μM Indole-3-butyric Acid Research Articles

In vitro plant regeneration from cotyledon-derived callus cultures of leguminous tree Gleditsia caspica Desf.

An in vitro regeneration system was developed using organogenic callus derived from in vitro grown cotyledonary explants of Gleditsia caspica Desf., an important leguminous tree. Murashige and Skoog (MS) basal medium augmented with 0.2 g L−1 myo-inositol and various concentrations of either 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid, or indole-3-butyric acid (IBA) alone as well as combined with cytokinins was used for callus induction. The highest frequency of organogenic yellowish-white and nodular callus (93 %) was obtained from explants grown on medium supplemented with 13.5 μM 2,4-D and 4.4 μM benzyladenine (BA). The yellowish-white and nodular callus when transferred to MS medium supplemented with BA (2.2–17.7 μM) or kinetin (KT; 2.3–18.8 μM) solely or in combination with 2.3 μM 2,4-D produced several microshoots after 5 weeks culture. The calli cultured on MS medium with 4.4 μM BA singly showed superior growth response and produced both maximum shoot regeneration (94 %) and the highest mean number (4.3) of microshoots per callus. Transfer of regenerated microshoots onto modified MS basal medium fortified with 5.8 μM gibberellic acid and 4.4 μM BA resulted in the maximum number of internodes per shoot and the highest shoot elongation after a period of 6 weeks. Optimum rooting of 90 %, an average 6.1 roots per shoot, and a mean root length of 3.6 cm was observed when half-strength MS medium was supplemented with 9.8 μM IBA and 0.92 μM KT. The regenerated healthy plants with well-developed shoots and roots showed a survival rate of 77 % after acclimatization and transplanting to garden soil for a 10-week hardening period under ex vitro conditions.

Effects of culture medium and auxins on growth of adventitious root cultures of Cuphea aequipetala Cav. and their ability to produce antioxidant compounds

Cuphea aequipetala Cav. (Lythraceae), a species highly valued for its medicinal properties, is threatened in the wild. To provide an alternative source of material for production of bioactive compounds, we established adventitious root cultures of C. aequipetala and determined their phenolic compounds contents and antioxidant activity. Cultures were initiated from root tips of in vitro C. aequipetala plantlets and were grown in B5 or SH culture medium containing either indole butyric acid (IBA) or α-naphthalene acetic acid at 0, 5 or 10 µM. The maximum root biomass (1.6 g/L dry mass (DM) per L medium) was recorded after 14 days of growth in B5 + 5 µM IBA. Roots in B5 medium remained green, whereas they tended to oxidize in SH medium. The highest contents of total phenolic compounds (9.1 ± 0.1 µg gallic acid equivalents/g DM) and flavonoids (37.5 ± 0.7 µg quercetin equivalents/g DM) were in roots grown in B5 + 5 µM IBA after 14 days of growth. Root cultures accumulated mainly flavan-3-ols, whereas roots or leaves from whole plants accumulated mainly flavonols. We analyzed the antioxidant properties of root extracts using in vitro assays. Roots grown in B5 medium showed stronger free-radical scavenging activity than that of roots grown in SH medium. Our results show that adventitious root cultures of C. aequipetala are a promising system for research on antioxidant compounds biosynthesis and for scaled-up production of useful biological materials.

Role of cytokinins and physical state of the culture medium to improve in vitro shoot multiplication, rooting and acclimatization of date palm (Phoenix dactylifera L.) cv. Boufeggous

