Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

Summary Chile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N⁶-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N⁶-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.     

Influence of nitrate concentration at the root surface on yield and nitrate uptake of kohlrabi (Brassica oleracea gongyloides L.) and spinach (Spinacia oleracea L.)

The objective of this study was to determine if plant roots have to take up nitrate at their maximum rate for achieving maximum yield. This was investigated in a flowing-solution system which kept nutrient concentrations at constant levels. Nitrate concentrations were maintained in the range 20 to 1000 μM. Maximum uptake rate for both species was obtained at 100 μM. Concentrations below 100 μM resulted in decreases in uptake rate per cm root (inflow) for both spinach and kohlrabi by 1/3 and 2/3, respectively. However, only with kohlrabi this caused a reduction in N uptake and yield. Thus indicating that this crop has to take up nitrate at the maximum inflow. Spinach, however, compensated for lower inflows by enhancing its root absorbing surface with more and longer roots hairs. Both species increased their root length by 1/3 at low nitrate concentrations.     

Effect of bicarbonate on root growth and accumulation of organic acids in Zn-inefficient and Zn-efficient rice cultivars (Oryza sativa L.)

Bicarbonate has been regarded as a major factor for inducing Zn deficiency in lowland rice, but the mechanisms responsible for this effect are not yet fully understood. The objective of the present study was to test whether early effects of bicarbonate (HCO₃ ⁻)are inhibition of root growth due to the accumulation of organic acids induced by HCO₃ ⁻. Solution culture experiments were conducted using two rice cultivars differing in susceptibility to Zn deficiency, and four bicarbonate concentrations (0, 5, 10, 20 mM). Bicarbonate (5–20 mM) strongly inhibited root growth of the Zn-inefficient cultivar within 4 days of treatments. In contrast, root growth of the Zn-efficient cultivar was slightly stimulated with bicarbonate at 5–10 mM and not affected at 20 mM. The inhibitory effect of bicarbonate on root growth in the Zn-inefficient cultivar was mainly that of impairment of new root initiation rather than suppression of elongation of individual roots. Bicarbonate (5–20 mM) increased the concentrations of malate, succinate and citrate in the roots of both cultivars, but to a greater extent for the Zn-inefficient than for the Zn-efficient cultivars. The results suggest that the impairment of root growth was likely to be the initial action of bicarbonate in inducing Zn deficiency in lowland rice, and the inhibitory effect of bicarbonate on root growth of the Zn-inefficient cultivar might result from high accumulation and an insufficient compartmentation of organic acids in the root cells.     

Fate of nitrate during initial nitrate utilization by nitrogen-depleted dwarf bean

The fate of nitrate and nitrogen-15 was followed during the apparent induction phase (6h) for nitrate uptake by N-depleted dwarf bean (Phaseolus vulgaris L. ev. Witte Krombek). Experiments were done with intact plants and with detached root systems. Qualitatively and quantitatively, xylem exudation from detached roots was a bad estimate of the export of NO^?₃ or NO^?₃-¹⁵N from roots of intact plants. In vivo nitrate reductase activity (NRA) agreed well with in situ reduction, calculated as the difference between uptake and accumulation in whole plants, provided NRA was assayed with merely endogenous nitrate as substrate (‘actual’ NRA). The majority (75%) of the entering nitrate remained unmetabolized. Both nitrate reduction and nitrate accumulation occurred predominantly in the root system. Some (< 25%) of the root-reduced nitrate-N was translocated to the shoot. Nitrate uptake occurred against the concentration gradient between medium and root cells, and probably against the gradient of the electro-chemical potential of nitrate. Part of the energy expended for NO^?₃ absorption came from the tops, since decapitation and ringing at the stem base restricted nitrate uptake.     

