Showing papers on "Murashige and Skoog medium published in 2002"

Rapid propagation of Holostemma ada-kodien Schult., a rare medicinal plant, through axillary bud multiplication and indirect organogenesis

Abstract: Efficient protocols of axillary bud multiplication and indirect organogenesis were established for Holostemma ada-kodien Schult. (Asclepiadaceae). Murashige and Skoog (MS) medium supplemented with 2.0 mg l–1 N6-benzylaminopurine (BAP) and 0.5 mg l–1 indole-3-butyric acid (IBA) induced an average of eight shoots per node and was the best for axillary bud proliferation. Subsequent cultures enhanced the number of shoots. The explant source of callus and the growth regulator inducing the callus exhibited significant influence on organogenesis. Callus developed from the basal cut end of the node explants differentiated more than 15 shoots on MS medium fortified with 1.5 mg l–1BAP. Callus from internode explants developed fewer shoots than callus from the basal cut ends of node explants. Leaf-derived callus did not undergo organogenesis. The abscission of leaves and shoot tips of the developed shoots was prevented by the addition of AgNO3 or CoCl2, but with a concomitant significant reduction in the number of shoots. Half-strength solid MS or liquid medium with 0.05 mg l–1 IBA exhibited the best in vitro rooting. Ninety percent of the rooted shoots survived in the field.

In vitro regeneration of Echinacea purpurea from leaf explants

Abstract: Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 μM) and NAA (0.054 μM) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.

Optimization of a mature embryo-based in vitro culture system for high-frequency somatic embryogenic callus induction and plant regeneration from japonica rice cultivars

Abstract: Optimization of the conditions for an efficient induction of somatic embryogenic calli and regeneration of plants from mature seeds of japonica rice cultivars was attempted The number, color, size, shape, and appearance time of the induced embryogenic calli varied among the rice cultivars depending on the type of basal medium (LS, MS, N6) Presence of adequate amount of sucrose in the medium was an absolute requirement for embryogenic callus formation and shoot induction Induction of the embryogenic calli, whose overall rates ranged from 30 to 56%, was most efficient in N6 medium supplemented with 30 mg l−1 of 2,4-D and 30 g l−1 of sucrose Agar concentration in the regeneration medium was also critical for the shoot induction Kinetin was found to be more effective for shoot regeneration compared with BA, while the highest shoot regeneration frequencies were observed when either cytokinin was combined with high concentration (20 mg l−1) of NAA The optimal concentration of kinetin for the highest shoot regeneration frequency (67∼77%) was different among the cultivars tested The embryogenic calli-derived shoots rooted on a plant growth regulator-free MS medium were successfully established in soil, producing fertile seeds

Rapid propagation of Phalaenopsis from floral stalk-derived leaves

Abstract: An efficient and rapid in vitro method was developed for regeneration of Phalaenopsis using leaf segments derived in vitro from flower stalk nodes. Leaf segments of four cultivars Tinny Sunshine ‘Annie’, ‘Taisuco Hatarot’, Teipei Gold ‘Golden Star’, Tinny Galaxy ‘Annie’ cultured on Murashige and Skoog medium supplemented with N6-benzyladenine (BA; 88,8 μM) and α-naphthaleneacetic acid (NAA; 5,4 μM) produced an average of 10–13 protocorm-like bodies (PLBs) after 12 wk. PLB proliferation was achieved on a modified Hyponex medium (1 gl−1 6.5N−4.5P−19K+20N−20P−20K+2gl−1 peplone +3% (w/v) potato homogenate +0.05% activated 1 gl−1 charcoal) and an optimal number of 13–18 PLBs developed from single PLB sections of different cultivars. Plantlet development was also achieved on a modified Hyponex medium. By repeated subculture of PLBs on a proliferation medium, and culturing them in the plantlet regeneration medium, plantlets could be produced continuously. Approximately 6 mo, were required from the initiation of culture to the production of plantlets for transplant to community pots.

