EFFECTIVE CACAO SOMATIC EMBRYO REGENERATION ON KINETIN SUPPLEMENTED DKW MEDIUM AND SOMACLONAL VARIATION ASSESSMENT USING SSRs MARKERS

Ajijah, Nur; Hartati, Rr. Sri; Rubiyo, Rubiyo; Sukma, Dewi; Sudarsono, Sudarsono

doi:10.17503/agrivita.v38i1.619

Cited by 21 publications

(39 citation statements)

References 17 publications

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“…SE has been reported in Arabidopsis thaliana (Gaj, 2001), Phoenix dactylifera L. (Naik and Al-Khayri, 2016), Theobroma cacao L. (Ajijah et al, 2016), Gladiolus hybridus (Wu J. et al, 2015), and Cassia occidentalis L. (Naz et al, 2015). However, the understanding of the mechanism underlying the formation of somatic embryos is still in its infancy, and SE remains the least understood pattern of regeneration (Fehér, 2015).…”

Section: Introductionmentioning

confidence: 97%

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

et al. 2017

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Plant somatic embryos are widely used in the fields of germplasm conservation, breeding for genetic engineering and artificial seed production. MicroRNAs (miRNAs) play pivotal roles in somatic embryogenesis (SE) regulation. However, their regulatory roles during various stages of SE remain unclear. In this study, six types of embryogenic samples of Lilium pumilum DC. Fisch., including organogenic callus, embryogenic callus induced for 4 weeks, embryogenic callus induced for 6 weeks, globular embryos, torpedo embryos and cotyledon embryos, were prepared for small RNA sequencing. The results revealed a total of 2,378,760 small RNA reads, among which the most common size was 24 nt. Four hundred and fifty-two known miRNAs, belonging to more than 86 families, 57 novel miRNAs and 40 miRNA*s were identified. The 86 known miRNA families were sorted according to an alignment with their homologs across 24 land plants into the following four categories: 23 highly conserved, 4 moderately conserved, 15 less conserved and 44 species-specific miRNAs. Differentially expressed known miRNAs were identified during various stages of SE. Subsequently, the expression levels of 12 differentially expressed miRNAs and 4 targets were validated using qRT-PCR. In addition, six samples were mixed in equal amounts for transcript sequencing, and the sequencing data were used as transcripts for miRNA target prediction. A total of 66,422 unigenes with an average length of 800 bp were assembled from 56,258,974 raw reads. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that 38,004 and 15,497 unigenes were successfully assigned to GO terms and KEGG pathways, respectively. Among the unigenes, 2,182 transcripts were predicted to be targets for 396 known miRNAs. The potential targets of the identified miRNAs were mostly classified into the following GO terms: cell, binding and metabolic process. Enriched KEGG analysis demonstrated that carbohydrate metabolism was the predominant pathway in Lilium SE. Thus, we performed systemic characterization, homology comparisons and profiling of miRNA expression, and we constructed an miRNA-target network during Lilium SE for the first time. Our findings establish a foundation for the further exploration of critical genes and elucidation of SE in Lilium.

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Section: Introductionmentioning

confidence: 97%

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

et al. 2017

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“…Immature flowers were stored in sterile basal DKW salts (Ajijah, Hartati, Rubiyo & Sukma, 2016) on ice during transportation and carried out to laboratory. Flower buds were surface sterilized following the protocol described by Urrea, Gallego & Atehortúa (2011), with modifications.…”

Section: Propagated Plant Materials By Somatic Embryogenesismentioning

confidence: 99%

Quality of cocoa (Theobroma cacao L.) DNA from foliar tissue at different stages of development

Ramírez¹,

Duque²,

Trujillo³

2018

Acta Agron.

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Quality of cocoa (Theobroma cacaoL AbstractTheobroma cacao L. and its products are consumed worldwide. Those products are of great research interest due to antioxidant properties of some of their polyphenolic constituents. The amount of these polyphenols and polysaccharides has shown that can interfere with the high quality and quantity of nucleic acids for molecular research. Therefore, cocoa DNA extraction protocols can require a large amount of plant material and optimization time according with plant material source. The aim of this study was to evaluate the quality and quantity of DNA isolated from field plant leaves at different developmental stages from TSH565 genotype using different DNA extraction protocols. In addition, DNA extraction protocol was evaluated for small amount of young leaf tissue collected from in vitro plantlets from CCN51 and TSH565 genotype. Subsequently, the selectivity of different polymerase enzymes for PCR amplification using the obtained DNA was evaluated. This study revealed that D stage of development in field leaves was efficient for extraction of high-quality genomic DNA using the PowerPlant® Pro Kit modified (183.80 ng.µL -1 (1.98 A260/A280-1.98 A260/A230)). Highest DNA concentrations were obtained for FPL with 128.68 ng.µL -1 and 114.42 ng.µL -1 for CCN51 and TSH565 respectively and with IVL, which was obtained 54.24 ng.µL -1 for CCN51 and 56.52 ng.µL -1 for TSH565 per 0.1 g of leaf tissue. Taq DNA Polymerase recombinant of Thermo Scientific® showed the highest performance specifically for this study, contributing to the undoubted amplification of molecular markers like microsatellites (SSRs). The results obtained have allowed improvements in genetic analyses and molecular studies using a reduced amount of plant tissue.