We investigated the effects of cytokinins and the physical state of the culture medium on in vitro shoot multiplication of date palm (Phoenix dactylifera L.) cv. Boufeggous. Organogenic cultures obtained from shoot tip explants were cultured on stationary liquid or semi-solid half-strength Murashige and Skoog medium (MS/2) supplemented with 3 μM indole-3-butyric acid (IBA) and with either N6-[2-isopentenyl] adenine (2iP), 6-benzylaminopurine (BAP), kinetin (KIN), or thidiazuron (TDZ) at 1, 2, 3, or 4 μM. After 3 months of culture, shoot multiplication, hyperhydricity, browning, and precocious rooting were significantly influenced by the type and level of cytokinins and the physical state of the culture medium. Generally, BAP and KIN stimulated shoot multiplication better than 2iP and TDZ, while semi-solid media supported morphologically superior shoots than stationary liquid media. The optimal medium for shoot multiplication was semi-solid MS/2 supplemented with 3 μM IBA and 3 μM BAP. The shoot multiplication rate increased with increasing BAP concentration, but more hyperhydricity was observed. Regenerated shoots were elongated and rooted on plant growth regulator-free MS/2 medium. After plantlets were transferred to a glasshouse, those that were previously cultured on semi-solid media showed higher survival rates, reaching 92.5 %. The protocol reported here will be useful for the rapid and efficient propagation of cv. Boufeggous, an elite date palm cultivar.

In vitro propagation, genetic and phytochemical assessment of Thymus persicus — a medicinally important source of pentacyclic triterpenoids

AbstractThymus persicus (Ronniger ex Rech. f.) Jalas is a valuable and endangered natural source of antitumor pentacyclic triterpenoids, i.e., betulinic acid, oleanolic acid and ursolic acid, which grows in northwest Iran. As the plant has a low propagation rate in nature, a suitable method for in vitro-propagation is needed. With the aim of identifying a suitable system for regenerating T. persicus via direct organogenesis, Murashige & Skoog (MS) medium supplemented with different plant growth regulators (PGRs) was tested. In vitro-grown shoot tips were exposed to the cytokinins 6-benzylaminopurine (BAP), kinetin (KN), and thidiazuron (TDZ), alone or in combination with the auxins 1-naphthalene-acetic acid (NAA), 2,4-dichlorophenoxyacetic (2,4-D), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). The highest shoot formation (7.1 ± 0.9) was obtained with a medium fortified with 8.9 μM BAP plus 2.7 μM NAA. Regenerated shoots were easily rooted on the different tested media, with the most abundant (16.6 ± 1.4) and strongest roots obtained on half-strength MS medium containing 2.5 μM IBA. The rooted plantlets were successfully acclimatized (76.6%) in a greenhouse before transference to natural conditions. Homogeneity and phytochemical productivity of the in vitro regenerated plantlets were confirmed by random amplified polymorphic DNA (RAPD) profiles and high performance liquid chromatography (HPLC), respectively.

Micropropagation of Agave americana

Agave species are economically important plants in tropical and subtropical desert ecosystems as ornamentals as well as potential bioenergy crops. However, their relatively long life cycles and the current lack of biotechnology tools hinder their breeding. In this study, an efficient system for micropropagation was developed for Agave americana L. by using basal stems as explants and grown on a modified Murashige and Skoog medium (MSI) or a 1/2 MSI medium supplemented with various concentrations of 6-benzylaminopurine (BA) for shoot proliferation. The highest number of shoots (18.5 shoots/explant) from basal stems was obtained on MSI supplemented with 13.32 μM BA. An efficient shoot regeneration system was also developed from leaf tissues. Combinations of auxin with cytokinin, basal media, and leaf regions were optimized for shoot induction. Adventitious shoot formation from leaf segments was induced and proliferated with combination ranging of 0.54 to 2.68 μM [α-naphthaleneacetic acid (NAA)] with 8.88 to 13.32 μM (BA), and the maximum frequency (≈69%) was obtained with 2.68 μM NAA plus 13.32 μM BA. MSI medium and the basal segment of leaf affected shoot induction. The highest rooting frequency and mean number of shoots occurred in 1/2 MSI containing with 4.92 μM indole-3-butyric acid (IBA) alone (90%, 3.4) or 1.48 μM IBA plus 1.61 μM NAA (92%, 5.2). Survival of in vitro plantlets after transfer and acclimatization to ex vitro conditions was 87%. This is the first complete protocol for micropropagation of A. americana.

