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).     

High efficiency plant regeneration and genetic fidelity of regenerants by SCoT and ISSR markers in chickpea (Cicer arietinum L.)

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l^?1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l^?1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l^?1 BAP along with 0.05 mg l^?1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l^?1), GA₃ (1.0 mg l^?1) and BAP (1.0 mg l^?1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l^?1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation.

     

Enhanced Efficiency of In Vitro Rootstock Micro-propagation Using Silica-Based Nanoparticles and Plant Growth Regulators in Myrobalan 29C (Prunus cerasifera L.)

Different experiments were conducted to establish and optimize an efficient in vitro micropropagation protocol for Myrobalan 29C rootstocks. Disinfection of initial explants with AgNPs (2.5%) reduced the needed amount of NaClO (5.0%) by half. The highest rates of induced active buds were obtained in the DKW (90.63%), MS (86.67%), modified MS (82.22%), and WPM (78.15%) culture media supplemented with BAP (2.22 μmol L^?1)?+?GA₃ (2.88 μmol L^?1)?+?IBA (0.05 μmol L^?1)?+?Fe-EDDHA (228.72 μmol L^?1). The highest quality of the proliferated shoots (5.0) was also achieved using DKW medium. Inclusion of GA₃ (5.76 μmol L^?1), Fe-EDDHA (114.36–228.72 μmol L^?1), or BAP (2.22 μmol L^?1) were also able to enhance the rate of shoot multiplication. Compared to the agar-solidified culture system, the established shoots proliferated more efficiently when immersed by bioreactor in the liquid DKW culture medium on a regular basis. Exogenous application of silica-based nanoparticles (NPs) including the chemically synthesized silica NPs (TSiO₂ NPs, 1.0 ppm), rice husk derived biogenic silica NPs (RSiO₂ NPs, 10.0 ppm), or amine modified silica NPs (ASiO₂ NPs, 10.0 ppm) to the multiplication medium increased the number of regenerated lateral shoots by 520%, 360%, and 349%, respectively. Proliferated shoots with well-developed root system were obtained from the rooting medium supplemented with 19.68 μmol L^?1 IBA. Our results indicated that the rootstocks of Myrobalan 29C could be efficiently propagated under in vitro condition providing proper culture medium and optimal concentrations of additives and plant growth regulators were adopted.

     

Efficient regeneration for enhanced steviol glycosides production in Stevia rebaudiana (Bertoni)

An efficient method of regeneration for antidiabetic plant (Stevia rebaudiana) has been established for healthy biomass and main steviol glycosides (SGs) production, using different PGRs and agar concentrations. Higher callus induction (93.3%) was recorded when leaf explants were placed on an MS medium supplemented with 3.5 gL⁻¹ agar and 2.0 mgL⁻¹ 2,4-D. The addition of 7.0 gL⁻¹ agar and BA (1.0, 2.0 and 4.0 mgL⁻¹) significantly (P < 0.01) influences shooting response (100%). A maximum mean shoot length (13.03 cm) and 28 shoots per explant were observed on a medium containing 1.0 mgL⁻¹ BA. However, the maximum number of leaves (132.67) was encouraged by the addition of BA (1.0 mgL⁻¹) and Kin (1.0 mgL⁻¹). Lower agar (3.5 gL⁻¹), IAA (2.0 mgL⁻¹), and NAA (2.0 mgL⁻¹) concentrations significantly influence the rooting percent (100%), the mean root length (2.9 cm), and the number of roots per plantlet (26.3). These plantlets were successfully acclimatized in the soil. The BA (3.0 mgL⁻¹) in combination with Kin (3.0 mgL⁻¹) and 3.5 gL⁻¹ agar increases dulcoside-A content (Dul-A; 71.8 μg/g-DW) in shoots compared to control (50.81 μg/g-DW). Similar PGRs with 7.0 gL⁻¹ significantly increases the production of steviosides (Stev. 82.48 μg/g-DW). A higher rebaudioside-A content (Reb-A; 12.35 μg/g-DW) was observed in shoots that underwent the addition of BA (1.0 mgL⁻¹) and 7.0 gL⁻¹ agar than in control (07.39 μg/g-DW). Hereby, we developed an efficient and cost-effective method for regeneration and major SGs production, which could be helpful for future studies on this species.     

