In vitro Clonal Propagation and Organogenesis in Spilanthes acmella (L) Murray: A Herbal Pesticidal Plant of North-East India

Multiple shoots were regenerated in MS medium using different concentrations of BAP and Kn and different combinations of BAP with IAA, NAA and IBA. Highest multiplication of shoots was obtained with BAP (0.75 mg l^?1) with 28.4 shoots per explant after 60 days of culture. Shoots rooted best on IBA (0.5 mg l^?1), numbering 48.8 per explant. Organogenesis was maximum in callus cultured on MS medium supplemented with BAP (2.0 mg l^?1) and IAA (1.0 mg l^?1).     

In Vitro Propagation and Conservation of Dendrobium lituiflorum Lindl Through Protocorm-Like Bodies

Axillary buds obtained from 5-month-old in vitro growing plants of Dendrobium lituiflorum Lindl were cultured in Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-D. Fastest initiation (13.3 days) of protocormlike bodies (PLBs) was observed in cultures containing MS medium supplemented with 0.5 mg l^?1 2,4-D. Maximum explant response of 83% was also observed in the same medium. PLBs obtained in MS medium containing 0.5 mg l^?1 2,4-D showed maximum regeneration potential of seedlings (19 explant^?1) when subcultured in MS medium. Well developed shoots and roots of the seedlings were obtained in the medium containing 0.5 mg l^?1 each of NAA and BAP, in combination. Encapsulated PLBs of D. lituiflorum could be stored at 8°C for 90 days with 80% regeneration. However, it was observed that regeneration potential of encapsulated PLBs reduced with further storage. Seventy seven per cent hardened plants survived and bloomed after 2.5 years of hardening.     

Embryogenesis,plant regeneration and cardiac glycoside determination in Digitalis ferruginea subsp. ferruginea L

The present study reports, for the first time, an efficient in vitro plant regeneration protocol for Digitalis ferruginea subsp. ferruginea L. (rusty foxglove). We have used different concentrations of gibberellic acid (GA₃) on Murashige and Skoog (MS) medium to assess the germination frequency of seeds. High frequency of germination was achieved on MS medium with 1.0 mg l^?1 GA₃. 6-Benzylaminopurine (BAP) combined with α-naphtaleneacetic acid (NAA) or 2, 4-dichlorophenoxy acetic acid (2, 4-D) in the induction MS medium induced both somatic embryogensis and shoot organogenesis. The highest percentage of callus growth (85 %) was obtained when hypocotyl explants were cultured on MS medium containing 0.5 mg l^?1 2, 4-D plus 1.0 mg l^?1 BAP. The maximum mean number of somatic embryos (7.3 ± 1.3 embryos) or shoots (12.0 ± 1.1 shoots) per callus was obtained when medium contained 0.25 mg l^?1 NAA plus 1.0 mg l^?1 BAP or 0.5 mg l^?1 NAA plus 2.0 mg l^?1 BAP. The regenerated shoots easily rooted on MS medium. Higher amounts of lanatoside C [13.2 ± 0.5 mg 100 g^?1 dry weight (dw)] and digoxin (2.93 ± 0.31 mg 100 g^?1 dw) accumulation were obtained when shoots were obtained by indirect regeneration. We also investigated derivatives of cardenolides, i.e., digitoxigenin (730 ± 180 mg 100 g^?1 dw), gitoxigenin (50 ± 20 mg 100 g^?1 dw) and digoxigenin (490 ± 170 mg 100 g^?1 dw) from natural samples.     

Optimizing micropropagation of drought resistant <Emphasis Type="Italic">Pyrus boissieriana</Emphasis> Buhse

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana, which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l^?1 BAP and 0.05 mg l^?1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l^?1 BAP and 0.07 mg l^?1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50–100 mg l^?1 PG with different concentrations of IBA or NAA at intervals of 30–60 days. Significant increase in shoot length was detected after 45–60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l^?1 IBA and 100 mg l^?1 PG. GA₃ negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l^?1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25–30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.     