<b>Plant regeneration and histological study of the somatic embryogenesis of sugarcane (<i>Saccharum</i> spp.) cultivars RB855156 and RB72454</b> - doi: 10.4025/actasciagron.v36i1.16342

The development of an efficient regeneration system is crucial for the genetic transformation of sugarcane. The aims of this study were to regenerate plants from somatic embryos and analyze the origin and division pattern of cells during the different embryonic developmental stages of the sugarcane cultivars RB855156 and RB72454. For both cultivars, the best results for embryogenic callus induction were obtained with explant cultures on culture medium containing 13.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by callus culture on solid medium with 4.5 µM 2,4-D. A higher rate of shoot induction was observed in RB855156 with the addition of 8.9 and 17.8 µM benzylaminopurine (BAP); this higher rate was also observed in RB72454 with the addition of 17.8 µM BAP. Murashige and Skoog (1962) (MS) medium containing 2.5 and 5.0 µM indolebutyric acid (IBA) for RB855156 and RB72454, respectively, was suitable for rooting. The plantlets were successfully acclimatized and the plant survival rate was 100% for both cultivars. Histological analysis revealed that the shoots in both cultivars originated from somatic embryogenesis

English

High frequency shoot regeneration from in vitro derived leaf explants of Wattakaka volubilis (L.f.) Stapf was achieved through callus mediated organogenesis. Organogenic calli were induced from 20 day old aseptic seedling explants on Murashige and Skoog medium fortified with various concentrations and combinations of plant growth regulators, benzylaminopurine (BAP), α-naphthaleneacetic acid (NAA), indole 3-butyric acid (IBA) and gibberellic acid (GA3). A mean of 8.6 shoots developed from organogenic callus induced from a 2 x 2 cm leaf explants on MS medium with 3% sucrose having 5.37µM NAA in combination with 2.22 µM BAP with 60% induction capacity. Further development of adventitious shoots could be achieved by sub culturing the callus to the same medium with 4.40 µM BAP and 0.288 µM GA3. Organogenesis could not be achieved from the calli of ex vitro derived leaf explants. The developed shoots were rooted on half-strength MS medium with 1% sucrose, 4.90 µM IBA and 0.93 µM kinetin at a frequency of 85%. Well rooted plantlets were then transplanted to vermiculite soil (3:1) mixture in polythene covered pots kept under culture room conditions. Approximately 60% plantlets survived and grew into whole plants. Key words: Adventitious shoots, caulogenesis, organogenic callus-histology.

Effects of plant growth regulators and carbon source on shoot proliferation and regeneration in date palm (Phoenix dactylifera L.) ‘16-bis’

SummaryThe aim of this study was to optimise in vitro culture conditions for the date palm (Phoenix dactylifera L.) cultivar ‘16-bis’. The effects of various combinations of plant growth regulators (PGRs) and sources of carbon in the growth medium on shoot proliferation and growth were evaluated. The PGRs used were indole-3-butyric acid (IBA), 2-naphthoxyacetic acid (NOA), 6-benzylaminopurine (BAP), and kinetin (KIN), each at 0, 2.5, or 5.0 µM. The carbon sources used were sucrose, sorbitol, or mannitol, each at 10, 20, 30, 40, or 50 g l-1. Explants cultured on 1.0× Murashige and Skoog (MS) medium supplemented with 2.5 µM IBA plus 2.5 µM BAP and 20 g l–1 sucrose showed the highest rate of shoot proliferation (22.3 shoots per organogenic cluster). Higher concentrations of PGRs increased the frequencies of hyperhydricity and precocious rooting. Carbon source and its concentration affected almost all of the parameters measured. The highest rate of shoot proliferation was on 1.0× MS medium containing 20 g l–1 sucrose. The frequency of precocious rooting increased with higher concentrations of carbon source. The proliferated shoots were transferred onto PGR-free 1.0× MS medium supplemented with 10, 20, 30, 40, or 50 g l–1 sucrose, sorbitol, or mannitol for elongation and rooting. Those grown on 1.0× MS medium containing 40 g l–1 sucrose showed the strongest growth and development, and the highest rate of survival (92.5%) after the plantlets were transferred to a glasshouse. These results will be useful for producing large numbers of ‘16-bis’ date palm plants to restore Moroccan palm groves decimated by bayoud disease.

