differently depending on the phenological stage. Treatment effects and their reversion revealed in vivo EPhen turnover. Finally, optical signatures of immature and mature leaf phenological stages with contrasting nitrogen and carbon economy were proposed, based on the relationship between the chlorophyll to EPhen ratio and the leaf nitrogen to carbon ratio.

Factors influencing somatic embryogenesis and regeneration ability in somatic tissue culture of spring and winter rye

Rye is an important crop in Northern and Eastern Europe. However, the application of various biotechnologies in rye breeding has been limited duo to its recalcitrant in tissue culture. In order to improve somatic tissue effi ciency, key factors affecting somatic embryogenesis and reproducible green plant regeneration of rye (Secale cereale L.) were evaluated and optimised. In this study, a total 27 rye genotypes including 10 spring and 17 winter genotypes were involved in the investigation. Genotype, culture medium, sugar, gel agent and auxin infl uenced somatic embryogenesis of immature embryo signifi cantly. One-two weeks cold pretreatment of young embryo enhanced somatic embryogenesis and green plant regeneration. In culture of immature embryos, infl orescences and leaf segments of the seedlings, explants signifi cantly infl uenced the culture effi ciency. Highest embryogenic callus yield resulted from rye immature embryo as explant compared to young infl orescence and leaf segment of seedling. Developmental stage of embryo played an important role in somatic embryogenesis. Late spherical coleoptile stage (embryo size 0.5–1mm in length) was optimal developmental stage of immature embryo for culture. Morphogenetic potential of embryogenic callus decreased with an increasing number of subcultures, and this ability could be maintained in vitro for a maximum of 8 months of culturing.;

Culm sucrose accumulation promotes physiological decline of mature leaves in ripening sugarcane

Photosynthetic activity in C 4 sugarcane has been suggested to be regulated by the demand for photoassimilate from sink tissues, including culm storage of sucrose and other components of the plant (e.g. roots and shoot primordia). This study examined the extent to which sink-demand influences leaf photosynthetic activity and controls leaf turnover in field-grown sugarcane. To increase sink-demand on selected leaves, plants were defoliated apart from the immature leaf before the first fully expanded leaf (2nd leaf) and the mature 8th leaf. Changes in leaf gas exchange and fluorescence characteristics were recorded for both leaves over a 28 d period. Variations in leaf and culm sucrose and hexose concentrations and allocation of 14C-labelled photosynthate were also measured. A decrease in culm internodal sucrose concentrations in partially defoliated plants was associated with significant increases in assimilation ( A) and electron transport rates (ETR) for both leaves 2 and 8. Conversely, accumulation of sucrose in the culms of control plants was related to a decline in photosynthetic rates in leaf 8 during the treatment period. Leaves 2 and 8 of defoliated plants (27 d) were characterised by an increase in partitioning of 14C to mature and immature internodes, respectively, compared to control plants. In addition, hexose levels in leaves of defoliated plants decreased significantly (36% and 48% decrease in leaves 2 and 8, respectively) compared to corresponding leaves of untreated controls over the duration of the experiment, indicating that the signaling mechanisms regulating the decline in leaf photosynthetic activity are likely hexose-mediated. It was concluded that leaf physiological ageing in sugarcane was promoted by sucrose accumulation during culm maturation as a consequence of decreased sink-demand for photosynthate.

