Green Globular Body (GGB) Induction and Differentiation in the Medicinal fern Drynaria Roosii

Background: The green globular body (GGB) of ferns is a special propagule induced in plant in vitro culture systems. Owing to its high proliferation efficiency, GGB is widely used in the in vitro propagation of important ornamental and medicinal ferns. In addition, propagation using GGB shows great development prospects in the conservation of rare or endangered ferns and the breeding of new fern varieties. However, due to the lack of systematic studies on GGB ontogenesis, the morphogenetic aspects of GGB during induction and differentiation remain unclear.Results: We characterized the response of five types of explants of Drynaria roosii to GGB inductive medium and further investigate morphological and anatomical changes of explants that developed GGBs. We found that the rhizome explants directly produced GGBs through cell proliferation of the shoot apical meristem and lateral meristem. The leaf and petiole explants produced GGBs indirectly through the proliferation of meristematic cells of somatic embryos derived from the epidermal cells of the explants. The root and gametophyte explants failed to produce GGB under our induction conditions. We further investigated the differentiation process of GGB. During GGB differentiation, shoot primordia and leaf primordia differentiate from meristematic cells on the epidermis, and the root primordia develop from an inner meristematic tissue with developing vascular tissue connecting all these primordia, which indicates the involvement of multiple organogenesis processes.Conclusions: Our results suggested that preexisting or reestablished meristematic cells were the direct source of GGB in D. roosii. Somatic embryogenesis and organogenesis were involved in GGB induction and differentiation, respectively. The comparison with other common propagules revealed that GGB in D. roosii was largely different from somatic embryos, callus, and protocorm or protocorm-like bodies.

PENGARUH UMUR EKSPLAN TERHADAP KEBERHASILAN PEMBENTUKAN KALUS EMBRIOGENIK PADA KULTUR MERISTEM JAHE (Zingiber officinale Rosc)

<p>ABSTRAK</p><p>Kendala dalam pengembangan jahe di Indonesia adalah terbatasnyabenih bermutu. Secara konvensional, budidaya jahe dilakukan denganmenggunakan bibit dari potongan rimpang. Dengan cara ini diperlukanbibit dalam jumlah yang banyak, antara 2-3 t/ha untuk jahe yang dipanentua dan 5-6 t/ha untuk yang dipanen muda. Kendala lain adalah penyakittular benih layu bakteri yang disebabkan oleh Ralstonia solanacearum.Salah satu upaya yang dapat dilakukan untuk mendapatkan benih jahebebas penyakit adalah perbanyakan melalui kultur jaringan. Penelitianbertujuan untuk mengkaji sumber eksplan dari tingkat umur panenrimpang yang berbeda terhadap kapasitas pembentukan kalus embriogenikpada kultur meristem jahe putih besar. Penelitian dilakukan di BalaiPenelitian Tanaman Obat dan Aromatik dari September 2007 sampaiMaret 2008, menggunakan rancangan acak lengkap dengan 20 kaliulangan. Bahan tanaman yang digunakan adalah meristem jahe putih besaryang diambil dari rimpang panen muda dan tua. Peubah yang diamatimeliputi: histologi jaringan, persentase kalus embriogenik yang terbentuk,bobot segar kalus, diameter kalus, dan morfologi kalus. Hasil penelitianmenunjukkan adanya daerah meristematik pada sayatan eksplan meristemjahe putih besar ukuran ± 0,25 cm. Persentase kalus embriogenik (92,1%)dan diameter kalus (0,59 mm) dari rimpang yang dipanen tua lebih tinggidari yang dipanen muda. Berat kalus (1,18 g) dan jumlah embrio somatikglobular (29,34) asal eksplan panen tua nyata lebih tinggi dari yangdipanen muda. Kalus embriogenik yang berasal dari eksplan rimpang yangdipanen tua mampu berkembang membentuk embrio somatik danberkecambah menghasilkan planlet normal.</p><p>Kata kunci : Zingiber officinale Rosc., umur rimpang, kalus embriogenik,embriogenesis somatik</p><p>ABSTRACT</p><p>Effect of explants age on the success of embryogenic calliformation in meristem culture of ginger (Zingiberofficinale Rosc.)</p><p>Constraint in ginger cultivation in Indonesia is the limited qualityof planting materials. In conventional cultivation, planting materials weretaken from a piece of rhizomes. By this technique, significant amount ofplanting materials is required, between 2-3 tons/ha for fully harvested and5-6 tons/ha for young harvested rhizomes. Another serious constraint isbacterial wilt disease infection caused by Ralstonia solanacearum. Effortfor obtaining free disease planting materials could be performed throughtissue culture mass propagation. In this study, different ages of rhizome asexplants sources was evaluated for their capacity in embryogenic calliformation on the meristem culture of ginger. The experiment wasconducted in Indonesian Medicinal and Aromatic Crops Research Institutefrom September 2007 to March 2008, using a completely random designwith 20 replicates. Plant material used was white ginger meristem takenfrom the fully and young harvested rhizomes. The observed variables wereexplant histology, percentage embryogenic calli formation (%), freshweight of calli, calli diameter, number of globular embryo, and callimorphology. The results showed a meristematic region at the incisionexplant big-white ginger meristem ± 0.25 cm in size. Percentage ofembryogenic calli formation from the fully harvested rhizome-explant(92.1%) and calli diameter (0.59 mm) were higher than that of the youngerone. Calli weight (1.18 g) and number of globular somatic embryos(29.34) from fully harvested rhizome-explants were significantly higherthan that of the younger one. Embriogenic calli derived from the oldharvested rhizome explants was able to grow well to form somaticembryos and then germinate to produce normal plantlet.</p><p>Key words : Zingiber officinale Rosc, age of rhizome, embriogeniccalli, somatic embryogenesis</p>

