Micropropagation of Lippia junelliana (Mold.) Tronc.

A method for the micropropagation of Lippia junelliana (Mold.) Tronc. from shoot tips or nodal segments was developed. Proliferating microshoot cultures were obtained by placing shoot tips or nodal segments on full strength Murashige and Skoog medium (MS) supplemented with 4.4 μM benzyladenine (BA) or 0.04 μM indolebutyric acid- (IBA) plus 4.4 μM BA. The rooting of shoots was better on full-strength MS medium without growth regulators. Rooted plantlets were successfully acclimatized to soil. The shoot cultures showed a lower essential oil accumulation in comparison with parent plants. Essential oil accumulation is closely related with growth and shows a negative correlation with shoot proliferation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation ofDalbergia sissoo from nodal explants of mature trees

A method for micropropagation ofDalbergia sissoo has been developed. Single node segments obtained from coppice shoots of a mature tree (20 – 25 year old) produced 3–4 shoots per explant on Murashige and Skoog (MS) medium containing 4.4 x 10⁻⁶ M benzylaminopurine (BAP) and 4.4 × 10⁻⁷ M of Β-naphthoxy acetic acid (NOA) (shoot multiplication medium) within 4 weeks. Thein vitro regenerated shoots were 3 – 4 cm in length and provided 2 to 3 culturable nodal segments which on shoot multiplication medium again produced 3–4 shoots. Following this procedure 18–24 shoots were produced from single nodal segment within 60 d. 80 % of the shoots directly produced five roots when they were firstly treated with MS medium supplemented with 10⁻⁵ M indole-3-butyric acid (IBA) and subsequently transferred to half strength liquid MS medium containing 1 % activated charcoal followed by half strength liquid MS free hormones, vitamins and activated charcoal. Thein vitro raised plants were hardened for survival after transplantation to soil by exposing them to various humidity conditions, gradually from higher to low, with nearly 100 % transplant success. Acknowledgement: Authors are grateful to CSIR and DST, New Delhi for financial assistance.     

Micropropagation and acclimatization of Hedeoma multiflorum

A method for the micropropagation of Hedeoma multiflorum Benth from shoot tips or nodal segments was developed. Proliferating microshoot cultures were obtained by placing shoot tips or nodal segments on half-strength Murashige and Skoog (1962) medium supplemented with 22.2 μM BA or 22.2 μM BA plus 0.05 μM NAA. Individual shoots were excised and transferred into rooting medium containing auxins (IBA, NAA or IAA). Rooting of shoots was better on half-strength MS medium containing 0.6 μM IAA than on half-strength MS medium without growth regulators. Rooted plantlets were successfully acclimatized to soil. Preconditioning at different sucrose concentrations prior to acclimatization had no effect on plant establishment, but influenced plant quality. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro micropropagation of Basella rubra L. through proliferation of axillary shoots

In vitro micropropagation protocol for Basella rubra regeneration was tried through proliferation of axillary shoots of the potted mature plant. The improved seed germination (70%) was recorded upon 2% urea treatment. The nodal shoot segments from matured potted plant were used to initiate the multiple shoot proliferation. The shoot segments exhibited 70% shoot initiation when cultured on Murashige and Skoog (MS) medium supplemented with Indole-3-acetic acid (IAA)?+?N₆ – Benzylaminopurine (BAP) (0.25?+?2.0 mg/L) and BAP?+?Kinetin (Kin) (2.0?+?0.5 mg/L) respectively. Multiple shoots (5–6) were obtained on MS medium supplemented with BAP?+?Kin and IAA?+?BAP respectively. When compared with silver nitrate (AgNO₃) (2–40 µM) and activated charcoal (AC) (0.1–1.0%), the MS medium devoid of any plant growth regulator showed good number of shoots (5.48?±?2.42), elongation (15.64?±?2.42 cm) and root length (14.52?±?2.78 cm). Upon transferring of regenerated microshoots to MS medium, simultaneous elongation of shoots with more shoot number, shoot length and rooting was achieved during four subcultures that carried out at 6 weeks’ interval. The regenerated in vitro shoots showed 100% rooting in MS medium and also in MS medium supplemented with 0.1–1.0% AC. Hundred percent survival of micropropagated shoots well rooted was established successfully under greenhouse condition and the plants were subsequently acclimatized and transferred to the field conditions wherein 90% success rate was noted.

