A Novel and Efficient In Vitro Organogenesis Approach for Ajuga lupulina Maxim

This work was aimed at establishing an effective approach for in vitro propagation of Ajuga lupulina Maxim, a medicinal and ornamental plant mainly found in eastern Xizang, in the western Sichuan region of China. We report an optimum response in the proliferation of axillary shoots from nodal segment explants (10.2 shoots/explant) on MS medium containing 3.0 mg L−1 of 6-benzyladenine (BA). When BA and TDZ individually or in combination with NAA were employed for adventitious shoot regeneration, shoots and embryo-like structures (ELSs) were noted in the callus from leaf explants. The maximum response of 26.4 shoots /explant (81.6%) and 12.0 ELSs/explant were ascertained on MS medium with 4.0 mg L−1 TDZ and 0.1 mg L−1 NAA. The leaf despite browning still demonstrated a high regeneration capacity. TDZ (2.0 mg L−1) and BA (2.0 mg L−1) along with NAA (0.01 mg L−1) were found to perform well for shoot regeneration via callus from shoot tip explants. The best for rooting was MS medium (half-strength) containing indole-3-butyric acid (IBA: 1.5 mg L−1) and (NAA: 0.5 mg L−1) with the maximum number of roots (25.8 per shoot) and the highest rooting frequency (81.71%). The survival of the plantlets in the greenhouse was 78.2% indicative of successful acclimatization. This work is the first report of a consistent, definitive, and unique protocol for A. lupulina regeneration, paving the way for the in vitro preservation of such significant genetic resources and also further allied systems based on such callus-based or embryo-based approaches.

Role of Biotechnology in Plant Preservation for Food Security

Overpopulation and the consequences of urbanization and reduction of agricultural lands represent the most important challenges that face scientists nowadays. In addition, extinction of specific species or reduction in their number occurs continuously in different places of the world at a rapid rate. These challenges urge scientists to use the biotechnological techniques to secure food and to alleviate the risk of loss of the genetic variability of cultivated plants as a result of environmental changes and human practices. These techniques are based on preservation of the genetic materials for long periods. Plants can be stored either in vivo or in vitro. The plant preservation includes in situ and ex situ. One form of the ex situ plant preservation is the in vitro plant preservation. There are different in vitro preservation techniques. However, the two main approaches of in vitro preservation of plants germplasm are slowing the growth and crysoperservation. The former technique could be achieved through either modifying the culture medium or reducing temperature and/or light intensity. The latter is taking place through storing the species between -79 and -196°C, the temperature of liquid nitrogen. Each approach includes several techniques that will be thoroughly discussed with examples in this chapter.

