Number Of Adventitious Shoots Research Articles

Enhancement of Pistachio (Pistacia vera L.) Propagation In Vitro Utilizing the Shoot and Embryo Culture Techniques

The effects of sugar type, activated charcoal, and naphthalene acetic acid on the propagation of pistachio (Pistacia vera) in vitro were investigated. Length of roots increased up to 30% when sucrose was used instead of maltose and up to four times when 2 g/L of activated charcoal was added to the medium. The number of adventitious shoots was enhanced (up to 3.2 shoots) when a Rugini and Verma medium supplemented with 0.75 mg/L naphthalene acetic acid, 0.75 mg/L indole acetic acid, and 2 mg/L 6-benzyl amino purine was used. To enhance rooting of shoots in vitro, we developed a technique to increase the amount of hormones available to the growing shoots. The hormones were delivered using a sterilized fine needle to the base of the growing shoots. This technique enhanced rooting up to 3 times (73%) especially when the hormone indole butyric acid was used as a growth regulator.

Multiple adventitious shoot formation in Spanish Red Cedar (Cedrela odorata L.) cultured in vitro using juvenile and mature tissues: an improved micropropagation protocol for a highly valuable tropical tree species

Cedrela odorata L. is a valuable tropical tree widely appreciated for its wood. This species confronts serious problems due to both overexploitation of its natural populations and its susceptibility to the Meliaceae borer Hypsipyla grandella, which destroys the apical meristems and produces structural deformations. The rapid introduction of new varieties through clonal forestry has been demonstrated to be the most effective way to improve the production of perennial plantation species. In this work, we report both a protocol for the rejuvenation of elite mature trees of C. odorata and the optimization of an in vitro culture system to scale up micropropagation. Several media formulations and the use of temporary immersion culture in bioreactors were evaluated. The addition of 20% coconut water to TY17 medium increased the number of adventitious shoots from hypocotyl segments to an average number of 4.68 shoots per explant. To replace coconut water and to define the culture medium, several cytokinins were tested at various concentrations; however, none of them produced the effect of coconut water. Rejuvenation of elite mature individuals was investigated by ex vitro grafting of mature tree twigs onto 3-mo-old juvenile trees. Although the grafting had a positive effect on the micropropagation of mature material, the multiplication rate of 1.5 new shoots per explant did not compare to the organogenic capacity of younger materials. Shoot and root elongation as well as acclimatization to ex vitro conditions were carried out in a temporary immersion culture of juvenile material using BioMINT® bioreactors. A 3.5-fold increase in shoot elongation and a 4-fold increase in root elongation were achieved compared to material cultured on semisolid media. Furthermore, this culture system allowed for 98% effectiveness in the soil adaptation of the in vitro-grown plants. The scaled-up multiplication capacity over a period of 6 mo calculated for the system is above 16,000 plants per mother plant with young materials but is only 125 with mature materials.

A scaled-up system for in vitro multiplication of thidiazuron-induced red raspberry shoots using a bioreactor

SUMMARYA procedure for the mass propagation of adventitious shoots regenerated from leaf explants of red raspberry (Rubus idaeus L.) using a liquid medium-containing bioreactor system combined with gelled medium has been described. Leaf segments of the raspberry cultivars ‘Festival’, ‘Heritage’, and ‘Latham’ produced multiple buds and shoots in 4.5 µM thidiazuron (TDZ)-containing shoot induction medium within 6 – 8 weeks of culture initiation. The raspberry cultivars differed significantly with respect to their shoot regeneration and multiplication rate, with ‘Heritage’ producing the highest number of adventitious shoots per explant.TDZ supported rapid shoot proliferation at low concentrations (1.2 – 2.3 µM) in the bioreactor system, but shoot elongation was improved in medium containing 4.4 µM 6-benzyladenine (BA). BA-induced, elongated shoots rooted in the bioreactor vessel containing the same medium, but without any plant growth regulators. In vitro-derived plantlets were acclimatised and eventually established in a greenhouse and in the field. These results suggest the possibility of large-scale multiplication of raspberry shoots using a bioreactorbased system.

