Showing papers on "Murashige and Skoog medium published in 2005"

Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron

Abstract: The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10μM) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5μM TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5μM indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.

An efficient micropropagation system for Eclipta, alba —A valuable medicinal herb

Abstract: An efficient rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Eclipta alba (Asteraceae) by enhanced axillary shoot proliferation in cotyledonary node segments was designed. The medium type, various carbon sources, plant growth regulators, and coconut water markedly influenced in vitro propagation of Eclipta alba. An in vitro plantlet production system has been investigated on Murashige and Skoog (MS) medium with the synergistic combination of benzyladenine (4.4μM), kinetin (4.6μM), 2-isopentenyladenine (4.9μM), gibberellic acid (1.4μM), 5% coconut water, and 3% sucrose which promoted the maximum number of shoots as well as beneficial shoot length: Subculturing of cotyledonary node segments on a similar medium enabled continuous production of healthy shoots with similar frequency. Rooting was highest (94.3%) on full strength. MS medium containing 9.8 μM indolebutyric acid. Micropropagated plants established in garden soil, farmyard soil, and sand (2∶1∶1) were uniform and identical to the donor plant with respect to growth characteristics as well as floral features. These plants grew normally without showing any morphological variation.

Overexpression of mtlD gene in transgenic Populus tomentosa improves salt tolerance through accumulation of mannitol.

Abstract: The mtlD gene encoding mannitol-1-phosphate dehydrogenase, which catalyzes the biosynthesis of mannitol from fructose, was cloned from Escherichia coli and transferred to poplar (Populus tomentosa Carr.) through Agrobacterium-mediated transformation. The transgenic plants were screened and selected on Murashige and Skoog (MS) medium containing 30-50 mg l(-1) kanamycin and verified by polymerase chain reaction (PCR) and Southern blotting. Expression of the gene led to synthesis and accumulation of mannitol in the transgenic plants. Gas chromatography and mass spectrometry (GC/MS) and capillary gas chromatography (GC) showed that transgenic plants accumulated much more mannitol in their tissues than the wild-type plants, whether cultured in vitro, or grown hydroponically or in the field. Increased salt tolerance of transgenic plants was observed both in vitro and in hydroponic culture. The transgenic buds rooted normally on MS medium containing 50 mM NaCl, whereas wild-type buds did not. In the 40-day hydroponic experiments, transgenic poplar plants survived in a 75-mM NaCl treatment, whereas the wild-type poplar plants tolerated only 25 mM NaCl. Under the same NaCl stress, stomatal conductance, transpiration rates and photosynthetic rates were all higher in transgenic plants than in wild-type plants, whereas cellular relative conductivity was lower. We demonstrated that the mtlD gene was expressed in transgenic poplar plants, resulting either directly or indirectly in mannitol accumulation and improved salt tolerance. The constant mannitol concentrations in transgenic plants during the NaCl treatments indicated that mannitol accumulation caused by the mtlD gene was not a consequence of NaCl stress. Height growth was reduced by about 50% in the transgenic plants compared with the wild-type plants in the absence of salt; however, relative growth rate was much less influenced by salt stress in transgenic plants than in wild-type plants. The stunted growth of the transgenic plants may in part explain their improved salt tolerance.

In vitro induction of tetraploid plants from diploid Zizyphus jujuba Mill. cv. Zhanhua

Abstract: Tetraploid plants of Zizyphus jujuba Mill. cv. Zhanhua were obtained with in vitro colchicine treatment. Shoot tips from in vitro-grown plants were treated with five different concentrations of colchicine (0.01, 0.03, 0.05, 0.1, 0.3%) in liquid MS medium (Murashige and Skoog 1962), and shaken (100 rpm) at 25 °C in darkness for 24, 48, 72 or 96 h, respectively. Tetraploids were obtained at a frequency of over 3% by using 0.05% colchicine (48 h, 72 h) and 0.1% colchicine (24 h, 48 h) treatment as determined by flow cytometry. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The chromosome number of diploid plants was 24 and that of tetraploid plants was 48. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata were reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The selected tetraploid plants were grafted onto mature trees of Z. jujuba Mill. cv. Zhanhua in the field, resulted in thicker stems, rounder and succulent leaves, larger flowers and a delay in florescence time (3–4 days later) than diploid plants.

Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators.

Abstract: A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and α-naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 μM BA and 1.0 μM NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 μM BA and 1.0 μM NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 μM indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.

Efficient plant regeneration via shoot tip explant in Jatropha curcas L.

