Stem Node Explants Research Articles

BAP and Kinetin Application for In Vitro Bud Induction of Vanilla (Vanilla planifolia Andrews.) from Stem Node Explants in the Initiation Stage

Due to the scarcity of seedlings, vanilla farming in Indonesia is not at its best. The goal of this study was to comprehend how BAP and Kinetin affected the introduction of vanilla buds as a sustainable seedling strategy. The study is carried out at Salatiga, Central Java, Indonesia, at the Plant Tissue Laboratory of the Plantation Seeds Center and the Plant Tissue Laboratory of Diponegoro University’s Physiology and Plant Breeding Laboratory. A completely randomized design (CRD) 5 x 3 with two replications was utilized in the study. The first factor is BAP concentration: B0 = 0 mg.L−1, B1 = 0.6 mg.L−1, B2 = 1.2 mg.L−1, B3 = 1.8 mg.L−1, and B4 = 2.4 mg.L−1. The second factor is Kinetin concentration: K0 = 0 mg.L−1, K1 = 1 mg.L−1, and K2 = 2 mg.L−1. The result shows addition of BAP is able to shorten the day of bud emergence at 8 days. The addition of BAP and Kinetin did not significantly affect the number of buds. Application of BAP 1.44 mg.L−1 was able to increase bud length. There is interaction between Kinetin 2 mg.L−1 and the addition of BAP 2.4 mg.L−1 resulting in the largest bud diameter with a linear regression form. There is interaction between Kinetin 0 mg.L−1 and BAP 1.2 mg.L−1 resulting in the highest number of roots.

Effect of explant and phytohormone on in vitro regeneration of Solanum indicum, an important medicinal weed

The aim of this study was to observe the effect of different explants viz. apical shoot, nodal stem and leaf of Solanum indicum and the concentration of phytohormones on tissue culture responses during the in vitro regeneration and development of micropropagation protocol. For primary establishment of explants, 0.1% HgCl2 solution for 5 minutes was found to be most effective for surface sterilization and Murashige and Skoog (MS) medium having 3 mg/l kinetin and 3 mg/l 6-benzylaminopurine was found to be the best for all the explants. Among all three explants, leaf explants showed the best primary establishment. The explants showed callus formation after 10 to 12 days of culture. The frequency of callus formation was higher on MS medium supplemented with 3 mg/l 3-indoleacetic acid and 5 mg/l 6-benzylaminopurine by the leaf explant. The highest frequency of shooting was recorded on MS medium with 3 mg/l kinetin and 2mg/l 6-benzylaminopurine. Apical shoot showed higher potential of shooting than nodal stem and leaf explants. The in vitro regenerated elongated shoots were then excised from shoot clumps and transferred to rooting medium containing Indole butyric acid at 1.5 mg/l. The in vitro regenerated plantlets were shifted to the polycups having mixture of sterilized soil and farm yard manure in 1:1 ratio. Initially, these plants were kept under high humidity and progressively acclimatized to reduced humidity for their hardening and acclimatization. Plantlets, thus developed, were successfully established and finally transferred to a greenhouse.

A New Aspect for In vitro Propagation of Jerusalem Artichoke and Molecular Assessment Using RAPD, ISSR and SCoT Marker Techniques

JERUSALEM artichoke is an important crop for a wide range of agricultural, medicinal and industrial purposes. Its tubers are rich in inulin and it is cultivated for food and animal feed. The current study aimed to develop an applicable protocol for micropropagation of three cultivars of Jerusalem artichoke (i.e., Balady, Fuza and Alba) using stem node explants. These explants were cultured on ½ MS medium augmented with different concentrations of kanamycin or cefotaxime. The lowest contamination correlated with highest survival percentages were recorded for ½ MS supplemented with 62.5mg L-1 cefotaxime. For shoot multiplication, the maximum number of shoots (11) were obtained for Alba cultivar cultured on MS fortified with 1mg L-1 BA + 0.1mg L-1 NAA + 50mg L-1 nano selenium. For rooting in vitro, the maximum values of rooting percent, root numbers/plantlet and length of roots were observed with ½ MS + 2mg L-1 IBA + 0.1mg L-1 NAA + 0.5mg L-1 KIN. Resulted in vitro regenerated plantlets, were acclimatized on perlite and peat moss mixture (1:1), which gave the highest percent of survival (100%) for Alba followed by Fuza and Balady (80 and 60%, respectively). Moreover, the molecular characterization based on RAPD, ISSR and SCoT techniques was carried out. The polymorphic percentages among in vivo shoots of the three cultivars recorded 38.09, 42.3 and 34.61%, respectively; 61.53, 67.8 and 50% between in vivo shoots, in vitro regenerates and stem derived calli of the three cultivars. The dendrogram analysis of combined techniques showed high similarity between Balady and Fuza followed by Alba.

