Preliminary Genetic Manipulation Studies On Muskmelon (Cucumis Melo L.) Using Protoplast Technology

In this study, parallel attempts have been made to initiate somatic embryos from two different explants on various hormone combinations and the results obtained were then applied to establish plant regeneration system for protoplastderived macrocalli. Simultaneously, optimisation of plant transformation system using electroporation techniques (direct uptake) were carried out to facilitate future research on the genetic improvement of muskmelon.Somatic embryos were successfully initiated from Birdie radicle region and Hami melon cotyledon using MS medium containing 1.0 mg/L 2,4-0, 2.0 mg/L NAA and 0.1 mg/L BAP and medium gave 2.0 mg/L NAA and 0. 1 mg/L BAP, respectively. The somatic embryos developed into normal plantlets after subculturing onto hormone-free M S media solidified with 0.4% (w/v) Gelrite®.Both Birdie and Hami melon protoplasts were successfully isolated using cotyledons of seven day in vitro seedlings incubated in different enzyme concentrations. Birdie melon explants were incubated in 0.05% (w/v) pectolyase Y23 and 1.0% (w/v) cellulase RS while Hami melon explants were incubated in 0.025% (w/v) Pectolyase Y23 and 1 .0% (w/v) Cellulase RS for 1.5 hours . . Subsequently, Birdie protoplast which were cultured onto semi-solid (0.09% Gelrite®) medium containing 1/2 strength MS supplemented with 0.1 mg/L NAA and 0.5 mg/L BAP gave the highest number of macrocolonies formation (60.96 number of colonies/field) after 30 days in culture, while Hami melon protoplast gave highest number of macrocolonies formation (30.48 number of colonies/field) in liquid culture containing 1/2 strength MS supplemented with 0.5 mg/L NAA and 1 .0 mg/L zeatin. Further experiments were conducted to establish the regeneration system for the macrocalli with addition of auxin and cytokinin, sucrose, growth regulators and amino acids. The results obtained showed that the macrocalli had potential to regenerate into plantlets on two types of medium (0.5 mg/L and 2.0 mg/L BAP). However. further studies must be performed to obtain a more suitable regeneration media from protoplast-derived macrocalli.In this study, genetic transformation of muskmelon protoplasts were carried out using electroporation system. Using pBI22 1 as a standard, various parameters for electroporation were optimised and the GUS activities were assayed. Pulse strength of 3.0 kV/cm, single pulse, 10.0 us pulse length, 25.0 ug/ml plasmid DNA and 15 hours incubation time were optimum for efficient DNA uptake and transient assay. Treatment of the protoplast with external stimulus such as UV irradiation in the presence of different plasmid constructs showed that the plasmid pYCN01 and pYCN02 containing different length of PSPAL promoter fragment gave higher GUS activity compared to the CaMV 35S promoter. The results indicate that ultra violet irradiation of the protoplasts was at least partly responsible for the activation of PAL promoter

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing α1-3 fucose and/or β1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%)

Application of cucumber green mottle mosaic virus vector as peptide presentation system

Recently plant viruses have been exploited as an alternative production method for pharmaceutically important peptides [1-9]. Antigenic peptides that were produced through this approach have been shown to be immunogenic

63 The Isolation and Expression Analysis of a Class I Chitinase from Developing Winged Bean Seed (Psophocarpus tetragonolubus)

Abstract. Chitinase catalyses the hydrolysis of β-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin and is involved in the inducible defenses of plants. The aim of this research was to isolate and clone chitinase cDNAs from the seed of winged bean. Chitinase gene fragments were isolated from a winged bean seed cDNA library using two sets of degenerate primers corresponding to the conserved regions of chitinase class I and IV. The poly A + mRNA was reversed transcribed and further amplified using RT-PCR. A 1.1 Kb fragment was selected, cloned and sequenced. A nucleotide sequence comparison identified the fragment as a Class I basic chitinase cDNA; this fragment was subsequently used as a probe to screen for a full length transcript from the cDNA library. Library screening resulted in the isolation of a 1324 bp clone designated CHRZP; encoding a polypeptide of 289 amino acids containing the diagnostic N-terminal cysteine-rich domain of class 1 chitinases. CHRZP showed 47 % similarity to a chitinase sequence from Rice and to another class I winged bean chitinase (ChitiWb1) at the amino acid level. RNA blot hybridization revealed that CHRZP mRNA accumulates to the highest level in leaves followed by tubers and pods. Southern hybridization analysis indicated that this gene is likely to be present as a single copy in the winged bean genome