Abstract: An efficient regeneration method via shoot tip explant has been developed for Jatropha curcas, which is a medicinally as well as economically important plant Shoot tips were proliferated on MS medium incorporated with BAP (20 mg l−1) and IAA (05 mg l−1) along with adenine sulphate, glutamine and activated charcoal In vitro produced shoots were induced to root on IBA (05–50 mg l−1) added to half strength MS medium The highest frequency of root induction was on the medium with 30 mg l−1 IBA Regenerated plantlets were successfully transferred to field after initial acclimatization

Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (Brassica juncea var. tsatsai)

Abstract: Cotyledon and leaf segments of stem mustard (Brassica juncea var. tsatsai) were cultured on Murashige and Skoog medium supplemented with various concentrations of different cytokinins [6-benzyladenine (BA), N-(2-chloro-4-pyridyl)-n-phenylurea (CPPU), 6-furfurylaminopurine (KT) and thidiazuron (TDZ)] in combinations with different levels of α-naphthalene acetic acid (NAA). The shoot regeneration frequency of cotyledon and leaf segment was dependent on the kinds and concentrations of cytokinins used in the medium, while in most cases cotyledon gave high regeneration frequency than leaf segment. TDZ proved to be the best cytokinin to induce shoot from both cotyledon and leaf segments compared to BA, KT and CPPU. The highest frequency of shoot regeneration was 61.3–67.9 % in cotyledon and 40.7–52.4% in leaf segment respectively when 2.27 or 4.54 μM TDZ was combined with 5.37 μM NAA. Next to TDZ, CPPU was also very suitable to induce shoot formation both in cotyledon and leaf segment. When 1.61 μM CPPU was combined with 2.69 μM NAA, shoot regeneration frequency was 45.0% in cotyledon and 36.4% in leaf segment, respectively. It was also shown that KT and BA affected shoot regeneration from cotyledon and leaf segment, the shoot regeneration was greatly increased when NAA was added together with cytokinins. The efficient and reliable shoot regeneration system was developed in both cotyledon and leaf segments. This regeneration protocol may be applicable to the improvement of this crop by genetic engineering in the future.

Micropropagation of zedoary (Curcuma zedoaria Roscoe)--a valuable medicinal plant

Abstract: Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l−1 sucrose and 5 g l−1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l−1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l−1 BA, and 0.5 mg l−1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l−1 sucrose and 8 g l−1 agar, supplemented with 20 (v/v) CW and 2 mg l−1 NAA. More than 95 of the rooted plants were established in pots after hardening.

In vitro regeneration of Bambusa balcooa Roxb.: Factors affecting changes of morphogenetic competence in the axillary buds

Abstract: Axillary buds from field-grown culms of Bambusa balcooa were used as explants to induce multiple shoots in liquid Murashige and Skoog’s (MS) medium supplemented with 11.25 μM of 6-benzylaminopurine (BAP) and 4.5 μM kinetin (Kn). A clump of at least 3 shoots was used for root induction in half strength MS medium with 1.0 μM of 3-indolebutyric acid (IBA). Morphogenetic competence of the axillary buds varied widely in different months of two consecutive calendar years. The highest morphogenetic potentials were observed in October. The major problem encountered was presence of systemic fungal contaminants. Perhaps, rainfall positively contributed to induce morphogenetic competence. A moderately high phenolic content of the nodal explant was also detrimental for in vitro morphogenesis. The morphogenetic competence of B. balcooa correlated with the season in which the explants were excised from the natural stands. To the best of our knowledge this is the first report on in vitro regeneration of B. balcooa from mature field-grown axillary buds.

Organogenesis, embryogenesis, and synthetic seed production in arnebia euchroma – a critically endangered medicinal plant of the himalaya

Abstract: This is the first report of simultaneous organogenesis and somatic embryogenesis in Arnebia euchroma, a highly valued, critically endangered medicinal plant of the Himalaya Root-derived callus showed only rhizogenesis, whereas leaf-derived callus showed simultaneous organogenesis and somatic embryogenesis Organogenesis was optimal (122 shoots per culture) in 1 μM indole-3-butyric acid combined with 25 μM 6-benzyladenine and induction of somatic embryogenesis (163 embryos per culture) occurred in 25 μM indole-3-butyric acid combined with 25 μM 6-benzyladenine Shoots rooted (100%) best in half-strength Murashige and Skoog (MS) medium supplemented with 20 μM indole-3-butyric acid Early cotyledonary-stage embryos encapsulated with 3% sodium alginate and calcium nitrate (100 mM for 25 min) showed 606% germination in MS medium Rooted shoots transferred to a mixture of sterile soil, sand, and peat (1:1:1 by volume) showed 72% survival ex vitro Application of these protocols would be helpful

Rosmarinic acid production by Coleus forskohlii hairy root cultures

Abstract: Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.

