In vitro plant regeneration from organogenic callus of <Emphasis Type="Italic">Curcuma kwangsiensis</Emphasis> Lindl. (Zingiberaceae)

A novel protocol for callus-mediated shoot regeneration was established for an important medicinal and ornamental plant native to South China, Curcuma kwangsiensis, using shoot base sections excised from seedlings in vitro as explant sources. The frequency of callus formation reached 91% for explants cultured on MS medium containing 1.4 μM TDZ, 4.4 μM BA and 2.3 μM 2,4-D. 8.2 shoots per callus was achieved on MS medium supplemented with 1.4 μM TDZ, 17.8 μM BA and 2.7 μM NAA. Single shoots transferred into MS medium free of plant growth regulator rooted well. Regenerated plants acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions.     

High frequency shoot organogenesis and somatic embryogenesis in juvenile and adult tissues of seabuckthorn (<Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.)

Seabuckthorn (Hippophae rhamnoides) is a multipurpose small tree with unique berries of high nutritional and pharmaceutical values. A clonally propagated plant originating from a 20-year-old tree of H. r. rhamnoides × mongolica hybrid cultivar Julia and seedling offspring of this cultivar were investigated regarding induction of shoot organogenesis in leaf explants and in roots of intact seedlings, and induction of direct somatic embryogenesis in explants from shoot tissue. The highest percentage of leaf explants showing shoot organogenesis was achieved (juvenile explants, 65%; adult explants, 75%) when incubated in Murashige and Skoog (MS) medium supplemented with either 4.5 μM of the phenylurea cytokinin thidiazuron (TDZ) or 2.25 μM TDZ plus 2.2 μM 6-benzyladenine (BA), for juvenile and adult explants, respectively, both supplemented with 0.53 μM α-naphthaleneacetic acid (NAA). Juvenile explants developed on average 18 shoots per explant in the MS medium supplemented with 4.5 μM TDZ, a four fold increase over those incubated on the medium supplemented with 2.25 μM TDZ and 2.2 μM BA. Adult leaf explants grown on medium containing 2.25 μM TDZ and 2.2 μM BA medium produced 12 shoots per explant, while those grown on medium containing 4.5 μM TDZ produced 5 shoots per explant. Shoot organogenesis was observed in roots of intact seedlings pre-cultured on plain medium lacking nutrients (PM) or woody plant medium (WPM) salts and then grown on WPM salts supplemented with 4.4 μM BA, 0.29 μM gibberrelic acid (GA3), and 57.0 μM indoleacetic acid (IAA). The number of shoots formed on each seedling root system was ten fold higher when the pre-culture was in WPM medium indicating a promoting effect of mineral nutrients in the pre-culture medium. Somatic embryogenesis was induced in both juvenile and adult leaf explants in 65 and 78% of the explants, respectively, in MS-based medium supplemented with 2.0 μM N-(2-Chloro-4-pyridyl)-N ¹-phenylurea (CPPU), 0.53 μM NAA and varying concentrations of BA. There was an interaction effect between MS salt strength and BA concentration. The most effective medium for inducing somatic embryogenesis in juvenile explants contained half strength MS salts and 2.2 μM BA and full strength MS salts and 13.2 μM BA for adult explants.     

Comparison of in vitro regeneration pathways in <Emphasis Type="Italic">Punica granatum</Emphasis> L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA₃). However, adding 24 μM silver nitrate (AgNO₃) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l⁻¹ sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l⁻¹ sucrose.     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.     

