Efficacy of DNA barcode internal transcribed spacer 2 (ITS 2) in phylogenetic study of Alpinia species from Peninsular Malaysia.

Alpinia belongs to a large genus with many species found in Peninsular Malaysia. Several species of Alpinia exhibit important medicinal potential. However, progressive studies on the genus Alpinia were hampered by difficulties encountered in species identification. With the advancement achieved in genomic technology, more sensitive tools such as DNA barcoding were developed, which can be used for species identification. Internal Transcribe Spacer 2 (ITS2) is a DNA barcode which has proven to be a promising tool for species identification. The criterions of ITS2 efficacy namely universality and efficacy for species identification were tested on Alpinia species collected from Peninsular Malaysia. The results showed that a success rate of 96.97% was achieved using ITS2 for screening 11 species of Alpinia and an outgroup sample (Zingiber specatabile). Combined with 15 additional sequences from the Genbank for five Alpinia species, ITS2 demonstrated high species identification efficacy with 88.2% of species identified using phylogenetic and distance analysis. The analysis was further improved with the use of ITS2 secondary structure. The results of both criterions demonstrated the ability of ITS2 to successfully discriminate Alpinia species, which will help to improve species identification of Alpinia species in Peninsular Malaysia.

Exogenous Cytokinin Induces Callus and Protocorm-Like-Bodies Formation in In Vitro Root Tips of Vanilla planifolia Andrews.

Vanilla is a popular flavouring essence derived from the pods of vanilla orchid plants. Due to the high demand for vanilla flavour, high yielding vanilla plantlets are necessary for establishing vanilla plantations. Clonal micropropagation is a viable technique for the mass production of high yielding vanilla plantlets. This study reports an efficient regeneration protocol by using cytokinin as the sole plant growth regulator to regenerate plantlets from the root tips of a commercial vanilla orchid species, Vanilla planifolia. Most studies to date have reported using seeds and nodes as starting explants for in vitro micropropagation of vanilla orchids. So far, regeneration from roots has not been very successful. Previous studies favoured the use of auxins only or high auxin to cytokinin ratios to induce callus, and sole cytokinins were used for direct shoot regeneration. However, it was sporadically observed in plantlets regeneration of V. planifolia that multiple shoots were regenerated from the tips of intact aerial roots submerged in media. This study therefore investigated the regeneration of excised vanilla root tips through the application of most commonly used auxins (1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid) and cytokinins (6-benzylaminopurine and thidiazuron). High auxin presence is known to promote callusing in in vitro plants. However, in this study, auxin treatment inhibits callusing in root tips. While cytokinin treatments, even at low levels, has promoted high rate of callusing. These callus cells regenerate into protocorm-like-body (PLB) shoots when cytokinin levels are increased to 0.5 mg/mL 6-benzylaminopurine (BAP) under light conditions. The findings of the study have the potential of providing large quantity of high yielding vanilla plantlets through clonal micropropagation.

The biology and in vitro propagation of the ornamental aquatic plant, Aponogeton ulvaceus.

Aponogeton ulvaceus Baker (Aponogetonaceae) is a commercially important ornamental aquatic plant species with traditional medicinal uses. Due to the low survival rate of seedlings, propagation by conventional means has been met with many difficulties. In this study, botanical aspects of A. ulvaceus were examined with regards to the morphology, anatomy and physiology of the plant and an efficient protocol for its in vitro propagation using immature tuber explants has been established. The existence of glandular trichomes on the leaves was discovered and the occurrence of circumnutation in A. ulvaceus has been demonstrated. Immature tuber segments with meristems were cultured on MS medium supplemented with various combinations (0, 1, 2, and 3 mg/L) of BAP and NAA for callus induction. The highest percentage of callus production (100 %) was obtained in two different treatments: 1 mg/L BAP and 3 mg/L NAA, and 2 mg/L BAP and 3 mg/L NAA. For shoot and root organogenesis, the combination of 1 mg/L BAP and 1 mg/L NAA was shown to be significant for A. ulvaceus regeneration when compared to control, which yields a mean shoot and root number of 22.50 and 29.50 respectively. The current protocol is the first reported successful establishment of in vitro clonal propagation of A. ulvaceus.