Agus Purwito

Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja

Somatic hybrid plants were produced after protoplast electrofusion between a dihaploid potato, cv. BF15, and a wild tuber-bearing relative, Solanum phureja, with a view to transferring bacterial wilt resistance into potato lines. A total of ten putative hybrids were selected. DNA analysis using flow cytometry revealed that six were tetraploids, two mixoploids, one amphiploid and one octoploid. In the greenhouse, the putative hybrids exhibited strong vigor and were morphologically intermediate, including leaf form, flowers and tuber characteristics. The hybrid nature of the ten selected plants was confirmed by examining isoenzyme patterns for esterases and peroxidases, and analysis of RAPD and SSR markers. Analysis of chloroplast genome revealed that eight hybrids possessed chloroplast (ct) DNA of the wild species, S. phureja, and only two contained Solanum tuberosum ct type. Six hybrid clones, including five tetraploids and one amphiploid, were evaluated for resistance to bacterial wilt by using race 1 and race 3 strains of Ralstonia solanacearum, originating from Reunion Island. Inoculations were performed by an in vitro root dipping method. The cultivated potato was susceptible to both bacterial strains tested. All somatic hybrids except two were tolerant to race 1 strain, and susceptible to race 3 strain. Interestingly, the amphiploid hybrid clone showed a good tolerance to both strains.

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.

Screening of pepper accessions for resistance against two thrips species (Frankliniella occidentalis and Thrips parvispinus)

Thrips are damaging pests in pepper worldwide. They can cause damage directly by feeding on leaves, fruits or flowers, and also indirectly by transferring viruses, especially tomato spotted wilt virus (TSWV). Although thrips are among the most damaging pests in pepper, until now there is no commercial variety with a useful level of resistance to thrips. This is at least partly due to the lack of knowledge on resistance levels in pepper germplasm of QTLs and/or genes for resistance, and of information about resistance mechanisms to thrips in pepper. This paper describes our research aimed at developing practical and reliable screening methods for thrips resistance in pepper and at identifying pepper accessions showing a strong resistance to thrips. Thirty-two pepper accessions from four species of pepper (Capsicum annuum, C. baccatum, C. chinense and C. frutescens) and two species of thrips (Frankliniella occidentalis and Thrips parvispinus) were used in this study. Our results indicate that the laboratory based leaf disc test and the detached leaf test can be used as reliable screening methods for thrips resistance in pepper. We observed a large variation for resistance to thrips in Capsicum that can be exploited in breeding programs.

Resistance factors in pepper inhibit larval development of thrips (Frankliniella occidentalis)

The western flower thrips [Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)] is a major pest in pepper cultivation. Therefore, host plant resistance to thrips is a desirable trait. The objectives of this study were to determine the effect of resistance on the development of thrips and to identify metabolite compounds related to the resistance. Three highly resistant, three medium resistant, and three susceptible pepper accessions were used in this study. Adult and pre‐adult survival, developmental time, and oviposition rate were assessed. Gas chromatography‐mass spectrometry was used to identify compounds that correlate with the level of resistance to thrips. Our results show that resistance of pepper accessions has a significant effect on oviposition rate and larval mortality. Seven compounds were identified that correlate with resistance to thrips and six compounds were identified that correlate with susceptibility to thrips. Some of these compounds, such as tocopherols, were previously shown to have an effect on insects in general. Also, some specific secondary metabolites (alkanes) seem to be more abundant in susceptible accessions and were induced by thrips infestation.

CHARACTERIZATION OF SOME MORPHOLOGY, ANATOMY AND PHYSIOLOGY OF THE WHEAT MUTANT (Triticum aestivum L.) DEWATA AND SELAYAR IN TROPICAL LOWLAND.

