Combinations Of Cytokinins Research Articles

Induction and multiplication of callus from endosperm of Cycas revoluta

The usage of medicinal plants in traditional medication has gained the attraction from global and local markets, mainly to cure diseases or simply for health maintenance. Callus cultures were initiated from the endosperm of the medicinal plant Cycas revoluta, cultured on half-strength Murashige and Skoog (MS) medium supplemented with 30 g/L sucrose and various concentrations (5, 10 and 20 iM) of 2,4- dichlorophenoxyacetic acid (2,4-D), 1-naphthalene acetic acid (NAA) and 4-amino-3,5,6- trichloropicolinic acid (picloram). Explants treated with various auxins formed calli with different morphologies. In the induction studies, 20 iM picloram was the most efficient formulation for callus formation. The callus was formed after 17.8 ± 0.5 days in the medium. However, callus was not formed in the control medium (MSO) and medium supplemented with 20 iM 2,4-D. Calli were successfully maintained in 10 iM picloram at normal photoperiod (16 h light, 8 h dark). The calli treated with10 iM picloram that incubated in 24 h dark condition was found to exhibit less browning effects. Addition of 1 g/L polyvinylpyrrolidone (PVP) aided in overcoming the browning effects by absorbing the phenolic compounds in the medium. The combination of auxin (10 iM picloram) and cytokinin (10 iM kinetin) was able to multiply more friable and yellowish-green calli.

In vitro propagation of seven Daphne L. species

Cultures of seven Daphne species: Daphne caucasica, D. cneorum, D. giraldii, D. retusa, D. jasminea, D. laureola and D. tangutica were established in vitro on MS/WPM based media. Five of the species responded best on MS-based media (D. tangutica, D. laureola, D. caucasica, D. retusa and D. giraldii), while the remaining two species performed best on WPM-based media (D. cneorum, and D. jasminea). Shoot proliferation was achieved from both apical and nodal explants. Shoots were sub-cultured from stock cultures, cut into nodal explants 3–5 cm long and place vertically on basal media supplemented with different concentrations and combinations of cytokinins and auxins. Individual species displayed different responses to the various cytokinins and auxins. Among species, D. jasminea produced the greatest proliferation rate with an average of 7.84 + 0.6 shoots per explant on WPM supplemented with 2.32 μM BA + 0.0045 μM TDZ + 0.054 μM NAA, while the best multiplication rate for the same species grown on the same media supplemented with a single cytokinin (BA) and no auxin was 2.60 + 1.3 shoots per explant. Following multiplication, new shoots transferred to the elongation trails and then 50–100 mm Shoots used for rooting experiments. Increased rooting efficiencies were observed on in vitro-generated shoots with the two-layer medium or dipping methods over when PGRs were uniformly incorporated into the medium. Maximum rooting frequencies (average) ranged from 59% in D. tangutica to 85% in D. jasminea. Following in vitro rooting, rooted shoots immersed in 0.01% solution of humates and planted into a standard horticultural substrate composed and watered weekly with a solution containing half-strength MS salts.

Genetic stability of micropropagated ginger derived from axillary bud through cytophotometric and RAPD analysis.

A protocol was developed for the in vitro propagation of ginger (Zingiber officinale) cv. Suprava using dormant axillary buds from unsprouted rhizomes. The dormant axillary buds embedded in the rhizome nodes were induced to sprout when cultured on MS medium supplemented with 6-benzyladenine (BA) alone (1-6 mg/1) or with a combination of BA (1-6 mg/1) and indole-3-acetic acid (IAA) (0.5, 1 mg/l). In vitro sprouted buds were transferred to the multiplication medium containing various combinations of auxins and cytokinins. MS basal medium supplemented with BA (1 mg/l), IAA (1 mg/l) and adenine sulfate (100 mg/l) was found optimum for the in vitro multiplication of shoots producing (8.2 +/- 0.2) shoots from a single explant within 30 days of culture. The multiplication rate remained unchanged in subsequent subcultures. Rooting of shoots occurred in the same multiplication media. Upon transfer of the in vitro culture to ex vitro in pots, 96% of plants survived and established successfully under natural conditions. Tissue culture-raised plantlets of ginger could be conserved in vitro through subculturing at an interval of 4 months. The genetic stability of micropropagated clones was evaluated at regular intervals of 6 months up to 24 months in culture using cytophotometric estimation of 4C nuclear DNA content and random amplified polymorphic DNA (RAPD) analysis. Cytophotometric analysis revealed a unimodal distribution of the DNA content with a peak corresponding to the 4C value (23.1 pg), and RAPD analysis revealed monomorphic bands showing the absence of polymorphism in all fifty regenerants analyzed, thus confirming the genetic uniformity among in vitro grown somaclones of Z. officinale. This study is of commercial significance as axillary bud explants are available throughout the year for initiating a fresh culture of the elite ginger cv. Suprava to be used as a source of true-to-type disease-free planting material thereby minimizing the adverse effect of repeated subculturing from the same explant source.