Genotype-Dependent in Vitro Regeneration Assessment from Decapitated Embryonal Axis and Stem-Node Explants among Selected Pigeonpea Varieties

ABSTRACT: The inherent regeneration ability among selected varieties of pigeonpea using decapitated embryonal axis and stem-node explants in the presence of different growth regulators were assessed for multiple shoot bud induction. Among three different hormones namely BAP, Kinetin and TDZ tested for in vitro regeneration at different concentration for decapitated embryonal axis explants, BAP was found to be comparatively better as evident from number of buds per explants. IPA-242 variety was found to be the best for direct organogenesis resulting in the formation of 10 buds when subjected to MS Medium supplemented with 2.0 mgL-1 of BAP. Under treatment with different concentration of TDZ, Pusa-9 revealed 10 shoot buds with 0.15 mgL-1 of TDZ. The overall response of these varieties at different concentration of kinetin was very poor. With stem-node explants under variable concentration of BAP, IPA-3088 performed best showing 17 buds per explants. Similarly Pusa-9 and IPA 3088 revealed maximum regeneration ability forming 10 and 8 buds under TDZ and kinetin respectively. NAA was found to be effective growth regulator for rooting of shoots regenerated both from decapitated embryonal axis and stem-node explants.

An efficient micropropagation protocol for Monochasma savatieri Franch. ex Maxim through seed germination and direct shoot regeneration

Monochasma savatieri Franch. ex Maxim is a valuable medicinal plant that is currently threatened due to human over excavation. Therefore, an efficient plant micropropagation protocol for M. savatieri through seed germination and direct shoot regeneration is described. The effects of different seed germination treatments, explant source on shoot induction, plant growth regulators on shoot multiplication, and survival rate of regenerated plants were examined. The highest M. savatieri seed germination rate was over 90% on Murashige and Skoog (MS) medium supplemented with 0.1 mg L−1 6-benzylaminopurine (6-BA) and 0.01 mg L−1 α-naphthaleneacetic acid (NAA), with the survival rate reaching 86.0% one-mo after transplanting to soil. Direct shoot proliferation was achieved with two explant types, but the use of nodal stem explants gave best results in comparison to shoot tip explants regarding shoot multiplication frequency and average number of shoots obtained per explant. For nodal segments, medium supplemented with 0.5 mg L−1 6-BA and 0.2 mg L−1 NAA gave the highest multiplication frequency of 90.0%. Among the various treatments tested for shoot multiplication, the best response occurred on MS medium containing 0.5 mg L−1 6-BA and 0.5 mg L−1 gibberellin (GA), with the number of shoots per explant reaching 27.6 and the average height of 4.25 cm. To obtain thick, elongated, and good quality buds, 6-BA at 0.1 and 0.5 mg L−1 along with 0.4 mg L−1 GA should be used interchangeably. Four-mo after transplantation, the survival rates were significantly affected by the types and concentrations of plant growth regulators used during plant tissue culture. The most effective combination of plant growth regulators 0.2 mg L−1 was 6-BA and 0.5 g L−1 ABT (mixture composed of 20% α-naphthaleneacetic acid content and 30% indole-3-acetic acid as active ingredients) with a survival rate of 85.5%. The in vitro propagation protocol for M. savatieri reported here will be useful for the rapid and large-scale propagation of this species.