Micropropagation and Polyploid Induction of Acer platanoides ‘Crimson Sentry

Protocols were developed for micropropagation and induction of autopolyploids in a fastigiate cultivar of Norway maple (A. platanoides L. ‘Crimson Sentry’). Murashige and Skoog (MS) medium, woody plant medium (WPM), and Quoirin and Lepoivre medium were supplemented with 2 μM 6-benzylaminopurine (BA), meta-Topolin, 6-(γ,γ-dimethylallylamino) purine, kinetin, or thidiazuron to evaluate microshoot proliferation. Murashige and Skoog medium with 2 μM BA yielded the most microshoots (3.2) and longest microshoots (30.6 mm) per subsample after 5 weeks. The influence of BA concentration on proliferation was evaluated at 0, 2, 4, 8, or 16 μM. Optimal multiplication rate was achieved at 2 or 4 μM BA producing approximately 2.8 microcuttings per subsample after 5 weeks. To induce in vitro rooting, half-strength WPM was supplemented with 0, 5, 10, 20, 40, or 80 μM indole-3-butyric acid (IBA). Optimal in vitro rooting (70%), number of roots (2.5), and root length (15 mm) per subsample were achieved with 10 μM IBA after 8 weeks. To induce polyploidy, microcuttings were pretreated for 7 days on MS medium with 4 μM BA alone or combined with 1 μM IBA, indole-3-acetic acid (IAA), or 1-naphthaleneacetic acid prior to treatment in liquid MS medium containing 15 μM oryzalin for 3 days. Homogenous tetraploids were only obtained from shoots pretreated with IAA. This research provides optimized protocols for micropropagation and autopolyploid induction of A. platanoides ‘Crimson Sentry’ and demonstrates the development of tetraploid lines for use in future improvement programs.

In vitro regeneration and transformation studies on Pelargonium graveolens (geranium) - an important medicinal and aromatic plant

Development of improved protocols for the regeneration of Pelargonium graveolens is important for their commercial utilization. Callus was induced from leaf explants on Murashige and Skoog (MS) medium supplemented with auxins and combinations of auxins and cytokinins. Micropropagation of nodal bud explants was achieved using medium supplemented with cytokinins. The regenerated explants were acclimatized and the transformation potential of geranium was demonstrated using Agrobacterium tumefaciens. Maximum proliferation was obtained in medium supplemented with combinations of 20 µM indole butyric acid (IBA) + 10 µM kinetin (KIN), 20 µM IBA + 10 µM benzyl amino purine (BAP) and 20 µM indole-3-acetic acid (IAA) + 10 µM KIN. Plantlets were regenerated when callus was given a photoperiod of 16 h light and 8 h dark conditions at 25°C. After being transferred to greenhouse conditions, the plantlets were successfully acclimatized and transferred to the field condition. The study has established an efficient protocol for in vitro regeneration and transformation of P. graveolens as an alternative method for vegetative propagation. Key words: Callus, geranium, in vitro regeneration, transformation.

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with s-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.

