Micropropagation of guava (Psidium guajava L.)

Summary

Guava (Psidium guajava L.) is difficult to propagate using conventional asexual techniques, with most growers using seedling planting stock. However, these seedlings are highly variable. We therefore developed an in vitro technique to clonally propagate guava. Various concentrations of BAP (6-benzylaminopurine) and TDZ (thidiazuron) were used to regenerate and micropropagate plants. Two explant sources were compared: greenhouse grown plants (GHRP) and in vitro-harvested axillary buds (IVDS). GHRP with BAP at 2 mg l^–1 gave 3.7 shoots per single node cutting with an average length of 0.7 cm. Shoots 3.0 cm long were obtained with 0.5 mg l^–1 BAP, however only 2.1 shoots per explant were produced. For IVDS, the largest number of shoots (3.9 per explant) was obtained with BAP at 0.25 mg l–1 , with an average shoot length of 1.6 cm. Generally, lower concentrations of BAP gave fewer but longer shoots. The highest number of roots and longest roots per shoot (5.4 and 2.0 cm, respectively) were obtained with 1 mg l^–1 indole-3-butyric acid (IBA). A protocol for producing clonal plants over eight weeks is described.     

Meristem culture and micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of oleoresin

Summary

Meristems of ginger with or without leaf primordia were induced to form shoots on three-quarter strength Murashige-Skoog’s (MS) medium containing sucrose 6%, coconut milk (CM) 20%, ascorbic acid (AA) 100 mg l^?1, glutamine (GL) 400 mg l^?1, activated charcoal (AC) 250 mg l^?1, 6-benzylaminopurine (BAP) 0.5 mg l^?1, indolebutyric acid (IBA) 0.4 mg l^?1 and agar 0.8%. Meristem-derived shoots exhibited consistent multiplication on three-quarter strength MS medium containing sucrose (3%), AA (100 mg l^?1), AC (100 mg l^?1), BAP (4–5 mg l^?1) and agar (0.8%). Liquid media (agitated or static) were less effective than a solid (agar-gelled) medium for micropropagation. Kinetin and naphthalene acetic acid (NAA) incorporated at various levels (0.01–0.8 mg l^?1) with or without added BAP and IBA neither improved plantlet formation nor enhanced shoot multiplication. The in vitro plants were successfully established in vivo and the rhizome yield was comparable with that of plants grown by conventional methods.     

The Effect of Soil pH and Manganese Toxicity Upon the Growth and Mineral Composition of the Hop Plant

Summary

The productivity of ornamental foliage plants is related to their capacity to increase their leaf number and leaf size. In Monstera deliciosa, a change in leaf shape is also a pre-requisite for successful marketing. The aim of this work was to describe the effects of different concentrations of exogenous 6-benzylaminopurine (BAP; 5, 50, 100, or 200 mg l^–1) on the control of both leaf size and leaf shape in M. deliciosa, and the impact of these changes on commercial plant productivity. We found an increase of between 15.4 – 23.1% in the rate of leaf appearance (RLA), which reflected a shortening of the phyllochron, and an increase of between 17.5 – 34.9% in the relative rate of leaf area expansion (RLAE) at most of the BAP concentrations tested. This resulted in higher biomass accumulation in both roots and shoots through an increase of between 5.4 – 7.9% in the relative growth rate (RGR), mainly associated with higher net assimilation rates (NAR; increases from 9.0-fold to 11.0-fold) and increased photoassimilate partitioning to the shoots. The most important result of this work was the early appearance of perforated leaf laminae in M. deliciosa plants sprayed with 50 – 200 mg l^–1 BAP, which made them ready for sale.     

High multiplication frequency and genetic stability for commercialization of the three varieties of micropropagated tea plants (Camellia spp.)

RAPD and microsatellite markers were used to determine the genetic fidelity of micropropagated plants from the three varieties of tea plant derived from explants of field grown mother bush as well as in vitro germinated seedlings. Rate of shoot multiplication was better from nodal explants than from shoot tips. A maximum of 32–33 shoots was observed in cotyledonary node in 1/2 MS medium with BAP (6 mg/l), GA₃ (1 mg/l) and IBA (0.5 mg/l). 90% of the in vitro derived microshoots were micrografted into rootstocks. The micropropagated plantlets showed both cytological and genetical stability. SSR primers showed complete stability among the regenerants. The results convinced that plants derived from axillary as well as adventitious mode of propagation can be genetically true to type. This cost effective technique would help in fast clonal propagation at a commercial scale.     

Determination of essential oil contents and micropropagation of Gaultheria fragrantissima,an endangered woody aromatic plant of India

Summary

Leaves of mature of Gaultheria fragrantissima Wall. plants (Indian wintergreen) were collected from various locations in the Eastern Himalayan region on the Indo-China border and were analysed by steam distillation and gas chromatography to identify an elite line that contained 1.79% (v/v) essential oils, 98% of which was methyl salicylate. Subsequently, a highly reproducible micropropagation protocol using adult shoot tips from this elite genotype was developed in order to conserve this highly-valued, endangered, woody oil-bearing aromatic shrub in India. Among several plant growth regulator (PGR) combinations tested for in vitro multiplication, up to 35 shoots per explant could be induced within 14 weeks of culture on woody plant medium (WPM) fortified only with 0.22 mg l^–1 thidiazuron (TDZ). These shoots could elongate on WPM containing 1.0 mg l^–1 kinetin. Rooted plantlets were acclimatised ex vivo, with 70% success. Random amplified polymorphic DNA (RAPD) analysis indicated the genetic uniformity of both the micropropagated plantlets and the donor plants. This is the first report on in vitro micropropagation of G. fragrantissima.     