Sterilization procedure and callus regeneration in black turmeric (Curcuma caesia)

Sterilization procedure, media composition, explants selection and control of physical environment are critical for successful cultures and callus induction with surface sterilization being very challenging in most plants. Five different sterilization methods were evaluated to come up with the best for subsequent use to establish an in vitro regeneration method for the induction of callus in Curcuma caesia using excised leaf and rhizome explants. Murashige and Skoog (MS) media supplemented with various concentration of 2,4-Dichlorophenoxy acetic acid (2,4-D)/Indole-3-acetic acid (IAA) (0.5- 5.0mg/L), singly or in combination with Benzyl aminopurine (BAP)/Kinetin (KIN) (0.1-5.0mg/L), 0.3% sucrose and 0.08% agar were used. The result of the sterilization procedures showed 15% NaHClO3 (5min) + 70% Ethanol (30s) + 0.1% HgCl2 (5min) to be the most effective in controlling contamination in C. caesia among all the treatments tested. The response to callus induction was found to depend on the type of explants used and growth regulators combination. Leaf explants gave the highest percentage of callus induction. Highest percentage of callus induction (66.70%) was obtained in the growth regulator combination of 2, 4-D (0.5mg/L) + BAP (0.1mg/L) and least (14.29%) in IAA (2.0mg/L) + BAP (0.5mg/L). Equal and higher concentration of 2, 4-D + BAP of 5.0mg/L each also provided better result (40.00%). No callus was obtained in all the single concentration of 2, 4-D used.

Organogenesis and Ultrastructural Features ofIn VitroGrownCanna indicaL.

An efficient protocol for micropropagation ofCanna indicaL., an economically and pharmaceutically important plant, was standardized using rhizome explants, excised from two-month-old aseptic seedlings. Complete plant regeneration was induced on MS medium supplemented with 3.0 mg/L BAP plus 1.5 mg/L NAA, which produced the highest number of shoots (73.3 ± 0.5%) and roots (86.7 ± 0.4%) after 2 weeks. Furthermore, the optimum media for multiple shoots regeneration were recorded on MS enriched with 7.0 mg/L BAP (33.0 ± 0.5%). Plantlets obtained were transplanted to pots after two months and acclimatized in the greenhouse, with 75% survival. In addition, ultrastructural studies showed that rhizomes ofin vitrogrown specimens were underdeveloped compared to thein vivospecimens, possibly due to the presence of wide spaces. Meanwhile, the leaves ofin vivospecimens had more open stomata compared toin vitrospecimens, yet their paracytic stomata structures were similar. Hence, there were no abnormalities or major differences betweenin vitroregenerants and mother plants.

Plant regeneration of Alstroemeria in vitro

The regenerative ability of explants from various organs of <em>Alstroemeria</em> plants was investigated. Rhizome apical and axillary tips cultured on the Murashige and Skoog medium with BA - 2. mgl^-1 and NAA - 0,5 mgl^-1 were the best among the tissue tested as initial explants. Five weeks after isolation the rhizome with 1-4 upright growing shoots were obtained. The types of rhizome explants influenced development and growth of lateral rhizomes and upright growing shoots. There were no significant differences in number of roots formed on various kind of rhizome explants. Rooting was strongly influenced by NAA. Subapical segments of vegetative stem, segments of flower pedicels and parts of ovary did not regenerate rhizome or roots but occasionally callus was formed on the medium with kinetin - 2 mgl^-1 and NAA - 2 mgl^-1. Segments excised from vegetative stem sporadically developed roots on the medium with NAA or IBA in concentrations 3 and 9 mgl^-1.

780 PB 398 BENZYLADENINE UPTAKE AND ENDOGENOUS FREE IAA LEVELS DURING SHOOT INDUCTION IN CYMBIDIUM cv. YUH HWA RHIZOMES

Long term exposure to high benzyladenine (BA) concentrations inhibited shoot elongation and root growth of Cymbidium cv. Yuh Hwa rhizome explants. Through transfer experiments, it was determined that the commitment to shoot induction occurred between 10 and 14 days of exposure to at least 2.5 μM BA. BA when supplied at 20 μM during the fit 14 days of culture was found to be sufficient to induce shoot formation. Both shoot elongation and root formation were greatly improved by decreasing the BA concentration. By using radiolabelled BA, adenine was found to be a major metabolite in the rhizome tissue. Free IAA levels, quantitated by GC-MS, did not correlate well with the organogenesis of rhizomes, nor did the levels of free IAA correlate well with the activities of peroxidase and IAA oxidase, indicating a complex relationship between hormone concentration and differentiation.