PENYIMPANAN IN VITRO TANAMAN PURWOCENG (Pimpinella pruatjan Molk.) MELALUI APLIKASI PENGENCERAN MEDIA DAN PACLOBUTRAZOL

<p>ABSTRAK</p><p>Purwoceng (Pimpinella pruatjan Molk.) adalah tanaman obatlangka asli Indonesia yang dikategorikan hampir punah. Konservasi in situtidak dapat diandalkan karena rusaknya habitat alami (hutan konservasi),sedangkan konservasi ex situ di lapang menghadapi kendala karenapurwoceng sulit dibudidayakan di luar habitat aslinya. Dengan demikian,konservasi in vitro merupakan alternatif yang dapat diterapkan untukmenghindari kepunahan tanaman purwoceng. Tujuan penelitian untukmengetahui efek dari kombinasi perlakuan pengenceran media dankonsentrasi paclobutrazol terhadap pertumbuhan kultur purwoceng, dayaregenerasi dan stabilitas genetik pasca penyimpanan. Penelitian dilakukanpada tahun 2004 di Laboratorium Kultur Jaringan, Balai Penelitian danPengembangan Bioteknologi dan Sumberdaya Genetik Pertanian, Bogorselama 9 bulan. Bahan tanaman yang digunakan bersumber dari koleksitanaman purwoceng di Kebun Percobaan Gunung Putri, Balai PenelitianTanaman Rempah dan Obat. Kegiatan penelitian mencakup: (1)Perbanyakan tunas in vitro purwoceng sebagai sumber eksplan denganmenggunakan regenerasi, yaitu media DKW + BA 1 ppm + Thidiazuron0,2 ppm + arginin 100 ppm, (2) Penyimpanan in vitro tunas purwocengdalam media DKW (1, ½, dan ¼ dosis) + paclobutrazol (0, 1, 3, dan 5ppm), (3) Regenerasi kultur purwoceng pasca penyimpanan in vitro padamedia regenerasi, dan (4) Evaluasi karakter sitologi kultur yang telahdisimpan melalui penghitungan jumlah kloroplas sel penjaga stomata.Rancangan percobaan disusun secara faktorial dalam lingkungan acaklengkap dengan 6 ulangan. Hasil penelitian menunjukkan tidak adanyainteraksi yang nyata antara pengenceran media dan konsentrasipaclobutrazol. Periode simpan kultur tidak dapat diperpanjang lebih dari 4bulan karena paclobutrazol mempunyai pengaruh penghambatan pertum-buhan yang sangat kuat sehingga sebagian besar kultur purwoceng mati.Efek residu paclobutrazol masih tampak pada jangka waktu lebih dari 4bulan pada tahap pemulihan, ditandai dengan adanya penampilan roset.Pengamatan ciri sitologi melalui penghitungan jumlah kloroplas selpenjaga stomata menunjukkan bahwa penggunaan paclobutrazol tidakmenyebabkan perubahan tingkat ploidi. Disimpulkan bahwa paclobutrazoltidak sesuai digunakan untuk penyimpanan in vitro purwoceng karenamenyebabkan pertumbuhan yang abnormal (roset) sekalipun pada tahapregenerasi pasca penyimpanan. Selanjutnya disarankan untuk mengguna-kan regulator osmotik, yang mampu meningkatkan potensi osmotik dalammedia dan memperlambat penyerapan nutrisi sehingga masa simpankemungkinan dapat diperpanjang tanpa menyebabkan pertumbuhan yangabnormal pada tahap regenerasi pasca penyimpanan.</p><p>Kata kunci : Pimpinella pruatjan Molk., penyimpanan in vitro, pengen-ceran media, dan paclobutrazol</p><p>ABSTRACT</p><p>Purwoceng (Pimpinella pruatjan Molk.) is an Indonesian medicinalplant categorized as endangered plant. In situ conservation is quiteimpossible since conservation forest has been damaged whereas ex situconservation in the field is difficult because the plant needs specificagronomical condition. In vitro conservation is therefore the only choice tobe applied. The objectives of the study were to find out the effects ofcombined treatment between media dilution and paclobutrazolconcentration to the growth of pruatjan cultures, the genetic regenerationand stability after preservation. The research was conducted at the TissueCulture Laboratory, the Indonesian Center for Agricultural Biotechnologyand Genetic Resources Research and Development for 9 months. The plantmaterials were taken from Gunung Putri. The activities included: (1)Propagation of in vitro shoots as explants source in DKW media + 1 ppmBA + 0.2 ppm Thidiazuron + 100 ppm arginin, (2) Preservation of in vitroshoots of pruatjan on DKW (full, half, and quarter strength) +paclobutrazol (0, 1, 3, and 5 ppm), (3) Regeneration of the cultures after invitro preservation, and (4) Evaluation of cytological character of preservedcultures through chloroplast guard cells counting. The experiment wasarranged factorially in Completely Randomized Design with 6replications. The result revealed that there was no interaction betweenmedia dilution and paclobutrazol concentration. Preservation period couldnot be prolonged more than 4 months because this compound stronglyinhibited the growth so that almost none of them could survive longer. Theresidual effect of paclobutrazol was still appeared more than 4 months inregeneration phase assigned by rossette performances. Observation ofcytological character through chloroplast guard cells counting revealedthat paclobutrazol could not change ploidy level of preserved pruatjancultures. It was concluded that paclobutrazol is not suitable for in vitropreservation of pruatjan since it causes abnormal growth on regenerationstep after preservation. Thus, it was suggested to use osmotic regulatorwhich can increase osmoticum potential in media and decrease nutritionabsorption so that preservation period may be prolonged without abnormaleffect on regeneration step after preservation.</p><p>Key words: Pimpinella pruatjan Molk., in vitro preservation, mediadilution, and paclobutrazol</p>

