Gentianella lutescens subsp. carpatica J. Holub.: Shoot Propagation In Vitro and Effect of Sucrose and Elicitors on Xanthones Production

In vitro shoot culture of the endangered medicinal plant Gentianella lutescens was established from epicotyl explants cultured on MS basal medium with 0.2 mg L−1 6-benzylaminopurine (BA) and evaluated for xanthones content for the first time. Five shoot lines were obtained and no significant variations in multiplication rate, shoot elongation, and xanthones profile were found among them. The highest rooting rate (33.3%) was achieved by shoots treated for 2 days with 5 mg L−1 indole-3-butyric acid (IBA) followed by cultivation in liquid PGR-free ½ MS medium for 60 days. HPLC analysis revealed the lower content of xanthones—mangiferin, bellidifolin, demethylbellidifolin, demethylbellidifolin-8-O-glucoside and bellidifolin-8-O-glucoside—in in vitro cultured shoots compared to wild growing plants. The increasing concentration of sucrose, sorbitol and abiotic elicitors salicylic acid (SA), jasmonic acid (JA) and methyl jasmonate (MeJA) altered shoot growth and xanthone production. Sucrose and sorbitol applied at the highest concentration of 233.6 mM increased dry matter percentage, while SA at 100 μM promoted shoot growth 2-fold. The increased sucrose concentration enhanced accumulation of xanthones in shoot cultures 2–3-fold compared to the control shoots. Elicitors at 100–300 μM increased the accumulation of mangiferin, demethylbellidifolin-8-O-glucoside, and bellidifolin-8-O-glucoside almost equally, while MeJA at the highest concentration of 500 μM enhanced amount of aglycones demethylbellidifolin and bellidifolin 7-fold compared to the control. The obtained results facilitate conservation of G. lutescens and pave the way for further research on large-scale shoot propagation and production of pharmacologically active xanthones.

GIS-Facilitated Effective Propagation Protocols of the Endangered Local Endemic of Crete Carlina diae (Rech. f.) Meusel and A. Kástner (Asteraceae): Serving Ex Situ Conservation Needs and Its Future Sustainable Utilization as an Ornamental

Conservation and sustainable exploitation of threatened endemic plants with medicinal and/or horticultural/ornamental value can be achieved through the development of effective propagation protocols. After unveiling the bioclimatic preferences of Carlina diae (Asteraceae) with geographic information systems (GIS), four propagation trials were conducted using seeds of this endangered local Cretan endemic for in vivo and in vitro germination, as well as seasonal vegetative propagation trials (softwood cuttings) and micropropagation (nodal explants). Seed germination was accomplished at a level of 77–90% in vivo (30 days) and 96% in vitro (10 days) using an MS medium with 2.9 μM gibberellic acid (GA3). The optimum treatments for cuttings’ rooting were 1000 and 2000 ppm indole-3-butyric acid (IBA) (11–16 roots, 2–3 cm long, 100% rooting) within 40 days in mist. In vitro shoot propagation exhibited a 2.8 proliferation rate after six successive subcultures on an MS medium with 2.9 μM GA3. Both ex vitro rooting and acclimatization were successful in 40 days, with 96% microshoot rooting and an equal survival rate. The GIS-facilitated effective species-specific propagation protocols developed in this study can consolidate the perspective of successful re-introduction of ex situ-raised material of C. diae into wild habitats and may serve its sustainable exploitation for high-added value ornamental products.

In vitro germination and bulblet and shoot propagation for wild edible Eremurus spectabilis M.Bieb.

