Callus induction, regeneration and transformation of sugarcane (Saccharum officinarum L.) with chitinase gene using particle bombardment

This study was carried out to optimize the conditions for introducing a chitinase gene into the sugarcane cv. Phil 66-07 calli by particle bombardment. Young leaves were cultured on the modified Murashige and Skoog (MS) medium supplemented with varied concentrations of 2,4- dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA), yeast extract and coconut water (CW). The maximum percentage of callus induction was obtained from the MS medium supplemented with 3 mg/l 2,4-D and 15% (v/v) coconut water. Multiple shoots were achieved by transferring sugarcane calli to the MS medium amended with 1 mg/l benzyl aminopurine (BA) and 0.5 mg/l indole-3-butyric acid (IBA). Additional experiments were also performed to determine the effect of antibiotics on regeneration of sugarcane. It was found that growth of sugarcane calli and plantlets were completely inhibited by hygromycin concentrations of 25 and 50 mg/l, respectively. The genetic transformation was achieved via particle bombardment with an optimal helium pressure of 900 psi and the stopping screen set at 9 cm. Sugarcane was transformed with either GUS or a chitinase gene and a gene for hygromycin selection. GUS transformed calli were produced to optimize the particle bombardment protocol. Using the optimized protocol, the chitinase gene was transformed into sugarcane and polymerase chain reaction (PCR) was used to verify the integration of a chitinase gene, 35S promoter and nitric oxide synthase (NOS) terminator in transgenic sugarcane.Key words: Sugarcane, genetic transformation, particle bombardment, chitinase gene

Repeated-batch ethanol fermentation from sweet sorghum juice by free cells of Saccharomyces cerevisiae NP 01

Low-cost inoculum preparation (IP) media for ethanol production were developed. It was found that sweet sorghum juice (SSJ) containing 100 g l-1 of total sugar without nutrient supplement could be used as the low-cost IP medium instead of the typical IP medium or yeast extract malt extract (YM) medium. Ethanol production from the SSJ (total soluble solids of 24 °Bx) by the inoculum of Saccharomyces cerevisiae NP 01 in batch and repeated-batch fermentations was then investigated. The fermentations were carried out under static condition in 500 ml air-locked Erlenmeyer flasks at 30°C and the initial yeast cell concentration was 1×108 cells ml-1. In the batch fermentation, the concentration (P), productivity (Qp) and yield (Yp/s) of ethanol were 110.09 ± 0.81 g l-1, 2.29 ± 0.01 g l-1 h-1 and 0.51 ± 0.02, respectively. In the repeated-batch fermentation, the yeasts could be used at least eight successive batches without a marked decrease in ethanol production. The repeated-batch fermentation with fill and drain volume at 75% of the working volume gave higher ethanol production efficiencies than those at 50% of the working volume in terms of total ethanol production rate (g h-1). The average P, Qp and Yp/s of the eight successive batches at 75% fill and drain volume in a 2 L bioreactor at the agitation rate of 100 rev min-1 were 93.30 ± 9.44 g l-1, 1.21 ± 0.43 g l-1 h-1 and 0.48 ± 0.03, respectively.Key words: Repeated-batch, ethanol fermentation, low-cost nutrient, sweet sorghum juice, Saccharomyces cerevisiae

Chemical and molecular characterization of fragrance in Black Glutinous Rice from Lao PDR

Fifty three accessions of lowland Black Glutinous Rice (BGR) and three white rice varieties from Lao PDR were evaluated for variations in 2-acetyl-1-pyrroline (2-AP), a main aroma compound present in fragrant rice and a 257 bp genomic fragment associated with a gene encoding 2-AP. The objective of this study was to characterize the variation in aromatic characters of Lao BGR by using chemical and molecular techniques. Thirty six out of the 56 accessions had aromatic properties, containing 2-acetyl-1-pyrroline (2-AP). The concentrations of 2-AP in unpolished samples were about 50% higher than those of the polished samples. The concentrations of 2-AP in BGR accessions were lower than those of white aromatic rice varieties in which 2-AP was highest in Kai Noi Leuang (0.688 ppm). Seventeen accessions and two of white rice (Kai Noi Leuang and TDK5) had a common 355 bp allele corresponding to that found in Calrose, a non-fragrant variety. A 257 bp allele was found in the 36 varieties of BGR and a local aromatic white rice variety, Hom Nang Nouan, sharing a common allele as in the fragrant variety KDML 105. Surprisingly, Kai Noi Leuang, a popular Lao local aromatic rice variety with the highest 2-AP concentration did not possess a 257 bp fragment which has been identified previously as a fragrant allele. This is the first study of the chemical and molecular aromatic properties of lowland BGR varieties of the Lao PDR and the data obtained will be valuable in screening other germplasm and in planning a breeding program in the country geared towards fragrant rices with superior agronomic properties