Efficient Plant Regeneration System From Leaf Discs of Zonal (Pelargonium x hortorum) and two scented (P. capitatum and P. graveolens) Geraniums

An efficient and rapid plant regeneration system was established for zonal and scented geraniums using leaf discs as explants. Several explants, medium and culture conditions were studied to optimize shoot induction. Leaf discs taken from 4–5 weeks old in vitro grown plants, whatever the genotype, were more effective for shoot regeneration than those taken from greenhouse grown plants. Darkness proved to be a stimulating factor for shoot regeneration and the combination between NAA and two cytokinins gave the best results. Direct shoot regeneration (100%) was obtained from leaf discs of P. capitatum on half-strength MS medium supplemented with 0.5 mg l⁻¹ NAA in combination with 1 mg l⁻¹ of BAP and zeatin in darkness (11.4 shoots per explant). In the same medium and culture conditions, all P. graveolens leaf discs also exhibited direct shoot regeneration (7.3 shoots per explant). For P. x hortorum, 100% of leaf discs underwent shoot regeneration on a MS medium supplemented with 0.2 mg l⁻¹ NAA in combination with 0.5 mg l⁻¹ of BAP and zeatin in darkness (8.8 shoots per explant) or under low light conditions with 0.2 mg l⁻¹ NAA and 1 mg l⁻¹ of BAP and zeatin (7.5 shoots per explant). For this species, the best results for shoot elongation were obtained on half-strength MS medium gelled with Phytagel 0.3% (v/v). Whatever the genotype, all shoots rooted readily when transferred to diluted MS medium (MS/2) containing 1 mg l⁻¹ IAA. Acclimatized plants grew normally and flowered in greenhouse conditions. Flow cytometry analysis made on leaves of acclimatized plants revealed that all the scented geranium plants are similar to mother plants while 71% of P. x hortorum plants which showed strong growth were tetraploid.     

Micropropagation of Anethum graveolens L Through Axillary Shoot Proliferation

A protocol is described for rapid and large-scale in vitro propagation of Anethum graveolens by enhanced axillary shoot induction that was dependent on BAP supply. The synergistic combination of 0.5 mg l^?1 BAP and 0.1 mg l^?1 IBA induced 100% shoot formation as well as shoot number (6.6 ± 0.48 per explant). Subculturing of shoot tips of in vitro plants on multiplication medium enabled continuous production of healthy shoots with similar frequency. Rooting of shoots was achieved on a medium with 1mg l^?1 IBA and 0.5 mg l^?1 Kn. Micropropagated plants established in garden were uniform and identical to the donor plant with respect to morphological and cytological characteristics.     

Efficient organogenesis and plantlet regeneration in the timber species <Emphasis Type="Italic">Cunninghamia lanceolata</Emphasis> (Lamb.) Hook

Two efficient regeneration systems were developed in Cunninghamia lanceolata, the most important conifer for industrial wood production in China. Cotyledons and hypocotyls derived from greenhouse-grown seedlings were used as initial explants in our research. A high frequency (95.1?±?1.84%) of adventitious buds were initiated directly from cotyledons cultured on Douglas-fir cotyledon revised (DCR) medium supplemented with 1 mg l^?1 benzyladenine (BA), 0.1 mg l^?1 α-naphthaleneacetic acid (NAA), and 0.004 mg l^?1 thidiazuron (TDZ) with a maximum mean number of adventitious buds per cotyledon explant of 3.76?±?0.08. In contrast, a high percentage (93.73?±?0.55%) of adventitious buds regenerated via callus produced from hypocotyls cultured on DCR medium supplemented with plant growth regulators with a maximum number of adventitious buds per explant (16.71?±?0.34). Adventitious buds elongated on DCR medium supplemented with 0.2 mg l^?1 BA and 0.02 mg l^?1 NAA. After liquid pretreatment with 50 mg l^?1 indole-3-butyric acid (IBA), over 95% of the shoots successfully rooted on ½ DCR medium supplemented with 0.3 mg l^?1 IBA. The innovated systems reported in this study will be useful tools for future genetic manipulation of C. lanceolata and may be adapted for large-scale propagation in other conifers.     

Callus Culture and Plant Regeneration from Seedling Explants of Pergularia daemia (Forsk) Chiov

Callus cultures were established from seedling explants of Pergularia daemia (Forsk) Chiov on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins. Optimal callus developed from leaf explants on MS medium supplemented with 2,4-D (2 mg l^?1) + 2iP (0.1 mg l^?1), was used for morphogenesis. Adventitious shoots were regenerated (70%) from the calli on MS medium supplemented with NAA (0.1 mg l^?1)+ BAP (2 mg l^?1). Individual shoots were rooted on half strength MS medium supplemented with 0.1 mg l^?1 IBA. Plantlets with well developed roots were successfully transferred to soil and 50% of the transferred plants survived.     