Efficient micropropagation protocol of <Emphasis Type="Italic">Spilanthes acmella</Emphasis> L. possessing strong antimalarial activity

Micropropagation has been achieved in a promising larvicidal asteraceous taxon Spilanthes acmella L. using seedling leaf explants. The explants were reared on a variety of growth regulators, namely 2,4-dichlorophenoxyacetic acid, 1-naphthalene acetic acid, Indole-3-butyric acid, N⁶-benzyladenine, and kinetin either alone or in combination on Murashige and Skoog’s (MS) medium. The best green and compact callus was obtained on 1 μM NAA and 10 μM benzyladenine (BA) in 15 d. The callus on subculture to the same but fresh medium after every 30 d differentiated an average of 12.90 ± 0.32 shoot buds in 50% cultures. Elongation in shoot buds occurred only if they were transferred to NAA lacking MS+BA medium. An average number of 4.22 ± 0.83 shoots and 15 ± 0.84 shoot buds per explant were obtained in 70.3% cultures on MS + 10 μM BA in 30 d. One hundred percent excised shoots rooted in MS(1/2) + 0.1 μM IBA within 2 wk. The plants were gradually hardened and established in soil where they flowered and set viable seeds. The regenerated plants were morphologically similar to the field grown plants and showed 100% larvicidal activity against malaria and filarial vectors.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the tennessee coneflower (<Emphasis Type="Italic">Echinacea tennesseensis</Emphasis>)

Summary Tennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed into plants that were morphologically identical to the source material.     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

Regeneration of <Emphasis Type="Italic">Aeschynanthus radicans</Emphasis> via direct somatic embryogenesis and analysis of regenerants with flow cytometry

Plant regeneration through direct somatic embryogenesis in Aeschynanthus radicans ‘Mona Lisa’ was achieved in this study. Globular somatic embryos were formed directly from cut edges of leaf explants and cut ends or on the surface of stem explants 4 wk after culture on Murashige and Skoog (MS) medium supplemented with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA), TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D), or 6-benzylaminopurine (BA) or kintin (KN) with 2,4-D. MS medium containing 9.08 μM TDZ and 2.68 μM 2,4-D resulted in 71% of stem explants producing somatic embryos. In contrast, 40% of leaf explants produced somatic embryos when induced in medium containing 6.81 μM TDZ and 2.68 μM 2,4-D. Somatic embryos matured, and some germinated into small plants on the initial induction medium. Up to 64% of stem explants cultured on medium supplemented with 9.08 μM TDZ + 2.68 μM 2,4-D, 36% of leaf explants cultured on medium containing 6.81 μM TDZ and 2.68 μM 2,4-D had somatic embryo germination before or after transferring onto MS medium containing 8.88 μM BA and 1.07 μM NAA. Shoots elongated better and roots developed well on MS medium without growth regulators. Approximately 30–50 plantlets were regenerated from each stem or leaf explant. The regenerated plants grew vigorously after transplanting to a soil-less substrate in a shaded greenhouse with more than a 98% survival rate. Three months after their establishment in the shaded greenhouse, 500 plants regenerated from stem explants were morphologically evaluated, from which five types of variants that had large, orbicular, elliptic, small, and lanceolate leaves were identified. Flow cytometry analysis of the variants along with the parent showed that they all had one identical peak, indicating that the variant lines, like the parent, were diploid. The mean nuclear DNA contents of the variant lines and their parent ranged from 4.90 to 4.99 pg 2C⁻¹, which were not significantly different statistically. The results suggest that the regenerated plants have a stable ploidy level, and the regeneration method established in this study can be used for rapid propagation of ploidy-stable Aeschynanthus radicans.     

Shoot regeneration from cotyledonary leaf explants of <Emphasis Type="Italic">Jatropha curcas</Emphasis>: a biodiesel plant

A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l⁻¹ activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.     

Somatic embryogenesis and shoot organogenesis from leaf explants of <Emphasis Type="Italic">Primulina tabacum</Emphasis>