The role of sucrose and nitrogen in adventitious root formation on cultured rose shoots

Cultured shoots ofRosa ‘Improved Blaze’ were used to determine the effects of sucrose and inorganic nitrogen on adventitious root formation. Shoots grown in media containing high sucrose concentrations (146.07–262.93 mM) produced more and longer roots than those grown in media containing 0–87.64 mM sucrose. This response to sucrose was related to the metabolism of sucrose rather than its osmotic properties since the use of mannitol and 3-O-methyl-α-D-glucopyranoside as osmotic substitutes did not reproduce the effect on rooting. The number and length of roots increased when the shoots were grown in media with the nitrogen concentration of the Murashige-Skoog (MS) salt formulation reduced from 60 to 7.5 mM. Neither nitrate (NO ₃ ⁻ ) nor ammonium (NH ₄ ⁺ ) alone at any of the concentrations tested had the effect on rooting that both had together in the ratio of the MS salt formulation. When the sucrose and nitrogen concentrations were both varied, the greatest rate of root initiation occurred on shoots grown in media with a high sucrose to nitrogen concentration ratio.     

Effect of boron supply on nitrate concentration and its reduction in roots and leaves of tobacco plants

Shoot and root mass of tobacco plants treated with only 0.05 μM boron was decreased by 25 and 50 %, respectively, when compared to plants sufficiently supplied with B (2 and 5 μM). Leaf B content of 0.05 μM B-treated plants decreased (about 80–90 %) when compared to 2 μM B treated plants; this drop of B content were not as marked (about 25–45 %) in roots. Leaf and root nitrate contents in B-deficient plants were 45–60 % and 35–45 % lower, respectively, than those from 2 and 5 μM B treated plants. It is suggested that B deficiency might decrease nitrate uptake rather than nitrate reductase activity in tobacco plants.     

Accelerated Nitrate Uptake in Wheat Seedlings: Effects of Ammonium and Nitrite Pretreatments and of 6-Methylpurine and Puromycin

The experiments reported herein had two objectives. One was to determine if the slow rate of nitrate uptake which occurs upon initial exposure of nitrogen-depleted wheat (Triticum vulgare cv. Knox) plants to nitrate was the result of insufficient reduced nitrogen. The second was to determine the impact of restrictions in ribonucleic acid or protein synthesis on both nitrate uptake and nitrate reduction. Pretreatments of 14-day-old seedlings for a few hours in ammonium or nitrite did not result in an enhancement of the initial slow rate of nitrate uptake. Growth for two additional weeks in ammonium also failed to eliminate the induction period. The evidence indicates that the presence of nitrate, rather than a product of its reduction, was required to initiate development of the accelerated rate of nitrate uptake. Puromycin (400 μg ml^?1) and 6-methylpurine (0.5 mM) prevented development of the accelerated phase of nitrate uptake. With both compounds, the relative restriction of nitrate uptake was greater than that of nitrate reduction as revealed by incorporation of ¹⁵N from labeled nitrate into reduced forms. The proportion of reduction which occurred in the root system under the imposed treatments could not be delineated precisely, preventing an unequivocal determination of the extent to which the two processes are coupled in the root system. The data nevertheless indicate nitrate reduction was closely associated with nitrate uptake. Accumulation of nitrate in the shoots was markedly restricted in presence of 6 methylpurine. This effect most likely was a result of a severe restriction in the translocation of nitrate into the xylem, rather than an increase in the reduction rate in the shoots.     

Differences in uptake kinetics of ammonium and nitrate in legumes and cereals

Influx isotherms were obtained for nitrate and ammonium from three legumes, Cajanus cajan (L.) Millsp., Cicer arietinum L. and Arachis hypogaea L. and three cereals, Sorghum bicolor (L.) Moench., Pennisetum glaucum L. and Zea mays L. The transition in influx isotherms for both nitrogen sources was found to be within the concentration range (0.05–2.5 mM) tested. There were significant differences in Km and Vmax for ammonium between legumes and cereals. The difference in the kinetic properties for nitrate uptake between the two groups of plants only became apparent at the higher concentration tested. Legumes translocated absorbed nitrate and ammonium to shoots more rapidly than cereals. Results show that there are significant differences in uptake and translocation of ammonium and nitrate between legumes and cereals.     