Improved germination of somatic embryos and plant recovery of European chestnut

For the mass production of chestnut trees with selected, hybrid, or genetically engineered genotypes, one potentially desirable propagation strategy is based on somatic embryogenesis. Although methods exist for the initiation of embryogenic cultures of Castanea sativa from immature zygotic embryos or leaf explants, the embryos produced have had low rates of conversion into plantlets. This study explored the possible benefits for somatic embryos that have already undergone maturation and cold treatments, of (a) partial slow or fast desiccation, and (b) of the addition of plant growth regulators or glutamine to the germination medium. Germination response was evaluated in terms of both conversions to plantlets and through embryos developing only shoots (shoot germination) that could be rooted following the micropropagation protocols developed for chestnut. Two or 3 wk slow desiccation in sealed empty Petri dishes resulted in a slight reduction in water content that nevertheless increased total potential plant recovery, shoot length, and the number of leaves per plantlet. However, best results were achieved by 2 h fast drying in a laminar flow hood, which reduced embryo moisture content to 57–58% and enhanced the potential plant recovery and quality of regenerated plantlets. Plant yield was also promoted by addition of 0.44 μM benzyladenine and 200–438 mg/l of glutamine to the germination medium, and plantlet quality (as evidenced by root, shoot, and leaf growth) by the further addition of 0.49 μM indole-3-butyric acid.

Direct organogenesis from leaf explants of Stevia rebaudiana and cultivation in bioreactor

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 µM of N 6-benzylaminopurine and kinetin ranging from 4.65 to 6.98 µM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm−3 sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 µM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm−3 sucrose resulted in a high biomass yield of 50.68 g dm−3 (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 µM, 15 g dm−3 sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.

Advanced data-mining strategies for the analysis of direct-infusion ion trap mass spectrometry data from the association of perennial ryegrass with its endophytic fungus, Neotyphodium lolii.

Direct-infusion mass spectrometry (MS) was applied to study the metabolic effects of the symbiosis between the endophytic fungus Neotyphodium lolii and its host perennial ryegrass (Lolium perenne) in three different tissues (immature leaf, blade, and sheath). Unbiased direct-infusion MS using a linear ion trap mass spectrometer allowed metabolic effects to be determined free of any preconceptions and in a high-throughput fashion. Not only the full MS(1) mass spectra (range 150-1,000 mass-to-charge ratio) were obtained but also MS(2) and MS(3) product ion spectra were collected on the most intense MS(1) ions as described previously (Koulman et al., 2007b). We developed a novel computational methodology to take advantage of the MS(2) product ion spectra collected. Several heterogeneous MS(1) bins (different MS(2) spectra from the same nominal MS(1)) were identified with this method. Exploratory data analysis approaches were also developed to investigate how the metabolome differs in perennial ryegrass infected with N. lolii in comparison to uninfected perennial ryegrass. As well as some known fungal metabolites like peramine and mannitol, several novel metabolites involved in the symbiosis, including putative cyclic oligopeptides, were identified. Correlation network analysis revealed a group of structurally related oligosaccharides, which differed significantly in concentration in perennial ryegrass sheaths due to endophyte infection. This study demonstrates the potential of the combination of unbiased metabolite profiling using ion trap MS and advanced data-mining strategies for discovering unexpected perturbations of the metabolome, and generating new scientific questions for more detailed investigations in the future.

First record of circinate vernation in bennettitalean foliage

The Bennettitales are Mesozoic gymnosperms with entire or pinnate leaves and complex reproductive structures. Although many forms superficially resemble cycads, particularly with regard to foliage macromorphology, bennettitaleans and cycads are not believed to be closely related at this time. Cuticular analysis of bennettitalean foliage from the Carnian (Upper Triassic) of Austria reveals that Pterophyllum filicoides displays circinate vernation in the form of inwardly coiled leaf segments, which are strikingly similar to immature leaf segments seen in extant Cycas species. This discovery represents the first record of circinate vernation in the Bennettitales and may suggest that bennettitalean leaves resemble those of cycads not only with regard to macromorphology but also development.

Nitrogen Uptake and Allocation in Sweet Viburnum During a Root Growth Flush

ABSTRACT Most woody ornamentals exhibit episodic growth flushes and nitrogen (N) uptake has also been demonstrated to be seasonal. However, there is little information on N uptake in relationship to plant growth cycle. In this study, N uptake and allocation of sweet viburnum during periods of low and high root elongation rates were studied. Plants were fertilized with ammonium nitrate (15NH4 15NO3) and after 6 d N absorption was determined. Significantly more N was absorbed by plants with low root elongation rate compared with plants with high root elongation rate. About 70% of the N absorbed by plants with low root elongation rate was allocated to the mature leaves compared to 35% on plants with high root elongation rate. It was evident that root growth activity influenced N absorption and allocation. Although only a small amount of the N absorbed by plants with low root elongation rates was allocated to the immature leaves, significantly more N was allocated to the immature leaves by plants with high root elongation rates. It is possible that the N necessary to support immature leaf growth, when root elongation rate is low, is provided by mobilization from other parts, possibly mature leaves.