Effect of genotype on callus induction and plant regeneration from leaf explants of sugarcane ( Saccharum sp.)

Abstract: Nine sugarcane genotypes (CP59-73, CP63-588, CP80-314, SP71-1081, F160, L62-96, CP70-321, CP57- 614 and Clone III) were evaluated for their callus induction capacity, embryogenic callus production and plant regeneration ability. Leaf cylinders were used as explants using Murashige and Skoog (MS) based medium supplemented with 3 mg l-1 2,-4 dichlorophenoxyacetic acid. Plant regeneration was accomplished on hormone free modified MS medium supplemented with casein hydrolyzate. The genotypes tested showed high callus induction percentage (69 to 95%) and high embryogenic callus percentage (60 to 100%). These genotypes also showed excellent regeneration capacities, with regeneration percentages ranged between 88 and 100%. Significant differences were observed between genotypes for callus induction capacity, embryogenic response and plant regeneration ability indicating that these criteria are genotype dependent. Plant regeneration ability is highly correlated with embryogenic callus production. The in vitro regenerated plants were successfully rooted and well acclimatised in growth cabinet conditions.

Efficient plant regeneration through direct somatic embryogenesis from leaf explants of phalaenopsis ‘little steve’

Abstract: Leaf segments of the orchid sp. Phalaenopsis ‘Little Steve’ were used as explants testing the effects of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.45, 2.26, 4.52 μM), 6-furfurylaminopurine (kinetin; 2.32, 4.65, 13.95 μM), N6-benzyladenine (BA; 2.22, 4.44, 13.32 μM), and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 2.27, 4.54, 13.62μM) on the induction of direct somatic embryogenesis. After 20–30 d of culture in darkness, clusters of somatic embryos formed from leaf surfaces and wounded regions of explants on half-strength Murashige and Skoog medium supplemented with BA and TDZ. However, kinetin had no response on direct embryo induction. In addition, 2,4-D highly retarded the frequency of embryogenesis that was induced by TDZ. Generally, adaxial surfaces near wounded regions had the highest embryogenic competency compared to other regions of explants. Histological sections revealed that somatic embryos mostly arose from epidermal cell layers of the explants. Secondary embryogenesis occurred at basal parts of embryos, and originated from outer cell layers. Following transfer of regenerated embryos onto growth regulator-free medium for 3.5–4 mo., plantlets with three to four leaves and several roots were obtained. This protocol provides a simple way to regenerate plants through direct somatic embryogenesis, and is suitable for further studies on embryo development and genetic transformation of Phalaenopsis.

Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds

Abstract: A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented. Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l(-1) TDZ followed by three cycles of selection on medium with 0.5 mg l(-1) BA and increasing concentrations of hygromycin (20-40-60 mg l(-1)). Selected shoot clusters were transferred to medium with 0.5 mg l(-1) BA for proliferation and 0.2 mg l(-1) BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium with 2.0 mg l(-1) NAA. The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny. Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene. This paper reports the first successful attempt at producing transgenic castor.

In vitro regeneration of the medicinal woody plant Phellodendron amurense Rupr. through excised leaves

Abstract: Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm2 sections of about 10-day-old leaves on MS medium enriched with 4.4 μM BAP and 1.0 μM NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 μM TDZ and 4.0 μM 2,4-D or 4.0 μM NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 μM BAP and 1.0 μM NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 μM IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.

Thidiazuron-induced high-frequency shoot organogenesis from leaf-derived callus of ia medicinal climber, Tylophora Indica (Burm. F.) merrill

Abstract: Tylophora indica (Burm. f.) Merrill is a threatened medicinal climber distributed in the forests of northern and peninsular India. An efficient and reproducible protocol for high-frequency callus regeneration from immature leaf explants of T. indica was developed. Organogenic callus formation from immature leaf pieces was obtained by using Murashige and Skoog (MS) medium supplemented with 7 μM 2,4-dichlorophenoxyacetic acid and 1.5 μM 6-benzyladenine. On this medium 92% explants produced callus. The optimal hormone combination for plantlet regeneration was 8 μM thidiazuron, at which shoot regeneration was obtained from 100% of the cultures, with an average of 66.7 shoots per culture. Histological studies of the regenerative callus revealed that shoot buds were originated from the outermost regions. For root formation, half-strength MS medium supplemented with 3 μM indole-3-butyric acid was used. Plants were transferred to soil, where 92% survived after 3 mo. of acclimatization.