Emetine and cephaeline production and regulation by in vitro propagation of Psychotria ipecacuanha (Brot.) Stokes in semi-solid media and temporary immersion bioreactor

Psychotria ipecacuanha, is a plant species with known medicinal properties that is critically endangered due to overexploitation of natural populations. Although the difficulties in conventional propagation by seed and by vegetative propagation are generally understood, the present study enhances our knowledge by describing efficient plant regeneration and root induction protocols for P. ipecacuanha while comparing alkaloid content (emetine and cephaeline) in in vitro-derived tissues. Stem node explants were cultured on MS medium MS supplemented with indolbutiric acid (IBA) in semi-solid media and the RITA® temporary immersion bioreactor. The highest root formation (81%) was in MS + 1.5 mg L−1 IBA in the bioreactor. After 24 months of acclimatization, the plants cultivated in MS + 0.50 and 1.0 mg L-1 of IBA had the highest number of roots (3), with mean values of 10.47 and 9.40 cm, respectively. The cultures coming from 1.0 mg L−1 and 0.5 mg L−1 IBA in the bioreactor contained higher cephaeline content, with a relative area of 14.2 and 14.9%, respectively. For emetine, the 1.0 mg L−1 IBA cultures in the bioreactor, 0.5 mg L−1 IBA and MS0 cultures contained higher content than the other treatments, with a relative area of 10.2, 10.2 and 10.1%, respectively.

Changes in Morphological Characteristics, Regeneration Ability, and Polysaccharide Content in Tetraploid Dendrobium officinale

Dendrobium officinale Kimura et Migo is a famous traditional Chinese medicinal plant. It produces various phytochemicals, particularly polysaccharides, which have nutraceutical and pharmaceutical values. To increase its biomass production and polysaccharide content, our breeding program has generated a series of polyploid cultivars through colchicine treatment of protocorm-like bodies (PLBs). The present study compared two tetraploid cultivars, 201-1-T1 and 201-1-T2, with their diploid parental cultivar, 201-1, in an established in vitro culture system. Tetraploid ‘201-1-T1’ and ‘201-1-T2’ had shorter leaves and shorter and thicker stems and roots, and they produced higher biomass compared with the diploid cultivar. The length and width of stomata significantly increased, but stomatal density decreased in tetraploid cultivars. The PLB induction rates from the stem node explants of the tetraploid cultivars were significantly higher than those of diploid. However, the PLB proliferation of tetraploids was lower than that of the diploid. The mean number of plantlets regenerated from tetraploid PLBs was also lower than that of the diploid after 4 months of culture. Polysaccharide contents in stems, leaves, and roots of 6-month-old tetraploid plantlets were significantly higher than those of diploids. The polysaccharide content in the stem of ‘201-1-T1’ was 12.70%, which was a 2-fold increase compared with the diploid cultivar. Our results showed that chromosome doubling could be a viable way of improving D. officinale in biomass and polysaccharide production.

IN VITRO PROPAGATION OF KORONAKI AND CORATINA OLIVE CVS.

Experiments carried out through 2016 revealed that a proper protocol for the in vitro propagation of Koronaki and Coratina olive cvs. as follows : Sterilization of stem node explants was carried out successfuly with copper sulphate at 50mg/l for Koronaki cv. And Ampicillin at 150mg/l for Coratina cv. both cultured on Olive medium.Multiplication on MS medium enriched with BAP at 1.5mg/l for both cvs. Rooting of Koronaki on half sterngth OM medium enriched with IBA at 0.5mg/l and Coratina on half sterngth MS medium enriched with NAA at 0.1mg/l. Acclimization of Koronaki plantlets derived from half strength OM rooting medium and acclimatized on peat : sand (1:1) and Coratina plantlets derived from MS rooting medium and acclimatized on peat :sand : vermiculite (1:1:1).