Nitrate and ammonium absorption by plants growing at a sufficient or insufficient level of phosphorus in nutrient solutions

Summary Absorption of nitrate and ammonium was studied in water culture experiments with 4 to 6 weeks old plants of barley (Hordeum vulgare L.), buckwheat (Fagopyrum esculentum L. Moench) and rape (Brassica napus L.). The plants were grown in a complete nutrient solution with nitrate (5.7±0.2 mM) or nitrate (5.6±0.2 mM) + ammonium (0.04±0.02 mM). The pH of the nutrient solution was kept at 5.0 using a pH-stat. It was found that phosphorus deficiency reduced the rate of nitrate uptake by 58±3% when nitrate was the sole N source and by 83±1% when both nitrate and ammonium were present. The reduction occurred even before growth was significantly impeded by P deficiency. The inhibition of the uptake of ammonium was less,i.e. ammonium constituted 10±1% of the total N uptake in the P sufficient plants and 30±5% in the P deficient plants. The reduction of nitrate absorption greatly decreased the difference between the uptake of anions and cations. It is suggested that P deficiency reduced the assimilation of NO ₃ ^– into the proteins, which might cause a negative feedback on NO ₃ ^– influx and/or stimulate NO ₃ ^– efflux.     

The physiological basis of increased biomass partitioning to roots upon nitrogen deprivation in young clonal tea (Camellia sinensis (L.) O. Kuntz)

Deprivation of nitrogen (N) increases assimilate partitioning towards roots at the expense of that to shoots. This study was done to determine the physiological basis of increased root growth of tea (sCammellia sinensis L.) under N shortage. Nine-month-old clonal tea (clone TRI2025) was grown in quartz sand under naturally lit glasshouse conditions. Three levels of N (0, 3.75 and 7.5 mM N) were incorporated in to the nutrient solution and applied daily. Plant growth, photosynthesis, root respiration and plant N contents were measured at 10-day intervals over a 45-day period. Root dry weight showed a sharp increase during the first 15 days after the plants were transferred to 0 mM N, whereas no such increase was shown in plants transferred to 7.5 mM N. In contrast, shoot dry weight increased at 7.5 mM N and was significantly greater than at 0 mM N, where no increase was observed. Due to the above changes, root weight ratio increased and leaf weight ratio decreased during the first 15 days of N deprivation. Leaf photosynthetic rates did not vary between N levels during the initial 15-day period. Thereafter, photosynthetic rates were greater at 7.5 mM and 3.75 mM N than at 0 mM N. Root respiration rate decreased at 0 mM N, whereas it increased at 3.75 and 7.5 mM N, probably because of the greater respiratory cost for nitrate uptake. Root respiratory costs associated with maintenance (R _m) and nitrate uptake (R _u) were calculated to investigate whether the sharp increase of root growth observed upon nitrogen deprivation was solely due to the reduced respiratory costs for nitrate uptake. The estimated values for R _m and R _u were 3.241 × 10^–4 mol CO₂ g^–1 (root dry matter) s^–1 and 0.64 mol CO₂ (mol N)^–1, respectively. Calculations showed that decreased respiratory costs for nitrate uptake could not solely account for the significant increase of root biomass upon N deprivation. Therefore, it is concluded that a significant shift in assimilate partitioning towards roots occurs immediately following N deprivation in tea.     

Effect of bicarbonate and root zone temperature on uptake of Zn,Fe, Mn and Cu by different rice cultivars (Oryza sativa L.) grown in calcareous soil

Pot experiments were conducted with a calcareous soil (Inceptisol) to elucidate the effects of bicarbonate (0 and 20 mM) and root zone temperature (15° and 25°C) on the uptake of Zn, Fe, Mn and Cu by "Zn-efficient" and "Zn-inefficient" rice cultivars. Bicarbonate decreased concentrations and total uptake of Zn in shoots of "Zn-inefficient" cultivars, especially of IR 26 at 25°C, but not in Zn-efficient cultivars. Bicarbonate decreased concentrations and uptake of Fe in shoots of Zn inefficient cultivars, particularly in IR 26. Concentrations and total uptake of Mn were lower in bicarbonate treatment in the Zn-inefficient cultivars at 15°C, and in all cultivars at 25°C. However, concentration and uptake of Cu were not affected by bicarbonate in all cultivars. Compared to the 25°C root zone temperature, the concentrations and total uptake of both Zn and Cu in shoots at 15°C were lower in Zn-inefficient than in the Zn-efficient cultivars. The results indicate that Zn-efficiency in rice is causally related to high tolerance of plant to elavated bicarbonate concentrations in soil solution.     