Temporal and Spatial Variation of Terpenoids in Eastern Hemlock (Tsuga canadensis) in Relation to Feeding by Adelges tsugae

The terpenoid content of eastern hemlock (Tsuga canadensis) foliage was measured over an annual cycle of development from bud opening, shoot elongation, shoot maturation, to bud-break at the start of the next growing season. The objective was to determine if variation in terpenoid composition is linked with spatial and temporal feeding preferences of the hemlock woolly adelgid (HWA; Adelges tsugae). The HWA has two periods of feeding over the course of 1 yr spanning two complete generations. There are two periods of feeding separated by a nonfeeding period where the adelgid estivates. HWA prefers to feed on mature, rather than young, expanding tissue. Feeding occurs in the leaf cushion at the base of the needle. The needle is the only tissue in hemlock with resin canals that store terpenoids. The needle and leaf cushion of both the current and previous years' growth were analyzed separately over a 1-yr period to examine the variation of terpenoid composition in space and time. Terpenoids were quantified by using headspace solid-phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS). New growth needles and leaf cushions do not resemble the previous year's growth either visually or in chemical composition until October/November, when the adelgid breaks estivation and begins feeding. Nearly all of the 23 terpenoids present exceeding 0.1% varied significantly either temporally or spatially, usually with complex interactions. Ordination and factor analysis revealed that terpenoids are less variable in mature leaf cushions than in young tissue. By entering a nonfeeding diapause during the late spring and summer, HWA avoids the unstable, variable levels of terpenoids in the immature leaf cushion and needles.

Plant Regeneration from Shoot Apex-Derived Calluses of Marigold (Tagetes erecta L.)

Marigold (Tagetes erecta L.) is an annual plant, native of Mexico, that belongs to the Asteraceae (Compositae) family. Its flowers are commercially cultivated, harvested, and processed on an important industrial scale as a source of high-value pigments of the carotenoid family. Marigold flowers are the most concentrated common source of carotenoids, with lutein accounting for 85% of their total carotenoid content (Barzana et al., 2002). The meal made from the flowers is used as a supplement in the poultry industry (for coloring skin and egg yolk). Industrial sources have stated that the increasing awareness of toxicity of synthetic products has paved the way for the use of pigments from natural sources such as marigold (DelgadoVargas et al., 2000). Bioactive extracts of different Tagetes tissues also exhibit nematocidal, fungicidal, and insecticidal activity (Vanegas et al., 2002). The development of suitable methods for in vitro regeneration is one of the main prerequisites for genetic improvement using biotechnologic means. Marigold plants have been in vitro regenerated from different sources, including immature unpollinated disc florets (Kothari and Chandra, 1984), leaf callus and suspension cultures (albeit with a loss of morphogenic potential as a result of subculturing) (Kothari and Chandra, 1986), hypocotyls (Belarmino et al., 1992), shoot tip proliferation from adult plants (Misra and Datta, 1999), and leaf segments (Misra and Datta, 2001; Vanegas et al., 2002). It is noteworthy to point out that, to our knowledge, there are no reports on regeneration through indirect organogenesis from shoot apex-derived calluses in Tagetes erecta. Genetic transformation, either by particle bombardment or Agrobacterium infection, is a single cell event. Agrobacterium transformation requires tissue wounding previous to bacterial exposure. This may limit the number of transformed plants to those that regenerate only from the wounded area. Because methods using callus tissue do not require wounding, we believe that this protocol would increase the chances to obtain transformed plants after exposure of calluses by immersion to a bacterial suspension and further plantlet regeneration. We report a consistent and reproducible protocol for the regeneration of plantlets from shoot apex-derived calluses of marigold without any loss of morphogenic potential resulting from subculturing. This protocol is based on the use of a modified Murashige and Skoog medium (MSB; Robert et al., 1987) containing different concentrations of benzyl adenine (BA) and indoleacetic acid (IAA). The highest mean number of shoots per explant was obtained with 70 mM BA and 10 mM IAA 10 weeks after the culture’s induction. When these shoots were excised and subcultured on MSB medium without plant growth regulators, they formed extensive root systems in vitro. Plantlets transferred to ex vitro conditions developed normally.

