In-vitro Regeneration from Direct and Indirect Organogenesis of Crescentia alata Kunth an Important Multipurpose Medicinal Tree

In this present work, an in-vitro regeneration protocol for Crescentia alata (C. alata) was developed using various explants on Murashige and Skoog (MS) medium augmented with different concentrations and combinations of plant growth regulators (PGRs) for direct and indirect regeneration. The direct organogenesis was established from nodes and internodes on MS medium supplemented with cytokinins and auxins. The indirect organogenesis via callus phase was obtained from leaf, nodes and internodes on MS medium supplemented with different concentrations of PGRs. The high frequency shoot organogenesis were achieved directly from nodal explants were cultured on MS medium supplemented with 3.0 mg/L BAP+0.5 mg/L KIN +1.0 mg/L NAA. Indirect organogenesis callogenic frequency was optimized at the concentration of MS medium containing 1.0 mg/L BAP + 5.0 mg/L IAA. The callus was obtained from all the explants were used, among these explants internodal explants gave best result on MS medium supplemented with different concentrations of cytokinins and auxins for indirect organogenesis experiment. Indirect organogenesis the highest number of shoot regeneration was obtained in MS Basal Medium with 4.0 mg/L BAP + 0.5 mg/L KIN + 2.0 mg/L NAA from internodal explants. For root formation the regenerative shoots which were sub cultured on MS medium containing different ratios of auxins. The rooted plantlets were transferred successfully to the pots containing sterilized soil and were successfully hardened at greenhouse condition for 20 days then exposed to the natural environment. This is the first successful micropropagation report of an efficient and rapid in-vitro clonal propagation protocol for C. alata by direct and indirect shoot organogenesis through various explants, which can be employed for conservation of this important medicinal tree species as well as the utilization of an biologically important active biomolecules. This protocol can be very useful to obtain plants from various explants, without the requirement of meristematic regions, enabling the obtainment of a higher number of plants in short period.

In vitro clonal propagation and efficient acclimatization with use of hydrogel of intensively sweet medicinal plant Lippia dulcis Trev.

Summary Introduction: The leaves of Lippia dulcis contain high amounts of hernandulcin. It is one thousand fold sweeter than sucrose, however, it hardly contains any calories. Objective: The aim of this research was to optimalisation of micropropagation and acclimatization of L dulcis Methods: The nodal explants were inoculated on phytohormone-free MS medium. After 6 weeks the explants were inoculated onto the MS medium with different plant growth regulators. Well-developed rooted plantlets were adapted to ex vitro conditions using hydrogel. Results: On the medium with BAP and NAA the highest number of shoots were produced. The higest average shoot length, number of the leaves and the leaf area were recorded on the medium with GA3. Adding IBA increased the number of roots. The addition of hydrogel enhanced the acclimatization efficiency. Conclusions: There was observed a positive, stimulating influence of growth regulators on mass propagation and increase in the number of leaves and the leaf area and influence of hydrogel on the development of plantlets during acclimatization.

CALLUS INDUCTION FROM LEAF EXPLANT OF FICUS DELTOIDEA VARKUNSTLERI

Ficus deltoidea or commonly known as ‘mas cotek’ is a herbal plant indigenous to Southeast Asia including Malaysia and Indonesia. This plant is popular for its medicinal values such as improve blood circulation, regain energy and enhance fertility naturally for both men and women. The main objective of this study is to develop in vitro clonal propagation method for rapid production of F. deltoidea using different concentrations of benzyl aminopurine (BAP) through shoot induction and multiplication, rooting and subsequent establishment in soil following acclimatization. Surface sterilization of the leaf explants was done using mercury chloride and ethanol as the disinfectants. Pre-treatment of the explants with carbendazim successfully reduced the occurrence of fungal contamination. At the end of the experiment, no shoot and root induction were observed but calli were successfully induced on MS medium containing 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l BAP, with calli induced from 3.0 mg/l BAP were bigger and healthier. In short, the higher the concentration of BAP used, the higher tendency for the explant to induce callus.

In vitro clonal propagation, organogenesis and somatic embryogenesis in Bacopa monnieri (L.) Wettst

Bacopa monnieri (L.) Wettst is a well-known medicinal herb in the Ayurveda. It is also used as laxative and curative for ulcers, inflammation, anaemia, scabies, leucoderma, asthma and epilepsy, enlargement of spleen, leprosy and others. In vitro propagation and regeneration through somatic embryogenesis of B. monnieri has played an important role in the production of healthy, disease-free plants with desirable traits. In B. monnieri, there are few reports which indicate rapid regeneration and somatic embryogenesis. For in vitro clonal propagation, the highest shoot formation was obtained when BAP 2 mg/ l used. The best response for rooting was obtained in IAA 1.0 mg/ l. The recorded survival rate of the plants was 70%. Plants were without any detectable phenotypic variations. Cytological study indicated that the chromosome number remain same (2n= 64) in in vitro and in vivo roots. A rapid, simple and efficient protocol for plantlet regeneration was achieved through embryogenic callus from leaf explants of B. monnieri. Callus induction and embryogenesis were significantly affected by presence/absence and type and concentration of growth regulators. Best organogenic callus induction was obtained in MS medium supplemented with BAP 5mg/ l. For induction of somatic embryogenesis, auxin (2, 4-D 1 mg/ l) was used in the culture medium subsequently in basal media for embryo maturation. Kn 0.2 mg/ l was the best for production of plantlet from embryo. Thus, this can be an easiest protocol for stable clonal propagation and plant regeneration through somatic embryogenesis in B. monnieri. The protocol used here for propagation and regeneration is much easier, low cost and reliable.