In vitro Propagation of Solanum sessiliflorum as Affected by Auxin and Cytokinin Combinations and Concentrations

In vitro Propagation of Solanum sessiliflorum as Affected by Auxin and Cytokinin Combinations and Concentrations

In vitro propagation of miracle berry (Synsepalum dulcificum Daniel) through embryo and nodal cultures

Miracle berry is an evergreen tropical shrub which modifies sour food to produce a sweet taste. Its propagation is, however, hindered by seed recalcitrance and difficulty of stem to root. Thus in vitro propagation was investigated through embryo and nodal explants using different levels and combinations of auxins and cytokinins in MS medium. Embryo was regenerated in MS medium supplemented with 0.1 mg/l NAA + 0.2 mg/l BAP. Lateral buds proliferation was induced on the germinated embryo with 0.6 - 3.0 mg/l BAP + 0.1 - 0.2 mg/l NAA in which 3.0 mg/l BAP + 0.1 mg/l NAA produced highest number of buds. Rooting of the embryo regenerated plantlets was achieved with 1.0 - 2.0 mg/l IBA + 0.1 mg/l BAP. Very low (5 - 10%) axillary and terminal buds formation was achieved from nodal cultures. Few of the nodal explants formed buds with 0.1 - 0.8 mg/l NAA + 0.2 - 1.0 mg/l BAP + 0.02 mg/l GA3 with 0.8 mg/l NAA + 0.2 mg/l BAP producing the best result. However, all efforts to induce rooting on the buds formed from nodal explants proved abortive.

Micropropagation of gerbera: lipid peroxidation and antioxidant enzyme activities during acclimatization process

Gerbera jamesonii H. Bolus ex Hook (Family: Asteraceae) has been successfully acclimatized from temperate to subtropical North Indian plains of Lucknow through in vitro propagation. Flower heads were collected from greenhouse, segmented into 4–16 pieces and cultured in Murashige and Skoog’s medium (MS) (Physiol Plant 15:472–497, 1962) supplemented with 2.87 μM indole-3-acetic acid (IAA) and 8.88 μM N6-benzyladenine (BA) for shoot regeneration. Shoots were subcultured on growth regulator free MS medium. Apical shoot meristems from in vitro plantlets of gerbera were tested in MS medium with different combination of cytokinins [BA, kinetin, and thidiazuron (TDZ)] alongwith 2.68 μM 1-naphthaleneacetic acid (NAA) for shoot multiplication. The optimum results were obtained with 8.88 μM BA. Regenerated plants with well-established root system were transferred to pots containing soil and sand (1:1 v/v) and were kept in humidity chamber with 80–90% relative humidity for 0, 5, 10, 15, 20, and 25 days before they were transferred to field (during October, 2005 to February, 2006). Survival percentage was higher when regenerated plantlets were kept under humidity chamber for 15 days. An attempt was made to obtain basic information on different biochemical changes during acclimatization process of in vitro raised plantlets. Increased lipid peroxidation and high H2O2 content in early stages of acclimatization process reflected a similar process of oxidative stress. Our work suggests that tissue-cultured plants develop antioxidant enzymatic protective system which determine the ability to survive in oxidative stress and up regulation of these enzymes would help to reduce the built up of reactive oxygen species (ROS).

Effect of plant growth regulators on direct somatic embryogenesis in camphor tree (Cinnamomum camphora L.) from immature zygotic embryos and embryogenic calli induction

A description of a successful direct somatic embryogenesis induction from immature zygotic embryos of a camphor tree (Cinnamomum camphora L.) is presented. After a subculture of 2–3 years, embryogenic calli could be derived from primary somatic embryos. Immature zygotic embryos were cultured on a Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins (BA) and auxins (2,4-D or NAA) for somatic embryo induction. Primary somatic embryos could be induced directly in almost all PGR combinations. A positive effect of 2,4-D on somatic embryogenesis from immature zygotic embryos of camphor tree was obtained. BA at appropriate concentrations ( 5 mg·L−1) of BA had the effect of restraining somatic embryo induction. NAA had a less positive effect on somatic embryogenesis than 2,4-D.