IN VITRO PRESERVATION OF RODENT TUBER (TYPHONIUM FLAGELLIFORME LODD.) PEKALONGAN ACCESSION WITH PACLOBUTRAZOL

Rodent tuber plant (Typhonium flagelliforme Lodd.), as one of the most potent medicinal plants, has to be developed as an active ingredient of degenerative drugs, including cancer drugs. However, this enormous potential must be supported by sustainable cultivation of the plant. The conventional preservation of rodent tuber can be done by planting various accessions in the field, but it will need land availability and intensive plant maintenance. Preservation through in vitro culture is an alternative method that can be used. The aim of this study was to determine the effect of paclobutrazol in suppressing the growth of rodent tuber cultures, and test the ability of culture regeneration after being preserved with paclobutrazol. The media formulation used was Murashige and Skoog (1962) (MS) with the addition of paclobutrazol at concentrations of 4, 5, 6, and 7 mg L-1. The results showed that using paclobutrazol at 5 mg L-1 was the best concentration that can inhibit the elongation of buds, seedling formation, leaf formation, and root elongation until 5 months. Cultures of paclobutrazol treatment at 5 mg L-1 had shoot heights, number of shoots, number of leaves, and root lengths of 0.49, 2.33, 7.23, and 0.3 cm, respectively. Paclobutrazol could inhibit the growth of in vitroculture of rodent tuber and prolong the shelf life of culture up to 5 months. The culture of rodent tuber from paclobutrazol treatment had normal growth and regenerative ability after transfer to the medium regeneration.

Konsentrasi Sukrosa Dan Agar Di Dalam Media Pelestarian In-Vitro Ubi Jalar Var. Sukuh

The objective of this research was to obtain suitable concentration of sucrose and agar in the sweet potato (Ipomea batatas (L) Lam) in vitro preservation medium, in order to make plant grow slowly and healthy. Experiment was done in Molecular Biologi Laboratory of PAU IPB. The experiment was arranged in factorial complete random design, using sweet potato var. Sukuh in order to know the effect of sucrose (40,50,60,70 and 80 g l-1) and agar (7 and 8 g l-1) which were added 1 g l-1 hyponex fertilizer 20-20-20. The experiment was replicated four times. Data were analyzed parametrically and non-parametrically. The result of these experiments showed the suitable conservation media was 1 g l-1 hyponex 20-20-20 + 60 g l-1sucrose + 7 g l-1 agar. Threfore, the media composition MS could produce high green inter nodes number, high root number and more than two green leaf number. This experiment also showed that MS media could be replaced by a cheaper and easily found conservation medium.

Pelestarian Secara In Vitro Melalui Metode Pertumbuhan Lambat Pada Beberapa Genotipe Ubi Jalar (Ipomea Batatas (L) Lam)

The objectives of this research were to estimate responses of several sweet potato genotypes in preservation media through in vitro slow growth preservation, and to achieve cheap and accessible media. Experiment was arranged in a completely randomized design with a single factor. The experimental factor was sweet potato genotype (Sukuh, 421.34,343.15 and 2040.8). These genotype were tested in a preservation media which consisted of 15% coconut water + 30 mg/l aspirin + 50 g/l sucrose + 7 g/l agar. Experiment was replicated four times. Data was analyzed parametrically and non-parametrically. The result showed that genotype gave high responses to preservation media. Genotype Sukuh produced highest leaves while genotype 421.34 yielded highest numbers of root and internode. In the preservation medium of Genotype Sukuh through in vitro slow growth preservation, aspirin could be added to inhibit growth by increased leaf senesence. This experiment also showed that Hiponex (20:20:20) could be used as the basic media for in vitro preservation of sweet potato