The effects of salinity on growth,nodulation and nitrogen fixation ofCasuarina equisetifolia

The growth, nodulation and nitrogen fixation ofCasuarina equisetifolia were compared at six levels (0–500mM NaCl) of salinity in sand culture. Dry weight of nodules, shoots and roots and N content of shoots increased at intermediate levels of salinity (50–100 mM) but decreased at 500 mM NaCl. Nodulation occurred at all NaCl levels, but at 500mM NaCl level, the nodule dry weight declined by 50% from the control. Increasing NaCl concentration of up to 200mM had little effect on the N₂-fixation rate, but at 500mM NaCl level the rate decreased to 40% of the control value.     

Large-scale production of Anogeissus pendula and A. Latifolia by micropropagation

Summary Success has been achieved in developing a complete protocol for mass propagation of Anogeissus pendula and A. latifolia, two important forest species found in India. Seeds cultured on plant growth regulator-free, semisolid Murashige and Skoog (MS) medium germinated within 5–6 wk and formed 4–6-cm long shoots. The shoots multiplied on MS+4.4 μM benzyladenine (BA)+5.7 μM indoleacetic acid (IAA) + casein hydrolysate (100 mgl⁻¹) + ascorbic acid (50 mgl⁻¹) + sucrose (3%) + agar (0.8%). A majority of the genotypes rooted with more than 90% efficiency when 5–6 cm individual shoots were cultured on 1/2MS (only major salts reduced to half strength)+2.3 μM IAA+2.5 μM indolebutyric acid (IBA) + sucrose (3%)+agar (0.8%) for 15 d. Those 10% (approx.) genotypes that did not root well on the above medium could be rooted with ease by increasing the concentration of IAA in the rooting media from 2.3 to 5.7 μM. The in vitro-raised plants were successfully transferred to the soil with a success rate of over 85%. Using this protocol, over 560 000 tissue-cultured plants of these two species have been produced and dispatched to various state forest departments for field trials and routine plantations.     

Responses of nitrate assimilation and N translocation in tomato (Lycopersicon esculentum Mill) to reduced ambient air humidity

The impact of low humidity in ambient air on water relations,nitrate uptake, and translocation of recently absorbed nitrogen,was investigated in 5-week-old tomato (Lycopersicon esculentumMill cv. Ailsa Craig) plants grown hydroponically in a completenutrient solution. Plants were subjected to dry air (relativehumidity 2–4% for 6 h. The transpiration rate increasedseveral-fold and the shoot water content decreased by almost20%, whereas root water content was unaffected. No effect onin vitro nitrate reductase (NR) activity was detected when usingan EDTA-contraining assay buffer. Replacement of EDTA with Mg²⁺revealed a significant decline in shoot NR activity, which suggestsphosphorylation of the enzyme during the stress treatment. Plantswere grown in a split-root system, in which one root half wasfed ¹⁵N-nitrate during the treatment, in order to determinenitrate uptake and translocation of recently absorbed nitrogenin the plants. Uptake of nitrate was substantially inhibited,but the proportion of absorbed ¹⁵N that was translocated tothe shoots was only slightly affected. In untreated plants,71% of the ¹⁵N recovered in roots had been retranslocated fromthe shoots, whereas in plants subjected to stress the deliveryof ¹⁵N from shoots to roots appeared to be completely inhibited.The data show that lowered humidity in air has significant effectson both uptake of nitrate as well as translocation of nitrogenwithin the plants. Some of these effects appear to be commonwith those observed in plants subjected to reduced water potentialsin the root environment and point to the possibility of theshoot water relations being highly influential on nitrogen uptakeand translocation. Key words: Air humidity, nitrate assimilation, nitrate reductase activity, nitrogen translocation, tomato, water stress     