Eremurus spectabilis M.Bieb is consumed as a vegetable because of its nutritious characteristics. The plants are also used for medicinal purposes, in the cut flower industry as an ornamental geophytes, and in industry as a natural adhesive. The aim of the present study was to improve the in vitro propagation protocol (in vitro germination and bulblet and shoot formation) of E. spectabilis. For this purpose, E. spectabilis seeds were in vitro germinated in four different nutrient media: Murashige and Skoog (MS), Gamborg (B5), White (WH), and Shenk and Hildebrandt (SH). To stimulate bulblet and/or shoot regeneration, hypocotyls of 35-40-day-old in vitro-germinated plantlets were cut into 0.5-1.0 cm pieces, and the resultant explants were cultured in MS media containing 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5, 1.0, 2.0, and 4.0 mg L-1) + Kinetin (0.5 mg L-1), Thidiazuron (TDZ) (0.5, 1.0, 2.0, and 4.0 mg L-1) + 1-Naphthylacetic acid (NAA) (0, 0.1, 0.5, and 1.0 mg L-1), and 6-Benzylaminopurine (BAP) (0.5, 1.0, 2.0, and 4.0 mg L-1) + 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.1, 0.5, and 1.0 mg L-1). The best outcomes for germination ratio (57.5%) were obtained from the B5 medium. In the third set of in vitro propagation experiments, 100% bulblet formation was achieved in TDZ (0.5 mg L-1) and NAA (0.5 and 0.1 mg L-1) combinations of MS media, and this was followed by 0.5 mg L-1 BAP-containing medium (81.3%). Shoot formation ratios with the same media combinations varied from 60-70%, and the number of shoots per explant varied from 1.4-2.4 shoots. Further in vitro propagation research is planned with larger bulb sizes to develop a protocol for rooting bulblets and/or shoots.

MIKROPROPAGASI TANAMAN TALAS BENENG (Xanthosoma undipes K. Koch) DENGAN PERLAKUAN BENZIL AMINOPURIN, TIAMIN, DAN ADENIN

Micropropagation of Beneng Taro (Xanthosoma undipes K. Koch) with Benzyl Amino Purine, Thiamine, and Adenine TreatmentABSTRACTConventional production of Beneng taro seeds (Xanthosoma undipes K. Koch) is constrained by the limited number of tubers, thus an alternative solution is needed such as in vitro propagation. This study was aimed to obtain a micropropagation technique of Beneng taro on MS media with BAP, thiamine, and adenine treatment, and to determine its growth at the acclimatization stage. This research consisted of shoot multiplication and acclimatization. Shoot propagation was carried out on MS media with 8 treatments, namely ½MS and MS without addition of growth promoting substance, and MS with 1, 2 and 3 mg×L-1 BAP, with or without addition of 1 mg×L-1 thiamine and 2 mg×L-1 adenine. Each treatment was replicated four times, each consisting of four shoots. Growth observation was made from 1st to 5th week on petiole length, and number of shoots, leaves and roots. Acclimatization was carried out on soil media, compost, and husks in a ratio of 1: 1: 1. The results showed that the best media for shoot multiplication was MS + 1 mg×L-1 BAP + 1 mg×L-1 thiamine + 2 mg×L-1 adenine with an average of 3.5 shoots, while the best medium for the petiole length was ½MS with an average value of 6.97 cm. The results of acclimatization showed that 100% planlets survived, and plantlets grown on MS media + 3 mg×L-1 BAP had the highest number of shoots with an average of 4.2.Keywords: adenine, Beneng taro, benzil amino purine (BAP), micropropagation, thiamineABSTRAKPenyediaan bibit talas Beneng (Xanthosoma undipes K. Koch) secara konvensional terkendala terbatasnya jumlah umbi, sehingga perlu solusi alternatif, diantaranya melalui perbanyakan in vitro. Tujuan penelitian ini adalah untuk mendapatkan teknik mikropropagasi talas beneng pada media MS dengan perlakuan BAP, tiamin, adenin, dan untuk mengetahui pertumbuhannya pada tahap aklimatisasi. Penelitian ini meliputi perbanyakan tunas dan aklimatisasi. Perbanyakan tunas menggunakan media MS dengan 8 perlakuan yaitu ½MS dan MS tanpa penambahan zat pengatur tumbuh (ZPT), serta MS dengan 1, 2 dan 3 mg×L-1 BAP dengan atau tanpa penambahkan 1 mg×L-1 tiamin dan 2 mg×L-1 adenin. Setiap perlakuan mempunyai empat ulangan, setiap ulangan terdiri atas empat tunas. Pertumbuhan diamati mulai minggu ke-1 hingga ke-5 terhadap panjang petiol serta jumlah anakan, daun dan akar. Aklimatisasi dilakukan pada media tanah, kompos dan sekam dengan perbandingan 1:1:1. Hasil menunjukkan bahwa media terbaik perbanyakan tunas adalah MS + 1 mg×L-1 BAP + 1 mg×L-1 tiamin + 2 mg×L-1 adenin dengan rata-rata 3,5 tunas, sedangkan media terbaik untuk panjang tangkai daun adalah ½MS dengan nilai rata-rata 6,97 cm. Hasil aklimatisasi menunjukkan bahwa 100% planlet hidup dan planlet yang ditumbuhkan pada media MS + 3 mg×L-1 BAP mempunyai jumlah anakan terbanyak dengan rata-rata 4,2.Kata Kunci: adenine, benzil amino purin (BAP), mikropropagasi, talas Beneng, tiamine