Oil derived from microalga has a big potential to substitute fossil fuel so that the oil extraction method needs to be developed. This study aims to compare the method for cell disruption in oil extraction of Spirulina sp. and Chlorella sp. microalgae. Spirulina sp. and Chlorella sp. were cultivated each in a pond with maximum capacity of 600 liters at Biotechnology Laboratory of Research and Development Center for Marine and Fisheries Product Processing and Biotechnology. Spirulina sp. were harvested by filtered it using satin. Chlorella sp. was harvested using coagulant NaOH, so it was needed to be neutralized to pH 7 with citric acid addition. The cell wall of Spirulina sp. and Chlorella sp. then was ruptured using sonicator and microwave, while other sample without disruption as control. The suspension then was macerated with n-hexane solvent, to extract the oil content. Oil content of Spirulina sp. which has been collected from this experiment gave result control: microwave: sonicator as 1.17%, 1.28%, and 1.97% respectively. Meanwhile, oil content of Chlorella sp. gave result from control, microwave, and sonicator as 0.93%, 1.20%, and 1.69% respectively. It was concluded that sonicator is the best method in oil extraction of cultured microalgae.The Bina Marga standard bridge has been implemented as bridge designing references in Indonesia. One of the standard bridge types is a composite bridge. This composite bridge is designed based on RSNI T-02-2005, an Indonesian bridge loading standard. This standard is based on Load and Resistance Factored Design (LRFD) method. LRFD method is treating statistical data on loads and strengths as random variables that have to be evaluated periodically using reliability index as the indicator. The purpose of this research is to evaluate the reliability of a 25 m simple span composite bridge structure due to statistical load data from weigh-in-motion (WIM) vehicular loads measurement in Pantura highway of Cikampek-Pamanukan, West Java in 2011 and RSNI T-02-2005 nominal vehicular load. The results of this research are maximum bending moment due to RSNI T-02-2005 nominal vehicular load is 526.55 kNm with a probability of exceedance of 4.5 x 10-7 and the reliability index of this composite bridge is 7.16, which is larger than reliability target of AASHTO of 3.50.

Globular Embryo Induction of Sugar Palm (Arenga pinnata (Wurmb) Merr.)

Abstract—The objective of this study was to get the best media to produce somatic embryos from zygotic embryos of sugar palm. This research was conducted at Tissue Culture Laboratory, Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University from October 2012 to April 2013. This research consisted of three phases which were callus induction, globular embryo induction, and globular embryo maturation. Sterilization using NaOCl 1.575 % and alcohol 96 % was able to provide the most sterile explants for treatment A1 (MS + 3 mg l -1 2,4-D + 2 mg l -1 NAA) of 89 % (16/18). During callus induction phase, embryogenic callus were mostly formed at treatment A5 (MS + 3 mg l -1 2,4D + 0.2 mg l -1 NAA + 100 mg l -1 biotin) which is 73.4 % after 8 weeks. All explants were able to form globular embryos on media B3 (callus A1 to media MS + 4 mg l -1 Kinetin), B6 (callus A1 to media MS + 1 mg l -1 Kinetin + 0.1 mg l -1 TIBA), B12 (callus A2 to media MS + 0.1 mg l -1 Kinetin + 1 mg l -1 TIBA), and B14 (callus A2 to media MS + 1 mg l -1 Kinetin + 1 mg l -1

Aplikasi 2,4-D dan TDZ dalam Pembentukan dan Regenerasi Kalus pada Kultur Anther Anthurium

