High efficient de novo root-to-shoot organogenesis in Citrus jambhiri Lush.: Gene expression, genetic stability and virus indexing

A protocol for high-frequency direct organogenesis from root explants of Kachai lemon (Citrus jambhiri Lush.) was developed. Full-length roots (~3 cm) were isolated from the in vitro grown seedlings and cultured on Murashige and Skoog basal medium supplemented with Nitsch vitamin (MSN) with different concentrations of cytokinin [6-benzylaminopurine, (BAP)] and gibberellic acid (GA3). The frequency of multiple shoot proliferation was very high, with an average of 34.3 shoots per root explant when inoculated on the MSN medium supplemented with BAP (1.0 mg L–1) and GA3 (1.0 mg L–1). Optimal rooting was induced in the plantlets under half strength MSN medium supplemented with indole-3-acetic acid (IAA, 0.5–1.0 mg L–1). IAA induced better root structure than 1-naphthaleneacetic acid (NAA), which was evident from the scanning electron microscopy (SEM). The expressions of growth regulating factor genes (GRF1 and GRF5) and GA3 signaling genes (GA2OX1 and KO1) were elevated in the regenerants obtained from MSN+BAP (1.0 mg L-1)+GA3 (1.0 mg L-1). The expressions of auxin regulating genes were high in roots obtained in ½ MSN+IAA 1.0 mg L-1. Furthermore, indexing of the regenerants confirmed that there was no amplicons detected for Huanglongbing bacterium and Citrus tristeza virus. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers detected no polymorphic bands amongst the regenerated plants. This is the first report that describes direct organogenesis from the root explant of Citrus jambhiri Lush. The high-frequency direct regeneration protocol in the present study provides an enormous significance in Citrus organogenesis, its commercial cultivation and genetic conservation.

OPTIMASI NAA DAN BAP TERHADAP PERTUMBUHAN DAN PERKEMBANGAN TUNAS MIKRO TANAMAN KANTONG SEMAR (Nepenthes mirabilis) SECARA IN VITRO

Conventional propagation of Nepenthes was difficult to do. To overcome the problems were required alternative method such as in vitro propagation. The objective of this research was to obtain the best treatment of BAP + NAA on shoot multiplication of Nepenthes through in vitro culture. The research design used Randomized Completely Design consist of seven treatments, e.g. 1) ½ MS0 (control); 2) ½ MS + 1 ppm BAP + 0.5 ppm NAA; 3) ½ MS + 1 ppm BAP + 1 ppm NAA; 4) ½ MS + 1.5 ppm BAP + 0.5 ppm NAA; 5) ½ MS + 1.5 ppm BAP + 1 ppm NAA; 6) ½ MS + 2 ppm BAP + 0.5 ppm NAA dan 7) ½ MS + 2 ppm BAP + 1 ppm NAA. The parameter observed were number of shoot, number of nodul, number of leafs, number of pitcher and number of root. The result of this research showed that treatment of ½ MS + 1 ppm BAP + 1 ppm NAA is the best treatment compared to others. At induction stage, this treatment can produce the number of shoot, number of nodul, and number of root were 1.6 shoots/explant, 10.8 nodul/explant and 3.6 root/explant, respectively. At subculture, this treatment can produce the number of shoot, number of leafs, and number of pitcher were 5.8 shoots/explant, 12.4 leafs/explant and 5.2 pitcher/explant, respectively.

Improving an in vitro propagation protocol for Cestrum nocturnum L.

The present study was carried out to assess the micropropagation of <em>Cestrum </em>(<em>Cestrum nocturnum<strong> </strong></em>L.) by using single nodes and shoot tips excised from soft cuttings using MS salts, 30 g × l^-1 sucrose, 7 g × l^-1 agar, and different concentrations of plant growth regulators in culture medium.<strong> </strong>The results revealed that the use of mercuric chloride (0.05%, HgCl₂) for 7 minutes was very effective in preventing contamination and gave the highest survival percentage (99%). The highest response (100%) was gained at initiation stage from lateral bud explants on MS medium supplemented with 1.5 mg × l^-1 of BA with most of NAA concentrations. However, in case of terminal buds, higher percentages of responses were resulted from the interaction of BA (1.5 mg × l^-1) with 0.2 mg × l^-1 NAA. The lateral buds also produced more new shoots as well as a higher number of leaves and length of new shoots on the medium supplemented with 1.5 mg × l^-1 BA as compared with those from terminal buds. Significant differences were observed at multiplication stage between the lateral buds and terminal buds, since the lateral buds produced a higher number of new shoots and leaves as well as longer new shoots. At rooting stage, the treatment with 1 mg × l^-1 IBA gave the highest percentage of rooting (100%), the highest number of roots (13.2 root/explant), and the longest roots (8.44 cm), respectively, on half strength MS medium. Plantlets obtained were transferred to pots and acclimatized with 90% success.

Bulblet regeneration from ex vitro root explant in lily hybrids

The influence of growth regulators on <i>in vitro</i> bulblet formation from <i>ex vitro</i> roots was studied in asiatic and oriental hybrids of <i>Lilium</i> The root segments (3–4 mm) isolated from the middle zone of 2–3 cm <i>ex vitro</i> root were cultured on Murashige and Skoog (MS) medium containing 1 or 1.5 mg/dm³ naphthalene acetic acid (NAA) and/or benzyladenine (BA). Bulblets were not produced in the presence of NAA and BA alone. A significant increase in the per cent explants producing bulblets was observed with 1.5 mg/dm³ NAA and 1 mg/dm³ BA. Maximum number of bulblets and average fresh weight per bulblet was observed with 2 mg/dm³ NAA and 1.5 mg/dm³ BA after 90 days of culture. No differences were found among cultivars in bulblet regeneration of explant or bulblet number although more weighty bulblets occurred in cv. Apeldoorn. About 82% bulblet survival was recorded in coco peat after 30 days of transfer to pots.