Morphogenic capability of Gentiana kurroo Royle seedling and leaf explants

Experiments have been carried out on seedling and primary leaf explants of Gentiana kurroo Royle. Morphogenic capacities of cotyledons, hypocotyls and roots were investigated using MS (1962) medium supplemented with 4.64 μM kinetin and 2.26, 4.52 or 9.04 μM 2,4-D. Percentage of callusing explants for each combination was inversely proportional to numbers of obtained embryos. Cotyledons showed the highest morphogenic capabilities. To assess the morphogenic potential of leaf explants, 189 combinations of auxin (NAA, dicamba and 2,4-D) and cytokinin (kinetin, BAP, zeatin, CPPU and TDZ) in different concentrations were tested. The presence of NAA with BAP and dicamba with zeatin produced the greatest number of differentiated somatic embryos. Microscopic analysis of responsive explants led to identifying rhizogenic centers, non-embryogenic and embryogenic cells. The best embryo conversion into germlings was obtained on MS medium containing 4.46 μM kinetin, 1.44 μM GA3 and 2.68 μM NAA or ½ MS. Both media were supplemented with 4.0% sucrose and 8.0% agar. Depending on explant origin and conversion medium, 55.8–71.0% of somatic embryos developed into germlings and plants.

In Vitro Propagation Through Nodal Explants in Helicteres isora L., a Medicinally Important Plant

Helicteres isora is medicinally important plant effective against asthma, diabetes, hypolipidemia, HIV, besides a good source of diosgenin. Seed dormancy and low rate of natural fruit production make this plant a perfect candidate for developing an in vitro method useful for its clonal propagation and further biotechnological developments. This is the first report on in vitro production of this plant. Nodal explants obtained from aseptically germinated seedlings were cultured on MS medium (Murashige and Skoog 1962) fortified with indole-3-acetic acid (IAA) (<TEX>$0.57-22.83\;{\mu}M$</TEX>), indole-3-butyric acid (IBA) (<TEX>$0.41-16.58\;{\mu}M$</TEX>), 6-benzylaminopurine (BA) (<TEX>$0.44-17.75\;{\mu}M$</TEX>) and kinetin (Kin) (<TEX>$0.46-13.94\;{\mu}M$</TEX>) either singly or in combinations of IAA + BA, IAA + Kin and BA + Kin. Combinations of cytokinins (BA and Kin) were most suitable for multiple shoot induction and <TEX>$13.94\;{\mu}M\;Kin\;+\;13.31\;{\mu}M\;BA$</TEX> was optimum (79% frequency) associated with high number of microshoots (7.1 shoots per explant) after 20 days of culture. Maximum shoot elongation and proliferation (10 shoots per explant with 4.8 cm average height) was achieved on MS media containing <TEX>$2.32\;{\mu}M\;Kin\;+\;2.22\;{\mu}M\;BA\;+\;2.85\;{\mu}M\;IAA$</TEX>. High rooting frequency (70%) was achieved on MS medium (1/2 basal strength) fortified with <TEX>$4.14\;{\mu}M$</TEX> IBA, while activated charcoal showed inhibitory effects on rooting. Hardening was done with 76% survival rate and these plants were growing without any visual defects and morphologically mimicking the naturally growing plants.

Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.

Production of multiple shoots and plant regeneration from leaf segments of fig tree (Ficus carica L.)

High frequency of multiple shoots and plant regeneration has been obtained from the leaf segments of fig tree (Ficus carica L.). Budbreak from dormant buds is highly dependent upon cultivar, so we chose cv. Seungjung Dauphine because it shows an excellent degree of budbreak. Tissue-browning can be an important limiting factor duringin vitro culture. This phenomenon could be substantially delayed or reduced by treating the tissues with 0.5 mM phloroglucinol, thus oxidizing the phenolic substances exuded from the segments. Wounded leaf explants cultured on MS medium supplemented with TDZ in combination with IBA produced more multiple shoots than did other combinations of auxin and cytokinin. For example, 2 mg L-1 IBA along with either 0.5 or 1.0 mg L-1 TDZ resulted in 8.1 or 10.8 multiple shoots per explant, respectively. We achieved a frequency of approximately 90% when tissues were first maintained under darkness in the culture medium for one week before being transferred to the light. Regenerated shoots rooted best in a full-strength MS basal medium.In vitro regenerated plant-lets were then successfully transferred to greenhouse conditions. Here, we have demonstrated a regeneration protocol that is suitable for use in conservation as well as genetic transformation studies of figs and related species.