An improved micropropagation protocol for teak

Abstract: An improved micropropagation protocol has been developed for teak (Tectona grandis) Nodal explants placed on MS medium supplemented with 222 μM benzylaminopurine and then serially transferred to fresh medium after 12, 24, 48 and 72 h gave maximum culture establishment (768%) Establishment was reduced when explants were retained in the initial culture medium longer than 12 h Explants collected in May showed maximum (768%) response Placement of the explants on MS medium supplemented with 222 μM benzylaminopurine and 057 μM indole-3-acetic acid resulted in the maximum average number of shoots In vitro raised micro shoots were rooted ex vitro by dipping in indole-3-butyric acid (98 mM) for 2 min followed by planting in polyethylene pots containing a soil:vermiculite (1:1 v/v) mixture This treatment resulted in 779% survival of the plantlets They were weaned in a glasshouse and finally moved to an agro-net shade house

Regeneration of banana (Musa spp. AAB cv. Dwarf Brazilian) via secondary somatic embryogenesis

Abstract: A method has been developed for the regeneration of the banana cultivar Dwarf Brazilian (Musa spp. AAB group). Primary somatic embryos were produced when explants of immature male flower buds were cultured on Murashige and Skoog (MS) medium plus 1 mg/l biotin, 100 mg/l malt extract, 100 mg/l glutamine, 4 mg/l 2,4-dichlorophenoxyacetic acid, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l α-naphthaleneacetic acid, 30 g/l sucrose and 2.6 g/l Phytagel, pH 5.8 (M1 medium) and then transferred to M1 medium plus 200 mg/l casein hydrolysate and 2 mg/l proline. Subsequent transfer to MS supplemented with 10% coconut water produced rapidly proliferating embryogenic callus that developed into secondary somatic embryos (SE2); these were subcultured on half-strength MS supplemented with 5 mg/l 6-benzylaminopurine (BA). Differentiated embryos were transferred to MS medium supplemented with 5 mg/l BA for development, and mature SE2 were isolated and cultured on hormone-free MS for germination and development into plantlets. Approximately 90% of the SE2 germinated and developed into plantlets, and these were subcultured onto MS medium plus 0.1% activated charcoal, 1 mg/l BA and 1 mg/l IAA where complete plantlets developed. Morphologically normal banana plants developed from all of the regenerated plantlets, the first of which were produced within 6 months of culture initiation.

An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture

Abstract: High-frequency protocorm-like body (PLB) formation directly from thin leaf sections of Doritaenopsis hybrid was achieved in order to develop a mass-scale propagation system. Concentrated efforts were made to study the effects of different cytokinins on in vitro PLB induction from thin leaf sections. Among the cytokinins tested, thidiazuron (TDZ) was found to be a more effective inducer of PLBs than benzyladenine and zeatin. A modified Murashige and Skoog medium supplemented with 9.0 µM TDZ was found to be the optimum concentration for PLB development from thin leaf sections of Doritaenopsis hybrid. Of the two different explant types used in the present experiment, the highest percentage of PLB formation (72.3%) and highest number of PLBs (18) per explant were observed on thin leaf sections (1 mm thick), while only 20% (4.3 per explant) of comparatively large leaf segments (5 mm thick) were able to produce PLBs under the same culture conditions. Light microscopy observations indicated that the initial cell divisions for PLB formation occurred on the region near the cut surface and that an intact epidermal layer appeared to play an important role in PLB formation. Proembryo initiation occurred from several cells just beneath the intact epidermal cell, and globular PLBs were clearly visible after 3 weeks of culture and subsequently developed into mature PLBs.

Somatic embryogenesis and plant regeneration in Eucalyptus globulus Labill

Abstract: Somatic embryogenesis was induced from juvenile explants of Eucalyptusglobulus Labill. Mature zygotic embryos, isolated cotyledons, hypocotyls, leaves and stems were cultivated at 24°C in darkness on Murashige and Skoog medium supplemented with 3% (w/v) sucrose and different growth regulator combinations. Callus was formed at the surface of the explant in all tested media containing sucrose but not in those containing mannitol. Calli were transferred to the same medium without growth regulators (MSWH) after 25 days. Somatic embryogenesis was observed in callus derived from cotyledon explants and from entire mature zygotic embryos in the presence of 3–15 mg l–1 α-naphthalene acetic acid (NAA) alone or in the presence of 1 mg l–1 NAA combined with 1 mg l–1 2,4-dichlorophenoxyacetic acid. Best embryogenic rates were obtained in the presence of 3–5 mg/l NAA, as approximately 30% of callus formed on these media produced somatic embryos. Exposure, for >1 week, to the highest NAA concentrations tested (15 mg l–1) failed to induce somatic embryogenesis. Addition of 500 mg l–1 casein hydrolysate and 500 mg l–1 glutamine to the induction medium increased the number of abnormal somatic embryos. Conversion of somatic embryos to plantlets (21%) was obtained when they were transferred to medium free of growth-regulators.