Micropropagation of clitoria ternatea linn. (fabaceae) – an important medicinal plant

Abstract: An efficient protocol was established for in vitro shoot multiplication from nodal explants of Clitoria ternatea on semisolid Murashige and Skoog (MS) basal medium supplemented with 8.9μM 6-benzylaminopurine (BA). Inclusion of 1-naphthaleneacetic acid (NAA) in the culture medium along with BA promoted higher rates of shoot multiplication than BA alone. The rate of shoot multiplication was maximum (5.21) after 4 wk of culture on MS basal medium supplemented with 8.9μM BA and 1.34μM NAA. The elongated shoots rooted within 7–8d in half-strength MS basal salts supplemented with 1.34μM NAA and 2% (w/v) sucrose. About 85% of the rooted plantlets were acclimatized and transferred to the greenhouse.

Influence of cultivar, explant source and plant growth regulator on callus induction and plant regeneration of cannabis sativa l.

Abstract: The effects of different combinations of plant growth regulators (PGRs) on callus induction and plant regeneration were investigated in five cultivars of Cannabis sativa L. Callus was induced from different explant sources (young leaves, petioles, internodes, axillary buds) on MS basal medium with various concentrations of PGRs (2,4-D, DICAMBA, KIN, NAA). The highest frequency of callus induction (avg. 82.7% of eight medium combinations) was exhibited by petiole explants of cv. Fibrimon-24. Plant regeneration was obtained from all studied cultivars. The highest number of plants was regenerated from callus tissue of petiole explants on MS medium containing DICAMBA. A total of 46 plants (1.35% of callus) were regenerated: 16 (0.47%) from cv. Silesia petioles, 7 (0.20%) from cv. Novosadska petioles, 6 (0.18%) from cv. Fedrina-74 petioles, 12 (0.35%) from cv. Fibrimon-24 axillary buds, and 5 (0.15%) from cv. Juso 15 internodes. Significant improvement of hemp plant regeneration in vitro was achieved.

Micropropagation of eclipta alba (l.) hassk – an important medicinal plant

Abstract: An efficient and reproducible protocol for mass propagation of Eclipta alba (L.) Hassk, an important medicinal plant, was standardized by culturing shoot tips and nodal segments taken from in vitro raised plants. Maximum shoot proliferation occurred when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 1 mg l−1 benzylaminopurine (BAP). The shoot buds formed were further multiplied and maintained on medium containing BAP (0.5 mg l−1) and gibberellic acid (0.5 mg l−1). Rooting was best achieved on MS medium supplemented with 1 mg−1 indole-3-butyric acid. Rooted plantlets attained maturity and flowered normally in the field.

Micropropagation of Hagenia abyssinica: a multipurpose tree

Abstract: An in vitro propagation protocol has been developed for Hagenia abyssinica using original material from both juvenile and mature trees. Juvenile explants were obtained from seedlings, as well as shoots and meristems from 5 to 7-month-old greenhouse grown plants. Shoots collected from stem bases of five genotypes were used to establish cultures from mature trees. Explants of seedling origin were used to optimize the multiplication medium and growth regulators concentration. The best result was obtained from shoots subcultured on either MS or WPM medium supplemented with 4.4 μM BAP and 0.49 μM IBA. The initiated shoots from all the different explants were multiplied on these media. Rooting of shoots was achieved using MS medium containing macronutrients at one-third strength supplemented with 4.9 μM IBA. The shoots were kept in the dark for 4 days and transferred to medium of the same composition but containing 0.3% activated charcoal without growth regulators. Up to 100% rooting was achieved depending on genotype. Shoots multiplied on MS medium rooted better than those multiplied on WPM. Plantlets were transferred to pots containing a mixture of soil and perlite in a 2:1 ratio, respectively, and were maintained in the greenhouse. Increased irradiance reduced stem and leaf lengths and increased branch number of micropropagated plants.