In vitro propagation of Psychotria ipecacuanha (Brot.) Stokes under different concentrations of Indoleacetic Acid

Psychotria ipecacuanha, é uma planta medicinal nativa da América com a maior área de ocorrência no Estado de Mato Grosso - Brasil, críticamente ameaçada devido à sobreexploração das populações naturais. Devido à dificuldades na propagação convencional, o objetivo deste estudo foi a avaliação do efeito de diferentes concentrações do regulador de crescimento ácido indolacético (AIA) na propagação in vitro de P. ipecacuanha. Segmentos nodais foram cultivados em meio de Murashige e Skoog (MS) sem reguladores de crescimento (controle) e suplementado com quatro concentrações (0,05; 0,5; 1,5 e 2,0 mg.L-1) de AIA, em meios semi-sólidos. Após 60 dias de cultivo, segmentos nodais (n=30) cultivados em meio de Murashige & Skoog (MS) suplementado com 0,05 mg.L-1 de AIA produziram, em média, 4,56 segmentos nodais por explante, cujas plântulas foram aclimatizadas com sucesso, sem exibição de qualquer anomalia morfológica ou variação.

Micropropagation protocol for Salvadora oleoides

Salvadora oleoides Decne. is a pharmaceutically important plant. Owing to poor seed formation, viability and, germination, and to anthropogenic disturbances, this species is on the verge of extinction. A reproducible micropropagation protocol to increase the population through tissue culture has been standardized and the results are reported here. Callus tissues were initiated from young leaves and stem explants. Leaf calluses proliferated with 1.5 mg/L BAP and 0.9 mg/L 2, 4-D with additives and continuous slow proliferation up to 15 weeks on 0.5 mg/L BAP and additives with 200 mg/L activated charcoal. Direct shoot initiation took place from stem node explants after 12 days; 4–5 shoots per node were produced in 30 days. Shoot clumps elongated and grew further on MS media supplemented with 2 mg/L BAP, 0.2 mg/L NAA and additives, which generated 20–23 shoots. The elongated shoots induced tap roots with 4 mg/L NAA and 200 mg/L activated charcoal in 12 days. In vitro raised plants produced secondary roots when transferred to pots containing vermiculite maintained at 28–35 °C. The plantlets successfully acclimatised in pots containing soil in natural conditions.

Agrobacterium-mediated transformation of a Eucalyptus camaldulensis × E. tereticornis hybrid using peeled nodal-stem segments with yeast HAL2 for improving salt tolerance

Genetic improvements of Eucalyptus require both breeding and gene transformation to generate desirable traits for forest industry. Because most of the current Eucalyptus transformation methods rely on juvenile seedling tissues as explants, the resulting transgenics are subjected to phenotypic segregation through the use of segregated seedlings. This is unsuitable for transformation of selected Eucalyptus breeding lines, especially the interspecific hybrids. Here, we developed an Agrobacterium-mediated transformation method using peeled nodal-stem explants from in vitro-propagated plantlets of a Eucalyptus hybrid. The explant was prepared by peeling off epidermal layers around the axillary buds using a pair of forceps, before co-cultivation with Agrobacterium tumefaciens. Transgenic shoots generated from the axillary buds were then obtained through kanamycin selection. The transformation efficiency was at most 24% as confirmed by PCR and GUS assay. Transformation of a E. camaldulensis × E. tereticornis F1 hybrid clone using yeast HAL2 showed that, out of 35, 11 transgenic plantlets had an increase in salt tolerance and grew and developed roots, though at a slow rate, on the medium supplemented with NaCl up to 300 mM concentration. This work demonstrates the efficiency of this transformation method for further genetic improvement of Eucalyptus clones selected through hybridization and breeding.

Trehalose 6-phosphate is involved in triggering axillary bud outgrowth in garden pea (Pisum sativum L.).

Trehalose 6-phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2-oxoglutarate. Supplying sucrose to stem node explants invitro triggered a concentration-dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot.