Growth, proline accumulation and ion content in sodium chloride-stressed callus of date palm

Abstract: Growth and physiological responses of date palm. Phoenix dactylifera L. cv. Barhee, callus to salinity stress were examined. Callus induced from shoot tips of offshoots was cultured on Murashige and Skoog medium supplemented with NaCl at concentrations ranging from 0 to 225 mM, in consective increments of 25 mM. Data obtained after 6 wk of exposure to salt have shown a significant increase in callus proliferation in response to 25 mM NaCl the lowest level tested, beyond which callus weight decreased. At 125 mM NaCl and higher, callus growth was nearly completely inhibited. Physiological studies on callus exposed to salt stress have shown an increase in proline accumulation in response to increased salinity. Proline accumulation was correlated to callus growth inhibition. Furthermore, increasing the concentration of NaCl in the culture medium generally resulted in a steady increase in Na+ and reduction in K+ concentrations. However, at 25 mM NaCl, the only level at which callus growth was significantly enhanced, an increase in K+ content was noted, in comparison to the NaCl free control. In response to increasing external NaCl level, the Na+/K+ ratio increased The Na+/K+ ratio was positively correlated to proline accumulation and hence callus growth inhibition. This study provides, an understanding of the response of date palm callus to salinity, which is important for future studies aimed at developing strategies for selecting and characterizing somaclonal variants tolerant to salt stress.

High frequency of bulblet regeneration from bulb scale sections of Fritillaria thunbergii

Abstract: Fritillaria thunbergii Miq. bulb-scale sections were cultured using Murashige and Skoog (MS) medium supplemented with NAA (1.62 μM) and KN/2iP/BA (0.47–23.23 μM).

Somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB) in liquid medium and scaled-up in a bioreactor

Abstract: The development of somatic embryos in liquid culture medium has a number of advantages for large-scale propagation of plants. This paper describes an improved system for the mass propagation via somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB). Explants from immature male flowers were used to form high frequency embryogenic tissue, this tissue was then used to establish embryogenic cell suspensions in a basic MS medium plus 1.0 mg l−1 biotin, 100 mg l−1 glutamine, 100 mg l−1 malt extract (Sigma), 1.0 mg l−1 2,4-D and 45 g l−1 sucrose. Secondary multiplication of somatic embryos was achieved in liquid media in rotary shaker and in bioreactors. The number of embryos per litre obtained with 80.0% DO2 and effects of pH were also studied. A high regeneration percentage of plants were obtained (89.3%) in only 1 month of culture, somatic embryos were then placed to germinate in temporary immersion systems and field testing of somaclonal variation.

Gum katira – a cheap gelling agent for plant tissue culture media

Abstract: Gum katira, an insoluble gum derived from the bark of Cochlospermum religiosum, has been successfully used as a gelling agent in tissue culture media for in vitro shoot formation and rooting in Syzygium cuminii and somatic embryogenesis in Albizzia lebbeck. The epicotyl segments, excised from in vitro grown seedlings of S. cuminii, developed shoots when cultured on MS medium (Murashige and Skoog, 1962), supplemented with 4% sucrose and 1 mg l−1 BA. The so-developed shoots were rooted on Knop's medium, augmented with 2% sucrose and 1 mg l−1 IAA. For somatic embryogenesis, hypocotyl segments derived from in vitro developed seedlings of A. lebbeck were cultured on B5 medium containing 2% sucrose. Media were gelled with either 3% gum or 0.9% agar. The quantitative response obtained on media fortified with either of the gelling agents was not significantly different. The media gelled with gum katira were almost as transparent as the liquid medium. However, viscosity of gum katira gelled medium was less than one-sixth of the viscosity of agar-gelled media, and therefore, shaking ofthe culture vessel often resulted in submersion of the explants. Nevertheless, even these submerged explants responded positively. To increase the firmness of the gum katira-gelled medium, various combinations of agar (0.2–0.6%) and gum (1–3%) were used. However, the viscosities of the media gelled with 3% gum katira as well as different concentrations of agar (0.2–0.6%) were lower than that of the medium containing only gum katira (3%). Moreover, the explant productivity obtained in neither of these combinations was more than those recorded on the control media, which were gelled either with 0.9% agar or 3% gum alone.