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.     

An Efficient Plant Regeneration System of <i>Hydrangea bretschneideri</i> Dipp via Stem Segments as Explants

Hydrangea bretschneideri Dipp is a highly popular ornamental plant for garden decoration. Genetic engineering technology has been successfully used in many plant species, but it is limited in Hydrangea. Here we established an efficient regeneration system by using stem segments as explants for the first time. In our study, the plant growth regulators (PGRs) were evaluated at the different regeneration processes, including axillary shoots regeneration and root induction. We found that the optimal concentration for axillary buds’ induction was 2.0 mgL⁻¹ 6-BA and 0.5 mgL⁻¹ 1 IAA, its highest induction rate was 70%. Moreover, the highest axillary shoots proliferation coefficient was 10.7 on the Murashige and Skoog (MS) medium with 2.0 mgL⁻¹ 6-benzyladenine (BA), 0.2 mgL⁻¹ indole-3-butyric acid (IBA), and 1.0 mgL⁻¹ gibberellin A3 (GA3). The highest frequency of root induction was 80.0 ± 0.06% by culturing the elongated shoots in 1/2 MS medium containing 0.1 mgL⁻¹ IBA. In summary, our study will provide an effective technology for large-scale propagation and important pathway for promoting the popularization and application of Hydrangea bretschneideri Dipp.     

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.     

In Vitro Propagation of Endangered Temperate Himalayan Medicinal Herb Picrorhiza kurroa Royle ex Benth using Leaf Explants and Nodal Segments

The present study describes the micropropagation of Picrorhiza kurroa, (commonly known as kutki) an endangered medicinal herb of the temperate Himalayas and a source of hepatoprotective picrosides. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segments and leaf tissue. For shoot regeneration, the hormone combinations kinetin (2.0 mg l^?1) and Kinetin + Indole-3-butyric acid (IBA) (2.0 mg l^?1 + 0.50 mg l^?1) with leaf explant was found superior. Interestingly, the basal MS medium gave 99.94 % response (direct proliferation) with nodal explant. The medium supplemented with IBA (1.0 mg ^?1) was found best for rooting of regenerated shoots. Nodal segments plated on the medium supplemented with TDZ + IBA (0.11 mg ^?1 + 0.50 mg ^?1) formed somatic embryos, however further regeneration could not be achieved. The in vitro raised plantlets were hardened and successfully established in the glass house conditions.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Soluble sugar,starch and phenolic status during rooting of easy- and difficult-to-root magnolia cultivars

The aim of the study was to determine the effect of indole-3-butyric acid (IBA) and exogenous sucrose concentrations on in vitro rooting, soluble sugar, starch and phenolic production, and ex vitro survival of four magnolia cultivars. There was a significant linear increase in rooting of most magnolia genotypes with an increase in IBA concentration in the medium from 1 to 6 mg L^?1. A further increase of IBA concentration to 10 mg L^?1 decreased (‘Elizabeth’, ‘Burgundy’) or had no effect on rooting frequency (‘Spectrum’). The effect of IBA on rooting of magnolia shoots was modified by sucrose supply. The three out of four magnolia cultivars showed the highest rooting efficiency in the presence of 6 mg L^?1 IBA and 30 g L^?1 of sucrose. Generally, decreasing and increasing the sucrose supply from 30 g L^?1 significantly lowered the rooting frequency. In ‘Yellow Bird’, sucrose at 40 g L^?1 totally blocked root formation. It has been found that the poor rooting ability of ‘Yellow Bird’ coincided with a low soluble sugar content, and high production of starch and phenolics in the shoot bases during the whole rooting period as compared to easy-to-root cultivars. After 5 weeks of the growth on IBA medium, rooted and unrooted shoots were transferred to ex vitro conditions. Both types of shoots showed a high survival and rooting rate (85.4–100%), but they differed in their growth activity and quality. Sucrose concentration in the rooting medium had a post-effect on ex vitro root formation and survival of magnolia plantlets. Ex vitro establishment (13.3%) of recalcitrant ‘Yellow Bird’ was obtained only when the shoots were taken from rooting medium containing the lowest level of sucrose (20 g L^?1).

