Pengujian Stabilitas Genetik Plantlet Citrumelo Hasil tTCL dari Kultur In Vitro Dengan Menggunakan Teknik Sekuen Berulang

<p>Protokol organogenesis untuk perbanyakan plantlet Citrumelo menggunakan metode transverse thin cell layer (tTCL) batang telah berhasil dikembangkan. Identifikasi stabilitas genetik tanaman hasil kultur jaringan mutlak diperlukan untuk menguji keberadaan off-type. Tujuan penelitian adalah untuk mengetahui potensi primer retrotransposon dan inter simple sequence repeat (ISSR) dalam mendeteksi stabilitas genetik tanaman Citrumelo dari periode kultur yang panjang. Penelitian dilaksanakan pada bulan Juni 2013 sampai dengan Oktober 2015 di Laboratorium Pemuliaan Tanaman, Balai Penelitian Tanaman Jeruk dan Buah Subtropika, Tlekung. Sebanyak empat penanda dengan urutan basa berulang, yaitu retrotransposon dan ISSR digunakan untuk menguji stabilitas genetik plantlet in vitro yang berumur 22 bulan dan untuk mengonfirmasi metode yang dapat diandalkan untuk perbanyakan jeruk Citrumelo yang true-to-type pada masa mendatang. Daun plantlet diseleksi dan diisolasi secara bulk. Amplifikasi dilakukan terhadap DNA dengan sistem bulk segregant analysis (BSA), dan kemudian dipisahkan menggunakan gel agarose. Tanaman in vitro yang sama secara morfologi dapat dibedakan oleh penanda INT-retrotransposon yang mendeteksi adanya kehilangan pita pada grup sampel dengan ukuran 550 bp. Keberadaan retrotransposon dalam genom berlimpah dan aktivasinya diinduksi oleh stres. Kondisi kultur jaringan berpotensi menginduksi aktivasi retrotransposon. Keragaman genetik diperoleh sebesar 2,6%, tetapi masih dapat diterima untuk plantlet yang dihasilkan dari kultur jangka panjang. Plantlet yang digunakan dalam penelitian ini adalah plantlet yang dikulturkan sejak awal tahun 2014 dan telah digunakan untuk mempelajari faktor media dan lingkungan kultur yang efisien pada Citrumelo selama periode 2014–2015. Aktivitas pengkajian variabilitas genetik plantlet yang dihasilkan melalui tTCL batang masih terus dilakukan. Kombinasi protokol dan deteksi berbasis penanda PCR menjadi sarana yang efektif untuk perbanyakan massa benih berkualitas hasil kultur jaringan untuk mendukung progam pemuliaan maupun perbenihan.</p><p>Assessment of genetic stability of long-term cultivation of plantlet derived tTCL Citrumelo using repetitive sequence primers. Regeneration of plantlet from organogenesis of stem transverse thin cell layer (tTCL) was achieved for Citrumelo, a valuable rootstock. Identification of the genetic stability of plant tissue culture is absolutely necessary. The aim of this study was to assess the potential retrotransposons and inter simple sequence repeat (ISSR) primers in detecting the genetic stability of the Citrumelo plantlet derived from tTCL technique. The research was conducted from Juni 2013 until October 2015 in Breeding Laboratory of Indonesian Citrus and Subtropical Fruits Research Institute. A four repetitive based sequences retrotransposon and ISSR marker assays were used to evaluate genetic stability of a group of 22 months old in vitro plantlets and to confirm the most reliable method for true-to-type propagation of Citrumelo. Leaves of plantlets were selected and isolated in bulk. Groups of DNA in bulk segregant analysis (BSA) were amplified and separated using agarose gel. Vitroplants that morphologically similar have been effectively distinguished by a selected primer INT- retrotransposon that detect an deletion band at 550 bp on a line a group of sample. Retrotransposon is abundance through the genome and its activation induced by stress condition. Tissue culture condition was reported potential to induce retrotransposon activation. The genetic variation of 2.6% was acceptable for the culture that produced from long-term. Plantlets used in this study were selected from population induced from early 2014, and employed for studying media as well as environment factors for efficiently organogenesis of citrumelo in period of 2014-2015. However, additional study is on going for evaluating genetic variability from a cycle plantlet production through tTCL of stems. This combination protocol of organogenesis and PCR based markers detection would be powerful tools for mass propagation of high quality seedling derived tissue culture for breeding or cultivation programs.</p>

Somatic embryogenesis from leaf transverse thin cell layer derived-callus of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.)

