Direct shoot regeneration from intact leaves of Arnebia euchroma (Royle) Johnston using thidiazuron

The present study highlights the importance of preculture time and concentration of TDZ (thidiazuron) for direct regeneration from in vitro leaves (attached to shoots) in Arnebia euchroma. Shoot buds proliferated to form multiple shoots on MS medium (Murashige and Skoog medium) with 5.0 μM Kn. Different additives viz. ascorbic acid, PVP (polyvinylpyrrolidone), PVPP (polyvinylpolypyrrolidone) or activated charcoal (50, 100 and 250 mg/l each) were used to check the phenolic exudations. Direct shoot regeneration was obtained when shoots were initially precultured for 40 days on medium with a higher concentration of TDZ (20.0 μM) and then transferred to a lower concentration (5.0 μM TDZ). The identity of shoot buds was confirmed by histological studies. Regenerated shoots were cultured for 30 days on medium containing Kn (5.0 μM) for proliferation and then transferred to IBA (0.25 μM)‐containing medium for rooting. Rooted plantlets were transferred to greenhouse with 45–50% survival.     

Micropropagation through excised root culture of Clitoria ternatea and comparison between in vitro–regenerated plants and seedlings

An efficient protocol has been developed for high‐frequency shoot regeneration and plant establishment of Clitoria ternatea – a potential medicinal legume. Adventitious shoots were regenerated from young excised root segments of aseptic seedlings on Murashige and Skoog (MS) medium supplemented with various concentrations of 6‐benzyladenine (BA), kinetin, α‐naphthalene acetic acid (NAA) or 2,4‐dichlorophenoxy acetic acid (2,4‐D) either singly or in various combinations. The highest frequency (100%) of shoot regeneration and maximum number (16.4 ± 0.24) of shoots per explant was obtained on MS medium supplemented with 20 μm BA and 2.0 μm NAA. Organogenic calli were produced on a medium containing 2,4‐D (10 or 20 μm ) and BA (5.0 μm ). The calli were differentiated into multiple shoots on MS medium supplemented with 2.5–10 μm BA and 2.0 μm NAA. The microshoots were rooted on half‐strength MS medium supplemented with 5.0 μm indole‐3‐butyric acid and transplanted successfully in field conditions. After 12 months of transfer to ex vitro conditions, the performance of micropropagated plants were evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo–grown plants of the same age. The sodium dodecyl sulphate polyacrylamide gel electrophoresis protein profile was same between regenerated and naturally growing shoots. Total soluble protein in aerial part as well as in seeds of in vitro–regenerated and in vivo–grown plants was almost the same. The mitotic study showed normal chromosomal movement and numbers (2x = 16).     

<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.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Plantlet regeneration from fascicular buds of seedling shoot apices of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> Sarg

Shoot apices of Pinus roxburghii Sarg were cultured on Murashige and Skoog’s medium (MS) supplemented with cytokinins [6-benzyladenine (BA), kinetin and N-benzyl-9-(2-tetrahydropyranyl) adenine (BPA)] alone and in combination with auxin, α-napthaleneacetic acid (NAA). Of the three cytokinins tested at varying concentrations, medium supplemented with 10 μM BA was found optimal in respect of explant responsiveness (97.22 %) and average number of buds induced per explant (7.42). The concentration of cytokinins in the induction medium had a profound effect on rate of elongation of induced buds on MS basal medium containing 0.5 % activated charcoal. Further, shoots induced on lower concentrations of BA increased up to 2.4 times in length in 4 weeks. Decapitation of the explant enhanced the rate of axillary bud elongation. Proliferating shoot cultures were established by sub-culturing the axillary shoots on MS supplemented with 10 μM BA. Shoots 2–3 cm in length were suitable for culturing as more buds were induced on them compared to longer or shorter shoots. Root primordia were induced on 70.83 % shoots when transferred to 1/2 MS medium supplemented with 5.0 μM NAA. Elongation of root primordia (60 %) was achieved in liquid 1/2 MS basal medium. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 20–22 weeks.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 μM BA and 1.59 μM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 μM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 μM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.     

Regeneration of <Emphasis Type="Italic">Aloe arborescens</Emphasis> via somatic organogenesis from young inflorescences

A system for in vitro regeneration of Aloe arborescens was developed using young inflorescences as explants. Different phytohormone combinations of N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ), benzyladenine (BA), 6-(γ,γ-dimethylallyl-amino)purine riboside (2iPR), zeatin ribozide (ZR), N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and kinetin (K), with or without ancymidol, were examined in order to induce plant regeneration. Efficient shoot regeneration was initiated on Murashige and Skoog (MS) medium supplemented with BA or TDZ. MS medium enriched with 19.6, 22.2 μM BA and 3.92 μM ancymidol (MSBA5/1 medium), promoted organogenesis enabling 87.3% of the explants to regenerate 6.04 ± 1.79 shoots/explant. Subsequent shoot elongation and plant regeneration were strongly affected by the medium composition used for shoot induction. Optimal elongation (three to four shoots per explant) was obtained when shoots, initiated on MSBA5/1 medium, were subsequently transferred onto MS containing only 4.4 μM BA. Rooting was performed on MS media lacking growth regulators. Histological analysis revealed that the initiated shoots originated from the receptacle tissue surrounding the residual vascular tissue of the flower buds.     

Rapid In Vitro Propagation of Eclipta alba (L) Hassk by Shoot Tip Culture

An efficient and rapid tissue culture system employing shoot tip explants has been developed for Eclipta alba (L) Hassk, an important medicinal plant of the family Asteraceae. The highest shoot regeneration frequency (95%) as well as the maximum number (32.2 ± 0.4) of shoots was recorded on MS medium amended with BA (5 μM) and NAA (0.5 μM). The regenerated shoots rooted best on MS medium supplemented with 0.2 μM IBA. The in vitro developed plantlets were acclimatized successfully with 100 % survival.     