     

In vitro regeneration using twin scales for restoration of critically endangered aquatic plant Crinum malabaricum Lekhak & Yadav: a promising source of galanthamine

This study intended to develop a significant in vitro regeneration protocol for sustainable propagation, conservation and re-establishment of critically endangered aquatic plant species Crinum malabaricum Lekhak & Yadav (Malabar river lily). This plant is the natural source of galanthamine, the drug to treat Alzheimer’s disease. We present a scientific understanding, emphasizing the use of twin scales (separated from the large parent bulb) in direct regeneration of new shoots and proliferation of bulblets assisted by nutrients supply. The meristematic region of the bulb plate, present between the scales was activated using cytokinins to produce shoots (maximum 12 shoots per twin scale) on full strength Murashige and Skoog’s (MS) medium augmented singularly with 2.0 mg L^?1 6-benzylaminopurine (BAP). Upon subculturing of shoots on diverse concentrations of plant growth regulators (BAP and IAA/NAA), BAP alone at 2.0 mg L^?1 was served optimum for the better proliferation of shoots (53 shoots). The regenerated shoots were rooted in vitro on half strength MS medium fortified with various types of auxins. Highest number of roots (11.6 within 4 weeks) and bulblets (after 3 months) resulted with 1.0 mg L^?1 Indole-3-butyric acid (IBA) under in vitro conditions. The rooted plants were hardened in the greenhouse and finally transferred to the natural stream with 83% survival rate. The SCoT (start codon targeted) and ISSR (inter simple sequence repeats) marker analysis of in vitro raised and mother plants confirmed the genetic stability of tissue cultured plants and the reliability of present protocol for C. malabaricum. It is the foremost report on in vitro regeneration and genetic fidelity analysis for restoration of this critically endangered aquatic plant using twin scale technique. The study could help in ex situ conservation, reintroduction and restoration of C. malabaricum population in its natural habitat.

     

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis.Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L⁻¹ 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L⁻¹ Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L⁻¹ each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.     

Technical Note: Phytoremediation of Hg and Cd from Industrial Effluents using an Aquatic Free Floating Macrophyte Azolla Pinnata

The level of heavy metal pollution in Singrauli, an industrial region in India, was assessed and the phytoremediation capacity of a small water fern, Azolla pinnata R.Br (Azollaceae), was observed to purify waters polluted by two heavy metals, i.e., mercury (Hg) and cadmium (Cd) under a microcosm condition. Azolla pinnata is endemic to India and is an abundant and easy-growing free-floating water fern usually found in the rice fields, polluted ponds, and reservoirs of India. The fern was grown in 24 40-L aquariums containing Hg²⁺ and Cd²⁺ ions each in concentrations of 0.5, 1.0, and 3.0 mgL^?1 during the course of this study. The study revealed an inhibition of Azolla pinnata growth by 27.0–33.9% with the highest in the presence of Hg (II) ions at 0.5 mgL^?1 in comparison to the control. After 13 days of the experiment, metal contents in the solution were decreased up to 70–94%. In the tissues of Azolla pinnata, the concentration of selected heavy metals during investigation was recorded between 310 and 740 mgKg^?1 dry mass, with the highest level found for Cd (II) treatment at 3.0 mgL^?1 containing a metal solution.     