Primulina tabacum is a rare and endangered species that is endemic to China. Establishing an efficient regeneration system is necessary for its conservation and reintroduction. In this study, when leaf explants collected from plants grown in four ecotypes in China are incubated on Murashige and Skoog (MS) medium containing 5.0 μM thidiazuron (TDZ) for 30 days, then transferred to medium containing 5.0 μM 6-benzyladenine (BA), adventitious shoots are then observed. Conversely, when leaf explants are incubated on medium containing 5.0 μM BA for 30 days, then transferred to medium containing 5.0 μM TDZ, somatic embryogenesis is induced. This indicates that somatic embryogenesis and shoot organogenesis could be switched simply by changing the order of two cytokinins supplemented in the culture medium. Histological investigation has revealed that embryogenic cells are induced within 30 days following incubation of explants in medium containing TDZ. Only if embryogenic cells were induced, TDZ could enhance somatic embryogenesis and BA could stimulate shoot organogenesis. When comparing explants from different ecotypes, leaf explants from Zixiadong in Hunan Province could induce low numbers (1–2) of either somatic embryos or adventitious shoots on medium containing either 5.0 μM TDZ or 5.0 μM BA, respectively. Whereas, leaf explants from plants collected from the other three ecological habitats could induce 50–70 somatic embryos/adventitious shoots per explant. Moreover, somatic embryos could induce secondary somatic embryogenesis and adventitious shoots on different media. All regenerated shoots developed adventitious roots when these are transferred to rooting medium, and over 95% of plantlets have survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite).     

Micropropagation of pitaya (<Emphasis Type="Italic">Hylocereus undatus</Emphasis> Britton et rose)

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

In vitro plant regeneration of Mammillaria elongata normal and cristate forms

In the normal form of Mammillaria elongata, shoots were regenerated in vitro, through callus, from tubercle explants excised from the upper part of the branch and cultured on Murashige and Skoog medium (MS) with 1.07 μM α-napthaleneacetic acid (NAA) and 22.20 μM 6-benzylaminopurine (BA). A high percentage of tubercles explants of the M. elongata cristate form, excised from the tip of the branch and cultured on MS with 0.54 μM NAA and 0.44 μM BA or 1.07 μM NAA, responded by initially forming an inflated cristate shoot, which gave cristate and normal shoots, without callus intervention, when transferred on basal MS. Callus formed on cristate tubercles explants gave both cristate and normal shoots when transferred onto basal MS. Normal and cristate shoots were rooted in vitro on MS with 9.84 μM or 0.98 μM indole-3-butyric acid, respectively, and established ex vitro. In both normal and cristate form, the differential response appeared to be associated with the site of the explant excision. The formation of shoots was influenced by the season of culture; i.e., explants excised in October had a higher shoot formation rate than those excised February. This revised version was published online in June 2006 with corrections to the Cover Date.     

An efficient regeneration system via direct and indirect somatic embryogenesis for the medicinal tree <Emphasis Type="Italic">Murraya koenigii</Emphasis>

A reproducible protocol for direct and indirect somatic embryogenesis was established in a small aromatic tree, Murraya koenigii. Embryogenic callus was obtained from 90% zygotic embryonic axis (ZE) and 70% cotyledon (COT) explants in Murashige and Skoog (MS) basal medium supplemented with 8.88 μM 6-benzyladenine (BA) and 2.675 μM α-naphthaleneacetic acid (NAA). Globular somatic embryos were induced and further matured from such embryogenic callus by subsequent culture on the same basal media containing thidiazuron (TDZ) (2.27–9.08 μM). The highest frequency of somatic embryos (14.58 ± 0.42) was recovered from ZE-derived callus after 6 weeks. The age and type of explant and concentration of TDZ played an important role in the development of somatic embryos. Explants excised from 60-day-old seed differentiated from 96.67% of ZE explants and 86.67% from COT explants when cultured on MS basal medium supplemented with 4.54 and 9.08 μM TDZ, respectively, after 4 weeks. The best result obtained for the average frequency of somatic embryos (11.28 ± 0.32) was from ZE explants, which was significantly higher than COT explants (7.34 ± 0.97). Most of the somatic embryos (above 95%), irrespective of their origin, germinated after 4 weeks in 1/2 MS basal media containing 2.32 μM kinetin (KN) and 1.07 μM NAA. Well-rooted plantlets were successfully acclimatized. Histological analysis and scanning electron micrographs confirmed the initiation, development, and germination of somatic embryos from both explants.     