Improvement of shoot proliferation and comparison of secondary metabolites in shoot and callus cultures of Phlomis armeniaca by LC-ESI-MS/MS analysis

Phlomis armeniaca Willd. is a medicinal plant in the Lamiaceae family endemic to Turkey. The present study describes efficient plant regeneration and callus induction protocols for P. armeniaca and compares phenolic profiles, total phenol and flavonoid contents, and free radical scavenging activity of in vitro-derived tissues. Stem node explants from germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with 75 plant growth regulator (PGR) combinations. The highest shoot number per explant, frequency of shoot proliferation, and frequency of highly proliferated, green, compact callus were obtained on MS medium containing 0.25 mg L−1 thidiazuron (TDZ) and 0.25 mg L−1 indole-3-acetic acid (IAA). The best root formation was on MS basal medium (control). Methanol extract of leaves obtained from regenerants contained higher total phenol and flavonoid contents than the callus extract. The callus extract showed stronger free radical scavenging activity than leaves with IC50 [concentration inhibiting 50% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical] values of 4.30 ± 0.08 and 2.21 ± 0.04 mg g−1 dry weight in leaves and callus, respectively. Apigenin, caffeic acid, p-coumaric acid, luteolin, rutin hydrate, vanillic acid, ferulic acid, salicylic acid, sinapic acid, and chlorogenic acid were detected by liquid chromatography–electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS/MS) analysis in in vitro-grown leaves and callus tissue. Rutin hydrate, p-coumaric acid, and vanillic acid were found at approximately tenfold higher levels in callus than in leaves. This new micropropagation protocol, the first for P. armeniaca, could be used in industrial production for new herbal tea and germplasm conservation.

In Vitro Screening of Stem Explants of In Situ Uv-B Irradiated Cowpea Varieties for Germplasm Storage

Even though seeds form the natural progenies of all sexually reproducing plants, change in climate makes it mandatory to device in vitro methods for selecting germplasm for conservation and regeneration. An experiment was conducted to induct callus in stem explants (nodal region from third node from top of canopy) harvested on 30 days after seed germination from in situ control and supplementary ultraviolet-B irradiated (UV-B = 2 hours daily @ 12.2 kJ m-2 d-1; ambient = 10 kJ m-2 d-1) three varieties of cowpea (Vigna unguiculata (L.) Walp.) viz. Gowmathi, Fola and NS-634 to assess their response to in vitro culture. Axillary bud initiation occurred only from GOWMATHI control stem explants, while callus induction occurred in GOWMATHI and FOLA control stem explants. Stem explants harvested from all the three UV-B stressed cowpea varieties did not proliferate callus. The nodal stem explants of the three varieties of cowpea are not the suitable materials for germplasm preservation for growing in UV-B elevated climate.Int J Appl Sci Biotechnol, Vol 4(1): 44-52

Callus induction, shoot proliferation and root regeneration of potato (Solanum tuberosum L.) stem node and leaf explants under long-day conditions

The present study investigated the effect of various concentrations and combinations of plant growth regulators on callus induction, shoot proliferation and root regeneration of three potato (Solanum tuberosum L.) cultivars by using potato nodal and leaf explants under long-day conditions. The minimum days for callus induction (8.33 d), the maximum callus formation percentage (87.50%), the largest callus diameter (1.90 cm) and the maximum callus weight (2.04 g) were recorded on 1.0 × Murashige and Skoog (MS) medium containing 3.0 mg L−1 benzyl amino purine (BAP) + 2.0 mg L−1 naphthalene acedic acid (NAA). Nodal explants demonstrated a better performance for callus induction compared to leaf segments. Nodal and leaf derived calli differentiated into shoot promordia when subcultured on MS media supplemented with different concentration of BAP, kinetin and thidiazuran as well as gibberellic acid (GA3) and NAA. The maximum shoot regeneration percentage (90.00%) and the maximum number of shoots (6.42) were obtained on 1.0 × MS medium containing 2.0 mg L−1 BAP + 0.25 mg L−1 GA3.

A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub.