PENGARUH WADAH KULTUR DAN KONSENTRASI SUMBER KARBON PADA PERBANYAKAN KENTANG ATLANTIK SECARA IN VITRO

The Effect of Culture Container and Carbon Source Concentration on In Vitro Shoot Propagation of Atlantic PotatoABSTRACTPotato is a food commodity that has the potential to support food diversification in Indonesia. There is an increasing demand for Atlantic potatoes as the raw material for processed potato products. The demand, which has not been met by the increased production, has been the cause of the ongoing potato import activities in Indonesia. The limitation of producing quality Atlantic potato seeds economically is one of the obstacles to increasing the production of Atlantic potatoes in Indonesia. The aim of this research was to study the effect of various table sugar concentrations as the carbon source and the type of the culture containers used for Atlantic potato shoot multiplication in vitro. The propagation was carried out in bioreactors and culture bottles with MS liquid medium + coconut water at a concentration of 150 mL/L medium, and 3 concentration levels of table sugar, namely 0; 7.5; and 15 g/L medium. The use of bioreactor significantly increased the height of the Atlantic potato plantlets. The use of bioreactor combined with table sugar addition decreased hyperhydricity level. The highest number of shoots, leaves, and roots were found at the table sugar concentration of 15 g/L medium in both containers.Keywords: bioreactor, micropropagation, shoot culture, Solanum tuberosum, sucrose ABSTRAKKentang merupakan komoditas pangan yang berpotensi mendukung program diversifikasi pangan di Indonesia. Peningkatan permintaan terhadap kentang Atlantik sebagai bahan baku kentang olahan yang tak diimbangi dengan peningkatan produksi kentang Atlantik menjadi penyebab masih berlangsungnya impor kentang Atlantik di Indonesia. Keterbatasan menghasilkan benih kentang Atlantik berkualitas yang ekonomis merupakan salah satu hambatan dalam meningkatkan produksi kentang Atlantik di Indonesia. Penelitian ini bertujuan mempelajari pengaruh variasi konsentrasi sukrosa teknis sebagai sumber karbon dan penggunaan jenis wadah terhadap perbanyakkan tunas kentang Atlantik secara in vitro. Perbanyakkan tunas kentang Atlantik menggunakan media MS cair + 150 mL/L air kelapa dalam wadah bioreaktor dan botol kultur dengan 3 taraf konsentrasi sukrosa, yaitu 0; 7,5; dan 15 g/L media. Penggunaan bioreaktor secara signifikan meningkatkan tinggi planlet kentang Atlantik yang dihasilkan. Penggunaan bioreaktor yang dikombinasikan dengan penambahan sukrosa teknis menurunkan tingkat hiperhidrisitas. Tunas, daun, dan akar terbanyak dihasilkan oleh perlakuan sukrosa teknis 15 g/L media dalam kedua jenis wadah.Kata Kunci: bioreaktor, kultur tunas, mikropropagasi, Solanum tuberosum, sukrosa

PENGARUH AUKSIN DAN SITOKININ TERHADAP PERBANYAKAN MIKRO TANAMAN BINAHONG (Anredera cordifolia (Tenore) Steenis)