The Production of Callus, Shoot, and Rooted Plantlets of Rhododendron catawbiense ‘English Roseum’ from Florets

The Rhododendron catawbiense (Michx.) varieties ‘Roseum Elegans’ and ‘English Roseum’ are the primary varieties of rhododendron sold in the northeastern United States; however, very little research has been published on their ability to produce shoots, roots, or plantlets in tissue culture. The suitability of two rhododendron varieties (including ‘English Roseum’) was examined using two different combinations of cytokinin and auxin in Anderson's rhododendron medium. It was determined that the variety ‘Scintillation’ performed poorly regardless of the media conditions used; however, ‘English Roseum’ produced callus and shoots using Anderson's medium and 4.0 mg·L− 1 IAA and 15 mg·L− 1 2iP. Rooted plantlets were produced using a rooting medium containing Anderson's medium and 4 mg·L− 1 NAA. The ability of ‘English Roseum’ to produce 35% successful plantlets under these conditions suggests that this variety is moderately suitable for micropropagation purposes.

Tissue culture in &lt;i&gt; Pinus caribaea &lt;/i&gt; mor. var. &lt;i&gt; hondurensis &lt;/i&gt; barr &amp; golf. 1. effects of two auxins and two cytokinins on callus growth and greening

Growth of callus explants was studied by manipulating the hormonal composition of Von Arnold and Eriksson's (AE) 1977 medium. Various auxin – cytokinin combinations were tested. Growth of callus on fresh weight basis, was best promoted by 2mgl-1 Naphthalene Acetic Acid (NAA) and also by the interaction between Indole Butyric Acid (IBA) at 2mgl-1 and 6- Benzyl Amino Purine (BAP) at 0.5mgl-1. Although there were no significant differences among the various combinations of NAA levels with kinetin levels and NAA levels with BAP levels with respect to fresh weight production, per cent increments over the controls were determined. Maximum increments were obtained from 1mgl-1 NAA x 0.5mgl-1 kinetin (406.5%) and 1mgl-1 NAA x 0.5mgl-1 BAP (198.06%). There were strong indications that high fresh weights were not always synonymous with dry matter production. Among the levels of NAA, 0mgl-1 and 0.5mgl-1 produced the highest percentages. In NAA x kinetin cultures, 0mgl-1 produced a value of 0.467% while 0.5mgl-1 in NAA x BAP cultures produced a value of 0.485%. For kinetin levels, 5mgl-1 produced the maximum percentage of 0.451% while for BAP levels, 0mgl-1 (0.471%) and 5mgl-1 (0.462%) produced the optimum percentages. With respect to the hormonal interactions, optimal percentages were obtained in 0mgl-1 NAA x 5mgl-1 kinetin (0.643%) and 0.5mgl-1 NAA x 0mgl-1 BAP (0.625%) cultures. Spectrophotometric studies revealed the existence of an association between the chlorophyll content of calluses and the grade of greening. Callus greening was best promoted by the main effects of kinetin (5mgl-1), BAP (5mgl-1) and NAA (2mgl-1). An unusual sign of physiological specialization (reddish pigmentation) was observed in some of the callus clumps. The implications of the results have been discussed.. Keywords: Totipotency, Explants, Auxins, Cytokinins, Callus Agro-Science Vol. 4(1) 2006: pp. 14-23

Comparison of Somatic Embryogenesis in Medicago sativa and Medicago truncatula

In this study, the regeneration through embryogenesis of two species of Medicago were studied. Seeds of Medicago sativa cv. Rehnani and M. truncatula line A17 were grown on MS medium. After 4-6 weeks, segments of leaf and stem from two species were transferred to MS medium containing 2 mg L(-1) NAA, 2,4-D and Kinetin. The results indicated that callus formation from leaf explants of M. sativa was higher than M. trancatula. In the next stage, media with different combinations of auxin, cytokinin or ethinyl estradiol were provided for regeneration. Then in two stages, explants of leaf and stem of two species were transferred on these media. Results after 3-6 weeks showed that in medium containing NAA and TDZ, stem pieces ofM. sativa produced shoots while leaf pieces on NAA and ethinyl estradiol formed roots. Leaf explants of M. truncatula in the medium containing NAA and BAP, produced somatic embryos. Also in media with auxin and ethinyl estradiol, somatic embryos were formed on calli of two species. Ethinyl estradiol and auxin together can induce somatic embryogenesis and root production on calli and stem or leaf explants.

Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.)

SummaryAn efficient in vitro regeneration procedure using thidiazuron (TDZ) has been developed to allow high frequency, multiple shoot induction from cotyledonary node explants of cluster bean (Cyamopsis tetragonoloba). Shoot bud induction occurred on Murashige and Skoog (MS) medium after 4 weeks in the presence of TDZ, followed by transfer onto shoot multiplication and elongation media containing MS salts, B5 vitamins, and different combinations of auxins and cytokinins. Multiple shoots were induced at all levels of TDZ in the medium, but the best proliferation capacity occurred at 5 µM TDZ. Combinations of auxins and cytokinins showed a stimulatory effect on shoot multiplication and also on the length of the newly formed shoots. Maximum shoot induction [i.e., the highest number of shoots (16.0 ± 0.94) per explant] was obtained on agar-solidified medium containing 5 µM benzyladenine (BA) with 0.5 µM indole-3-acetic acid (IAA). Rooting of in vitro-regenerated shoots was achieved in ex vitro conditions by a pulse treatment with 300 µM indole-3-butyric acid (IBA) for 15 min. Rooted plantlets were transferred to soil where 70 – 75% attained sexual maturity and produced viable seeds under greenhouse conditions. The present regeneration system is efficient and can be used in various in vitro manipulation studies.

The Influences of Growth Regulators and Culture Medium Composition on Shoot-Tip Cultures of Edible Canna

Explants obtained from the terminal buds of edible canna (Canna edulis Ker-Gawl.) were aseptically cultured with different combinations of auxins (NAA, IBA), cytokinins (BA, KN, 2ip), and culture media (B5, 1/2MS, MS), to establish a tissue culture system for this species. The combination of NAA or IBA with BA induced optimal explant growth, and when the explants grew substantially, TIBA promoted lateral shoot growth. For explant growth, the optimal concentration of NAA was 0.1∼0.5 mg L-1, irrespective of the BA concentration adopted, and the optimal concentration of BA was 0.5∼1 mg L-1. Some of the explants formed callus-like or protocorm-like tissues. Among the three culture media, B5 gave the highest survival rate of explants (92%), followed by 1/2 MS (72%) and MS (52%). In these combinations, micronutrients followed by macronutrients and vitamins, were found to be most important components for explant survival. Gellan gum at 2.5 g L-1 was a superior supporting/gelling material for explant growth compared to a filter paper-bridge with liquid medium or 4 g L-1 agar. Our results indicate that multiple plantlet propagation can be performed routinely, while callus formation remains a time-consuming endeavor.

Heat Resistance Enhanced by Trinexapac-Ethyl and Benzyladenine Combination in Creeping Bentgrass