An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments of mature plant were used as explants for culture establishment. Multiple shoots were optimally differentiated from the nodal stem explants through bud breaking on Murashige and Skoog (1962) medium containing 3.0mg l(-1) benzyladenine (BA). The effect of different plant growth regulators and minerals were studied on different stages of micropropagation procedure (i.e., explant establishment, shoot multiplication/growth and ex vitro rooting). Additionally, for enhancing shoot multiplication during subculture, MS medium was modified (MMS) with higher levels of magnesium, potassium and sulphate ions. Out of these, MMS3 medium containing 0.25mg l(-1) each of BA and Kin (N6-furfuryladenine), with 0.1mg l(-1) NAA (α-naphthalene acetic acid) was found the best for shoot multiplication (42.45 ± 3.82 per culture vessel). The in vitro regenerated shoots were rooted under ex vitro conditions on treating the shoot base with 500mg l(-1) of IBA (indole-3 butyric acid) for 3min on sterile Soilrite®. The ex vitro rooted plants were hardened in the greenhouse and transferred to the field with ≈85% survival rate. There were not any visual differences between wild and micropropagated plants in the field, although the later underwent significant changes during acclimatization. Micromorphological changes on leaf surface characters from in vitro to acclimatized plantlets were studied in terms of development of glandular trichomes, changes in vein spacing and vein structure in order to understand the nature of plant responses towards environmental conditions. The method developed and defined can be applied for commercial cultivation, which may be important for extraction of bioactive compounds and may facilitate conservation of this multipurpose endangered medicinal shrub.

PERBANYAKAN TUNAS Gyrinops versteegii (Gilg.) Domke

Gyrinops versteegii (Gilg.) Domke) is a member of Thymellaceae having an important role as agarwood source that is potential to develop. Agarwood is a non timber forest product (NTFP) with a high economic value. Gyrinops versteegii (Gilg) Domke was an endemic and rare plants, however, it is cultivated on the island of Lombok. The study of shoot multiplication from single node stem and cotyledon node explants to shoot produced was conducted to find out the best method to shoot multiplication. Modification of Murashige and Skoog (MS), Gamborg (B5) dan Woody Plant Medium (WPM) supplemented with 8.0 g/l agar, 30 g/l sucrose and vitamin (0.1 mg/l Thyamine, 0.5 mg/l Nicotinic acid, 0.5 mg/l Pyridoxine dan 2 mg/l Glysin) and 10 g/l myoinositol were used as growth medium treatments. The experiment was conducted on two stages i.e. germination and shoot multiplication. Shoot induction was conducted on modification of MS medium supplemented with Benzyl Adenin (BA) 0.5; 1.0 and1.5 mg/l. The multiplication of shoot conducted on modification of MS medium, WPM and B5 medium supplemented with 0.5; 0.75 dan 1.0 mg/l Benzyl Adenin (BA). For every treatment there were ten samples (bottles) with one explants in each bottle. The observation was conducted on shoot induction percentage, number of shoot and visual performance of culture. The result showed that modified MS + BA 0.5 mg/l was the best for shoot induction from single node stem explants with the average of 1.6 shoots on 12 weeks. The treatment of modified MS + BA 0.75 mg/l was the best for shoot multiplication from single node stem explants with the average of 5.7 shoots on 12 weeks. The treatment of modified WPM + 0.5 mg/l BA was the best for shoot multiplication from cotyledonery node explants with the average of 4.6 shoots on 12 weeks. The visual performance of shoot resulted was normal.