In vitro screening for increased drought tolerance in rice

Abstract: In vitro screening at the cellular level was performed with mature seed-derived callus from five rice varieties, viz. IR 18351-229-3, IR 3185-6-3-3-2, SR 26-B, Nona Bokra, and C 14-8 of diverse geographical origin and with differential drought resistance at the in planta level. Callus was induced from mature seeds on Murashige and Skoog medium supplemented with 2.0 mgl−1 (9 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0, 10.0, and 15.0 gl−1 of high molecular weight polyethylene glycol (PEG, 6000) as stressing agent to create chemical drought. Simultaneous efforts were also made to assess the effects of chemical drought in altering morphogenetic response in different varieties under in vitro culture. Seed germination was almost unaffected in SR 26-B and C 14-8, unlike in other varieties where germination was seriously affected. In general, seed germination was found to be decreased in three genotypes, viz. IR 18351-229-3, IR 3185-6-3-3-2, and Nona Bokra, with increased PEG concentrations. All genotypes displayed callus induction percentage in decreasing order with increased PEG concentrations supplemented in the callus induction medium (CIM), except SR 26-B and Nona Bokra. Callus induction was found to be more on CIM fortified with 5.0 gl−1 PEG. In general, embryogenic callus induction and plantlet regeneration was found to be indirectly proportional to increased PEG concentrations used in CIM. Considering all characters, C 14-8 was found to be most appropriate in developing drought-tolerant lines under in vitro culture conditions followed by SR 26-B and Nona Bokra. A number of putative drought-tolerant plants were developed in C 14-8, SR 26-B, Nona Bokra, and IR 18351-29-3, and forwarded for field evaluation. In the majority of the progenies, a monogenic inheritance pattern for the drought tolerance character was observed.

Transgenic herbicide- and disease-tolerant carrot ( Daucus carota L.) plants obtained through Agrobacterium -mediated transformation

Abstract: Transgenic carrot (Daucus carota L.) plants expressing a rice thaumatin-like protein (tlp), phosphinothricin acetyltransferase (bar) and the hygromycin phosphotransferase (hpt) genes were obtained by Agrobacterium-mediated transformation. Petiole and hypocotyl segments of three carrot cultivars were used as the explant sources. Following infection, selection was achieved on Murashige and Skoog medium with 1 mg/l phosphinothricin or 25 mg/l hygromycin B, which was increased after 2 weeks to 10 mg/l phosphinothricin and 100 mg/l hygromycin B. The presence of the tlp and bar transgenes was confirmed by polymerase chain reaction and Southern blot analyses, and the expression of the thaumatin-like protein was demonstrated by Western blot analysis. Among 45 primary transformants, 13 were selected for assessment of herbicide and/or disease tolerance. The transgenic plants showed varying levels of tolerance to the herbicide phosphinothricin, depending on the transformation events in different lines. Four transgenic lines also showed significantly enhanced tolerance to the foliar and root pathogen Botrytis cinerea or Sclerotinia sclerotiorum when inoculated under controlled environment conditions. Two lines had significantly enhanced tolerance to the herbicide phosphinothricin as well as to both pathogens. These results demonstrate the feasibility of introducing two potentially useful agronomic traits into carrot through genetic engineering.

Micropropagation of the Mediterranean tree Ceratonia siliqua

Abstract: An in vitro propagation protocol based on axillary bud proliferation has been developed for mature female trees of Ceratonia siliqua L. `Galhosa' and `Mulata'. Browning and contaminants were the major obstacles for culture establishment. Shoot culture initiation was greatly influenced by explanting season, with the highest survival percentage observed in spring. The cultivar, cytokinin type and concentration were the most important factors affecting shoot multiplication. The best multiple-shoot response was obtained with `Mulata' on Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine or 4.56 μM zeatin. Rooting was achieved on growth-regulator-free medium after basal dipping of shoots in indole-3-butyric acid (4.9 mM). Plantlets were successfully acclimatized (80–85%) under high relative humidity and then moved to the glasshouse. A field trial was established to follow their agronomic behaviour.