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“…Media induksi kalus primer terdiri dari media dasar DKW atau WPM, tergantung perlakuan, dengan penambahan glutamin 250 mg/l, glukosa 20 g/l, pemadat phytagel 2 g/l, serta zat pengatur tumbuh (ZPT) dichlorophenoxyacetic acid (2.4-D) 9 μM dan kinetin 1,16 μM. Media induksi kalus sekunder terdiri dari media dasar WPM dengan penambahan air kelapa 50 ml/l, glukosa 20 g/l, pemadat phytagel 2,2 g/l, serta ZPT dan kinetin masing-masing 2.4-D 9 μM dan 1,16 μM (Ajijah, Hartati, Rubiyo, Sukma, & Sudarsono, 2016). Modifikasi yang dilakukan meliputi penggantian thidiazuron pada media induksi kalus primer dengan kinetin dan penurunan konsentrasi kinetin pada media induksi kalus sekunder dari 1,40 μM menjadi 1,16 μM.…”

Section: Uji Pengaruh Media Dasar Dan Jenis Eksplan Terhadap Pembentuunclassified

“…Sebaliknya, mahkota bunga menunjukkan respons pembentukan kalus lebih tinggi (62%) dan berbeda nyata dibandingkan dengan staminoid (23%) apabila digunakan media WPM (Gambar 1). Adanya perbedaan respons pembentukan kalus di antara organ bunga kakao juga telah dilaporkan sebelumnya (Avivi, Hardjosoedarmo, & Hartanto, 2012;Ajijah, Rubiyo, & Sudarsono, 2014;Ajijah et al, 2016). Perbedaan kemampuan setiap jenis eksplan (hipokotil, kotiledon, dan akar) dalam membentuk kalus juga ditunjukkan pada tanaman Raphanus sativus L. (Aazami & Pouraghdam, 2009).…”

Section: Hasil Dan Pembahasan Pengaruh Media Dasar Dan Jenis Eksplan unclassified

“…Pada tanaman kakao, respons embriogenesis somatik dari eksplan mahkota bunga dan staminoid sangat bervariasi tergantung genotipe. Berdasarkan hasil penelitian Ajijah et al (2016), respons embriogenesis somatik yang lebih tinggi pada klon kakao Sca 6, UIT 1, ICS 13, DR 2, dan Cimanggu 1 ditunjukkan oleh eksplan staminoid, sedangkan klon Pa 300, GC 7, ICCRI 2, dan Cimanggu 2 ditunjukkan oleh eksplan mahkota bunga. Dari hasil penelitian ini menunjukkan bahwa eksplan mahkota bunga lebih direkomendasikan untuk digunakan di dalam menginduksi embriogenesis somatik pada kakao klon ICRRI 4.…”

Section: The Number Of Somatic Embryos Per Explant Formed From Callunclassified

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Pengaruh Komposisi Media Dasar dan Jenis Eksplan terhadap Pembentukan Embrio Somatik Kakao

Ajijah¹

2016

J. Tanaman Industri dan Penyegar

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ABSTRAKKomposisi media dasar sangat menentukan keberhasilan proses regenerasi di dalam kultur in vitro. Tujuan penelitian adalah mengetahui pengaruh komposisi media dasar pada media induksi kalus primer dan jenis eksplan terhadap pembentukan embrio somatik kakao. Penelitian dilaksanakan di Laboratorium Kultur Jaringan Unit Pengembangan Benih Unggul Pertanian Balitbangtan, Bogor, mulai bulan Juni 2014 sampai Desember 2015. Induksi kalus primer dari eksplan mahkota bunga dan staminoid kakao klon ICCRI 4 menggunakan dua jenis media dasar, yaitu media Driver dan Kuniyuki (DKW) + 2,4-D 9 μM + kinetin 1,16 μM dan woody plant media (WPM) + 2,4-D 9 μM + kinetin 1,16 μM. Setelah 14 hari, kalus disubkultur pada media induksi kalus sekunder (WPM + 2,4-D 9 μM + kinetin 0,58 μM), kemudian pada media DKW tanpa zat pengatur tumbuh untuk menginduksi pembentukan embrio somatik. Penelitian disusun dalam rancangan faktorial dua faktor dengan 5 ulangan. Faktor pertama adalah jenis media dasar pada media induksi kalus primer (DKW dan WPM), sedangkan faktor kedua adalah jenis eksplan (mahkota bunga dan staminoid). Hasil penelitian menunjukkan terdapat pengaruh interaksi yang nyata antara jenis media dasar dan jenis eksplan terhadap pembentukan kalus dan embrio somatik kakao. Persentase pembentukan kalus paling tinggi diperoleh pada penggunaan media dasar DKW dengan eksplan staminoid (92,5%). Akan tetapi, persentase pembentukan embrio somatik dan jumlah embrio somatik per eksplan paling tinggi diperoleh pada perlakuan media dasar DKW dengan eksplan mahkota bunga (36,5% dan 2,30). Dengan demikian, penggunaan media dasar DKW dan eksplan mahkota bunga lebih direkomendasikan untuk menginduksi pembentukan embrio somatik kakao klon ICCRI 4. Kata kunci: DKW, eksplan,embrio somatik, kalus, WPM ABSTRACT

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EFFECTIVE CACAO SOMATIC EMBRYO REGENERATION ON KINETIN SUPPLEMENTED DKW MEDIUM AND SOMACLONAL VARIATION ASSESSMENT USING SSRs MARKERS

Cited by 21 publications

References 17 publications

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Quality of cocoa (Theobroma cacao L.) DNA from foliar tissue at different stages of development

Pengaruh Komposisi Media Dasar dan Jenis Eksplan terhadap Pembentukan Embrio Somatik Kakao

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