ABSTRAK. Beberapa kultivar lengkeng toleran dataran rendah telah diintroduksi ke Indonesia termasuk lengkeng cv. Diamond River. Kultivar tersebut telah dibudidayakan secara komersial di daerah Kalimantan Barat. Namun, pengembangannya menghadapi kendala dalam hal penyediaan bibit. Dalam rangka memperoleh bibit lengkeng dalam jumlah yang berlimpah, perlu penerapan teknik kultur in vitro. Penelitian ini bertujuan untuk menginduksi dan meregenerasikan kalus embriogenik lengkeng cv. Diamond River. Induksi kalus dilakukan menggunakan daun muda sebagai eksplan. Regenerasi kalus embriogenik dilakukan dalam 4 tahap. Pada tahap pertama digunakan air kelapa pada konsentrasi 5 dan 10%. Pada tahap kedua, diuji pengaruh auksin (IBA dan NAA) serta sitokinin (BA dan kinetin) masing-masing pada taraf 0,5 ppm. Pada tahap ketiga, diuji pengaruh auksin IBA dan NAA pada taraf 0,1; 0,5; dan 1 ppm. Pada tahap keempat diuji perlakuan sukrosa pada taraf 2 dan 3% dengan atau tanpa auksin (IBA dan NAA) masing-masing pada taraf 0,5 dan 1 ppm. Hasil penelitian menunjukkan bahwa regenerasi melalui embriogenesis somatik berpeluang diterapkan pada tanaman lengkeng cv. Diamond River. Respons kalus embriogenik lebih dominan ke arah pembentukan akar daripada tunas. Penggunaan media yang mengandung NAA 1 ppm mampu meningkatkan pembentukan tunas hingga mencapai lebih dari 30%, sedangkan penggunaan sukrosa 3% tanpa auksin mampu meningkatkan pembentukan planlet hingga mencapai 12%. Persentase keberhasilan aklimatisasi adalah sebesar 14%. ABSTRACT. Roostika, I., V.N. Arief, and N. Sunarlim. 2009. Regeneration of Lowland Longan cv. Diamond River through Somatic Embryogenesis. Several low-land longan cultivars have been introduced to Indonesia, including cultivar of Diamond River. This cultivar has been planted commercially and produced well in West Kalimantan. Unfortunately, the development of this cultivar was facing a problem on the availability of planting materials. In order to provide large number of Diamond River seedlings, tissue culture technique was used. The aim of the study was to induce and regenerate embryogenic calli of longan cv. Diamond River. A research on callus induction was conducted using young leaves as explants source. Regeneration of embryogenic calli was conducted in 4 steps. The first, coconut water at the rate of 5 and 10% were used. The second, the auxin (IBA and NAA) and cytokinin (BA and kinetin) at the level of 0.5 ppm, respectively were tested. The third, the IBA and NAA at the level of 0.1, 0.5, and 1 ppm were used. The fourth, the sucrose at the level of 2 and 3% with or without addition of IBA and NAA at the level of 0.5 and 1 ppm were used respectively. The results showed that somatic embryogenesis regeneration was potentially applied to longan cv. Diamond River. The root formation was more dominant than the shoot formation. The use of 1 ppm NAA could increase the shoot formation up to more than 30% whereas the use of 3% sucrose without auxin could increase the plantlet formation up to 12%. The 14% of plantlet produced by this technique grew well during acclimatization period.ABSTRAK. Tanaman jeruk mengandung metabolit sekunder flavonoid, karotenoid, dan limonoid yang banyak terdapat dalam daun, kulit buah, biji, dan pulp. Penelitian bertujuan mengetahui kandungan flavonoid dan limonoid pada berbagai fase pertumbuhan tanaman jeruk Kalamondin dan Purut serta mendapatkan informasi kandungan limonoid pada fase embrio dan planlet hasil perbanyakan in vitro melalui embriogenesis somatik. Penelitian dilakukan di Balai Penelitian Tanaman Jeruk dan Buah Subtropika (Balitjestro) sejak bulan Mei sampai dengan Desember 2009. Ruang lingkup penelitian terdiri atas (1) identifikasi metabolit sekunder yaitu flavonoid dan limonoid pada berbagai fase pertumbuhan tanaman jeruk Kalamondin dan Purut dan (2) identifikasi limonoid pada fase embrio dan planlet tanaman jeruk Kalamondin yang diperbanyak dengan metode embriogenesis somatik secara in vitro. Analisis kandungan metabolit sekunder dilakukan di Unit Layanan Pengujian, Fakultas Farmasi, Universitas Airlangga. Hasil penelitian menunjukkan bahwa kandungan flavonoid dan limonoid dapat diproduksi dari berbagai bagian tanaman, seperti pada pulp, biji, kulit buah, dan daun pada berbagai fase pertumbuhan jeruk Purut dan Kalamondin. Kandungan flavonoid pada jeruk Purut dan Kalamondin tertinggi terdapat pada buah tua, masing-masing 18,8 ppm. Kandungan limonoid pada jeruk Purut hanya terdeteksi pada daun pendukung buah tua (1 ppm) dan biji (61 ppm), sedangkan pada jenis Kalamondin hanya terdeteksi pada biji yaitu sebesar 74 ppm. ABSTRACT. Devy, N.F., F. Yulianti, and Andrini. 2010. Flavonoid and Limonoid Contents in Every Growth Phase of Kalamondin (Citrus mitis Blanco) and Purut (Citrus hystrix Dc.). Citrus contains secondary metabolites such as flavonoid, carotenoid, and limonoid, which can be found in the leaf, peel of fruit, seeds, and pulp. The aims of this research were to determine flavonoid and limonoid contents in every growth phase of Kalamondin and Purut and the limonoid contents in embryo and plantlet phases derived from in vitro somatic embryogenesis. The research was conducted in Indonesian Citrus and Subtropical Fruits Research Institute (ICISFRI) from May to December 2009. The research consisted of two activities as follows: (1) analyses of flavonoid and limonoid contents in every growth phase of Kalamondin and Purut and (2) analyses of the limonoid contents in embryos and plantlet proliferated from somatic embryogenesis culture. Flavonoid and limonoid contents were analyzed at the Assessment Service Unit, Faculty of Pharmacy, Airlangga University. The results showed that flavonoid and limonoid compounds could be produced in all parts of plant i.e, such as pulp, seeds, peel of fruit, and leaves from every growth phase of Kalamondin and Purut. In Purut and Kalamondin, the highest flavonoid content was obtained from ripen fruit, with concentration 18.8 ppm. Liminoid content in Purut was detected only in leaf supporting ripen fruit (1 ppm) and seeds (61 ppm), and in Kalamondin was only in seeds with concentration 74 ppm.