Somatic embryogenesis and its conver- sion into plantlets in a multipurpose bamboo, Dendrocalamus hamiltonii Nees et Arn. Ex Munro

Abstract: New sprouts from the nodal segments of the fieldselected, mature, elite bamboos were used for the induction of embryogenic callus. Murashige and Skoog medium with BA and 2,4-D (1.0 mg/l each) was essential for culture establishment and callusing. Subsequently, elimination of 2,4-D and a corresponding increase in BA concentration induced embryogenesis. Upon transfer to sucrose -enriched medium without PGRs, these embryos matured and germinated into plantlets within 21 days, with a conversion rate of about 80%. The protocol thus established showed a low frequency of albinos when embryos were matured on a high sucrose (8%) containing medium under diffuse light conditions (5 μmol m –2 s –1 ). Rhizome formation took place upon prolonged culturing in the same medium and served as excellent propagules for multiplication. The plantlets were hardened and up to 78% survival rate was achieved in soil under greenhouse conditions. BAMBOOS form the backbone of rural economy of many southeast Asian countries. In recent years, bamboos have become important in social forestry programmes due to their short rotation cycles, fast growth and the possibility to be progressively harvested on a sustainable basis. Dendrocalamus hamiltonii is one such species which is popular for its strong culms that are used as structural support in local buildings, and the leaves serve as nutr itive fodder for milch cattle during winter months. Natural regeneration is dwindl ing due to the anthropogenic pressure on the local habitats. The traditional propagation method by ‘offsets’ limits the number of propagules, and even use of nodal segments for propagation is cumbe rsome and labour-intensive for large-scale establishment of bamboo plantations. Therefore, it is imperative to adopt alternative methods for rapid multiplication, and tissue culture techniques come handy. Although multiple shoot formation is accomplished with comparative ease, rooting of such shoots is still incon sistent in D. hamiltonii, and only up to 25–30% shows rooting 1 . In this con

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

Abstract: An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 125–50 μM 6-benzyladenine Addition of α-naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 125, 25 or 50 μM combined, respectively, with 25, 125–50 or 50 μM α-naphthaleneacetic acid Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 25 μM indole-3-butyric acid Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate The survival rate of plantlets under ex vitro conditions was 100% after 3 months The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies

Rapid clonal propagation of nothapodytes foetida (wight) sleumer - a threatened medicinal tree

Abstract: Nothapodytes foetida (Wight) is a small evergreen tree and the extract from this tree is used to make the antileukaemia and antitumoral compound camptothecin. Due to exploitation of this resource, efficient methods for rapid propagation of N. foetida are highly desirable. Multiple shoots were induced on hypocotyl segments of 20–25-d-old seedlings of N. foetida cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of cytokinins. The highest shoot multiplication was achieved on MS medium containing thidiazuron (TDZ) at the concentration of 2.2 μM. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to medium containing 2.2 μM benzylaminopurine which produced healthy shoots after three additional subcultures. The production of shoots was further promoted by repeated subculturing of original explants on fresh multiplication medium after each harvesting of the newly formed shoots. In vitro rooting was best induced (87%) in shoots excised from proliferated shoot cultures on one-fourth MS medium augmented with 5.7 μM indole-3-acetic acid and 2.4 μM indolebutryic acid (IBA). In vitro-developed shoots were also rooted ex vitro by dipping in 49 μM IBA for 10 min. In vitro- and ex vitro-rooted plants were successfully acclimatized and established in greenhouse conditions.

Effective Protocol for in Vitro Shoot Production Through Nodal Explants of Simmondsia Chinensis

Abstract: Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoog's medium supplemented with 20 μM N6-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 μM indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 μM IBA + 0.5% activated charcoal + 1 μM BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.

Artemisinin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium

Abstract: Murashige & Skoog medium was modified for enhancing artemisinin production in Artemisia annua hairy root cultures by altering the ratio of NO 3 − /NH 4 + and the total amount of initial nitrogen. Increasing ammonium to 60 mM decreased both growth and artemisinin accumulation in hairy root cultures. With NO 3 − /NH 4 + at 5:1 (w/w), the optimum concentration of total initial nitrogen for artemisinin production was 20 mM. After 24 days of cultivation with 16.7 mM nitrate and 3.3 mM ammonium, the maximum artemisinin production of hairy roots was about 14 mg l−1, a 57% increase over that in the standard MS medium.