     

An improved protocol for micropropagation of elite genotypes of Simmondsia chinensis (Link) Schneider

An efficient micropropagation protocol was developed for elite male and female genotypes of Simmondsia chinensis using nodal segments. Bud initiation was found to be best on Murashige and Skoog’s (MS) medium supplemented with 4.44 μM 6-benzylaminopurine (BAP) and 88.8 μM adenine. Upon sub-culture, 10–15 shoots per explant were obtained when 4.44 μM BAP and 74.0 μM adenine were incorporated in the medium. Increase in KNO₃ concentration in the medium improved shoot multiplication rate and in vitro flowering in 20 % of male cultures. Elongated shoots were harvested, pulse treated for 48 h on liquid medium supplemented with 49.0 μM indole-3-butyric acid, 5.40 μM α-naphthaleneacetic acid and 5.71 μM indole-3-acetic acid for root induction and rooting (92 %) was achieved on hormonal free half-strength MS medium supplemented with 1.37 μM chlorogenic acid, 1 % activated charcoal and 2 % sucrose. After successful hardening, plantlets were transferred to greenhouse with 99 % establishment.     

Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant

A rapid and efficient method for the large-scale propagation of a highly valuable medicinal plant, Andrographis paniculata Nees, through in vitro culture of nodal explants obtained from 15-d-old aseptic seedling has been developed. High frequency direct shoot proliferation was induced in nodal explants cultured on Murashige and Skoog’s medium supplemented with 6-benzylaminopurine (BAP). Amongst the various cytokinins tested (BAP, kinetin, thidiazuron and 2-isopentyl adenine), BAP proved to be the most effective. The shoot forming capacity of the nodal explants was influenced by the BAP concentration (1–12.5 μM), and the optimal response was observed at 10 μM BAP, which induced an average of 34 shoots in 94% of the cultures within 4 wk. Significant differences were recorded in terms of average number of shoots per explant (8.6–34.1) among the different concentrations of BAP investigated. Concentrations of all cytokinins tested reach a level that can be considered above the optimum level, as marked by a reduced frequency of shoot proliferation. The multiple shoots obtained on various concentrations of BAP failed to elongate even after transfer to hormone-free MS medium. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 μM GA₃ within 2 wk. A proliferating shoot culture was established by repeatedly subculturing the original nodal explants on shoot multiplication medium after each harvest of the newly formed shoots. The explants retained their morphogenic potential even after three harvests. Therefore, in 90 d, about 60–70 shoots were obtained from a single nodal explant and the nodal explants from primary shoots further regenerated equivalent number of shoots, depicting their high frequency regeneration potential in A. paniculata. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 μM indole-3-butyric acid (IBA), within a wk. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. The system is rapid: the initiation of shoot buds to the transplanting of regenerants to soil is completed in 8–9 wk.     

An improved micropropagation of <Emphasis Type="Italic">Spilanthes acmella</Emphasis> L. through transverse thin cell layer culture

An efficient and improved in vitro propagation system for Spilanthes acmella L. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration from tTCL nodal segments was affected by concentrations of plant growth regulators and orientation of the explant. MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium with 5.0 mg dm⁻³ BAP was optimal for shoot regeneration. Upon this medium, the explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (about 97%), and the highest number of shoots (31.5) per explant. The intact node (1.0–1.5 cm) cultured on the same medium had significantly lower shoot multiplication ability with only 4.5 shoots per responsive explant. As compared to BAP alone, the combination of BAP and Kin or NAA did not have positive effects on shoot multiplication from tTCL nodal segments. Rooting of shoots was achieved on growth regulator free full-strength MS medium. Plantlets were transplanted into soil with 90–100% survival rate.     

