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.     

In vitro propagation of Petasites hybridus (Asteraceae) from leaf and petiole explants and from inflorescence buds

In vitro shoot regeneration from sterile leaf and petiole explants and from in-situ-collected inflorescence buds of Petasites hybridus was achieved by a simple two-step protocol. Murashige and Skoog (MS) nutrient medium was supplemented with 17.6 μm benzyladenine (BA)+0.54 μm naphthaleneacetic acid (NAA) to induce shoots. After 5 weeks of culture, 40% of the petiole and 27% of the leaf explants produced shoots compared to 76% of the inflorescence buds. Single shoots were excised and subcultured on MS medium supplemented with various cytokinins (N⁶-(Δ²-isopentenyl)adenine, BA, kinetin and thidiazuron). A concentration of 8.8 μm kinetin+0.54 μm NAA performed best in terms of shoot multiplication rate, average shoot length and spontaneous root induction. Received: 20 August 1997 / Revision received: 29 December 1997 / Accepted: 5 February 1998     

Multiple shoot induction and plant regeneration of the endangered species <Emphasis Type="Italic">Crepis novoana</Emphasis>

In vitro culture is a useful tool in the ex situ conservation of rare, endemic, and threatened plant species. Crepis novoana (Compositae) is an endangered endemic in northwestern Spain. Use of in vitro culture tools is necessary due to the poor conservation status of populations of the species. The systems of in vitro propagation developed for this species in the present study were caulogenesis from leaf explants and growth of axillary buds from shoots. Explants were produced by placing fragments of leaves on Murashige and Skoog medium (MS) supplemented with 2.22 μM 6-benzyladenine (BA) and 2.69 μM naphthaleneacetic acid (NAA); caulogenesis was induced in 80% of explants, with development of a mean number of 2.48 shoots per explant. Axillary bud development from shoots was highest with MS supplemented with 4.44 μM BA and 0.54 μM NAA, resulting in production of a mean number of 49.77 shoots per explant. Immersion of the basal side of shoots in a solution of 5.37 mM NAA for 30 s yielded 90% success in the production of rooted shoots. Plantlets were well acclimatized, and almost 100% of plants transferred to soil recovered successfully.     

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.     

In vitro culture of Safflower L. cv. Bhima: initiation,growth optimization and organogenesis

Callus induction and in vitro plantlet regeneration systems for safflower (Carthamus tinctorius L.) cv. Bhima using root, hypocotyl, cotyledon and leaf explants were optimized by studying the influence on organogenesis of seedling age, media factors, growth regulators and excision orientation. Supplementation of the medium with an auxin: cytokinin ratio < 1 enhanced the growth rate of callus cultures; however, for 2,4-D the ratio was > 1.34–11.41 μM concentrations of growth regulators (IAA, NAA, BA and Kinetin) in the medium were found effective for callus induction and regeneration in all explants. The calli could be maintained over 32 months. BA (4.43 μM) combined with casein hydrolysate (10 mg l-1) yielded the highest rate of shoot production on hypocotyl (3–6) and cotyledon (5–7) explants and cotyledonary derived callus (4–8). More shoots were produced on explants cut from the most basal region of cotyledons from 5 to 7-day-old seedlings than from older seedlings or more distal cut sites. Apolar placement of explants, inhibited shoot regeneration. The shoot regeneration potential remained upto 7 months in calli developed on NAA + BA. Of three media tested, MS was superior to SH-M and B5. Rooting of shoots was not efficient; 42% of the shoots were rooted on MS medium containing sucrose (7–8%) + IAA (2.8–5.7 μM). Capitula induction was observed in both callus mediated shoots on cotyledons and shoots on rooting medium with sucrose, IAA, NAA and IBA. Well developed plantlets were transferred to the field with a 34% success rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

Effect of 1,2-benzisoxazole-3-acetic acid on adventitious shoot regeneration and in vitro rooting in apple