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.     

Morphogenic response of the alginate-encapsulated axillary buds from in vitro shoot cultures of six mulberries

Axillary buds obtained from in vitro shoot cultures of six mulberries (Morus alba L., M. australis Poir., M. bombycis Koidz., M. cathyana Hemsl., M. latifolia Poir., and M. nigra L.) were encapsulated in calcium alginate hydrogel containing Murashige and Skoog (1962) nutrients (MS) and 4.4 μM benzyladenine (BA). Morphogenic response of encapsulated buds to various planting media such as MS medium + 4.4 μM BA, MS basal medium, soilrite mix + half-strength MS medium, garden soil + half-strength MS medium, soilrite mix + tap water and garden soil + tap water was evaluated. Encapsulated buds of M. alba, M. bombycis, M. latifolia and M. nigra exhibited shoot development in each of the six media tested whereas that of M. australis and M. cathyana responded only to the first four media. Analysis of variance revealed that the planting medium exhibited the greatest influence on shoot development. Of the six planting media evaluated, shoot development was highest in MS medium containing 4.4 μM BA and lowest in garden soil moistened with water. Of the six Morus species studied, one-step regeneration, i.e. both shoot and root formation, was recorded in M. alba, M. bombycis and M. latifolia. Rooted shoots were retrieved from encapsulated buds of these species on all planting media tested except the one that contained BA. Root development was significantly affected by the planting medium and the plant species with planting medium contributing the maximum amount (82%) of the total variation observed. Of the five planting media tested, the percentage of root development was highest in MS basal medium. Of the six Morus species studied, the best shoot and root development was observed in M. alba. Encapsulated buds of M. bombycis, M. latifolia and M. nigra stored for 90 days and those of M. alba, M. australis and M. cathyana for 60 days at 4 °C still regenerated shoots. Plants regenerated from the encapsulated buds were hardened off and transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient Regeneration of Tetraploid Isatis indigotica Plants via Adventitious Organogenesis from Hypocotyl Explants

An efficient in vitro plant regeneration system via hypocotyl segments of tetraploid Isatis indigotica Fort. was established. Murashige and Skoog's (MS) and Gamborg's (GB₅) media were found to be superior to White medium for promoting shoot regeneration. The highest shoot regeneration (92 %) was achieved from hypocotyls cultured on MS medium containing 8.9 M benzyladenine (BA) and 2.7 M naphthaleneacetic acid (NAA), with an average of 4.2 shoots developed per explant. Plant regeneration was also improved when the explants were cultured in MS basal medium containing 3 % (m/v) sucrose and grown under a 12-h photoperiod. The developed shoots were well rooted in a half-strength MS medium supplemented with 0.5 M indole-3-butyric acid (IBA) and were morphologically normal after transfer to soil.     

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

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

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.     

Enhanced multiplication and improved <Emphasis Type="Italic">ex vitro</Emphasis> acclimatization of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>

The proposed work describes a protocol for high-frequency in vitro regeneration through nodal segments and shoot tips in Decalepsis arayalpathra, a critically endangered medicinal liana of the Western Ghats. Nodal segments were more responsive than shoot tips in terms of shoot proliferation. Murashige and Skoog’s (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA) was optimum for shoot initiation through both the explants. Among different combinations of plant growth regulators and growth additive screened, MS medium added with 5.0 μM BA + 0.5 μM indole-3-acetic acid + 20.0 μM adenine sulphate effectuated the highest response: 11.8 shoots per nodal segment and 5.5 shoots per shoot tip with mean shoot length of 9.2 and 4.8 cm, respectively. Half-strength MS medium with 2.5 μM α-naphthalene acetic acid was optimum for in vitro root induction. The plantlets with the well developed shoot and root were acclimatized in Soilrite? with 92 % survival rate in the field conditions. During acclimatization, chlorophyll content, net photosynthetic rate, stomatal conductance, and transpiration rate were gradually changed in dependence of formation of new leaves. Further, the changes in activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) as well as activity of carbonic anhydrase were also observed: a continuous rise in SOD activity, but a rise and fall in the activities of CAT, APX, and GR were also noticed. Maximum fresh mass (3.1 g plant^-1), dry mass (0.35 g plant^-1) of roots and 2-hydroxy-4-methoxybenzaldehyde content of 9.22 μg cm^-3(root extract) were recorded after 8 weeks of acclimatization.     

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.     

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytokinins,donor plants and time in culture affect shoot regenerative capacity of American elm leaves

Cytokinins, donor plants and their time in vitro as well as basal media were investigated for their influence on shoot regenerative capacity of American elm (Ulmus americana L.) leaves. Leaves excised from six 2-year-old seedlings formed adventitious shoots when placed on Driver and Kuniyuki Walnut (DKW) medium supplemented with 7.5, 15 or 22.5 M of benzyladenine (BA) or thidiazuron (TDZ). Thidiazuron induced significantly higher regeneration percentages on elm leaves than BA, regardless of concentration used. Donor plant also affected the efficiency of shoot regeneration, with certain seedlings having 1.5 to 7 times more explants forming shoots as compared to other seedlings tested. By subculture 15, the average number of shoots per regenerating explant increased at least 3-fold for leaves on media with BA or TDZ for the one donor plant that survived continued subculturing. Leaf explants from donor plants with the highest regenerative capacity had a higher percentage of shoot formation on DKW than MS medium. Explants from productive donor plants should be placed on DKW medium supplemented with TDZ to improve shoot regeneration efficiency from American elm leaves.