Enhanced production of bioactive compound in the callus culture of Glycyrrhiza glabra L. with the improved biomass accumulation

Licorice ( Glycyrrhiza glabra ) of Fabaceae family is known to have wide range of medicinal properties due to the metabolites found in the plant’s tissue. Callus cultures from Glycyrrhiza glabra were previously initiated in vitro using leaf as an explant. The current study was designed in a way to examine the possible role of different elicitors concentrations on the stimulation of biomass and their effects on different metabolite content such as total carbohydrates, protein, proline, phenol, alkaloid, flavonoid and glycyrrhizin. Elicitation with different concentration of elicitors increased the biomass and metabolite content in callus culture of G. glabra at different rates. The optimum concentration of adenine sulphate for maximum biomass accumulation (16.79 g/flask) was found to be at 50 mg/l on the incubation of 20 th days. Adenine sulphate as well as putrescine was also found to stimulate the different metabolite content to 3-4 folds in the callus cultures as compared to that of control. Results showed that the metabolite content and antioxidant enzyme of Glycyrrhiza glabra can be enhanced by appropriate forms of elicitation.

OPTIMASI DAN EVALUASI METODE KRIOPRESERVASI PURWOCENG

<p>ABSTRAK<br />Optimasi dan evaluasi metode kriopreservasi perlu dilakukan dalam<br />menentukan protokol standar untuk penyimpanan jangka panjang biakan<br />purwoceng. Penelitian ini bertujuan untuk mengetahui pengaruh kombinasi<br />perlakuan pratumbuh, prakultur, dan formulasi media pemulih terhadap<br />daya tumbuh dan daya regenerasi tunas in vitro dan kalus embriogenik<br />serta untuk mengevaluasi metode kriopreservasi melalui observasi<br />morfologi, anatomi, dan sitologi. Penelitian dilakukan di Laboratorium<br />Kultur Jaringan Kelompok Peneliti Biologi Sel dan Jaringan BB Litbang<br />Biogen pada tahun 2008-2009. Teknik kriopreservasi yang digunakan<br />adalah vitrifikasi (untuk apeks) dan enkapsulasi-vitrifikasi (untuk kalus<br />embriogenik). Pada teknik vitrifikasi, tunas pucuk diberi perlakuan<br />pratumbuh dengan sukrosa (3, 4, 5, dan 6%) selama 1 dan 2 minggu,<br />perlakuan prakultur dilakukan pada media yang mengandung sukrosa 0,3<br />M selama 1 dan 3 hari, perlakuan dehidrasi dengan PVS2 diberikan selama<br />15 dan 30 menit, dan media pemulih yang diujikan adalah media dasar MS<br />atau DKW dengan dan tanpa penambahan adenin sulfat 20 ppm. Pada<br />teknik enkapsulasi-vitrifikasi, kalus embriogenik dienkapsulasi terlebih<br />dahulu dengan Na-alginat 3%, perlakuan dehidrasi dengan PVS2 diberikan<br />selama 0, 30, dan 60 menit. Evaluasi metode teknik kriopreservasi<br />dilakukan melalui pengamatan morfologi secara visual, anatomi meristem<br />dengan scanning electron microscope (SEM), pengujian viabilitas dengan<br />fluorescein diacetate (FDA), dan analisis ploidi secara flowcytometry.<br />Hasil penelitian menunjukkan bahwa teknik enkapsulasi-vitrifikasi lebih<br />baik daripada teknik vitrifikasi untuk kriopreservasi purwoceng. Walaupun<br />persentase keberhasilan kriopreservasi rendah (10%), kalus embriogenik<br />purwoceng mampu berproliferasi dan beregenerasi menjadi ribuan embrio<br />somatik dewasa. Evaluasi metode kriopreservasi dengan SEM dan FDA<br />dapat diterapkan untuk memperkirakan keberhasilan teknik kriopreservasi<br />secara dini sedangkan analisis flowcytometry dapat diterapkan untuk<br />menguji stabilitas genetik bahan tanaman pasca-kriopreservasi.<br />Kata kunci: Pimpinella pruatjan Molk., kriopreservasi, SEM, FDA,<br />flowcytometry</p><p>ABSTRACT<br />Optimization and evaluation of cryopreservation methods should be<br />conducted to obtain standard protocol for long term conservation of<br />pruatjan. The objective of this study was to evaluate the effect of<br />combined treatments of pregrowth, preculture, and recovery media to the<br />survival and regeneration rate of in vitro shoots and embryogenic calli and<br />to evaluate the cryopreservation methods by observing the morphological,<br />anatomical, and cytological characters. The techniques of vitrification (for<br />apex) and encapsulation-vitrification (for embryogenic calli) were applied<br />in this study. On vitrification technique, the apical shoots were pregrown<br />on media containing of 3, 4, 5, and 6% sucrose for 1 and 2 weeks,<br />precultured on media containing of 0,3 M sucrose for 1 and 3 days,<br />dehydrated by PVS2 solution for 15 and 30 minutes, and planted on<br />recovery media (MS or DKW basal media supplemented with 20 ppm<br />adenine sulphate). On encapsulation-vitrification technique, embryogenic<br />calli were encapsulated by 3% Na-alginate, dehydrated by PVS2 solution<br />for 0, 30, and 60 minutes. The evaluation of cryopreservation methods was<br />done through visual observation, SEM analysis, viability test, and<br />flowcytometry determination. The result showed that encapsulation-<br />vitrification was better than vitrification technique for cryopreservation of<br />pruatjan. The successful rate of this method was low (10%) but the<br />embryogenic calli could proliferate and regenerate into thousands mature<br />somatic embryos. The evaluation by SEM and FDA can be applied as<br />early detection to estimate the successful of cryopreservation, whereas<br />flowcytometry analysis may determine the genetic stability of<br />cryopreserved materials.<br />Key words: Pimpinella pruatjan Molk., cryopreservation, SEM, FDA,<br />flowcytometry</p>

