Indonesian Journal of Agricultural Science
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THE POTENTIAL USE OF SSR MARKERS TO SUPPORT THE MORPHOLOGICAL IDENTIFICATION OF INDONESIAN MUNGBEAN VARIETIES
Mungbean varieties were mainly characterized based on morphological traits. Molecular genetic approach is expected to help the breeder in identification of mungbean varieties in more detail and to protect intellectual property right. This study aimed to identify Indonesian mungbean varieties based on DNA fingerprint profile using a marker set to support morphological characters. A total of 22 Indonesian mungbean accessions were characterized based on 21 morphological traits and 55 simple sequence repeats (SSRs) primers. Of the total 22 mungbean varieties used in the present study, 16 varieties were improved varieties and remaining six varieties were local varieties originated from Java, Nusa Tenggara and Sulawesi collected in GeneBank of ICABIOGRAD. The results showed that the 21 morphological characters were not sufficient to differentiate 22 mungbean varieties, while SSR analysis revealed that eight multi-alleles markers and high polymorphic information content (PIC) values have been successfully selected for varietal identification. The selected markers enabled to differentiate each mungbean variety according to their genetic marker with the lowest distance of 0.125, demonstrating the robustness of the selected marker set as a tool to identify a specific DNA fingerprint profile as a varietal identity (ID). The genetic identity of a variety was shown by digital barcoding which represented a series of alleles produced by corresponding markers. The DNA fingerprint profile of each variety would be beneficial as reference identities of a mungbean variety.
EFFECT OF GAMMA IRRADIATION ON THE GROWTH AND DEVELOPMENT OF SAGO PALM (Metroxylon sagu Rottb.) CALLI
The application of gamma irradiation on plant materials may increase the genetic variation of the offspring with useful traits. The experiment was conducted to determine the effect of irradiation dosage of gamma ray on growth and development of sago palm (Metroxylon sagu) calli. Friable calli of sago palm derived from suspension culture were used as a material source. The primary calli were initiated from apical meristematic tissues of sago palm suckers of Alitir variety from Merauke, Papua. The treatments used were dosage of gamma ray irradiation at 0, 5, 10, 15, 20 and 25 Gy. The treated calli were then subcultured on modified Murashige and Skoog (MMS) solid medium containing 3% sucrose and 0.1% activated charcoal and added with 1 mg l-1 2,4-D and 0.1 mg l-1 kinetin. The results showed that at all irradiation dosages, calli biomass increased significantly. The highest proliferation of calli biomass of 5.33 folds from the initial culture after 4 weeks was achieved at gamma irradiation of 25 Gy, whereas the lowest proliferation of calli biomass of 3.4 folds was achieved at control. The best development of embryogenic calli was obtained at 10 Gy that produced 100% somatic embryos, whereas the lowest somatic embryo formation at 0% was obtained at 0 and 25 Gy after one subculture. High response of somatic embryo induction to gamma irradiation at 10 Gy may increase production of somatic embryos. These results can be used in in vitro breeding of sago palm via mutagenesis to create new elite varieties
DEVELOPMENT OF EST-SSR MARKERS TO ASSESS GENETIC DIVERSITY OF BROCCOLI AND ITS RELATED SPECIES
Development of Expressed Sequence Tag-Simple Sequence Repeat (EST-SSR) markers derived from public database is known to be more efficient, faster and low cost. The objective of this study was to generate a new set of EST-SSR markers for broccoli and its related species and their usefulness for assessing their genetic diversity. A total of 202 Brassica oleracea ESTs were retrieved from NCBI and then assembled into 172 unigenes by means of CAP3 program. Identification of SSRs was carried out using web-based tool, RepeatMasker software. Afterwards, EST-SSR markers were developed using Primer3 program. Among the identified SSRs, trinucleotide repeats were the most common repeat types, which accounted for about 50%. A total of eight primer pairs were successfully designed and yielded amplification products. Among them, five markers were polymorphic and displayed a total of 30 alleles with an average number of six alleles per locus. The polymorphic markers were subsequently used for analyzing genetic diversity of 36 B. oleracea cultivars including 22 broccoli, five cauliflower and nine kohlrabi cultivars based on genetic similarity matrix as implemented in NTSYS program. At similarity coefficient of 61%, a UPGMA clustering dendrogram effectively separated 36 genotypes into three main groups, where 30 out of 36 genotypes were clearly discriminated. The result obtained in the present study would help breeders in selecting parental lines for crossing. Moreover, the novel EST-SSR markers developed in the study could be a valuable tool for differentiating cultivars of broccoli and related species
BIOMASS PRODUCTION AND FORMULATION OF Bacillus subtilis FOR BIOLOGICAL CONTROL
Bacillus subtilis is a widespread bacterium found in soil, water, and air. It controls the growth of certain harmful bacteria and fungi, presumably by competing for nutrients, growth sites on plants, and by directly colonizing and attaching to fungal pathogens. When applied to seeds, it colonizes the developing root system of the plants and continues to live on the root system and provides protection throughout the growing season. The study on biomass production and formulation of B. subtilis for biological control was conducted in the laboratory of Department of Plant Pathology, College of Agriculture, University of the Philippines Los Baños (UPLB-CA), College, Laguna from May to July 2005. The objective of the study was to determine the optimum pH and a good carbon source for biomass production of B. subtilis and to develop a seed treatment formulation of B. subtilis as biological control agent. Results showed that the optimum pH for growth of B. subtilis was pH 6 (1.85 x 109 cfu/ml). In laboratory tests for biomass production using cassava flour, corn flour, rice flour, and brown sugar as carbon sources, it grew best in brown sugar plus yeast extract medium (6.8 x 108 cfu ml-1 in sterile distilled water and 7.8 x 108 cfu ml-1 in coconut water). In test for bacterial biomass carriers, talc proved to be the best in terms of number of bacteria recovered from the seeds (3.98 x 105 cfu seed-1)
MORPHOLOGICAL CHANGES DURING THE DEVELOPMENT OF SOMATIC EMBRYOS OF SAGO (Metroxylon sagu Rottb.)