An improved micropropagation and assessment of genetic stability of micropropagated Salvadora oleoides using RAPD and ISSR markers

An improved micropropagation method has been developed for Salvadora oleoides, a valuable tree species of alkaline and arid regions. Nodal explant obtained from a mature tree (30- to 35-year-old) responded optimally (80.0 %) on BAP (2.0 mg l^?1) and produced (4.56 ± 0.52) shoots. Shoots were further multiplied by subculturing the in vitro excised shoots and transferring them to MS medium containing either BAP (0.0–2.0 mg l^?1) alone or in combination with lower concentrations of an auxin (IAA or NAA 0.05–0.4 mg l^?1). Among all the PGRs combination tested, MS medium supplemented with BAP (0.5 mg l^?1) and IAA (0.1 mg l^?1) formed the maximum number of shoots (68.40 ± 2.74 per culture bottle) with an average height (6.59 ± 0.30 cm), after 6 weeks of culture. Rooting in regenerated shoots was achieved by ex vitro methods and about 92.5 % of shoots were rooted with 5.25 ± 0.64 roots per shoot and an average length of 2.76 ± 0.53 cm after 3 weeks of incubation in the green house. More than (80 %) of hardened plantlets survived in the field conditions. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD and ISSR. Of the 10 RAPD primers finally selected, a total of 42 bands (out of 43) were monomorphic and one polymorphic, whereas from 10 ISSR primers selected, all the 43 bands were monomorphic revealing a high level of genetic homogeneity in the regenerated plants and the donor plant. In the present investigation, we achieved significantly more number of shoots during multiplication, which are higher than all previous reports and further evaluated the genetic fidelity of protocol for the first time in S. oleoides, which concludes the clonal (true-to-type) nature of micropropagated plantlets.     

Simple and Effective Regeneration of Mungbean (Vigna radiata (L) Wilczek) using Cotyledonary Node Explants

Mungbean (Vigna radiata (L) Wilczek cv ML — 267) is a recalcitrant grain legume species. Direct multiple shoots were developed from the cotyledonary node explants of 2-day-old in vitro grown seedlings of mungbean. Maximum number of shoots (an average 12.1 shoots per explant) was obtained on a medium containing MS salts, B5 vitamins and 5.0 mg l^?1 BAP. A medium with lower BAP concentration appeared suitable for rapid shoot elongation. The elongated shoots were rooted on 0.2 mg l^?1 NAA. The rooted plants were acclimatized under field conditions. The survival of the plants in the greenhouse was 90 %. Plants flowered and set seed normally.     

In Vitro Regeneration of Achillea millefolium L from Shoot-tips and Root Segments of Seedlings

This report describes, for the first time, an efficient plant regeneration system for Achillea millefolium L (yarrow), a medicinal plant, via shoot multiplication from shoot-tips and adventitious shoot regeneration from root segments. Higher numbers of shoots were obtained when shoot-tips were cultured on MSMO medium supplemented with 3.0 mg l^?1 BA and 0.5 mg l^?1 IAA, or 5.0 mg l^?1 KIN and 1.0 mg l^?1 IBA, producing 17.3 and 17.0 shoots per explant at 100% frequency, respectively. For adventitous shoot regeneration, only root segments developed shoots when cultured on medium containing a combination of 1 mg l^?1 TDZ, 0.5 mg l^?1 IAA and 0.5 mg l^?1 GA3 (18.9 shoots per explant at 100% frequency), while other types of explants (i.e., cotyledons, leaf lamina and petiole segments) or hormonal combinations tested were found ineffective. Regenerated shoots rooted readily on MSMO medium containing different concentrations of IAA, IBA, NAA or 2,4-D, however, NAA at 0.5 mg l^?1, or IBA at 0.5 or 1.0 mg l^?1 were found to be the most productive. Nearly all of the regenerated plants (98%) survived through the hardening process when the rooted plantlets were kept at 55–65% relative humidity for 2 weeks, which were then planted in pots containing potting soil and kept at 25–35% humidity.     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