Somatic Embryogenesis and Plantlet Regeneration in the Carica papaya L. cv. Eksotika

A highly efficient protocol for regeneration of Carica papaya L. cv. Eksotika somatic embryos from immature zygotic embryos was developed. This study was designed to overcome the obstacles in regeneration of somatic embryos from immature zygotic embryos of “Eksotika”, especially problems associated with formation of better root quality and callus formation at the base of somatic embryos. Somatic embryos were generated by incubation of immature zygotic embryos in half-strength salt Murashige and Skoog (MS) medium with full-strength vitamins supplemented with 7.5 mg L−1 2,4-D, 100 mg L−1 L-glutamine, 50 mg L−1 myo-inositol, 45 mg L−1 adenine sulphate, 0.33% gelrite, and 6% sucrose, followed by transfer to maturation medium consisting of ½ MS medium supplemented with 5 mg L−1 phloroglucinol, 100 mg L−1 L-glutamine, 100 mg L−1 myo-inositol, 68 mg L−1 adenine sulphate, 0.38% gelrite, and 3% sucrose. After that, well-formed somatic embryos were transferred to MS medium containing 3% sucrose and 0.8% agar for shoot production. The embryos were elongated in MS medium supplemented with 1 mg L−1 gibberellic acid, 0.5 mg L−1 indole-3-butyric acid, 100 mg L−1 myo-inositol, and 3.76 mg L−1 riboflavin. Root regeneration was achieved on MS medium containing 7.9 mg L−1 phloroglucinol and supported with vermiculite after 4 days of cultivation on ½ MS medium with 2 mg L−1 indole-3-butyric acid. After the rooting phase, in vitro plantlets were acclimatized in peat moss soil.

Axillary Shoots Derived from Thin Cell Layer and Adenine Sulphate Application in in vitro Mass Propagation of Gerbera [Gerbera jamesonii (H. Bolus ex Bolus f.)]