 The effect of 1,2-benzisoxazole-3-acetic acid (BOA), compared to 1-naphthaleneacetic acid (NAA), on adventitious shoot formation in leaf portions and compared to indolebutyric acid (IBA), on in vitro rooting in the apple (Malus domestica Borkh) cultivars McIntosh and Gala, and one rootstock, Jork 9, was investigated. BOA at 43.0 μm or 2.7 μm at NAA in combination with 17.8 μm benzyladenine (BA), induced the highest number of explants to produce adventitious shoots in Jork 9. In Gala, the combination of 21.5 μm BOA with 1.0 μm thidiazuron (TDZ) or with 22.0 μm BA induced the highest regeneration percentages, 58 and 54%, respectively, giving more satisfactory results than NAA (where only 42% of leaf explants exhibited shoot formation). In McIntosh, the highest percentage of regeneration was obtained with 1.3 μm NAA and 22.0 μm BA, while 51% was the highest response obtained with the BOA treatment. The combination of BOA with TDZ completely inhibited regeneration activity in leaf portions of this cultivar. The shoots of all the genotypes obtained with the most morphogenetic NAA or BOA treatments were excised, multiplied and successfully rooted and hardened. The results demonstrate that the synthetic auxin BOA is active in inducing shoot regeneration from leaf explants of apple and that the activity of BOA in plant regeneration is genotype dependent. When BOA was used to induce rooting in apple microcuttings, lower rooting percentages were obtained than with IBA, showing that the effect of BOA in inducing root formation is very low and that it cannot be used routinely to replace IBA in the in vitro rooting of microcuttings. Received: 18 June 1998 / Revision received: 4 January 1999 / Accepted: 29 January 1999     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

An efficient in vitro regeneration protocol for Tagetes minuta

Tagetes minuta is a source of secondary products which are used as pharmaceuticals, pesticides and as flavour components in the food industry. Cotyledons and hypocotyls of T. minuta were cultured on MS medium with combinations of IAA or NAA and BA. Hypocotyl-derived callus developed adventitious shoots which failed to develop further. Cotyledon-derived callus, cultured on medium with IAA, regenerated adventitious shoots which developed into plantlets on MS medium or half-strength MS with 2.85 μM IAA. Cotyledons cultured on medium with 5.71 μM IAA + 44.4 μM BA and transferred to MS medium for shoot growth yielded the highest number of shoots. Nodal segments from developing shoots were micropropagated on half-strength MS medium with 2.58 μM IAA and 95% of plantlets produced adapted successfully to greenhouse conditions. In vitro plants micropropagated from nodes had many shoots whereas plants regenerated from shoot tips had only a single main stem. This difference in morphology was retained after two months growth in a greenhouse. There were no significant differences in leaf and shoot fresh and dry weights among the regenerated plants after two months growth. After six subcultures of cotyledon-derived callus on medium with IAA and BA all explants lost their ability to regenerate except those cultured on medium with 17.23 μM IAA and 44.4 μM BA. The methods of regeneration developed will facilitate selection of T. minuta plants more tolerant of environmental stress, their micropropagation, and the in vitro production of secondary products. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

In vitro regeneration from leaf explants of Neoregelia cruenta (R. Graham) L.B. Smith,an endemic bromeliad from Eastern Brazil

An efficient plant regeneration system was developed for the induction of direct shoot formation from leaves derived from seedlings of Neoregelia cruenta, an endemic Bromeliaceae of Eastern Brazil. Shoot differentiation occurred directly from the leaf bases. In vitro responses were influenced by seedling age and growth regulator combinations. Highest regeneration rates were obtained from explants excised from 7-week-old seedlings cultured in the presence of 22 μM BA and 2.5 μM NAA. Shoot conversion to whole plants was most effective in shoots formed in response to 4.4 or 8.8 μM BA combined with 2.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Shoot organogenesis and plant regeneration in <Emphasis Type="Italic">Metabriggsia ovalifolia</Emphasis>

An efficient propagation and regeneration system via direct shoot organogenesis for an endangered species, Metabriggsia ovalifolia, was established. High activity cytokinins [6-benzyladeneine (BA) and thidiazuron (TDZ)] and low activity auxins [α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA)] could directly induce adventitious shoots from leaf or petiole explants within 5 weeks. Cytokinins (TDZ or BA) combined with auxin (NAA) in the induction media induced more adventitious shoots than when auxins or cytokinins were used alone. Adventitious shoots could be induced and also mass-propagated on media containing 2.5–5.0 μM TDZ (or BA) and 0.25–0.5 μM NAA. Adventitious roots differentiated at the proximal end of shoots on rooting media containing half-strength MS salts and 0.5 μM IBA, 0.5 μM NAA, 0.1% activated charcoal or no plant growth regulators. Over 90% of plantlets survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite) in basins.     

