In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

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.     

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.     

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.     

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.     

Establishment and in vitro regeneration studies of the potential oil crop species Camelina sativa

Camelina sativa was successfully established in vitro and systems for the regeneration of shoots from leaf explants developed. Methods for the surface-sterilisation of seeds were used which gave 95% germination, though the in vitro grown seedlings failed to develop beyond 28 days culture. In a micropropagation system, the rooting response of nodal explants was increased from a control level of 26.4% to 46.7% by the addition of 5.4 μM NAA. Leaf explants were more efficient for the regeneration of root and shoots than hypocotyls. For regeneration from leaf tissue the use of auxin (NAA) alone in the medium above a level of 0.54 μM resulted in root or callus growth. Cytokinin, in the form of BA alone failed to induce regeneration, but a combination of 4.44 μM BA and 0.54 μM NAA induced shoot regeneration at rates over 10.0 shoots per explant. Regenerated shoots were successfully transplanted to soil and flowered and set seed normally. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

In vitro plant regeneration from leaf explants of Ophiorrhiza japonica

An efficient in vitro plant regeneration system from leaves of Ophiorrhiza japonica Blume was established for the first time. Callus formation rate was more than 90.4 % from leaf segments on Murashige and Skoog (MS) supplemented with either α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzyladenine (BA). The highest shoot regeneration (78.9 %) was achieved on MS medium containing 2.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA, with an average of 9.4 shoots developed per leaf segment. Shoot regeneration was also improved when the leaf explants were cultured in MS basal medium supplemented with 0.5 % (m/v) polyvinylpyrrolidone (PVP). The leaf explants from seedlings with age of about 18–27 d showed the highest shoot regeneration. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA), which averagely produced 24.8 roots per shoot. The plantlets were transferred to soil, where 100 % survived after 1 month of acclimatization.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Rosa damascena</Emphasis> Mill.

Summary A protocol for in vitro propagation using direct induction of shoot buds from leaf explants of in vitro-raised shoots of Rosa damascena var. Jwala is reported. The present study is the first report on direct shoot regeneration in scented roses. Elite plants raised from nodal explants and maintained for over 2yr in vitro on a static liquid shoot multiplication Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 3% sucrose were used. Petioles from fully developed young leaves, obtained after 4 wk of pruning of old shoots, were found to be ideal for regeneration of shoots. Initially the explants were cultured in an induction medium [half-strength MS+3% sucrose+6.8μM thidiazuron+0.27 μM α-naphthaleneacetic acid (NAA)+17.7 μM AgNO₃] and subsequently transferred to the regeneration medium (MS+2.25 μM BA+0.054 μM NAA) after 7, 14, 21, 28, and 35d. The highest shoot regeneration response (69%) was recorded when shoots were kept in the induction medium for 21 d and later transferred to regeneration medium. Histological studies revealed direct formation of shoot buds without the intervening callus phase. In vitro rooting of micro-shoots was accomplished within 2wk on half-strength MS liquid medium supplemented with 10.0 μM IBA and 3% sucrose for 1 wk in the dark and later transferred to hormone-free medium and kept in the light. Plantlets, remaining in the latter medium for 5–6 wk when transferred to soil, showed 90% survival.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

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.     

An efficient plant regeneration system for Mucuna pruriens L. (DC.) using cotyledonary node explants

Summary The purpose of this study was to develop an efficient micropropagation system for Mucuna pruriens, an important medicinal plant in India. A range of cytokinins was investigated for multiple shoot regeneration with cotyledonary node explants from 7-d-old aseptic seedlings. Of all the cytokinins, 6-benzyladenine (BA), kinetin (KIN) and 2-isopentenyl adenine (2-iP) tested in Murashige and Skoog medium (MS), BA was the most effective and 5.0 μM was found to be optimum for inducing maximum shoots. Medium types, medium strength and pH were also investigated for induction and proliferation of shoots. The highest efficiency of shoot proliferation was observed in 5.0 μM BA and 0.5 μM α-naphthalene acetic acid (NAA) in half-strength MS medium at pH 5.8. The best condition for rooting was half-strength MS medium solidified with agar and with 2.0 μM indole-3-butyric acid (IBA). After rooting, the plantlets were transferred to plastic pots filled with sterile soilrite where 90% grew and all exhibited normal development.     

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.     

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.     

High-frequency plant regeneration through adventitious shoot formation in castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

An efficient plant regeneration protocol was established for castor (Ricinus communis L.). Hypocotyl tissue from zygotic embryo axis produced adventitious shoots when treated with either thidiazuron (TDZ, 1 μM) or 6-benzylaminopurine (BA, 20 μM). TDZ resulted in more than a threefold higher rate of shoot induction (a maximum of 24.2 shoots per explant) than BA (6.8 shoots). Our results also showed that the pretreatment of explants in the dark increased the number of shoots regenerated per explant by 82% and 36% with TDZ and BA, respectively. The elongation of hypocotyl tissue in the dark appears to be the primary cause of the increase. Comparable rates of rooting were achieved on the media supplemented with either indole-3-butyric acid (IBA, 84.3%) or 1-naphthaleneacetic acid (NAA, 87.4%) at 5 μM. However, IBA was more efficient in promoting root and shoot development, resulting in a higher rate of establishment (93.5%) in the soil, compared to the rate with NAA (39.5%). Histological analysis showed the adventitious induction of the shoot buds originated from the cortex of the hypocotyl tissue.     

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.     

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume

In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%) formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage of GUS⁺ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR⁺ plants.     

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.     

Adventitious shoot regeneration from leaf explants of almond (Prunus dulcis mill.)

Summary A method has been developed to facilitate shoot formation from leaf explants of almond. Leaves were dissected from micropropagated shoot cultures of the commercial cultivars Nonpareil and Ne Plus Ultra, and sections incubated on Almehdi and Parfitt's (1986) basal medium (AP) with varied plant growth-regulator conditions. Three auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA), in combination with two cytokinins, benzylaminopurine (BA) and thidiazuron (TDZ), were tested at various concentrations along with casein hydrolysate (CH) to determine, the conditions most conducive to adventitious shoot regeneration. Response to the tested plant growth-regulator conditions varied with genotype. Of the three auxins tested, NAA and IBA induced adventitious shoots from Ne Plus Ultra explants, but only IBA was effective for Nonpareil. For the cytokinins, shoot development from Ne Plus Ultra occurred in the presence of either BA or TDZ, whereas for Nonpareil only TDZ was effective unless CH was incorporated in the basal medium. The inclusion of CH (0.1% w/v) improved callus morphology, and increased regeneration frequencies for both cultivars. Maximum regeneration frequencies for Ne Plus Ultra (44.4%) and Nonpareil (5.5%) were achieved on AP basal salts supplemented with CH, IBA (9.8 μM), and TDZ at 22.7 and 6.8 μM, respectively.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.