A new route of in vitro mass propagation protocol of Gerbera jamesonii (H. Bolus ex Bolus f.) derived from application of thin cell layers (TCL) and adenine sulphate (AS) was successfully developed and established. Shoot tip explants and half-strength MS medium containing 0.25 mg/l N6-benzylaminopurine (BAP), 20 g/l sucrose and 7 g/l Swallow agar were used as explant source and basic medium. Different TCL of transversal TCL (tTCL) and longitudinal TCL (lTCL) in four slicing positions of 1, 2, 3 and 4; varieties and clones i.e. G. jamesonii ‘Black Jack’, ‘Carambole’, ‘Nuance’, ‘Violente’, 01.098 and 11.46 clone; AS concentrations viz. 0, 20, 40, 60, 80 and 100 mg/l were tested in the study. Each step of in vitro culture established had unique and specific results. In the initiation stage, first slicing position of ‘Black Jack’ shoot tip tTCL was the most optimal combination treatment to produce 7.0 shoots per explant with 13.5 leaves. The first slicing position on shoot tip explants of 01.098 clone tTCL and 20 mg/l AS in half-strength MS medium containing 0.25 mg/l BAP were the most optimal combination treatment in obtaining the highest number of shoots produced per shoot up to 9.4 shoots per shoot with 34.1 leaves and 2.37 cm length of leaves in the proliferation stage, however the treatment did not give significant effect compared to control. Under periodical subcultures on the basic medium, number of shoots and leaves increased gradually from the initial culture with 3-6 shoots per shoot and 9.4-11.6 leaves till the fourth subculture with 6-11 shoots per shoot and 16.7-28.8 leaves and declined thereafter. Subculturing of shoots in accordance to produce qualified shoots for planting materials could be carried out till sixth to seventh subculture. The highest shoot multiplication rate (SMR) was established on 01.098 clone with as high as 7.3. The well shoots were easily rooted on half-strength MS medium supplemented with 0.1 mg/l BAP, 0.05 mg/l NAA and 1.5 g/l AC. Plantlets were then transferred to ex vitro condition for acclimatization on a mixture of burned-rice husk and organic manure (1:1, v/v) with 85-100% survivability. The ‘Black Jack’ and 11.46 clone were the best genotypes on the acclimatization stage with 100% survivability of plantlets. Results of the study have implication that first slicing position of shoot tip tTCL can be applied in establishing of in vitro propagation protocol for other gerberas.

PERBANYAKAN IN VITRO PISANG KEPOK var. UNTI SAYANG TAHAN PENYAKIT DARAH MELALUI PROLIFERASI TUNAS

In Vitro Propagation of Kepok Banana var. Unti Sayang Resistant to Blood Disease through Shoot ProliferationABSTRACTKepok is a popular banana variety but sensitive to blood disease caused by Ralstonia solanacearum (Smith). The discovery of a natural mutant of Kepok banana var. Unti Sayang from Sulawesi which male bud falls naturally, is a shortcut to bypass the chains of the spread of blood disease, since the disease is transmitted by insects through the wounds of the male buds. The superior mutant needs to be mass propagated and disseminated to endemic areas to inhibit the spread of blood disease. To achieve that goal, an efficient and effective techniques of in vitro shoot proliferation needs to be developed. Shoot proliferation was performed by addition of BAP, thidiazuron and adenine sulphate. The results showed that the best medium for shoot multiplication was B2T5A (MS+2 mg/L BAP+0,5 mg/L TDZ+20 mg/L adenine sulphate), and for shoot growth was B4A (MS+4 mg/L BAP+20 mg/L adenine sulphate). Rooting was induced on MS medium without hormones. Acclimatization of plantlets on mixed soil, compost and husks with a ratio of 1:1:1 resulted in 92,35% survival rate.Keywords: blood disease, in vitro shoot, male budless, natural mutant, var. Unti Sayang ABSTRAKPisang kepok merupakan varietas yang digemari tetapi sangat peka terhadap penyakit darah yang ditimbulkan oleh bakteri Ralstonia solanacearum (Smith). Ditemukannya mutan alami pisang kepok yang jantungnya gugur secara alami yaitu varietas Unti Sayang dari Sulawesi, merupakan jalan pintas untuk memotong rantai penyebaran penyakit darah, mengingat penyakit ini ditularkan oleh serangga melalui luka bekas bunga jantan pada jantung. Mutan unggul tersebut perlu diperbanyak secara massal dan disebarluaskan ke daerah endemik untuk menghambat penyebaran penyakit darah. Untuk mencapai tujuan tersebut, perlu dikembangkan teknik perbanyakan in vitro pisang kepok Unti Sayang yang efektif dan efisien melalui proliferasi tunas. Proliferasi tunas dilakukan dengan penambahan BAP, thidiazuron dan adenin sulfat. Hasil penelitian ini menunjukkan bahwa media terbaik untuk multiplikasi tunas adalah B2T5A (MS+2 mg/L BAP+0,5 mg/L TDZ+20 mg/L adenin sulfat), media terbaik untuk pertumbuhan tunas adalah B4A (MS+4 mg/L BAP+20 mg/L adenin sulfat). Akar dapat diinduksi pada media MS tanpa hormon. Aklimatisasi planlet pada media campuran tanah, kompos dan sekam dengan perbandingan 1:1:1 menghasilkan 92,35% planlet hidup.Kata Kunci: penyakit darah, tunas in vitro, tanpa jantung, mutan alami, var. Unti Sayang