The influence of the cooling of donor cultures on the in vitro adventitious regeneration and carbohydrate metabolism of four dwarfing apple rootstocks

We examined the effects of cooling applied for 4 to 20 weeks on donor cultures of four dwarfing apple rootstocks (P16, P22, P59 and M26). Our aim includes increasing their competence for in vitro adventitious shoot regeneration from the leaves. Donor cultures were maintained on a shoot multiplication medium at 4°C in the dark for 4 months, followed by subculture on a fresh medium for 4 weeks. The cooling of the cultures caused an increase in the adventitious shoot number and a decrease in the starch content and an increase in the soluble sugar content (monosaccharides, raffinose and stachyose). The accumulation of stachyose in response to cold is a new observation, and it suggests that raffinose and stachyose play important role in the acclimation of dwarf apple rootstocks to low temperatures.

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l −1 BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l −1 BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.

Microenxertia interespecífica ex vitro em maracujazeiros

Este trabalho teve como objetivo avaliar o efeito da espécie de Passiflora usada como porta-enxerto no pegamento e desenvolvimento da microenxertia interespecífica ex vitro e caracterizar anatomicamente a região em que ela é realizada. Ápices caulinares de Passiflora edulis f. flavicarpa foram enxertados em hipocótilo de porta-enxertos de P. alata, P. cincinnata, P. edulis e P. setacea. Foram avaliados o desenvolvimento da microenxertia, a ocorrência de brotações adventícias do porta-enxerto e o percentual de microenxertos com folhas expandidas. Os porta-enxertos foram submetidos à avaliação anatômica e histoquímica. O desenvolvimento dos microenxertos e o número de brotações adventícias foram maiores quando se utilizou porta-enxertos de P. edulis e menores com porta-enxertos de P. setacea e P. alata. Porta-enxertos de P. edulis, P. cincinnata, P. alata e P. setacea proporcionaram, respectivamente, 30,3, 10, 1,6 e 0% de microenxertos com folhas expandidas. As avaliações histológicas evidenciaram maior espessura da região cortical em porta-enxertos de P. edulis, enquanto em P. setacea foi observado menor número de camadas corticais, maior quantidade de fibras floemáticas e de alcaloides, características que influenciaram negativamente o resultado da microenxertia.

Enhancement of in vitro organogenetic capacity of rose by preculture of donor shoots on the medium with thidiazuron

Rose genotypes can be recalcitrant in in vitro regeneration. A lack of competence for regeneration results in unsatisfactory numbers of adventitious shoots, which diminishes achievements in use of genetic engineering in rose modern breeding. Among the factors which can partially overcome this problem is the use of thidiazuron (TDZ) in the induction phase of regeneration. We studied the use of TDZ in concentrations 0.05 and 1.0 mg l−1, in the medium for the culture of donor shoots with the aim of increasing the regeneration competence of five rose cultivars differing in their regeneration ability. All the tested genotypes increased their regeneration potential when the donor shoots were cultured on TDZ media for 15 or 30 days. The strongest effect, doubling to quadrupling the number of shoots, was obtained in the cultivars characterized by the lowest regeneration potential. This enhanced regeneration ability was maintained through two or three subsequent 4-week long regeneration passages.

In vitro regeneration of cocona (Solanum sessiliflorum, Solanaceae) cultivars for commercial production