Regeneration of plants from leaflet explants of tissue culture raised safed siris (Albizia procera)

Shoot bud regeneration was obtained from isolated leaflets of Albizia procera cultured on MS medium with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). The highest numbers of adventitious buds were obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. The replacement of 7 g l-1 Difco bacto agar with 2.6 g l-1 Phytagel in the medium enhanced adventitious bud regeneration. Further, addition of 15 μM silver nitrate promoted callus-free shoot regeneration from leaf explants. The regenerated shoot buds were elongated on MS medium containing 0.01 μM BA and 1 μM NAA. Rooting was obtained on modified MS medium supplemented with 2 μM IBA. To our knowledge this is the first report of direct regeneration of shoots from leaflet explants in A. procera, and should help facilitate genetic transformation in this species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organogenesis plant regeneration from leaf explants of Exacum Styer Group

Exacum Styer Group plantlets were regenerated through direct organogenesis from leaf explants. Four genotypes were evaluated on MS media supplemented with combinations of BA (0, 0.44, 2.22, 4.44, or 8.88 μM) and NAA (0, 0.05, 0.54, or 2.69 μM) for direct shoot organogenesis without an intervening callus phase. Regression analyses were used to analyze and interpret the data. There were significant genotype, media, and genotype × media interactions for several variables. Genotypes 01-09-01 and 01-37-61 had the highest number of shoots per explant across media (10.2 and 6.6, respectively) while the 4.44 μM BA plus 0.54 μM NAA treatment induced the greatest number of shoots among the genotypes evaluated.     

Factors affecting plantlet regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured immature embryos and cotyledons of <Emphasis Type="Italic">Prunus mume</Emphasis> “Xue mei”

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.     

High frequency in vitro propagation of <Emphasis Type="Italic">Trichopus zeylanicus</Emphasis> subsp. <Emphasis Type="Italic">travancoricus</Emphasis> using branch–petiole explants

Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 μM 2,4-dichlorophenoxyacetic acid upon subculture to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot morphogenesis. Medium with 13.3 μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower concentrations of BA (4.4, 6.6 and 8.8 μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 μM BA containing medium, but with reduced number of shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6 μM IBA, the whole branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium containing 2.7 μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix: a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar to that of the source plants flowered normally and set fruits.     

In vitro propagation for the conservation of a rare medicinal plant <Emphasis Type="Italic">Justicia gendarussa</Emphasis> Burm. f. by nodal explants and shoot regeneration from callus

Justicia gendarussa is a valuable medicinal plant and various parts of this plant are pharmaceutically used for the treatment of different diseases. In vitro regeneration of shoot buds was obtained from culture of nodal cuttings as well as shoot regeneration from callus. The nodal cuttings differed in shoot proliferation in terms of percentage of explants that responded and average shoot length with various concentrations (4.4, 8.9, 13.3, 17.7, 22.2 μM) of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron. In all treatments, one shoot was invariably present. Optimum 87% of cultures responded with an average shoot length of 4.4 cm on Murashige and Skoog (MS) medium supplemented with 17.7 μM BA. Callus was induced from the mature leaf segments on MS medium supplemented with Kn (4.7, 13.9, 23.2 μM) alone or in combination with 2, 4-dichlorophenoxyacetic acid (2, 4-D; 2.3 μM, 4.5 μM). Optimum callus induction (78%) was obtained on MS medium supplemented with 14 μM Kn and 4.5 μM 2, 4-D. When the callus was subcultured on MS medium fortified with BA (8.9, 17.7, 26.6 μM) or Kn (9.3, 18.6, 27.9 μM) alone or in combination with α naphthalene acetic acid (NAA; 2.7, 5.4 μM), shoot regeneration was obtained. The highest response (92%) was observed on MS medium containing 17.7 μM BA and 5.4 μM NAA. On this medium, an average number of 12.2 shoots were obtained per responding callus. The shoots obtained from callus and nodal cuttings were rooted with a frequency of 73% on MS medium augmented with 9.8 μM indole-3-butyric acid. The rooted shoots were successfully transplanted to soil and sand mixture (1:1) with 90% survival rate. The protocol standardized for shoot proliferation and regeneration in J. gendarussa from nodal cuttings and leaf-derived callus is suitable for micropropagation and conservation of this essential medicinal plant.     

High frequency adventitious shoot regeneration in sainfoin

A procedure is described for the rapid and efficient adventitious shoot regeneration from leaflets, petioles and stems of field-grown sainfoin plants. All explants formed shoots on a range of media supplemented with 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). Stem explants appeared to have better regeneration capacity than leaflet and petiole explants in most media tested. The highest frequency of shoot regeneration was achieved from stem segments on a medium containing 20 μM BA and 0.5 μM NAA. Regenerated shoots rooted in half-strength Murashige and Skoog medium containing 5 μM indole-3-butyric acid and later established well under greenhouse conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.