No report on plant regeneration via somatic embryogenesis of P. vietnamensis has been previously published. In the present study, somatic embryogenesis via callus formation from cultures of leaf transverse thin cell layers (tTCLs) of Vietnamese ginseng (Panax vietnamensis Ha et Grushv.) was investigated. α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA) and thidiazuron (TDZ) were added separately and in combination into the culture media. Explant necrosis or low callogenesis rates were observed when 1-mm wide leaf tTCLs were cultured on media with TDZ, BA, 2,4-D or NAA. On the other hand, calli were successfully induced from the tTCL explants cultured on medium supplemented with either 2,4-D and BA or 2,4-D and TDZ. Callogenesis was observed under both light and dark conditions. The highest callogenesis rate (100%) was obtained on Murashige and Skoog (MS) basal medium supplemented with 1.0 mg l-1 2,4-D in combination with 0.1 mg l-1 TDZ in darkness after eight weeks of culture. White calli were cut into small pieces (1.0 x 1.0 cm dimension) and placed on MS media containing 1.0 mg l-1 2,4-D, 0.5 mg l-1 NAA and TDZ at various concentrations (0.01; 0.1; 0.2; and 0.5 mg l-1), and the best callus proliferation was recorded on medium containing 1.0 mg l-1 2,4-D and 0.2 mg l-1 TDZ. Somatic embryogenesis, with a success rate of 53.3% and 35 embryos per explant, was achieved when calli were subcultured onto MS medium supplemented with 1.0 mg l-1 2,4-D, 0.5 mg l-1 NAA and 0.2 mg l-1 TDZ.

High Frequency Plant Regeneration System from Transverse Thin Cell Layer Section of In vitro Derived ‘Nadia’ Ginger Microrhizome

An efficient and reproducible procedure is outlined for rapid in vitro multiplication of Zingiber officinale var. ‘Nadia’ through high frequency shoot proliferation from transverse thin cell layer (tTCL) sections of in vitro derived microrhizome. In vitro derived microrhizome of size 500 μm in thickness was used as initial explants for induction of somatic embryos. Among the different phytohormones tested, tTCL explants shows maximum calli proliferation in medium containing 2 mg/L 2,4-Dichlorophenoxyacetic acid (88.30±0.11%). Reduced concentration of 2,4 Dichlorophenoxyacetic acid was supplemented with different cytokinins for regeneration of callus. Among the different medium tested, optimum redifferentiation of somatic embryos were observed in medium containing 0.2 mg/L 2,4 Dichlorophenoxyacetic acid and 6.0 mg/L BAP (141.08±0.25). Clump of regenerated plantlets were further subculture and transfer into microrhizome inducing medium containing high sucrose concentration (8%). Plantlets with well developed microrhizome were successfully acclimatized and eventually transferred to the field. The application of studying embryo section for regeneration of plants might be useful alternative to ginger improvement programme. Histological analysis showed formation of somatic embryos and regenerated adventitious shoot.

Micropropagation of Rosa damascena Mill. through transverse thin cell layer culture

A basic factor underlying the success of large‐scale micropropagation and genetic transformation of any plant species is regeneration. In order to regenerate propagules of Rosa damascena Mill. on a large scale, an efficient and improved in vitro propagation system has been established using transverse thin cell layer culture (tTCL). By optimizing the position of the tissue and applying an improved selection procedure, in vitro shoots were elongated in 8 weeks of culture. Modified Murashige and Skoog (1962)(MS) medium fortified with 4.0 mg l‐1 6‐benzylaminopurine (BAP) and 0.4mg l‐1 anaphthalene acetic acid (NAA) gave optimal shoot regeneration. The explant was inoculated on this medium in the upright position and exhibited a high frequency of shoot regeneration (~96.66%), and it also gave the highest number of shoots (22.33/explant). The horizontally placed explant on an average 7.66 shoots/explant. Our experiments indicate that explant orientation strongly influences the organogenesis response. The frequency of shoot initiation and the number of multiple shoots produced from each explant were significantly dependent on the plant source, concentration of plant growth regulators and the orientation of the explants and contributed significantly to in vitro regeneration. Rooting of well developed shoots was achieved on hormone free ¼ strength MS medium with 4% sucrose.