Studi Kualitas Regeneran Phalaenopsis Hasil Kultur In Vitro dari Eksplan Tangkai Infloresen, Tunas Pucuk, dan Empulur (The Quality Study of Phalaenopsis Regenerants from In Vitro Propagation of Inflorescence, Shoot Tip, and Pith Explants)

<p>Anggrek <em>Phalaenopsis</em> memiliki nilai komersial yang tinggi, karena keindahannya dapat dinikmati sepanjang tahun. Hal tersebut berdampak pada kebutuhan benih tanaman yang semakin meningkat. Salah satu cara penyediaan benih secara massal adalah melalui perbanyakan klonal secara in vitro sehingga perlu dilakukan studi kualitas regeneran hasil perbanyakan klonal untuk menjamin ketersediaan benih dengan kualitas baik. Penelitian bertujuan menguji kualitas regeneran yang dihasilkan dari perbanyakan klonal secara in vitro beberapa varietas Phalaenopsis dengan menggunakan eksplan yang berbeda. Penelitian dilaksanakan di Laboratorium Kultur Jaringan, Kebun Percobaan Segunung, Balai Penelitian Tanaman Hias (Balithi) sejak bulan Januari 2014 hingga Mei 2015. Penelitian menggunakan dua faktor, yaitu varietas (Ayu Lestari, Ayu Pratiwi, dan Karindra) dan jenis eksplan (tangkai infloresen, tunas pucuk, dan empulur). Percobaan disusun menggunakan rancangan acak kelompok pola faktorial dan setiap perlakuan diulang tiga kali. Hasil penelitian menunjukan tidak terjadi interaksi yang nyata antara faktor jenis eksplan dan varietas yang diujikan pada semua tahap percobaan. Respon terbaik diperoleh pada eksplan empulur dengan 42,85% eksplan berhasil membentuk kalus pada minggu ke-8 dan hampir 100% kalus tersebut dapat beregenerasi menjadi tunas pada minggu ke-24 dengan tingkat multiplikasi tunas 1,87 kali. Pada minggu ke-32 terbentuk rata-rata 3,13 daun per planlet dengan 2,47 cm panjang daun, 1,36 cm lebar daun, 1,52 akar per planlet, dan panjang akar per planlet mencapai 1,26 cm. Kerapatan stomata memiliki korelasi negatif dengan tingkat abnormalitas planlet. Planlet dengan kerapatan stomata tertinggi dan abnormalitas yang rendah diperoleh pada var. Karindra dan planlet yang berasal dari eksplan empulur dan tunas pucuk. Setelah 8 minggu tahap aklimatisasi, tingkat keberhasilan hidup tertinggi (92%) diperoleh pada tunas yang berasal dari eksplan empulur. Penelitian membuktikan bahwa perbedaan varietas tidak memiliki pengaruh nyata pada tingkat abnormalitas regeneran dan dari eksplan empulur diperoleh jumlah regeneran tertinggi dengan kualitas baik (tingkat abnormalitas rendah).</p><p><strong>Keywords</strong></p><p>Kultur jaringan; Kualitas regeneran; <em>Phalaenopsis</em>; Jenis eksplant</p><p><strong>Abstract</strong></p><p><em>Phalaenopsis</em> orchids have a high commercial value, because of its beauty and it can be enjoyed throughout the year. This condition gives the impact on the increasing demand of the seeds. One of the ways of providing mass seeds is through in vitro clonal propagation. However, it is necessary to study the quality of regenerants of clonal propagation products to ensure the availability of qualified seeds. The aimed of this study was to test the quality of regenerants obtained from in vitro clonal propagation of Phalaenopsis using inflorescence stalk, shoot tips, and pith explants. This research was conducted at Tissue Culture Laboratory, Segunung Experimental Station, Indonesian Ornamental Crops Research Institute (IOCRI) from January 2014 to May 2015. The study used two treatments, varieties (Ayu Lestari, Ayu Pratiwi, and Karindra) and type of explant (inflorescence stalk, shoot tips, and pith). Experiments were prepared using a randomized complete block design with two factors and each treatment was replicated three times. The results showed there were no significant interaction between types of explants and varieties tested in all experiment stages. The best response was obtained using pith explants with 42.85% callus formation in the week eighth and nearly 100% callus can regenerate into shoots at week 24th with the rate of shoot multiplication up to 1.87 times. At week 32th the cultures formed planlets with an average number of leaves of 3.13 and an average size of 2.47 cm x 1.36 cm (length x width) and an average number of roots of 1.52 with average length reached 1.26 cm. Stomatal density has negative correlation with plantlet abnormality rate. Plantlets with the highest stomatal density and low abnormality were obtained in var. Karindra and plantlet derived from explant pith and shoot buds. After 8 weeks of acclimatization stage, the highest survival rate (92%) was obtained on the shoot originating from pith explant. This study proved that varietal differences did not have a significant effect on regenerant abnormalities, and the highest number of regenerant with good quality (low abnormality rate) was obtained from pith explant.</p>