Cocona (Solanum sessiliflorum Dunal) is a solanaceous shrub native to the Amazon region that produces an edible fruit. This species has numerous advantages, particularly a high nutritional value and productivity. However, due to irregular germination and rapid loss of seed viability, there are few plantations for production on a large scale. Development of alternative propagation strategies is essential for the production of homogeneous seedlings of genotypes with superior agronomic performance. We developed techniques for in vitro regeneration of the cocona varieties Santa Luzia and Thaís for large-scale production of healthy plantlets. Twenty days after seeding, seedling segments germinated in vitro were used as explant sources. Three successive experiments were performed: one to test the effect of the explant source and combinations of two growth regulators, auxin (indole acetic acid, IAA) and kinetin (KIN), on the morphogenetic response; another to investigate the effect of the combination of growth regulators on the morphogenetic response of hypocotyl segments, and another to evaluate how sucrose concentration affects the development of adventitious shoots. The best shoot induction was obtained using hypocotyl segments and stem apices, while rhizogenesis was greatest in leaves with a petiole. The number of adventitious shoots per explant on hypocotyl segments increased with 10 and 20 mg/L KIN, combined with 0.02 mg/L IAA in the variety Santa Luzia. Sucrose combined with these growth regulator levels increased the average number of calli; these were optimally produced when 45 g/L sucrose and 0.01 mg/L IAA + 20 mg/L KIN were applied. Only sucrose concentration influenced shoot proliferation in the two S. sessiliflorum varieties, with a maximum at 17.5 g/L.

Adventitious Shoot Regeneration and in vitro Flowering of Anthemis xylopoda O. Schwarz, a Critically Endangered Turkish Endemic

A protocol has been developed for the direct induction of adventitious shoots from leaf explants of in vitro raised Anthemis xylopoda O.Schwarz, a critically endangered Turkish endemic. The Murashige and Skoog (MS) media supplemented with N^6-benzyladenine (BA), kinetin (KIN), and thidiazuron (TDZ) were used in regeneration experiments. BA at 0.5 mg L^{-1} concentration was chosen as the optimal treatment because it yielded the maximal number of adventitious shoots with the best quality (6.70 ± 1.05 shoots/explant). The highest mean of maximum shoot length was observed in MS media containing 0.2 mg L^{-1} BA (4.30 ± 0.48 cm). Regenerated shoots rooted best on ½MS medium containing 0.5 mg L^{-1} indole-3-butyric acid (IBA). Flower buds also occurred during rooting. For this study, the existence of auxins in the medium was essential for in vitro flower bud induction. IBA was found to be more efficient than indole-3-acetic acid (IAA) in the induction of flower buds of A. xylopoda. The maximum flowering percentage was obtained when the shoots were cultured on ½MS medium containing 1.0 mg L^{-1} IBA. Flowers produced in vitro were morphologically normal and yellow.

An Improved 2-step Liquid Culture System for Efficient In Vitro Shoot Proliferation of Sundew (Drosera rotundifolia L.)

An efficient procedure for in vitro shoot production of the medicinal plant sundew (Drosera rotundifolia L.) was developed. Of three cytokinins tested, zeatin (ZEA) at a concentration of 2 μM resulted in the formation of large numbers of adventitious shoots on leaf explants. A larger size of the small shoots was achieved by combining a 2-weeks preculture with ZEA (step 1: shoot induction) with a 5-weeks main culture without plant growth regulators (step 2: shoot elongation). Liquid media were superior to semisolid media: An average of 27.4 shoots per leaf explant, and 53.3 shoots per shoot explant were achieved. The 2-step culture system with liquid media permits a comparatively cheap micropropagation of sundew as well as in vitro biomass production, with potential for scale-up.

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash ( Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.

In Vitro Shoot Multiplication of Echinacea purpurea

ABSTRACT Cytokinin growth regulators, 6-benzylaminopurine (BAP), kinetin (KIN), and 2-isopentenyladenine (2-iP), induced in vitro shoot multiplication from shoot explants of Echinacea purpurea (Eastern purple coneflower). Murashige and Skoog (MS) medium supplemented with 17.76 μM 6-benzylaminopurine and 0.269 μM naphthalene acetic acid produced the greatest number of adventitious shoots, although all cytokinin treatments with or without naphthalene acetic acid were effective in inducing shoots. Subsequent rooting and growth of the induced shoots in a potting media in a greenhouse demonstrated the new plants developed normally.

Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.