In vitro propagation and assessment of genetic integrity of Talinum triangulare (Jacq.) Willd: a valuable medicinal herb

Talinum triangulare is a medicinally important herb and various parts of the plant are used pharmaceutically for the treatment of different diseases. In our study, a rapid and efficient protocol for micropropagation has been developed from shoot tip and nodal explants of T. triangulare. High shooting frequency (93.33?%) was achieved with shoot tip explants when cultured on Murashige and Skoog??s (MS) medium supplemented with 1.0?mg/L 6-benzyl amino purine (BAP) producing an average of 12.50?±?0.23 shoots and 5.07?±?0.02?cm shoot length per explant. A combination of 0.5?mg/L BAP and 0.5?mg/L kinetin was found to be more effective by producing 15.67?±?0.25 shoots and 6.22?±?0.02?cm shoot length per explant. The microshoots were excised and cultured on half-strength MS and full-strength MS medium containing different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA) for root induction. More number of roots (45.10?±?0.96) with an average length of 5.46?±?0.08?cm was obtained on half-strength MS medium supplemented with 0.5?mg/L IBA. The rooted shoots were successfully transplanted from different planting substrates to the field with a 100?% survival rate. Random amplified polymorphic DNA analysis was carried out using four random decamer primers. The amplification products were monomorphic in the micropropagated plants and were similar to the mother plant. Absence of polymorphism revealed that no variation was induced, thus maintaining the genetic integrity of the micropropagated plants of T. triangulare.     

Effect of Explant Source on Axillary Shoot Multiplication During Micropropagation of a Rare Medicinal Plant — Wattakaka volubilis (L f) Stapf

Nodal explants of in vivo plants and in vitro seedlings of Wattakaka volubilis were cultured on Murashige and Skoog medium fortified with various concentrations of cytokinins — BA (0.5–5 mg l^?1), KN (0.5–10 mg l^?1),TDZ (0.05–1 mg l^?1) either singly or in combination with NAA (0.1 mg l^?1). KN proved best for inducing healthy shoots in both in vitro and in vivo derived explants. Maximum number of shoots (14.1±0.84) with 80% regeneration frequency was obtained from nodal explants of seedlings cultured on 5 mg 1^?1 KN + 0.1 mg l^?1 NAA. In vivo nodal explants produced a maximum of 4.2 shoots on MS medium fortified with 2 mg l^?1 BA+0.1 mg l^?1 NAA. The differentiated shoots from both could be rooted with 85% frequency on 1/2 strength MS medium (1% sucrose) with 0.6% agar + 1 mg l^?1 IBA + 0.2 mg l^?1 KN. Rooted shoots were transplanted to vermiculite-soil (3:1) mixture in polyethylene covered pots with 45% transplantation success. Peroxidase isozymes (native PAGE) analysis helped to verify the variation in regenerated plants.     

Biotransformation of Externally Added Vanillin Related Compounds by Multiple Shoot Cultures of Vanilla planifolia L

The multiple shoots and callus cultures of Vanilla planifolia obtained from the nodal explant on MS medium supplemented with 6-benzylaminopurine (BAP) 2 mg l^?1 and α-naphthalene acetic acid (NAA) 2 mg l^?1 were maintained by regular subculturing every 30 days and also cultured liquid MS medium of the same hormonal combination. Shoots were transferred to the MS basal medium for rooting. Different explants along with vanilla pods and in vitro cultures were analyzed using HPLC for the presence of vanillin and related compounds. When the amount of these compounds was determined in explants and in in vitro cultures after precursor feeding and curing process, explants showed different profile after precursor feeding and after undergoing curing process. During further investigations we have applied a novel approach for curing in vitro tissues as done for vanilla beans. Curing of in vitro shoots resulted in a significant change in the aromatic compound profile.     