Transformasi Genetik Tanaman Kentang cv. Atlantik Dengan Mengintroduksikan Gen Hordothionin untuk Mendapatkan Ketahanan terhadap Penyakit Bakteri

Hordothionins are small anti-bacteria proteins present in barley endosperm, To reveal the potential of this proteins for engineering bacterial disease resistance into potato, a semi-synthetic hordothionin gene construct was introduced in potato cv. Atlantic via Agrobacterium tumefaciens strain LBA 4404, under the control of a cauliflower mosaic virus (CaMV) 358 promotor. The in vitro grown stem (internodus) was used in this research. After 6 weeks in regeneration medium and 2 weeks in rooting medium there were 22 regenerated plants that were screened in kanamycine containing medium. PCR analysis using spesific primer from CaMV 358 promotor showed the presence of amplified T-DNA in 4 transgenic lines from 22 putative transgenic plants were tested, The in vitro toxicity against Ralstonia solanacearum tested from transgenic lines showed variation in resistance level, There were only 2 of the transgenic lines were tolerant, while one of them was moderate tolerant even one of them was susceptible. Key words: Potato, Hordothionin gene, Disease resistance

Studi RegeDerasi daD Produksi Protoplas Mesofil Daun Beberapa KlOD TanamaD Kentang (Solanum tuberosum L.) .

The objectives of these experiments were to obtain medium composition to enlarge leaf size for protoplast production and to obtain medium composition for plant regeneration. The result showed that the best medium to produce the larger leaves was medium MS with double concentration of macronutrients without hormone supplemented with Morel vitamins, 3% (w/v) sucrose and. 7 g/l agar. This medium produced leaves with diameter of 1.44 cm comparing to control medium MS with 0.67 cm in diameter. Medium MS containing 0.1 mg/l 1AA, 0.5 mg/l Zeatin and 0.5 mg/l GA3 was able to regenerate vigorous shoots of 7 clones. Protoplast isolation of 5 clones using enzyme composition containing 0.5 % cellulase Onozuka RS, 0.05 %, pectolyase Y-23, 0.05 % MES, 9.1 % mannitol and pH 5.5, without CP W medium produced protoplast with variable yield from 10.50 x 1oJ protoplast/g leaves for Atlantic to 46.58x 1oJ protoplast/gforBF15. Keyword: Leaves size, Plant regeneration, Potato, Protoplast i

Perakitan Kultivar Kentang Unggul Indonesia secara Cepat dengan Metode Turunan Klonal Biji Tunggal dan Pra - Evaluasi Secara In Vitro

At least ten years needed to abtain new potato cultivar through sexual hybridization, somatic hybridization or through genetic transformation, To short cut this process, Laboratory of Biotechnology, Department of Agronomy, IPB employed a strategy so called single seed in vitro clonal decent ( SSICD) by usings elected parental lines for TPS (True Potato Seed) production. This breeding consist of in vitro pre evaluation for resistance wilt, fusarium wilt, black leg, rot knot nematode and maturity. Using the same number of bacterial cell / (10 9 cell/ml), there were positive correlation between in vitro test for disease resistance through dripping test or dripping test with greenhouse test through direct inoculation of Ralstonia solanancearum. Resistant clones to fusarium wilt and verticillium were also resistant to bacterial wilt. In vitro tuberization could be use to evaluate maturity of potato cultivar. Key words: Potato, SSICD