In Vitro Propagation of Alexandrian Laurel (Danae racemosa L. Moench), a Valuable Ornamental Plant

To overcome the limitations of traditional propagation, this research was initiated to develop an alternative means for efficient production of Alexandrian laurel (Danae racemosa L. Moench). An in vitro propagation protocol has been developed for Danae racemosa L. Moench using seeds as a source of material for culture initiation. Seedlings were produced after seeds were cultured for 3 month on MS (Murashige and Skoog, 1962) medium. Shoot multiplication occurred on MS medium with or without 6-benzylaminopurine (BAP) with 100% multiplication percentage. However, shoot number was significantly increased from an average of 2.8 to more than six with the addition of 5 or 25 μM BAP. Among two indole-3-butyric acid (IBA) treatments tested for rooting of seedlings, incorporation of 5 μM IBA in MS medium significantly increased rooting percentage to 86.4% compared with 71.2% without IBA. The greatest number of roots (three) was produced by 5-minute IBA pulse. However, both IBA treatments significantly reduced root length. The longest root (12.8 mm) was observed on MS medium without any IBA treatment and the shortest (6.1 mm) was produced by IBA pulse. In vitro-propagated plantlets grew well after transfer to a substrate of peat and pine bark (1:1) in the greenhouse. No morphological variation was observed.

Regeneración de plantas a partir de cultivos de callos de <i>Vitex trifolia</i> (Lamiales: Lamiaceae): una planta medicinal potencial

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25 +/-2 degrees C under a light intensity of 61 micromol/m2s from cool white fluorescent lamps and a 16 h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10 microM BAP in combination with 0.54 microMNAA. Addition of 135.74-271.50 microM adenine sulphate (Ads) and 0.72-1.44 microM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10 microM BAP in combination with 0.54 microM NAA, 271.50 microM Ads and 1.44 microM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23 microM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae)

In vitro propagation for Mesomelaena pseudostygia a difficult-to-propagate dryland sedge species (Cyperaceae) endemic to Western Australia is described. Multiple avenues to in vitro propagation were investigated: shoot culture, organogenesis and somatic embryogenesis, with zygotic embryos as initiation material. The highest multiplication rate for shoots was 3.4 ± 1.0 after 6 wk on basal medium (1/2 strength Murashige and Skoog) with 2.5 μM kinetin and 0.5 μM 6-benzylaminopurine. Shoots achieved peak rooting (83%) following a pulse treatment on basal medium containing 10 μM indolebutyric acid and 2 μM α-naphthaleneacetic acid for 7 wk, followed by transfer to medium (without growth regulators) for a further 7 wk. Alternatively, in vitro grown shoots were pulse treated on basal medium with both 100 μM indolebutyric acid and 20 μM α-naphthaleneacetic acid for 1 wk then placed in Rockwool plugs (under propagation house conditions) for another 7 wk resulting in 63% root induction. Rooted plantlets were also successfully transferred to potting mixture either in Rockwool plugs or bare rooted and maintained in propagation house conditions with ≥95% survival after 7 wk. These results indicate that micropropagation of M. pseudostygia is feasible for small to medium scale restoration purposes. The highest frequency of callus induction was from cultured zygotic embryos on basal medium with 5 μM α-naphthaleneacetic acid, whereas 2,4-dichlorophenoxacetic acid (2 or 5 μM) produced the largest callus sizes. A low frequency of shoot regeneration occurred in zygotic callus tissues in basal medium treatments containing cytokinin (kinetin or thidiazuron at 1 μM). A small proportion (<20%) of zygotic embryo callus explants from 2,4-dichlorophenoxyacetic acid treatments were found to be embryogenic, firstly developing embryo-like structures after 2 wk on basal medium (minus plant growth hormones), that continued to develop with approximately one in twenty germinating after a further 4 wk on basal medium to form small plantlets. Further optimisation is needed to improve somatic embryogenesis efficiency for mass propagation.

Differences in hypericin synthesis between experimentally induced seedling shoot cultures of Hypericum hookerianum Wight &amp; Arn.