In vitro regeneration in Sarcostemma acidum (Roxb.) –an important medicinal plant of semi-arid ecosystem of Rajasthan, India

An efficient regeneration protocol for Sarcostemma acidum – an important medicinal plant has been established. Callus initiated from nodal explant on MS medium with 2.0 mg?L^?1 of NAA + additives. Callus initiated was subcultured on MS medium containing various concentrations of NAA or 2,4-D. Out of these combinations, MS medium +1.0 mg?L^?1 of NAA + additives was found to be effective for the multiplication of callus. Subculture was done after an interval of 20–22 days. For differentiation of callus BAP or Kinetin alone was found to be less effective. Maximum frequency of shoot regeneration recorded on MS medium +1.0 mg?L^?1 of BAP?+?0.5 mg?L^?1 of Kinetin and 0.1 mg?L^?1 of NAA + additives. The in vitro differentiated shoots were excised and inoculated on 1/4 strength MS medium +2.0 mg?L^?1 of IBA?+?0.02 % activated charcoal for in vitro rooting. Maximum response (90 %) was recorded on this medium. In vitro differentiated shoots were inoculated on autoclaved soilrite® after treatment with root inducing auxins. Ex vitro rooting in this plant species has been reported for the first time. Eighty five percent of the shoots rooted under ex vitro conditions. Both in vitro and ex vitro rooted plantlets were hardened in a green house.     

Identification of somaclonal variants in proliferating shoot cultures of Senecio cruentus cv. Tokyo Daruma

A protocol for in vitro propagation of cineraria (Senecio cruentus) was developed. The highest frequency of shoot proliferation was obtained from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with 2.0?mg L^?1 6-benzyladenine (BA) and 0.5?mg L^?1 ??-naphthalene acetic acid (NAA), with a mean number of 14 shoots per explant. A high concentration of BA (4.0?mg L^?1) and repeated subcultures resulted in hyperhydric shoots. Decreasing the BA concentration to 1.0?mg L^?1 in the culture medium eliminated hyperhydricity. The concentration of ammonium nitrate (NH₄NO₃) and temperature had marked effects on somaclonal variation. Variation was observed when the cultures were maintained at 15?°C but not at 25?°C. Variants with blue-colored leaves and stems were identified; whereas, normal plants maintained their green-colored leaves and stems. The highest frequency of variation (67.5?%), with a mean number of 3.0 variant shoots per explants, was obtained on shoot proliferation medium (MS?+?2.0?mg L^?1 BA and 0.5?mg L^?1 NAA) devoid of NH₄NO₃. The best rooting (100?%), with the highest number of roots per shoot (10.8) and the greatest root length (6.8?cm) was obtained on medium supplemented with 0.1?mg L^?1 NAA. In vitro-grown plantlets were successfully acclimatized in a greenhouse, and transferred to the field.     

Efficient plant regeneration of the endangered medicinal orchid, <Emphasis Type="Italic">Coelogyne cristata</Emphasis> using protocorm-like bodies

An efficient method of Coelogyne cristata mass propagation was developed using segment of protocorm-like bodies (PLBs) (3 mm² in size). It was observed that ½ MS medium showed to be more effective to induce shoots through PLBs segment. The explants when cultured on ½ MS media containing TDZ and CP showed relatively superior effect on shoot regeneration as compared to the media containing TDZ alone or in combination with BP. Addition of BP and CP to the medium containing NAA and BA combinations proved distinctly better for shoot multiplication than that of the medium with NAA and BA combinations alone. The highest percentage of explants producing shoots, with a maximum average of 8.1 per explant, was induced on the medium supplemented with 1.0 mg l^?1 NAA and 0.5 mg l^?1 BA with CP. Shoots produced an average of 15 roots per explant on ½ MS medium supplemented with 2.0 mg l^?1 IBA and BP. The 4 cm height plantlets with well-developed roots were successfully acclimatized. The results suggest that CP and BP can be used effectively to initiate shooting and rooting of Coelogyne cristata. Ploidy analysis of regenerated plants using flow cytometry revealed the same ploidy level (diploid). This efficient and reliable protocol could be useful for mass multiplication and germplasm conservation of the wild medicinal orchid.     

In vitro Regeneration of Gerbera jamesonii Bolus from Leaf and Petiole Explants

Regeneration of adventitious shoots from leaf and petiole pieces of Gerbera jamesonii has been obtained on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins and cytokinins. About 75’77 per cent of the calli from both types of the explants produced 12’15 shoots per callus with 3 mg l^?1 SAP. Auxins and kinetin, separately failed to produce shoots. The shoots regenerated on the callus induction medium (elM). The regenerated shoots multiplied with 1 mg l^?1 SAP, were rooted on MS medium containing 1mg l^-1 BAP + 0.1 mg l^-1 IAA. The plants obtained were transferred to pots and acclimatized with 60’70 per cent success.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.