PERBANYAKAN TUNAS Gyrinops versteegii (Gilg.) Domke

Gyrinops versteegii (Gilg.) Domke) is a member of Thymellaceae having an important role as agarwood source that is potential to develop. Agarwood is a non timber forest product (NTFP) with a high economic value. Gyrinops versteegii (Gilg) Domke was an endemic and rare plants, however, it is cultivated on the island of Lombok. The study of shoot multiplication from single node stem and cotyledon node explants to shoot produced was conducted to find out the best method to shoot multiplication. Modification of Murashige and Skoog (MS), Gamborg (B5) dan Woody Plant Medium (WPM) supplemented with 8.0 g/l agar, 30 g/l sucrose and vitamin (0.1 mg/l Thyamine, 0.5 mg/l Nicotinic acid, 0.5 mg/l Pyridoxine dan 2 mg/l Glysin) and 10 g/l myoinositol were used as growth medium treatments. The experiment was conducted on two stages i.e. germination and shoot multiplication. Shoot induction was conducted on modification of MS medium supplemented with Benzyl Adenin (BA) 0.5; 1.0 and1.5 mg/l. The multiplication of shoot conducted on modification of MS medium, WPM and B5 medium supplemented with 0.5; 0.75 dan 1.0 mg/l Benzyl Adenin (BA). For every treatment there were ten samples (bottles) with one explants in each bottle. The observation was conducted on shoot induction percentage, number of shoot and visual performance of culture. The result showed that modified MS + BA 0.5 mg/l was the best for shoot induction from single node stem explants with the average of 1.6 shoots on 12 weeks. The treatment of modified MS + BA 0.75 mg/l was the best for shoot multiplication from single node stem explants with the average of 5.7 shoots on 12 weeks. The treatment of modified WPM + 0.5 mg/l BA was the best for shoot multiplication from cotyledonery node explants with the average of 4.6 shoots on 12 weeks. The visual performance of shoot resulted was normal.

Multiple Shoot Induction of Asparagus (Asparagus Officinalis Var. Uc-157) Through Nodal Stem Culture Technique

Multiple shoot induction is a powerful tool for mass propagation of promisingclones and is essential to asparagus due to its dioecious nature. Along this line anexperimental research conducted to determine the most effective plant growth regulators(PGRs) that would induce multiple shoot formation from asparagus nodalstem explants using agar-solidified Murashige and Skoog (MS) basal medium. Fourcombinations of kinetin or benzyl amino purine (BAP) and naphthalene acetic acid(NAA), arranged in Randomized Complete Block Design, served as treatments withhormone-free MS medium as control. Treatment means were compared using Duncan’sMultiple range Test (DMRT). Only medium supplemented with PGRs initiatedshoots. Among the treatments, the medium containing 0.5 mg/L kinetin and0.1 mg/L NAA, or 0.5 mg/L BAP and 0.1 mg/L NAA significantly induced thehighest percentage (100%) shoots from cultured explants after 15.27 and 22.97 days,respectively. The same PGR combinations produced an average of 3-4 shoots per explantsafter eight weeks of culture. The medium supplemented with 0.5 mg/L kinetinand 0.1 mg/L NAA produced longer shoots (4.64 cm) of which 10% produced roots.This PGR combination is recommended since produced the best shoot attributes andlikewise shorten the duration of in vitro culture.Keywords: Botany, plant tissue culture, multiple shoot induction, experimental design,Cebu City, Philippines

In Vitro Culture and Rooting of Diospyros virginiana L.

The hard, strong, very close-grained wood of common persimmon (Diospyros virginiana L.; Ebenaceae) is desirable for specialty products such as golf club heads, percussion sticks, billiard cues, and for wood turnery. The edible fruit of cultivated varieties is sold as pulp for use in puddings, cookies, cakes, and custards. Persimmon is usually propagated by grafting. Own-rooted clonal persimmon could offer several advantages to specialty fruit growers such as elimination of grafting, graft incompatibility issues, and improved rootstocks for variety testing. Four mature, grafted (male and female) persimmon genotypes and one hybrid were used for nodal explant culture. Nodal stem explants were cultured on Murashige and Skoog (MS) medium containing 10 μM zeatin, 3% (w/v) sucrose, and 0.7% (w/v) Bacto agar. Explants were routinely transferred to fresh medium every 3 weeks until shoot cultures were established. All nodal explants excised from grafted greenhouse plants produced at least one viable shoot. For in vitro rooting of microshoots, half-strength MS medium with 0, 5, 10, or 15 μM indole-3-butyric acid (IBA), 0.1 g·L−1 phloroglucinol, 3% (w/v) sucrose, and 0.7% (w/v) Bacto agar were tested with a 10-day dark culture treatment followed by culture in the light. Best rooting (14% to 87%) was achieved on medium containing 5 μM IBA for the common persimmon genotypes with means averaging from 0.5 to 3.9 roots per shoot. Ninety-one percent rooting with 5.3 ± 2.6 roots per shoot was achieved for the hybrid persimmon. Rooted plants were successfully acclimatized to the greenhouse.