An improved micropropagation of Terminalia bellirica from nodal explants of mature tree

An efficient and improved in vitro propagation method has been developed for Terminalia bellirica, a medicinally important tree from nodal explants of 10-year-old mature tree. Shoot multiplication was influenced not only by cytokinin types, their concentrations and their interaction with auxin but also by successive transfer of mother explants for different passages, subculture of excised shoots on fresh medium and different medium composition. MS medium containing 2.22 μM BAP was found to be the best for shoot multiplication in a single step. After excision of newly formed shoots, mother explants successively transferred to the same medium produced maximum shoots per explant after IV passage. Further enhancement in morphogenetic response occurred when excised shoot clumps (2–3 shoots) were subcultured on MS medium supplemented with 2.22 μM BAP, 1.16 μM Kn and 0.57 μM IAA. Half-strength MS medium supplemented with 24.60 μM IBA and 100 mg l⁻¹ AC was most effective for rooting of the shoots. To reduce labor, cost and time, an experiment on ex vitro rooting was also carried out and it was observed that highest percent shoots rooted ex vitro when treated with 2,460 μM IBA for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. In comparison to plantlets developed from in vitro rooted, percent survival of plants those rooted ex vitro was significantly higher. Use of ex vitro rooting technique for plant production serves as a more economical option; therefore, present method can be used for large-scale commercial production of this medicinally important tree.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

Micropropagation of <Emphasis Type="BoldItalic">Embelia ribes</Emphasis> Burm f. through proliferation of adult plant axillary shoots

Micropropagation of Embelia ribes was achieved through proliferation of axillary shoots obtained from mature plants. Nodal shoot segments, collected March–May, exhibited high-frequency (75%) shoot initiation when cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) at 1.13 μM and indole-3-butyric acid (IBA) at 0.49 μM. Subculture of sprouted shoots from the original explants on medium containing TDZ (1.13 and 0.45 μM) during the first and second subcultures was found essential for further shoot proliferation, while inhibition of shoot elongation by TDZ could be overcome by transferring shoot cultures onto MS medium containing 6-benzylaminopurine (BAP; 11.10 μM) for the third subculture. Treating the explants with an antioxidant mixture of 568 μM ascorbic acid, 119 μM citric acid, and 307 μM glutathione prior to inoculation, coupled with subculture at 2-wk intervals onto fresh medium, both helped to reduce browning of the explants and facilitated production of five to six shoots/explant. MS medium supplemented with BAP (4.44 μM) and IBA (0.49 μM) induced shoot multiplication, producing five to six shoots/explant with a shoot length of 3 to 4 cm over a 4-wk culture period. Shoots of 3 to 4 cm in length exhibited 100% rooting within 4 wk after transfer to media containing half the nutrient salt concentration of MS medium with 3.69 μM IBA. Ex vitro rooting in the greenhouse from the in vitro shoots treated with 4.93 μM IBA for 30 min exhibited 95% rooting in soilrite™ medium in a 4-wk period. About 85% of micropropagated plants were established successfully in root trainers. Three-month-old, hardened plants could further be successfully established in the field. In 1 yr, by using the above protocol, 3,200 plants could be produced from a single shoot and 2,700 could be established in the field.     

High frequency in vitro propagation of Isodon wightii (Bentham) H. Hara

A protocol for in vitro propagation of Isodon wightii (Bentham) H. Hara from nodal segments was developed. Multiple shoots were successfully established on half strength MS medium supplemented with 4.4 μM BA. Enhancement of shoot multiplication and elongation was achieved on half strength MS medium supplemented with 4.4 μM BA and 1.4 μM GA₃. The regenerated shoots were rooted successfully on half strength MS medium supplemented with 4.9 μM IBA. Acclimatization of in vitro rooted shoots was successful. The in vitro regenerated plants grew well in the greenhouse without any phenotypic changes.     

Rapid clonal propagation of Vitex trifolia

This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2-isopentenyladenine (2-iP) (0.25 – 10.0 μM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 μM BAP. Shoot cultures was established repeatedly subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 μM naphthaleneacetic acid (NAA). Rooted plantlets transferred to pots containing autoclaved soil and vermiculite mixture (1:1) showed 90 % survival when transferred to outdoor.     

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

Padar (Stereospermum personatum, family Bignoniaceae) is a well-known medicinal tree. Its complete regeneration occurred through shoot bud culture in vitro. The seeds germinated sequentially on plastic trays and polyethylene bags for 21 days served as explants source. Nodal segments from the seedlings were established on MS medium supplemented with 4.44 μM BA, in which 86.6% nodes showed shoot bud elongation. Then, nodal segments from the developed shoots were cultured on MS medium with several BA concentrations; best shoot multiplication was obtained with 0.44 μM BA. In a second experiment where PVP was added to proliferation medium, nodal segments from developed shoots produced maximum 2.78 shoots per node. The nodal segments showed shoot multiplication up to seventh subculture on. Finally, shoots were rooted on MS medium with 2.46 μM IBA. The plants transferred to net pots containing coco-peat were acclimatized in green house, where more than 80% plants survived and grew normally.     