High efficiency <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration from epicotyl explants of Ponkan Mandarin (C<Emphasis Type="Italic">itrus reticulata</Emphasis> Blanco)

Ponkan mandarin (Citrus reticulata Blanco) is one of the most important commercial cultivars of mandarin orange in China. This study reports an improved and efficient protocol for in vitro plant regeneration of Ponkan mandarin. Epicotyl segments, which were cut longitudinally into two halves, were used as explants. The shoot regeneration frequency was significantly increased by longitudinal cutting. A 100% shoot regeneration frequency and 13.2 shoots per explant were obtained when cultures were maintained in darkness for 20 d before being transferred to light conditions, with bud induction by indirect organogenesis. A 72.5% shoot regeneration frequency and 7.8 shoots per explant were obtained when explants were incubated under a 16-h light photoperiod continuously with buds differentiating directly from the cutting wound surface. The optimal medium for shoot formation was Murashige and Tucker basal medium supplemented with 2 mgL⁻¹ BA and 30 gL⁻¹ sucrose both under light conditions. The addition of the auxin NAA reduced the frequency of regeneration. A “filter-paper bridge” technique was used for rooting in this study. The basal portion of regenerated shoots was dipped into 1,000 mgL⁻¹ IBA solution for 15 min before placement on a filter-paper bridge that was maintained in 1/2 MS liquid medium supplemented with 10 gL⁻¹ sucrose. Eighty percent of the shoots rooted, and an average of 2.0 roots per shoot were achieved. Survival rate through acclimatization was 100%.     

Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Spilanthes acmella</Emphasis>

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm⁻³ 6-benzyladenine (BA) and 0.1 mg dm⁻³ α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm⁻³ BA and 1.0 mg dm⁻³ IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm⁻³ IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.     

Thidiazuron-induced Shoot Multiplication and Plant Regeneration in Bamboo (Dendrocalamus strictus Nees)

Thidiazuron (TDZ) stimulated shoot proliferation from different seedling explants (i.e., shoot, basal node, node and apical segment) of bamboo (Dendrocalamus strictus) when incorporated in half-strength Murashige and Skoog (MS) medium having 2% (w/v) sucrose. All the concentrations of TDZ (0.01 to 1.0 mg l^?1) tried were effective in shoot proliferation. Maximum shoots (14.8 ± 1.0) were obtained from the shoot explants cultured in 0.5 mg l^?1 TDZ supplemented halfstrength MS liquid medium for 21 days and subsequently transferred to the same medium devoid of TDZ. The longer culture period (i.e. 28 and 35 days) in TDZ medium caused reduction in shoot proliferation. The shoots regenerated with lower concentrations of TDZ treatment (i.e. 0.01 to 0.1 mg l^?1) rooted in half-strength MS liquid medium. The shoots formed with 0.5 mg l^?1 TDZ treatment did not root in basal medium and required auxin supplementation in the medium for rooting and about 55% shoots produced roots in 1.0 mg l^?1 IBA supplemented medium. The shoots formed with 1.0 mg l^?1 TDZ did not root even after auxin treatment. The well rooted shoots transplanted to plastic pots filled with sand and garden soil (1:1) mixture showed 98% establishment.     

In vitro study on the effect of cytokines and auxins addition to growth medium on the micropropagation and rooting of Paulownia species (Paulownia hybrid and Paulownia tomentosa)