Plant regeneration from in vitro leaf tissues of <Emphasis Type="Italic">Viburnum dentatum</Emphasis> L

Shoots were regenerated from in vitro leaf tissues of two genotypes of Viburnum dentatum, a popular shrub species for landscape use. Adventitious shoots were induced when leaf tissues were cultured on woody plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Effects of cytokinin concentration, indole-3-butyric acid (IBA), and dark treatment on shoot regeneration were investigated. Dark treatment for the first 4 weeks of leaf explants cultured in the regeneration medium significantly increased the frequency of regeneration. The highest frequency of shoot regeneration (70%) for ‘Synnesvedt’ was obtained when leaf tissues were cultured in the medium with 40 μM BA or 8 μM TDZ with 4 weeks dark treatment. The highest frequency of shoot regeneration (90%) for ‘MN34’ was found in the 4 μM TDZ medium with 4 weeks dark treatment. Addition of IBA significantly enhanced shoot regeneration. Ethyl methanesulfonate (EMS) treatment inhibited callus proliferation, particularly in the early stage of callus recovery; however, no significant difference in shoot regeneration among different treatments was observed, indicating that the inhibitory effect of EMS was minimal after calluses re-acquired their capacity to grow and regenerate in the regular medium. Regenerated shoots (>1.5 cm) were rooted in the half-strength MS medium containing 5-10 μM IBA or naphthalene acetic acid (NAA). Rooted plants were transferred to the potting medium and grown in the greenhouse.     

Direct shoot regeneration from leaf explants of Jatropha curcas in response to thidiazuron and high copper contents in the medium

Leaf explants of Jatropha curcas cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ; 0.90 μM) in combination with indole-3-butyric acid (IBA; 0.98μM) produced adventitious shoot buds directly on the surface of the explants without formation of intervening callus while shoot bud formation was accompanied with callus formation on medium supplemented with 6-benzylaminopurine (BAP; 13.3 μM) and IBA (2.46 μM). TDZ treatment resulted in more than twice higher rate of shoot bud induction than BAP. Shoot buds were multiplied and elongated following repeated transfers to medium containing BAP (2.22 μM) and gibberellic acid (GA₃; 1.44 μM). The effect of copper sulphate on differentiation of shoot buds from leaf segments was also investigated. Both shoot induction and multiplication media were supplemented with different levels of CuSO₄ (0–5 μM). Significant improvement in shoot bud induction was observed when the concentration of CuSO₄ was increased to 10 times the normal MS level. Healthy elongated shoots were rooted on half strength MS medium supplemented with IBA (2.46 μM). Rooted plantlets were transferred to field and survived. Histological analysis revealed direct formation of shoot buds from leaf explants.     

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented <Emphasis Type="Italic">Pelargonium capitatum</Emphasis> cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively.     

Adventitious shoot regeneration of oriental lily (Lilium orientalis) and genetic stability evaluation based on ISSR marker variation

A regeneration system was developed for oriental lily (Lilium orientalis) based on both leaf and bulb scale. Adventitious shoots were regenerated from leaves of in vitro cultures on Murashige and Skoog medium containing thidiazuron (TDZ) or 6-benzylaminopurine (BA) and naphthaleneacetic acid (NAA). The highest percent regeneration from leaf explants was 74.2%, being observed on medium containing 10.8 μM TDZ and 0.54 μM NAA. The highest mean number of shoots generated was 4.4 and was obtained from bulb scale explants on medium containing 0.54 μM TDZ and 0.54 μM NAA. Adventitious shoots were successfully rooted at rates ranging from 79.2% to 100%. The rooted plantlets survived after acclimatization in the greenhouse. The effect of kanamycin concentration on adventitious shoot regeneration was also evaluated, a value of 100 mg l⁻¹ being suggested as a lethal dose for lily transformation. Eighteen ISSR markers were employed to determine the genetic stability of the regenerated shoots in comparison to their mother plant. Eleven primers in total produced 70 clear and reproducible bands. Genetic similarity indicators among the clonal derivatives and the mother plant ranged from 0.92 to 1.0. All 15 micropropagated progenies and the mother plant could be grouped together in one major cluster with a similarity level of 92%. The somaclonal variation rate across the plantlets was estimated as 4.2%, indicating that direct shoot formation from explant regeneration is a safe method for multiplication of “true-to-type” plants.     

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.