Rapid clonal multiplication of Lavandula viridis l'Hér through in vitro axillary shoot proliferation

Abstract: In vitro clonal propagation of native Mediterranean Lavandula viridis was obtained from a mature field-grown plant. Single node explants were successfully established on Murashige and Skoog medium supplemented with 0.44 μM of 6-benzyladenine. The highest multiplication rate (11.69 shoots/node) was obtained with 0.67 μM 6-benzyladenine in Murashige and Skoog medium with macronutrients at half-strength. Shoots were easily rooted on Gresshoff and Doy medium. Increasing sucrose concentration from 58.4 to 87.6 mM resulted in a significant increase in rooting frequency. Eighty per cent of plantlets were successfully acclimatised to ex vitro conditions, exhibiting a normal development.

Micropropagation of pitaya (Hylocereus undatus britton et rose)

Abstract: A procedure for micropropagation of pitaya using thidiazuron (TDZ) and naphthaleneacetic acid (NAA) is described. Explants were excised from young joints of mature plants and cultured on Murashige and Skoog medium (MS) containing 0.5 μM NAA and 0.5 μM TDZ. Shoots produced from these first explants were cut up to produce secondary explants, either by decapitation or by longitudinal division into three parts. The decapitated and longitudinal explants were cultured on MS supplemented with 0.5 μM NAA and either 0.01, 0.09, 0.5, or 0.9 μM TDZ. Decapitated explants produced more shoots at higher frequency that the longitudinal explants. For both types of secondary explants, most shoots were developed from the distal parts. Shoots produced from secondary explants were rooted in MS and then transferred to soil where they produced normal plants.

Germination of synthetic seeds of pineapple ( Ananas comosus L. Merr.)

Abstract: Tufts of multiple shoots were produced from dormant, axillary buds of pineapple in vitro. Tiny shoots (2–5 mm) isolated from the tuft of multiple shoots were encapsulated in 3% sodium alginate prepared using hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose. The encapsulated shoots represented synthetic seeds that germinated and formed roots in vitro after subculture onto one of the following media solidified with 0.8% agar: (1) hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose (Pin1), (2) Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol, 0.06 M sucrose, 9.67 µM 1-naphthalene acetic acid, 9.84 µM indole-3-butyric acid and 9.29 µM kinetin (Pin2), and (3) White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 0.54 µM 1-naphthalene acetic acid and 1.97 µM indole-3-butyric acid (Pin3). Pretreatment of shoots in either liquid Pin3 or Pin4 medium (White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 10.8 µM 1-naphthalene acetic acid and 39.4 µM indole-3-butryic acid) was required for development into plantlets with roots after culture on either Pin1, Pin2 or Pin3 media. One hundred percent germination of synthetic seeds to plantlets occurred after pretreatment of shoots in liquid Pin4 medium for 12 h followed by culture of synthetic seeds on Pin2 medium. Synthetic seeds stored at 4°C remained viable without sprouting for up to 45 days. Plantlets produced in vitro from synthetic seeds were successfully established in soil. The protocol provides an easy and novel propagation system for pineapple, an otherwise vegetatively propagated fruit crop.

Somatic embryogenesis through pseudo-bulblet transverse thin cell layer of Lilium longiflorum

Abstract: Somatic embryogenesis was achieved directly from pseudo-bulblettransverse thin cell layers (tTCL) of Lilium longiflorum.Embryo-like structures (globular embryos) were obtained from different sizeexplants of pseudo-bulblet tTCLs after 45 days culture on Murashige and Skoog,1962 (MS) medium containing 5.4 μM naphthalene acetic acid(NAA)and 1.1 μM thidiazuron (TDZ). The embryo-like structures werethen isolated and mass proliferated on MS medium, containing 5.4μM NAA and 0.4 μM TDZ, every 45 days. A0.8–1.0 mm thick explant was shown to be optimal forobtaining the highest number of embryo-like structures. For plant regenerationthese structures were transferred to hormone-free MS medium with 30g/l sucrose. All of these structures formedplantlets after 90 days culture.