Critical evaluation of thein situ nitrate reductase assay

Anin situ method, derived from anin vivo method, was used to determine nitrate reductase activity (NRA) in:i) excised barley and corn shoots and excised soybean leaves during a N-depletion experiment and; ii) roots and shoots of N-depleted barley and corn seedlings during induction of nitrate, reductase (NR). Nitrate reduction, calculated from thesein situ RNA measurements, was compared with estimates of each organ's nitrate reduction in light aerobic conditions from NO ₃ ⁻ consumption and a¹⁵N model (Gojonet al., 1986b). Thein situ RNA of roots strongly underestimated their¹⁵NO ₃ ⁻ reduction. In contrast, in barley and corn shoots and in the first trifoliolate leaves from 26-day-old, soybean, thein situ NRA assay gave a fair approximation of the true NO ₃ ⁻ reduction rate (relative differences ranging from −14 to +32%). In young soybean leaves (from 20-day-old plants), however, thein situ NRA strongly underestimated the actual NO ₃ ⁻ reduction. The physiological significance of thein situ NRA assay in shoots and roots, and its value for field studies are discussed from these results.     

The toxicity of cadmium to barley plants as affected by complex formation with humic acid

An ‘alternating solution’ culture method was used to study the effects of chloride ions and humic acid (HA) on the uptake of cadmium by barley plants. The plants were transferred periodically between a nutrient solution and a test solution containing one of four levels of HA (0, 190, 569 or 1710 μg cm⁻³) and one of five levels of Cd (0, 0.5, 1.0, 2.5 or 5.0 μg cm⁻³) in either a 0.006M NaNO₃ or 0.006M NaCl medium. Harvest and analysis of shoots and roots was after nineteen days. The distribution of Cd in the test solutions between Cd²⁺, CdCl⁺ and HA-Cd was determined in a separate experiment by dialysis equilibrium. In the nitrate test solutions Cd uptake was clearly controlled by Cd²⁺ concentration and was therefore reduced by HA complex formation. In the absence of HA, chloride suppressed Cd uptake indicating that Cd²⁺ was the preferred species. However complex formation with Cl⁻ enhanced uptake when HA was present because of an increase in the concentration of inorganic Cd species relative to the nitrate system. The ratio root-Cd/shoot-Cd remained at about 10 across a wide range of shoot-Cd concentrations, from about 3 μg g⁻¹ (sub-toxic) up to 85 μg g⁻¹ (80% yield reduction). The ability of the barley plants to accumulate ‘non-toxic’ Cd in their roots was thus very limited. Humic acid also had no effect on Cd translocation within the plant and the root/shoot weight ratio did not vary with any treatment. At shoot-Cd concentrations in excess of 50 μg g⁻¹, K, Ca, Cu and Zn uptake was reduced, probably the result of root damage rather than a specific ion antagonism. The highest concentration of HA also lowered Fe and Zn uptake and there was a toxic effect with increasing HA concentration at Cd=0. However the lowest HA level, comparable with concentrations found in mineral soil solutions, only reduced yield (in the absence of Cd) by <5% while lowering Cd uptake across the range of Cd concentrations by 66%–25%.     

Growth and development of seminal and crown root systems in N-limited barley,and their contributions to nitrate acquisition during vegetative and generative growth