High-frequency shoot multiplication in Artemisia vulgaris L. using thidiazuron

An in vitro propagation system for Artemisia vulgaris L., a traditional medicinal plant, has been developed. The best organogenic response, including adventitious shoot number and elongation, was obtained when hypocotyl segments were cultured onto MS medium supplemented with 4.54 μM TDZ (N-phenyl-N′-(1,2,3-thidiazol-yl) urea). Up to 28 shoots formed per explant for an optimal duration of exposure of 48 days. Regenerated shoots formed roots when subcultured onto a medium containing 8.56 μM IAA (indole-3-acetic acid). Healthy plantlets were transferred to a garden soil:farmyard soil:sand (2:1:1) mixture for acclimatization, which was successful, and subsequent maturity was achieved under greenhouse conditions over a six-month period. The survival rate of the plantlets varied under acclimatization. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of A. vulgaris. This optimized protocol has been successfully employed for genetic transformation studies in A. vulgaris, which are currently underway in our laboratory.

INCREASE IN THE QUALITY OF RASPBERRY CULTURES UNDER THE INFLUENCE OF FEEDDHA

The quality of raspberry shoot cultures was far from optimal because of leaf chlorosis increasing with the duration of culture. We, therefore, added iron in the form of the chelate FeEDDHA (50 mg/L) to the multiplication medium from which leaves for adventitious regeneration were collected or to the both - multiplication and regeneration medium. The FeEDDHA supplementation of multiplication medium eliminated leaf chlorosis and increased the number of adventitious shoots in three cultivars - 'Beskid', 'Canby' and 'Veten'. The supplementation of regeneration medium increased the number of regenerating leaves of 'Canby', 'Malling Seedling' and 'Norna' and the number of the adventitious shoots of 'Veten'. The highest level of regeneration was observed on the regeneration medium containing 78.8 mg/L FeEDDHA as the sole source of iron.

Optimisation of regeneration and maintenance of morphogenic callus in pear ( Pyrus communis L.) by simple and double regeneration techniques

The purpose of our work was to improve the regeneration capacity of leaf explants and the maintenance of shoot morphogenesis in callus of six pear cultivars: Abate Fetel, Conference, Dar Gazi, Harrow Sweet, Kaiser and Williams, by altering the composition of both regeneration and proliferation media of explant donor shoots, and choosing the right type of explant. Regeneration capacity of leaf explants collected from in vitro shoots has been improved in the majority of cultivars also due to shoot preconditioning. For the first time, long term morphogenic callus production and maintenance have been established in some cultivars by a “double regeneration”. Using this technique, morphogenic callus of two cultivars, ‘Dar Gazi’ and ‘Conference’, was maintained for several subcultures but only when they were initiated from small leaflets – less than 2–3 mm long – which had been collected from the neoformed adventitious buds. MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plant. 15, 473–497] proved to be an efficient regeneration medium by stimulating adventitious buds, while the explants of all cultivars, except for Kaiser, showed a high regeneration capacity when they were collected from shoots proliferated on modified QL medium [Quoirin, M, Lepoivre, P., Boxus, P., 1977. Un premier bilan de dix annees de recherche sur les cultures de meristemes et la multiplication in vitro de fruitiers ligneux. Compte rendu des recherches, Station des Cultures Fruitieres et Maraicheres de Gembloux (1976–1977), 93–117]. This medium conferred leaf expansion, overcoming 90% of regeneration in explants of cv Dar Gazi and Williams. Well expanded leaves were obtained and collected by rooting the shoots, while regeneration percentage was not improved and the number of adventitious shoots was increased in most cultivars, reaching up to 10 shoots per explant. When cefotaxime at 200 mg/l, which is normally effective in controlling Agrobacterium, was used for genetic transformation, regeneration percentage and number of shoots per explant (in leaf explants collected from rooted shoots) were increased and a uniform bud regeneration on all the leaf surface was promoted.

The influence of FeEDDHA in red raspberry cultures during shoot multiplication and adventitious regeneration from leaf explants

We examined the effect of FeEDDHA on multiplication via axillary branching and adventitious shoot regeneration from leaf explants in five red raspberry cultivars. When applied during multiplication, FeEDDHA reduced chlorosis, increased content of chlorophyll and iron but had no effect on the number of side shoots in four of the five cultivars. Addition of FeEDDHA to regeneration medium increased the percentage of regenerating leaves in some cultivars and the number of adventitious shoots in all five cultivars.