This study represents an efficient preliminary protocol for in vitro mass production of two Paulownia species (Paulownia hybrid and Paulownia tomentosa) seedlings by using seed explant. Different concentrations of benzyladenine (BA) or Kinetin (Kin) (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L) were tested during multiplication stage. The number of shoots/explants was significantly increased with increasing either BA or Kin concentration; however, the shoot length significantly decreased. Data show that media fortified by BA (10 mg/L) combined with indole butyric acid (IBA) at 1.0 or 1.5 mg/L recorded the highest number of shoots/explant (9.13 and 9.25, respectively). After six weeks during the multiplication stage, data cleared that media fortified by benzyladenine (10 mg/L) combined with IBA at 0.5 mg/L recorded the highest shoot length (3.23 cm). The inclusion of indole butyric acid (IBA) or naphthalene acetic acid (NAA) at 1.0–1.5 mg/L to the medium significantly increased the number of roots/plantlets and the highest root length. The results indicated that IBA supplementation was more effective than NAA for in vitro rooting of both Paulownia species. The best treatment for multiplication was 10 mg/L and 8.0–10 mg/L BA for P. hybrid and P. tomentosa, respectively. Peat moss and sand (1:1, v/v) or peat moss and sand (1:2, v/v) were investigated as soil mixture during the adaptation stage. The results referred that Paulownia species plantlets were successfully survived (100 %) in soil mixture contained peat moss: sand (1:2, v/v). This mixture recorded the highest values of plantlet height and number of leaves/plantlets.     

Axillary shoot proliferation and growth of Populus deltoides shoot cultures

Shoot cultures of four genotypes of Populus deltoides Bartr. ex Marsh. were established from adventitious shoots regenerated from internodal stem explants. Stable shoot cultures for all four genotypes were maintained in a continuous culture regime for over one year. The stable shoot cultures were used as explants to investigate the effects of zeatin concentration and genotype on axillary shoot production and growth. The concentration of zeatin significantly affected the production of axillary shoots, with 1.0 mgL^–1 zeatin producing the greatest number of shoots (31.0 shoots per culture vessel) while 0.25 mgL^–1 zeatin produced the greatest growth (5.9 mg per axillary shoot) when measured by dry weight accumulation per shoot. Genotypic differences were significant in the production and growth of axillary shoots.Abbreviations DKW Driver and Kuniyuki Walnut medium - PAR Photosynthetically Active Radiation Journal Series No. 9111, Agricultural Research Division, University of Nebraska     

Rapid micropropagation of Woodfordia fruticosa (L.) Kurz (Lythraceae), a rare medicinal plant

A rapid propagation method comprising initiation of in vitro shoot tip culture from field-grown flowering plants and reculture of the nodal segments of regenerated shoots in Schenk and Hildebrandt (1972) medium was developed for Woodfordia fruticosa (L.) Kurz., a rare medicinal shrub. A medium supplement of 6-benzylaminopurine (0.2 mg.l^–1) induced high frequency (88%) development of axillary shoot buds (3.2) in 4–5 weeks. Subculture of the explants with multiple new shoots in fresh medium for 30 days yielded an even larger number (9.7) of shoots. Highest multiplication (26–35 shoots) was recorded when using culture initiation media with 0.5 mg.l^–1 each of BAP and NAA followed by subculture in 0.2 mg.l^–1 BAP. The shoot multiplication rate was further accelerated by reculturing 0.4–0.6 cm nodal segments of regenerated shoots in media with 1.0 mg.l^–1 BAP. Shoot cuttings (3.5–7.0 cm) were rooted in 0.2 mg.l^–1 IAA. Regenerated plants displayed uniform morphological, growth and flowering characteristics.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - SH Schenk and Hildebrandt (1972) medium     

<Emphasis Type="Italic">In vitro</Emphasis> propagation,micromorphological studies and <Emphasis Type="Italic">ex vitro</Emphasis> rooting of cannon ball tree (<Emphasis Type="Italic">Couroupita guianensis</Emphasis> aubl.): a multipurpose threatened species

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l^?1 indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l^?1 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l^?1 each of BAP and Kinetin (Kin) + 0.5 mg l^?1 α-naphthalene acetic acid (NAA) with additives (50 mg l^?1 of ascorbic acid and 25 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l^?1 IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l^?1 of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7–8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.