During seed germination trials of Hypericum hookerianum, seedlings of Lake View accession from Palni hills of Southern India segregated into green- (97.44 %) and red-pigmented (2.56 %) types. Seedlings cultured in Murashige and Skoog (1962) basal medium developed into fast growing green and slow growing red plant types in 6 weeks, the latter showing increased concentrations of total phenols, anthocyanins and flavonoids and 19-fold higher concentration of hypericin. Hypocotyls/cotyledons of red seedlings cultured using 2.325 μM kinetin (KIN) produced hypericin-rich (4.38 ± 0.18 mg/g DW), stunted (0.5–1.2 cm) shoots which ceased to grow in 8 weeks. Segments (4–6 mm) of these shoots sub-cultured in the dark for 4 weeks followed by 2-week light exposure and repeated subculture enabled mass multiplication of productive (3.93 ± 0.06 mg hypericin/g DW) shoots. Green hypocotyls and cotyledons subjected to 4 + 2 weak dark–light treatment also produced 9.18 ± 2.44 and 4.25 ± 0.96 comparable hypericin-rich (3.73 ± 0.21 mg/g DW) shoots. Red and green seedling explants cultured in basal medium in the dark produced 6.82 ± 0.75 cm etiolated shoots with reduced leaves which synthesized 2.27 ± 0.15 mg hypericin/g DW on illumination. Green cotyledons cultured in the dark using 2.45 μM indole-3 butyric acid (IBA) formed calluses which on illumination formed 12.64 ± 3.8 productive (3.86 ± 0.31 mg hypericin/g DW) 0.5- to 1.5-cm-long shoot clusters. Phenotypic segregation of seedlings, the ability of both red and green seedling explants to multiply in the dark and produce hypericin on illumination, and IBA-induced indirect shoots producing significant amounts of metabolite compared to wild plants (0.35 ± 0.09 mg/g DW) and green shoot cultures (0.91 ± 0.03 mg/g DW) are new to Hypericum.

In Vitro Culture and Rooting of Diospyros virginiana L.

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

In Vitro Micropropagation of Jatropha curcas L. from Bud Aggregates

Entire plants were regenerated from nodes explants of Jatropha curcas L. following a procedure of bud aggregate induction on MS (Murashige and Skoog) medium supplemented with 25 mg.l-1 citric acid, 12.2 mg.l-1 adenine sulfate, 15 mg.l-1 L-arginine, 2.46 µM IBA (indole-3-butyric acid), 30 g.l-1 sucrose and 7 g.l-1 of agar, and enriched with different balances of BA (benzyladenine) and L-glutamine. The histological studies performed on aggregates showed that the buds result from both the development of axillary buds and adventitious budding starting from underlying tissues of the explant. The culture medium containing 6.65 µM BA and 25 mg.l‑1 L-glutamine gave the best results with an average of 64 buds per aggregate after three weeks for all accessions tested. The buds developed into shoots when placed in an MS medium supplemented with 2.21 µM BA, 5.70 µM IAA (indole-3-acetic acid) and 15 mg.l-1 L‑arginine. These shoots were isolated and then rooted in MS containing 2.46 µM of IBA, 2% sucrose and 0.7% agar. The entire process took 13 weeks with a 98% survival rate in terms of plantlets acclimatization. We obtained a multiplication rate of 13 buds per explant and per subculture which is the double of those obtained in other recent works based on the micropropagation of J. curcas from node explants. This protocol is economically more profitable.

Efficient plant regeneration from petiole explants of West Indian gherkin (Cucumis anguria L.) via indirect organogenesis

