Tri Joko Santoso

Evaluasi Sifat Daya Tembus Akar dan Identifikasi Mutan Stabil pada Populasi Penanda Aktivasi

Abstract Drought stress is one of the important limiting factors in increasing rice production in Indonesia. Development of rice varieties with increased tolerance to drought is needed to meet the rice production challenge. Some transgenic Nipponbare rice lines that carry activation tag have been generated from the previous study. The purpose of this study was to evaluate the root penetration ability and the stability of Ds element in the T1 generation of activation tag rice lines. Materials used in the study were T1 seeds of 47 transgenic lines, drought tolerant check varieties (Cabacu and IRAT112), and sensitive check varieties (IR64 and wild type Nipponbare). T1 seeds were prescreened by germination in Basta herbicide solution to eliminate T1 individuals that did not carry activation tag element. Root penetration ability was evaluated using wax-petrolatum layers as a proxy for compacted soil layers. The presence of bar gene and the absence of hpt gene as detected by PCR were used to identify T1 stable mutants. Out of 47 transgenic lines tested, 38 lines showed better root penetration ability than non transformed Nipponbare. PCR analysis identified four stable mutants, namely M-Nip-12.12, M-Nip-19.8, M-Nip-19.9, and M-Nip- 20.13. One stable mutant, M-Nip-20.13, showed better root penetration ability than tolerant check varieties. This mutant is a good candidate for isolation of drought tolerance gene. Abstrak Cekaman kekeringan merupakan salah satu faktor pembatas penting dalam peningkatan produksi padi di Indonesia. Perakitan varietas unggul padi toleran kekeringan diperlukan untuk menjawab tantangan tersebut. Beberapa galur padi Nipponbare transgenik yang membawa penanda aktivasi telah dihasilkan pada penelitian sebelumnya. Tujuan penelitian ini adalah mengevaluasi daya tembus akar dan stabilitas elemen Ds pada galur-galur penanda aktivasi generasi T1. Bahan yang digunakan dalam penelitian ini adalah benih T1 47 galur transgenik, varietas cek toleran kekeringan (Cabacu dan IRAT112), dan varietas cek peka (IR64 dan Nipponbare tipe liar). Benih T1 ditapis terlebih dahulu dengan perkecambahan pada larutan herbisida Basta untuk mengeliminasi individu yang tidak membawa elemen penanda aktivasi. Daya tembus akar dievaluasi menggunakan lapisan lilin sebagai tiruan lapisan tanah yang padat dan keras (hardpans). Keberadaan gen bar dan ketiadaan gen hpt yang dideteksi dengan PCR digunakan untuk mengidentifikasi mutan-mutan stabil. Dari 47 galur yang diuji, 38 galur menunjukkan daya tembus akar yang lebih baik daripada Nipponbare non transforman. Analisis PCR mengidentifikasi empat mutan stabil, yaitu M-Nip-12.12, M-Nip-19.8, M-Nip-19.9, dan M-Nip-20.13. Satu mutan stabil, M-Nip-20.13, menunjukkan daya tembus akar yang lebih baik daripada varietas cek toleran. Mutan ini menjadi kandidat yang baik untuk isolasi gen toleran kekeringan.

Efficiency of Genetic Transformation via Pollen-Tube Pathway of Jatropha (Jatropha curcas L.) Based on Histochemical and Molecular Analysis