Aspen micropropagation: use for phytoremediation of soils

Toxic pollution of soils is a major environmental problem in the Czech Republic. Most contemporary remediation approaches do not provide acceptable solutions. For environmental clean-up, the use of specially selected and engineered plants capable of effective restoration and stabilization of contaminated sites is an emerging technology called phytoremediation. Aspen (Populus spp.) trees represent optimal plants for absorption, accumulation, storage, and degradation of environmental pollutants such as heavy metals, pesticide residues, and other waste products. The first aim of this study was to verify possibilities of micropropagating selected elite European aspen (Populus tremula) and hybrid aspen (Populus tremula × Populus tremuloides) mature trees growing in polluted areas. The primary cultures were established from 24 years old trees from provenance plots. Dormant buds were sampled from selected trees in spring. After sterilization of dormant buds, the shoot tips were extirpated and put on nutrient media. MS medium with a higher concentration of BAP (1.0 mg/l) and IBA (0.1 mg/l) showed to be suitable for induction of organogenesis. MS medium with lower concentration of BAP (0.2 mg/l) and higher concentration of glutamine (100 mg/l) in agar medium was used for multiplication. A high number of adventitious shoots (20–30) was produced per multi-apex culture. The losses during rooting and acclimatization were minimal, around 2%. Currently, the plantlets grow on the outside bed of the experimental nursery. Twenty-five clones were established in the gene bank. Secondly, standardization of the efficient transformation of hybrid aspen was investigated with the aim to facilitate production of transformed hybrid aspen with valuable genes for increased capability of phytoremediation in the near future. Four gene constructs of Agrobacterium tumefaciens comprising different promoters (bearing two genes, NPTII coding resistance to kanamycin, and GUS coding glucuronidase synthesis allowing histochemical and fluorometrical identification) were used for transformations of aspen hybrid stem segments. A promoter suitable for transformations was selected on the basis of GUS activity assay. The highest activity was estimated in transformants with construct 148 controlled by promoter CAMV 35S.

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.

Perbanyakan Ruskus (Ruscus hypophyllum L.) secara In Vitro

These experiments were aimed to obtain optimum medium for micropropagation of Ruscus. There were two experiments consist of in vitro shoots proliferation, shoot elongation and rooting. The experiment of shoot proliferation performed by inducing adventitious shoots from explant in the Murashige and Skoog (1962)(MS) basal medium supplemented with combination of plant growth regulators BAP (0.0, 0.5, 1.0, 2.0, 4.0 and 6.0 mg/l) and IAA (0.0, 0.1, 0.2 and 0.4 mg/l). The elongation and rooting of plantlets were induced in the different concentration of the MS basal medium (0.5, 1.0 and 2.0 strength) combined with IBA (0.0, 1.0, 2.0, 3.0 and 4.0 mg/l). Both experiments were arranged as completely randomized design with 15 replications. Adventitious shoots were produced in all medium supplemented with BAP with or without IAA. However MS medium supplemented with BAP 1 mg/l or 2 mg/l combined with IAA 0.2 mg/l were the best. The number of adventitious shoots in these medium were 9.2 and 9.4 shoots after 8 weeks cultured respectively. Increasing concentration of BAP more than 4 mg/l decreased number and size of adventitious shoots. The plantlets produced in the proliferation medium were then transferring to the next treatments for elongation and rooting. The best medium for elongation and rooting were medium with half strength of MS with or without IBA. Acclimatization conducted by transferring the rooted plantlets on the medium containing sterilized soil and rice husk charcoal (1:1). After 4 weeks acclimatization, 60-100 percent of plantlets were survived and growth, depend on treatments. Key words: Acclimatization, adventitious shoot, micropropagation, rooting, Ruscus hypophyllum L.