Creeping bentgrass (Agrostis stolonifera var. palustrisHuds) is one of the most widely used turfgrasses for golf greens and fairways. The optimum temperature for creeping bentgrass ranges between 15 to 24 C for shoot growth and 10 to 18 C for root growth (Beard, 1973). However, temperatures during summer months often exceed the optimal ranges for prolonged periods. High-temperature stress is one of the major factors limiting use of creeping bentgrass in transitional and warmer climatic regions (Beard, 1997; Beard and Daniel, 1966; Carrow, 1996; Wang et al., 2003). Typically, grasses are exposed to high temperatures more than 35/30 C (day/night) for 20 to 30 d during the summer in many areas of China. Such prolonged high temperatures severely limit shoot and root growth, and survival of plants. Many physiological factors are involved in heat stress injury. Heat stress reduces photosynthesis (Carrow and Duncan, 2003; Paulsen, 1994; Xu and Huang, 2001), and increases respiration, which exhausts carbohydrate reserves (Xu and Huang, 2003). Carbohydrate metabolism is among the key physiological processes controlling plant growth. High-quality turf is often cultivated under a frequent mowing program. Clipping removal is one of the main concerns in reduction of energy reserves and leaf area required for photosynthesis in creeping bentgrass. Trinexapac-ethyl (TE) is a plant growth regulator that inhibits gibberellin (GA) synthesis by blocking the transformation of GA20 to GA1 (Rademacher, 2000). TE reduces vertical growth of many turfgrass species, which results in decreased clipping weights (Fagerness et al., 2004; McCullough et al., 2004, Tegg and Lane, 2004). TE has been shown to reduce sod heating through reducing plant respiration, resulting in enhanced stress resistance in Kentucky bluegrass (Poa pratensis) (Ervin and Koski, 2001a,b; Heckman et al., 2001), indicating its effects on energy conservation from respiratory consumption. Cytokinin, a phytohormone, may regulate plant response to high-temperature stress. A period of heat stress reduced the level of cytokinins in roots in many plants (Itai et al., 1973; Liu et al., 2002; Udomprasert et al., 1995). Application of cytokinins (Liu and Huang, 2002) or products containing cytokinins (Zhang et al., 2003) to the root system or shoot (Zhang and Schmidt, 2000) could enhance the heat resistance of turfgrasses. The positive effects of cytokinins on stress resistance of plants are related to protection from oxidative stress by preventing the formation of free radicals or by the regulation of antioxidant enzyme activities (Caers et al., 1985; Liu and Huang, 2002; Wang et al., 2003; Zhang and Schmidt, 2000). Although many plant growth regulators are commercially available for regulating plant growth and production, few of them have been reported to have positive effects on improving plant heat resistance. Cytokinins and TE are two chemicals that have shown potential beneficial effects for plant heat resistance in previous reports. However, no work has been conducted to determine the interactive effects of TE and 6-benzyladenine (BA). The objectives of this study were to investigate 1) whether a TE and cytokinin combination can enhance heat resistance of creeping bentgrass and 2) the morphological and physiological effects of TE and BA in the regulation of turf summer performance and heat resistance in creeping bentgrass. Materials and Methods

The role of sucrose and different cytokinins in the in vitro floral morphogenesis of rose (hybrid tea) cv. “First Prize”

Shoots of rose (hybrid tea) cv. “First Prize” were induced to flower in vitro on Murashige and Skoog (MS) medium containing various sucrose concentrations (15, 30 or 45 g l−1) and different phytohormone combinations of different cytokinins [N6-benzyladenine (BA); thidiazuron (TDZ) and zeatin] with α-naphthaleneacetic acid (NAA). Results indicate that sucrose is the key factor in floral morphogenesis while cytokinin increases the flowering percentage and helps the normal development of floral buds. From the three cytokinins that were used, BA and zeatin were considered to be more suitable as inductive flowering agents than TDZ. Reduced inorganic and organic salt concentration in MS media had a positive effect on in vitro flowering. The morphology of shoots bearing floral buds varied with different cytokinin treatments. The highest percentage (45%) of flowering was obtained on MS medium supplemented with 3.0 mg l−1 BA, 0.1 mg l−1 NAA and 30 g l−1 sucrose.

Rapidin Vitro propagation and enhanced stevioside accumulation inStevia rebaudiana Bert

A very rapid and efficient regeneration method has been established using mature expiants ofStevia rebaudiana Bert. Adventitious shoots were induced from nodal expiants of field-grown plants on four basal media supplemented with various combinations of auxins and cytokinins. The best performance (23.4 ± 2.1 shoots per expiant) was obtained on a Murashige and Skoog (MS) medium containing 2 mg L-1 IAA and 0.5 mg L-1 kinetin. Roots were then produced when thesein vitro- regenerated shoots were transferred to an MS medium supplemented with 3% sucrose and 2 mg L-1 IBA. When acclimatized to soil, the rooted plants had a 98.4% survival rate. Following transplantation in the field, stevioside contents were similar between the regenerated plants (10.68 mg g-1 dry weight) and the mother plants (12.01 mg g-1 dw).

Transformation and Plant Regeneration from Leaf Explants of Solanum tuberosum L. cv. ‘Shepody’

As part of a large-scale genomics project focused on understanding and improving the Shepody potato, we have increased the regeneration and transformation rates for this cultivar. Using combinations of auxins and trans-zeatin, leaf and stem explants were evaluated for callus induction and shoot formation. Several plant growth regulator combinations resulted in higher plant regeneration rates over a previous method. Using the best combination of auxin and cytokinin in combination with Agrobacterium-mediated transformation, we regenerated independent putative transformants from 59.5% of the total explants plated. We ran PCR on a sample of the plants to confirm transformation and 47.1% were nptII positive; giving a confirmed transformation rate of 28.0%.