Effect of medium composition and explant size on embryogenic calli formation of cassava (Manihot esculenta Crantz) local genotypes

Cassava (Manihot esculenta Crantz) is an important tropical crop species used for human consumption, feed and raw material for various industries. Genetic transformation through embryogenic tissues is known as an effective method for cassava genetic improvements. Objective of this study was to obtain a suitable medium and length of explants to induce embryogenic callus on friable embryogenic callus (FEC) as a target for genetic transformation. Immature leaf lobes (1-3 mm, 3-5 mm and larger than 5 mm in length) of local genotypes of cassava (Adira 4. Menti, Iding, Gebang, Rawi and Timtim-29) cultured in vitro were used as explants. The explants were incubated for 2 and 4 weeks on MS (Murashige-Skoog) or GD (Greshooff & Doy) semi solid medium containing 10 mg/L picloram, 6 mg/L NAA supplemented with 4% sucrose and 4 μM CuSO4. Results showed that the highest percentage (100%) of embryogenic calli formation for 4 weeks obtained by culturing Iding of 3-5 mm length on GD semi solid medium, whereas the lowest (33%) one obtained by incubation 5 mmleaf lobe of Timtim-29 on the same medium. The most suitable medium for callus induction was GD, whereas the optimum length of explants was 5 mm or larger. Further study needs to be done to obtain friable embryogenic calli (FEC) by employing different concentration of picloram and varying other critical factors.© 2006 Jurusan Biologi FMIPA UNS SurakartaKey words: Manihot esculenta Crantz, cassava, medium, embryogenic, callus

First Report of Lethal Yellowing Disease of Coconut Palms Caused by Phytoplasma on Nevis Island.

Coconuts (Cocos nucifera) are an important small-holder's crop in many tropical countries and are used to enhance esthetics of coastal areas. Lethal yellowing (LY) is the single most important plant disease affecting the coconut industry in Jamaica. It affects many palm species in Jamaica, Florida, and Guatemala. This coconut disease was first recorded in Grand Cayman Island in 1834 and Jamaica in 1884. Symptoms of LY disease include premature nut fall, necrosis of the inflorescence, yellowing of the leaves, and defoliation. Thirty-eight coconut palms displaying symptoms indicative of LY disease were sampled in April, 2005 at several locations in Nevis. Immature leaf tissues (leaf bases adjacent to the apical meristem) and nondestructive (boring with a bit and braces) samples were collected from disease and healthy control coconut trees. DNA was extracted (2). The first round of polymerase chain reaction (PCR) with phytoplasma universal primer pair P1/P7 (1,3) resulted in an rDNA fragment of 1.8 kb, and a subsequent nested PCR using LY16-23Sr/LY16Sf primers yielded an amplicon of 1.74 kb (4). Purified product was cloned for sequencing. Sequences obtained were analyzed with Vector NTI Software Suite. The sequence of LYN 18-3 was entered in Genbank and Accession No. DQ378279 was assigned. LYN 18-3 has approximately 99% homology with LY Phytoplasma U18747 from Florida (Manila palm [Veitchia merrillii]). The disease-associated phytoplasma was reliably detected in immature tissues and trunk phloem at the onset of foliar symptoms in palms by PCR. On the basis of the results obtained from this study, it is clear that LY phytoplasma (16SrIV group) was found in the samples collected from Nevis. To our knowledge, this is the first report on lethal yellowing disease in Nevis.