In vitro clonal propagation of holarrhena antidysenterica (l.) wall. through nodal explants from mature trees

Abstract: In vitro clonal propagation of 18–20-yr-old Holarrhena antidysenterica tress has been achieved by employing nodal explants. The tree explants showed marked seasonal variation in their morphogenic response under in vitro conditions. Maximum response was obtained from the beginning of May to the end of July, followed by a gradual decline, finally dropping to zero from October to February. The explants induced multiple shoots only on cytokinin-containing medium. Several cytokinins [N6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2ip), 6-furfuryl aminopurine (Kn), and adenine sulfate (Ads)] were assayed. The best response was achieved with 15 μM BA in which 62.5% of cultures produced 2.75±0.2 shoots per explant with 3.56±0.2 cm average length. Amongsth the three heavy metals assayed, silver nitrate (AgNO3) significantly improved the response. This compound enhanced both the percentage of responding cultures (86.6%) and the average shoot number (4.73±0.2) at a concentration of 20mgl−1. Further improvement in the morphogenic response occurred when explants from in vitro shoots were employed instead of mature trees. In this case, the percentage of morphogenic cultures was increased to 100% at the third subculture with an average of 11.45±0.3 shoots per explant. Regenerated shoots were rooted in half-strength Murashige and Skoog medium with 10 μM indole-3-acetic acid. The plantlets were successfully acclimatized in soil.

An efficient adventitious shoot regeneration system for Zhanhua winter jujube (Zizyphus jujuba Mill.) using leaf explants

Abstract: The direct induction of adventitious shoots from leaf explants obtained from adult plants of Zhanhua winter jujube, an elite variety of Zizyphus jujuba Mill., is reported. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when 10-day-old leaves were explanted onto Woody Plant Medium and maintained initially in the dark. The plant growth regulator thidiazuron (TDZ) was effective in stimulating shoot regeneration from leaf explants of Zhanhua winter jujube. The highest efficiency of shoot formation was observed with a 20-day culture in the dark on WPM containing 4.54 μM TDZ and 2.85 μM indoleacetic acid (IAA). The regenerated shoots were transferred to MS medium containing 0.89 μM benzyladenine and 5.77 μM gibberellic acid for growth. When the shoots were about 2 cm in height, they were transferred to Nitsch medium supplemented with 1.14 μM IAA and 2.46 μM indolebutyric acid to induce rooting. This system of adventitious shoot production from leaf explants of adult plants could be useful for the genetic engineering and polyploidization of winter jujube.

In vitro regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. (Rosaceae) from leaf explants

Abstract: A protocol for shoot regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. has been developed using leaf explants originating from in vitro seedlings and mature material. The explants were cultured on Murashige and Skoog medium containing various concentrations of alpha-naphthaleneacetic acid and thidiazuron (TDZ). Concentrations of TDZ lower than 1.0 microM promoted direct shoot regeneration, but higher concentrations promoted callus induction. Around 96-100% regeneration was obtained between 1.0 and 10 microM TDZ. The average number of shoots per explant at 1.0 microM TDZ was 8.4+/-4.8. Among the different explants used, the highest percentage of regeneration and shoots per explant was obtained from complete leaf explants. A significant (P< or =0.05) difference in regeneration capacity was observed among the five genotypes examined. The resulting shoots were multiplied on multiplication medium, rooted and acclimatised in a greenhouse.

Agrobacterium-mediated transformation of bottle gourd (Lagenaria siceraria Standl.).

Abstract: We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance (bar) gene and the β-d-glucuronidase (GUS) reporter gene. The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6–8 days on co-cultivation medium supplemented with 0.1–0.001 mg/l l-α-(2-aminoethoxyvinyl) glycine (AVG). The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2–3 weeks of culturing on selection medium containing 2 mg/l dl-phosphinothricin. These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l dl-phosphinothricin. Transgenic plants were obtained at frequencies of 1.9%. Stable integration and transmission of the transgenes in T1 generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses. Genetic segregation analysis of T1 progenies showed that transgenes were inherited in a Mendelian fashion. To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

Rapid micropropagation of Curcuma longa using bud explants pre-cultured in thidiazuron-supplemented liquid medium

Abstract: Multiple shoots of Curcuma longa were induced by culture of bud explants for 1 week in Murashige and Skoog (MS) liquid medium supplemented with 72.64 μM thidiazuron (TDZ) prior to culture on MS gelled medium without growth regulator for 8 weeks. The regeneration rate was up to 11.4 ± 1.7 shoots/explant. Rooting was spontaneous and the regenerated plants were successfully transferred to soil. This protocol can be an alternative for rapid micropropagation of C. longa used for phytomedicine raw material production.