The genetic transformation via pollen-tube pathway is an alternative method to overcome the constraints imposed by genotype specificity in transformation and regeneration in jatropha (Jatropha curcas L.) tissue culture. Therefore, it is necessary to establish important parameters for efficient genetic transformation of jatropha via pollen-tube pathway. The objective of the research was to study the efficiency of direct transformation of jatropha via pollen-tube pathway based on histochemical and molecular analysis. Solution of purified pCAMBIA1301 DNA plasmid carrying a hptII marker gene and a gus reporter gene with concentration level of 0.05, 0.25, 0.50 µg µl-1 were applied to stigma of flowers at 1, 2, 4, 7, 10 h after pollination. Seedling of IP3A, IP3P and JcUMM18 jatropha’s genotypes derived from 15 combination treatments of plasmid DNA concentration and application time, also wild type was subjected to histochemical and molecular analyses. Based on those analyses, the efficiency of transformation via pollen-tube pathway of three jatropha genotypes ranged from 1.5-16.7%. PCR analysis showed that a number of positive plants were identified by using specific primers hptII and gus, i.e. 1-3 and 3-7 plants of the 15 combined treatments, respectively. It indicated that the transformation efficiency via the pollen-tube pathway varied in each jatropha genotype. Keywords: Jatropha curcas L., pCAMBIA1301, plasmid DNA, stigma-drip

Deteksi Gen HptII dan Keragaan Agronomis pada Populasi BC1F1 Tanaman Padi Transgenik

Rice varieties tolerant to drought stress are needed to stabilize rice production under drought stress condition. We developed transgenic rice cv. Nipponbare carrying hptII gene that might also contain OsDREB1A gene. OsDREB1A gene responsible to drought tolerance trait need to be transferred into cultivated rice in order to obtain new local rice variety tolerant to drought stress. The aims of this research were to detect the presence of hptII gene in the F1 and BC1F1 transgenic rice and to observe the agronomic performace of those populations and their plant physiology. F1 population was developed by crossing transgenic Nipponbare, as donor parent, with Batutegi, Code, Ciherang, and Konawe genotypes, as recipient parents. BC1F1 population was developed by backcrossing F1 transgenic line with recipient parents, respectively. The presence of hptII gene was analyzed by PCR using a pair of primers for hptII. The observation of agronomic performance was carried out in the green house, meanwhile the observation of stomata was done using microscope. The result of PCR analysis showed that BC1F1 Batutegi trans, BC1F1 Code trans, BC1F1 Konawe trans1, BC1F1 Konawe trans3, dan BC1F1 Konawe trans4 were detected carrying the hptII gene. Agronomic data showed that BC1F1 transgenic rice lines yielded panicles, filled grains, and total grains higher than those of recipent parents. Comparing to the recipient parents, BC1F1 Konawe trans1 and BC1F1 Konawe trans3 had less stomata on the lower side of the leaf, but had more stomata on the upper side of the leaf.

Identification of Aroma Gene (Mutated badh 2) and Properties of Aroma on Aromatic BC 5 F 2 Ciherang

Aromatic rice varieties have some weaknesses such as low productivity, and less resistant to pests and diseases. This study aimed to obtain homozygous strain of BC 5 F 2 Ciherang aromatic through the identification of aroma gene (mutated badh2) and properties of the aroma. Ciherang paddy (nonaromatic paddy) was used as the female parent, whereas Mentik Wangi paddy (aromatic paddy) was used as the male parent. The experiment was conducted in BC 5 F 2 because it is expected to generate plants with properties 98.4% close to female parent. The DNA from five strains of paddy plants BC 5 F 2 Ciherang X Mentik Wangi was isolated by a modified CTAB method. The concentration of DNA was determined by measuring absorbance at 260 nm wavelength, while its purity was determined from the ratio of the absorbance at a wavelength of 260/280 nm. PCR-based molecular selection was done by using the Bradbury primers. PCR results showed that of the 250 samples, there were 66 samples had DNA fragment of the same size as that of Mentik Wangi, i.e. 257 bp, 67 samples had the same size as the DNA fragment of Ciherang, i.e. 355 bp, and 117 samples had the same size with the both of DNA fragments, i.e. 257 bp and 355 bp. Plants with amplified 257 bp DNA fragment was subjected to leaf aroma test using 1.7% KOH. The results showed that 42 positive samples, out of 66 samples. Samples positive on leaf aroma test were tested again on rice aroma test. Rice aroma test results showed the majority (85.4%) samples that are positive on leaf aroma test is also positive on the rice aroma test.