Micropropagation of <Emphasis Type="Italic">Pongamia pinnata</Emphasis> through enhanced axillary branching

A complete protocol is presented for the first time for the micropropagation of Pongamia pinnata, a biofuel tree, using cotyledonary nodes derived from axenic seedlings. Multiple shoots were induced in vitro from nodal segments through forced axillary branching. Murashige and Skoog (MS) medium supplemented with 7.5 μM benzylaminopurine (BAP) induced up to 6.8 shoots per node with an average shoot length of 0.67 cm in 12 d. Incorporation of 2.5 μM gibberellic acid (GA₃) in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Single use of GA₃ during the first subculture eliminated the need for prolonged culturing on BAP medium. Further use of GA₃ in the medium was not useful. Shoot culture was established for at least two subcultures without loss of vigor by repeatedly subculturing the original cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 16–18 shoots were obtained in 60 d. Shoots formed in vitro were rooted on full-strength MS medium supplemented with 1.0 μM indole butyric acid (IBA). Plantlets were successfully acclimated, established in soil, and transferred to the nursery.     

High-frequency in vitro propagation of the endangered species <Emphasis Type="Italic">Tuberaria major</Emphasis>

A novel protocol suitable for the micropropagation of the endangered species Tuberaria major using seedlings as explants is reported. Using this protocol, we studied the effects of explant type (apical shoots and nodal segments) and cytokinins [6-benzyladenine (BA), kinetin, and zeatin (ZEA)] on shoot proliferation. Explant type significantly influenced the proliferation frequency and mean number of shoots, with nodal segments showing a higher proliferation capacity. The mean number of shoots was significantly higher when the explants were cultured in half-strength (1/2) MS medium supplemented with 0.2 mg l⁻¹ BA (6.83 ± 0.77 shoots) or ZEA (6.55 ± 0.71 shoots). The shoots showed a great rooting capacity that was significantly influenced by the concentration of MS macronutrients but not by the concentration of auxins. The highest rooting frequencies (97–100%) were obtained in 1/2 MS medium with or without plant growth regulators. The plants obtained were easily acclimatized to ex vitro conditions, with 97% surviving after 6 weeks. The micropropagated plants were successfully reintroduced into their natural habitat and exhibited normal development. In conclusion, our culture protocol, with efficient seed germination, subsequent multiplication of nodal explants using ZEA at 0.2 mg l⁻¹, and successful ex vitro establishment of well-rooted plantlets on 1/2 MS medium, provides a simple and reliable methodology for the large-scale propagation of T. major, thereby contributing to germplasm preservation of this endangered species.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

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.     

A micropropagation protocol forCinnamomum camphora

Summary A micropropagation protocol was developed forCinnamomum camphora (L.) Sieb., using as initial explants 3–5-mm shoot tips from newly emerged laterals of 2-yr-old trees. Performance of small shoot tips was compared with that of 2.0-cm nodal segments during subculture. Murashige and Skoog medium (MS) supplemented with different concentrations of N₆-benzyladenine (BA) or thidiazuron (TDZ) was used to examine shoot proliferation. In separate experiments, MS was supplemented with 1-naphthaleneacetic acid (NAA) for rooting of shoots, and the commercial preparation EM₂ for prevention of hyperhydricity. BA stimulated shoot formation and callus development, whereas TDZ promoted only callus development. Both cytokinins induced hyperhydricity when small shoot tips were used, with severity being directly related to concentrations. Hyperhydricity was avoided in subcultures by using larger nodal segments. EM₂ did not alter degree of hyperhydricity but suppressed callus development and strongly promoted shoot multiplication. The number of new shoots after a 6-wk subculture was 9 per nodal segment when supplemented solely with 4.4 μM BA and 18 per segment when further supplemented with 1000 mg EM₂ per I. Rooting of shoots occurred best when supplemented solely with 0.54 μM NAA, averaging 7 roots per shoot in 4 wk. Ninety percent of rooted shoots survived transfer to the greenhouse.     

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.