EFFECT OF AUXIN AND CYTOKININ ON MICROPROPAGATION OF BINAHONG (Anredera cordifolia (Tenore) Steenis)Binahong (Anredera cordifolia (Tenore) Steenis) is known as miraculous medicinal plant for its potential to cure for various kinds of diseases such as diabetes, stabilizing blood pressure and circulation, accelerating wound healing, and preventing stroke. In order to provide high quality seedlings of this medicinal plant continuously in large amount, the study on binahong micropropagation was performed. Plant growth regulators of auxins and cytokinins were applied in single or in combination so as to observe their effect on the growth of binahong explants. The results showed that 2,4-D induced callus formation in large diameter on all treatments. Nevertheless, this plant growth regulator had a negative effect on growth and development of shoot and leaves. In the combination treatments between IAA and BAP, it revealed that the higher the concentration of BAP in the media, the lower the number of leaves initiated on shoot explants. Increasing the concentration of IAA upto 1.5 ppm influenced the increasing of shoot tallness and the number of internodes. Our results can be useful for improving the binahong shoot propagation efficiency, as well as callus culture studies.Keywords: Auxin, cytokinin, callus, micropropagation, medicinal plantABSTRAKBinahong (Anredera cordifolia (Tenore) Steenis) dikenal sebagai tanaman obat ajaib karena dapat digunakan untuk pengobatan berbagai macam penyakit seperti diabetes, melancarkan peredaran dan tekanan darah, mempercepat penyembuhan luka, mencegah stroke. Dalam mendukung ketersediaan bibit tanaman obat herbal yang berkualitas secara berkelanjutan maka dilakukan kajian tentang perbanyakan bibit tanaman binahong. Zat pengatur tumbuh auksin dan sitokinin dalam bentuk tunggal maupun kombinasi diaplikasikan pada penelitian ini untuk melihat pengaruhnya terhadap berbagai eksplan binahong. Hasilnya menunjukkan bahwa 2,4-D merangsang pembentukan kalus dengan ukuran yang besar pada semua perlakuan. Namun demikian zat pengatur tumbuh ini memberikan pengaruh negatif terhadap pertumbuhan dan perkembangan tunas dan daun. Dari perlakuan kombinasi zat pengatur tumbuh IAA dan BAP, pertambahan konsentrasi BAP di dalam media menurunkan jumlah daun yang terbentuk pada eksplan pucuk binahong. Demikian pula dengan pertambahan konsentrasi IAA hingga 1,5 ppm sangat berpengaruh terhadap pertumbuhan meninggi tunas dan pertambahan jumlah ruas. Hasil dari studi ini dapat dimanfaatkan untuk studi lanjutan dalam meningkatkan efisiensi perbanyakan tunas serta kultur kalus binahong.Kata kunci: Auksin, sitokinin, kalus, perbanyakan mikro, tanaman obat herbal

Somatic embryogenesis and in vitro shoot propagation of Gentiana utriculosa

Study describes protocols for

MICROPROPAGATION AND ACCLIMATIZATION OF Aegiphila verticillata Vell.: AN ENDANGERED WOODY SPECIES

The objective of this work was to establish an efficient protocol for in vitro multiplication and rooting, as well as ex vitroacclimatization of Aegiphila verticillata, a woody species found in Brazilian rocky fields. Aseptic cultures were established by seeds and two multiplication analyses were performed. In the first, we employed 6-benzylaminopurine (BAP – 0, 2.5, 5 and 7.5 μM) + α-naphthalene acetic acid (NAA – 0, 0.2, 0.4 and 0.6 μM) and, in the second, were studied adenine sulfate, kinetin and thidiazuron (0, 5, 7.5, 10 and 12.5 μM). After 90 days, we assessed the quantitative and qualitative shoot propagation. There were more than 90% seed germination and low contamination (2%). In multiplication phase, the culture medium that promoted the best quantitative and qualitative culture development was supplemented with 7.5 μM BAP + 0.4 μM NAA. In the rooting assay, were used NAA, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) (0, 0.1, 0.2, 0.3 or 0.4 μM). After 90 days, the root number and rooting quality were evaluated. In this analysis, differences were not found between the control and the other treatments. Rooted plantlets were acclimatized in styrofoam trays for 30 days, after which they were transferred to pots in the greenhouse. Only 3% of the plants subjected to initial acclimatization died and 70% of the plants transferred to the field conditions survived and showed normal development. The results founded in this work are the first involving in vitro propagation and ex vitroacclimatization of Aegiphila verticillata and provide a continuous supply of this medicinal native species, endangered due anthropogenic activities.