Development of somatic embryos of sago (Metroxylon sagu Rottb.) on agar-solidified medium are highly varied producing heterogeneous seedlings. Understanding of this phenomenon may help in improving the cultural procedures and conditions of sagosomatic embryogenesis to obtain uniform seedlings in a large scale. This experiment was conducted at the laboratory for plant cell culture and micropropagation, Indonesian Biotechnology Research Institute for Estate Crops from January to March 2006 to examine morphological changes i.e. color and development stages of sago during their somatic embryo development on an agar-solidified medium. Twenty single globular somatic embryos of sago with specific color (yellowish, greenish, and reddish) were cultured in a Petri dish supplemented with a solid medium. The medium was a micronutrients-modified MS (MMS) with half strength of macronutrients containing 0.01 mg l-1 ABA, 2 mg l-1 kinetin, 20 g l-1 sucrose, 0.5 g l-1 activated charcoal, and 2 g l-1 gelrite. Parameter observed was the percentage of embryo’s number based on color and developmental stage. The result showed that at the end of 6-week culture passage, most originally greenish (80.8%) and reddish (95.8%) embryos remained unchanged in their colors, whereas almost half of the originally yellowish embryos turned to greenish and only 30%remained yellowish. At the same time, single globular embryos have changed gradually into the next developmental stages, although not all of the embryos were germinated. The initial color of embryo affected the rate of the developmental stage changes. Yellowish and greenish globular embryos developed more rapidly into cotyledon or germinant stages at 58% and 55% respectively, in 6 weeks than the reddish ones (41%). Therefore, the yellowish and greenish embryos are the best sources of material for in vitro mass propagation and synthetic seed production of sago
SELECTION RESPONSE FOR INCREASED GRAIN YIELD IN TWO HIGH OIL MAIZE SYNTHETICS
Selection for increased oil level in maize showed the increase was associated with decrease in starch concentration, kernel weight, and grain yield. The study was conducted with the objectives: (1) to evaluate response to six cycles for increased grain yield in the high oil maize Alexho Elite (AE: 60-90 g kg-1 oil concentration) and Ultra High Oil (UHO: 100-140 g kg-1 oil concentration) using inbred tester B73; (2) to measure responses to selection for increased grain yield with changes in yield components; and (3) to determine a suitable tester. Previously the two synthetics had been selected for oil concentration. After six cycles, the six genotypes i.e. AE C0, AE C3, AE C6, UHO C0, UHO C3, and UHO C6 were testcrossed to B73, LH185, and LH202 inbreds (40 g kg-1 oil concentration) to a total of 18 testcrosses. Two field experiments were used to evaluate selection in AE and UHO testcrosses. The study showed selection using inbred tester B73 in AE and UHO was effective in increasing grain yield of AE testcrosses without changing (i.e. decreasing) oil and protein concentrations. AE testcrosses produced higher grain yield and greater selection response for grain yield than UHO testcrosses. LH185 was best for grain yield in AE and UHO testcrosses. Increase in grain yield in most of the testcrosses was associated with increases in starch concentration, kernel weight, kernel number, and grain weight
EFFECT OF PERMEABLE VESSEL CLOSURE AND GELLING AGENT ON REDUCTION OF HYPERHYDRICITY IN IN VITRO CULTURE OF CARNATION
Hyperhydricity, an abnormal morphological appearance and physiologicalfunction, is an important problem in carnation tissue culture. The problem causes premature flowering, high occurrence of abnormal shoots, difficulty in transferring hyperhydric plantlets to soil, and low survival rate of plantlets. High relative humidity and the water potential are considered as the key factors involved in the abnormality. Furthermore, permeable culture vessel and gelling agent were assured to be high potential treatment to eliminate it. Objective of this research was to reduce hyperhydricity in regenerants of carnation using different permeable vessel closures and gelling agents and to assess the multiplication and acclimatization abilities of recovered shoots. Experiment was arranged in randomized complete block design with four replications. First factor was different types of closure, i.e. cotton wool, plastic wrap, parafilm and aluminium foil, while second one was gelling agents, i.e. bacto agar, phytagel, swallow agar, and Type 900 agar. The recovered shoots were then multiplied, rooted, and acclimatized. The results showed that hyperhydricity was successfully reduced by applying permeable closure (cotton wool and plastic wrap) in combination with Type 900 agar. The combination of plastic wrap and Type 900 agar was the most appropriate treatment in reducing hyperhydricity and producing good quality shoots. The treatment reduced the problem down to 23% of total condition of hyperhydricity (100%) and increased leaf chlorophyll content from 0.0883 to 0.1288 mg mg-1. The plastic wrap was easily applied and cheaper material compared to cotton wool. The recovered shoots were able to produce 1-3 healthy axillary shoots and easily rooted on half-strength MS. The recovered plantlets were simply acclimatized with survival rate up to 100% on kossas peat + soil (1:1, v/v) and flowered 4-5 months after acclimatization with decreasing in number and size of flower