Barley (Hordeum vulgare L., cvs Golf and Laevigatum) was grown under nitrogen limitation, controlled by the relative rate of nitrate-N addition (RA), in solution culture. The seminal and crown root systems were kept apart, but in contact with the same nutrient solution throughout culturing. Growth, nitrate uptake, and in vitro nitrate reductase (NR) activity in the different root parts were studied at plant ages from 40 (late vegetative stage) to 110 (mid grain-filling) days. The RA was during this time interval stepwise decreased from 0.08 day^–1 to 0.005 day^–1. The ratio between seminal root dry weight and total plant dry weight decreased drastically during post-anthesis growth, whereas the contribution by crown roots remained unchanged. Tissue nitrogen concentrations in seminal roots did not change with time, but decreased in crown roots after day 80. The NR activity decreased with age in both seminal and crown roots. The V_max for net nitrate uptake decreased throughout the experiment in the seminal root system, but not in the crown root system. The kinetic properties (V_max and K_M) were used to calculate the nitrate concentration required to maintain a relative rate of nitrate-N uptake that equals the relative addition rate. These concentrations (2 to 5 mmol m^–3) were found to closely match actually measured nitrate concentrations in the nutrient solution (1 to 6 mmol m^–3). From uptake kinetics, it was deduced that the contribution by seminal roots to total nitrate uptake at these concentrations decreased from more than 50% in vegetative plants, to about 20% just after main shoot anthesis, and to less than 5% during grain-filling. ei]Section editor: H Lambers     

Effect of nodulation on the nitrate assimilation in vegetative soybean plants

Summary Nitrate assimilation in the first trifoliate leaf of vegetative soybean plants (Glycine max L. Merr, cv Hodgson) was studied in relation to nodulation. Nodulated and non-nodulated plants were grown in a nitrate medium (4 mM). As a control nodulated plants were grown in a nutrient medium without combined nitrogen. This study included measurements of the acetylene reduction activity of the whole plant and of thein vitro nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities in the first leaf and of the nitrate concentration. Nitrate accumulation and nitrate reductase activity were depressed in nodulated plants; root growth was decreased in the presence of nitrate. The relationships between nitrate assimilation and nodulation are discussed.     

Inhibition of nitrate uptake by aluminium in maize

Experiments with two maize (Zea mays L.) hybrids were conducted to determine (a) if the inhibition of nitrate uptake by aluminium involved a restriction in the induction (synthesis/assemblage) of nitrate transporters, and (b) if the magnitude of the inhibition was affected by the concurrent presence of ambient ammonium. At pH 4.5, the rate of nitrate uptake from 240 μM NH₄NO₃ was maximally inhibited by 100 μM aluminium, but there was little measurable effect on the rate of ammonium uptake. Presence of ambient aluminium did not eliminate the characteristic induction pattern of nitrate uptake upon first exposure of nitrogen-depleted seedlings to that ion. Removal of ambient aluminium after six hours of induction resulted in recovery within 30 minutes to rates of nitrate uptake that were similar to those of plants induced in absence of aluminium. Addition of aluminium to plants that had been induced in absence of aluminium rapidly restricted the rate of nitrate uptake to the level of plants that had been induced in the presence of aluminium. The data are interpreted as indicating that aluminium inhibited the activity of nitrate transporters to a greater extent than the induction of those transporters. When aluminium was added at initiation of induction, the effect of ambient ammonium on development of the inhibition by aluminium differed between the two hybrids. The responses indicate a complex interaction between the aluminium and ammonium components of high acidity soils in their influence on nitrate uptake. ei]{gnA C}{fnBorstlap}     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>, a critically endangered ethnomedicinal plant

Summary This study reports a protocol for successful micropropagation of Decalepis arayalpathra (Joseph and Chandras) Venter. (Janakia arayalpathra Joseph and Chandrasekhran; Periplocaceae), a critically endangered and endemic ethnomedicinal plant in the southern forests of the Western Ghats which is overexploited for its tuberous medicinal roots by the local Kani tribes. Natural regeneration is rare and conventional propagation is difficult. Conservation of the species through micropropagation was attempted. The nodal explants of greenhouse-raised plants, were more desirable than cotyledonary nodal explants of aseptic seedlings. The basal nodes (73%) of 12–16-wk-old greenhouse-grown plants cultured in Murashige and Skoog (MS) medium containing 12.96 μM 6-benzyladenine (BA), 2.48 μM 2-isopentenyladenine (2-ip) and 2.68 μM α-naphthaleneacetic acid (NAA) formed 16–17 cm long unbranched robust solitary shoots in 8 wk. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8±0.3 nodes from 18.0±0.6 cm long shoots within 5–6 wk. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while the 2.5–3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Field-established plants showed uniform growth and phenotypic similarity to parental stock.