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature petiole explants of West Indian gherkin (Cucumis anguria L.). Calluses were induced from immature petiole explants excised on 7-day-old in vitro seedlings and mature petiole explants of 40-day-old in vivo plants. The maximum frequency of immature petiole explants (98.0 %) and mature petiole (91.5 %) produced green, compact organogenic callus in Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l−1 sucrose, 8.0 g l−1 agar and 4.0 μM naphthalene acetic acid (NAA) with 2.0 μM benzyl amino purine (BAP) after two successive subculture at 11 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MSB5 medium supplemented with 3.0 μM TDZ, 1.0 μM NAA and 0.05 mM L-glutamine with shoot induction frequency of immature petiole 45 shoots and mature petiole 40 shoots per explant. The shoots were excised from callus and elongated in MSB5 medium fortified with 3.0 μM gibberellic acid (GA3). Then elongated shoots were rooted in half strength MSB5 medium supplemented with 3.0 μM indole 3-butyric acid (IBA). Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Plantlets with well-developed shoot and root systems were successfully acclimatized (95 %) in winter season and exhibited normal morphology and growth characteristics. The survival percentage differed with seasonal variations.

Erratum to: Establishment of dedifferentiated callus of haploid origin from unfertilized ovaries of tea (Camellia sinensis (L.) O. Kuntze) as a potential source of total phenolics and antioxidant activity

This is the first report of induction of haploid callus with significant antioxidant activity from unpollinated ovary cultures of tea. Out of the five cultivars tested, TV18 gave the highest percentage of callus induction. Within 1 wk of induction, ovules swelled to almost double their original size, and white, friable callus emerged. A high cytokinin/auxin ratio, provided by 8.5 μM benzyl adenine and 4.5 μM 2,4-dichlorophenxyacetic acid, and high-temperature treatment (33°C) for 10 d in the dark promoted maximum callus induction. Callus was maintained on MS medium containing 22.2 μM benzyl adenine and 9.8 μM indolebutyric acid (callus line RM 1) in the light at 25°C. Well-developed tracheids were formed within 4 wk in callus subcultured on MS medium containing 1.8 μM thidiazuron and 5.0 μM 2,3,5-triiodobenzoic acid (line RM 2). Flow cytometric analysis revealed that most cells were haploid. Both RM 1 and RM 2 produced phenolic compounds with significant antioxidant capacity. Phenolic content showed a positive linear correlation with antioxidant activity. The total phenolic content of RM 1 was 3.47 ± 0.21 gallic acid equivalents (GAE) mg/g dry weight and that of RM 2 was 2.39 ± 0.12 GAE mg/g dry weight. Antioxidant activity was measured using IC50, a measure of inhibitory concentration; a lower IC50 value reflects greater antioxidant activity. The IC50 value of RM 1 was 2,530 μg/ml and that of RM 2 was 3,170 μg/ml. The results suggested that the phenolic compounds contributed significantly to the antioxidant capacity of the in vitro cell lines.

PLANT GROWTH REGULATORS ENHANCE GOLD UPTAKE IN BRASSICA JUNCEA

The use of plant growth regulators is well established and they are used in many fields of plant science for enhancing growth. Brassica juncea plants were treated with 2.5, 5.0 and 7.5 μM auxin indole-3-butyric acid (IBA), which promotes rooting. The IBA-treated plants were also sprayed with 100 μM gibberellic acid (GA3) and kinetin (Kin) to increase leaf-foliage. Gold (I) chloride (AuCl) was added to the growth medium of plants to achieve required gold concentration. The solubilizing agent ammonium thiocyanate (1 g kg−1) (commonly used in mining industries to solubilize gold) was added to the nutrient solution after six weeks of growth and, two weeks later, plants were harvested. Plant growth regulators improved shoot and root dry biomass of B. juncea plants. Inductively Coupled Plasma Optical Emission Spectrometry analysis showed the highest Au uptake for plants treated with 5.0 μM IBA. The average recovery of Au with this treatment was significantly greater than the control treatment by 45.8 mg kg−1 (155.7%). The other IBA concentrations (2.5 and 7.5 μM) also showed a significant increase in Au uptake compared to the control plants by 14.7 mg kg−1 (50%) and 42.5 mg kg−1 (144.5%) respectively. A similar trend of Au accumulation was recorded in the roots of B. juncea plants. This study conducted in solution culture suggests that plant growth regulators can play a significant role in improving phytoextraction of Au.