MOSAIC DISEASE OF MAIZE CAUSED BY SUGARCANE MOSAIC POTYVIRUS IN SULAWESI
Mosaic disease of maize and grasses is commonly found in Sulawesi. The symptoms resemble the common mosaic symptoms of virus infection, but the pathogen has not been identified. The objective of this study was to identify the causal agent of the mosaic disease of maize and grasses in Sulawesi. Transmissions of the virus were studied by mechanical inoculation and the insect vector aphid. Serological study was done by using enzyme linked immunosorbent assay (ELISA). Results of mechanical inoculation showed that the disease was caused by a virus which was transmitted from diseased maize and grasses to healthy sweet corn seedlings. The disease was also transmitted by aphid (Rhopalosiphum maidis). Serological study indicated that the virus was closely related to the sugarcane mosaic virus (SCMV). Based on these results, it can be concluded that the maize and grass mosaic disease was caused by SCMV
BIOLOGICAL CONTROL OF BANDED LEAF AND SHEATH BLIGHT DISEASE (Rhizoctonia solani KUHN) IN CORN WITH FORMULATED Bacillus subtilis BR23
Rhizoctonia solani Kuhn. causing banded leaf and sheath blight diseases is one of the important fungi of corn world wide. The fungus is commonly controlled by using fungicide because no resistant variety available. The objective of the study was to develop a seed treatment formulation of the selected Bacillus subtilis to control R. solani in corn. The study was conducted in the Department of Plant Pathology, College of Agriculture, University of the Philippines Los Bañòs, College, Laguna from May 2004 to August 2005, using sweet corn var. IPB Supersweet as test plant. Corn seeds were surface sterilized for 10 minutes in 1% sodium hypochlorite solution and 5% ethanol, washed thrice with sterile distilled water and air-dried. The seeds were coated with formulated B. subtilis BR23 and used for several experiments, such as evaluation for their germination and growth in the laboratory, effectively on R. solani in the baked and nonbaked field soil under greenhouse condition, and in the microplots artificially infested with R. solani. The treatment was compared with other standard seed treatment of synthetic fungicides such as captan (10 g per kg seeds) and metalaxyl (10 g per kg seeds). The experiments were designed in a completely random design with three replications. Parameters observed were seed germination, plant height, disease scores, and plant yield. Laboratory formulated B. subtilis BR23 used as seed treatment had no detrimental effects on seed germination and seedling vigor. In microplots artificially infested with a selected highly virulent R. solani, seed treatment with the same formulation increased grain yield by 27% compared to that of the control captan seed treatment with 14.4%. The studies showed the potential of B. subtilis BR23 for commercialization as a seed treatment for the control of banded leaf and sheath blight disease (R. solani) in corn
IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS ON CATTLE BREEDS IN INDONESIA USING BOVINE 50K
Single nucleotide polymorphisms (SNPs) abundant in bovine genome influence genetic variation in biological mechanism. The study aimed to identify SNPs on Indonesian cattle breeds and analyze their genetic diversity using Bovine 50K SNP chip. Twenty eight "Ongole Grade" (OG) beef cattle and 20 "Holstein Friesian" (HF) dairy cattle were used for the Infinium II assay test. This assay included amplification of genomic DNA, fragmenta-tion, precipitation, resuspension, hybridization, processing bead chip for single-base extension, and imaging at iScan. Data and clusters were analyzed using GenomeStudio software. The Bovine 50K SNP chip containing 54,609 SNPs was observed spanning all chromosomes of bovine genome. Genotyping for the total SNPs was successfull based on Call Rate, GeneCall and GeneTrain scores. Most SNP markers had alleles that shared among the individuals or breeds, or had specific alleles at distinctive frequencies. Minor allele frequency (MAF) spreads equally with intervals of 0-0.5. The breeds of OG and HF tended to be separated in different clusters without considering their genetic history and twin or normal. This result suggests that most individuals are closely related to one another, regardless of the same breed. Some genes identified on chromosomes 3, 4, 5, 7, 13, 17 and 18 were located in the loci/regions that contained SNPs with specific alleles of either HF or OG breed. These SNPs were more powerful for differentiation of beef cattle and dairy cattle than among individuals in the same breed. These SNP variations and genetic relatedness among individuals and breeds serve basic information for cattle breeding in Indonesia