Large-scale in vitro propagation of giant reed (Arundo donax L.), a promising biomass species

Summary

Giant reed (Arundo donax L.), a promising energy crop, is vegetatively-propagated from fragments of stems and rhizomes. This may limit large-scale cultivation, since it is time-consuming and involves considerable cost and effort. Tissue culture is an alternative to conventional methods of vegetative propagation and may represent a useful tool for large-scale propagation of plants for biomass production programmes. This report describes a protocol for the large-scale in vitro propagation of giant reed by adventitious bud formation. Stem nodes with dormant buds proved to be the most effective to initiate in vitro cultures giving the highest percentage of differentiated shoots (77%) compared to the other plant fractions tested. A sterilisation procedure using 5 g l^–1 HgCl₂ enabled the production of sterile explants. Moreover, early results indicated that late Autumn excision dates not only gave a higher percentage of well-developed shoots ( > 80%), but also a lower level of bacterial contamination (15 – 20%). 6-Benzylaminopurine (BAP) at 3.0 mg l^–1 was most effective in promoting shoot multiplication when added to a basal medium containing Murashige and Skoog (MS) macro- and micro-nutrients, Morel’s vitamins, 30 g l^–1 sucrose, and 7.0 g l^–1 bacteriological agar supplemented with 1.0 mg l^–1 indole-3-acetic acid (IAA) and 0.05 mg l^–1 gibberellic acid (GA₃). Rooting was successfully induced on the same basal medium used for proliferation, modified by halving the MS macro-nutrients and replacing BAP and the other growth regulators with 2.0 mg l^–1 1-naphthaleneacetic acid (NAA). Successful acclimatisation (> 95% survival) of plantlets was carried out, even in late Winter, in a cold greenhouse or under simpler facilities such as shade nets.     

Comparison of seed-derived with micropropagated male-sterile plants of Tagetes erecta L. for F1 hybrid seed production

Summary

To multiply large number of male-sterile marigold plants for F₁ hybrid seed production, an efficient protocol for in vitro cloning of field-grown differentiated male sterile plants has been developed. A comparative field performance study of tissue culture and seed-derived male sterile plants of two marigold genotypes was undertaken to test the possibility of using micropropagated plants in hybrid seed production. Tissue culture raised plants of both genotypes had superior field performance to the seed-derived counterparts. These plants were more vigorous in growth, i.e. in terms of plant height, number of secondary branches and number of leaves and plant spread, while the leaf chlorophyll contents were equal to that of seedling plants. Flowering was earlier by 2-3 weeks and the number of flowers per plant was also higher in such plants. Repeated hand pollination of sterile flowers with bagged flowers of cv. Pusa Narangi Gainda showed that seed set and bold seed yield were higher or almost comparable with the seed-derived plants. The results clearly indicate that the tissue culture can be adopted for the successful cloning of male-sterile plants, which could then be utilized for producing F₁ seeds with higher quantities of bold seeds with better storability.     

Viability of excised embryos,shoot proliferation and in-vitro flowering in a species of rattan Calamus thwaitesii Becc.

Summary

92% of embryos excised from fresh mature unripe fruits of Calamus thwaitesii germinated in a modified Y3 medium with 0.05 mg l^±1 of 6-benzylaminopurine (BAP). This was higher than the 72% germination obtained with ripe seeds sown in soil. Stored seed lost viability within two weeks due to dehydration of embryos. Germination commenced with the differentiation of the haustorium and the cotyledonary sheath, observable in embryos germinating in vitro. This was followed by the development of the plumule. The first eophylls were simple and lanceloate. Decapitation of the in-vitro seedlings and transfer to a medium with higher levels of BAP at 5 or 8 mg l^±1 resulted in the production of multiple shoots after 4±5 months, initially from buds that developed around the collar region. Repeated subculture resulted in the development of a clustering habit similar to that of field clumps with a rhizome, axillary shoots and dormant buds. Two axillary meristems were induced to develop precociously into inflorescences. Incorporation of activated charcoal and alpha-naphthaleneacetic acid (NAA) with 5 or 8 mg l^±1 BAP reduced multiple shoot formation and brought about root development. Single shoots or clusters developed roots in a Y3 medium with reduced macro elements and supplemented with NAA (5.mg.l^±1) and activated charcoal. Nursery establishment with 65% survival of plantlets was possible. In-vitro culture of excised embryos could be recommended, as propagules could be made available whenever desired by rooting proliferated shoots. It also allowed the safe transport of germplasm.     

In vitro propagation of ‘Troyer’ citrange from epicotyl segments

Isolated epicotyl, root meristem and root segment tissues of ‘Troyer’ citrange [Poncirus trifoliata (L.) Rat. × Citrus sinensis (L.) Osbeck] were established in continuous culture to compare their regeneration potential. Callus was obtained from these explants on a Murashige—Skoog (MS) medium containing NAA (10 mg l^?1) and BAP (0.1–10 mg l^?1). Formation of shoots from root segments was direct without callus formation on MS medium containing BAP (10 mg l^?1) and NAA (1 mg l^?1). Shoot formation from epicotyl callus occurred on MS medium containing 0.25 mg l^?1 BAP and 0.1 mg l^?1 NAA. Formation of shoots from epicotyl segments occurred on MS medium containing BAP (0.5 mg l^?1) and NAA (0.1–1.0 mg l^?1), while rooting of regenerated shoots occurred in treatments containing 2.0 mg l^?1 NAA alone. This system provides a rapid method for propagation of ‘Troyer’ citrange.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

Micropropagation of red raspberry and the influence of phloroglucinol

Micropropagation of 12 raspberry seedling selections and the cultivar ‘Malling Jewel’ has been achieved. A basic culture medium (Linsmaier and Skoog, 1965) supplemented with benzylaminopurine (BAP), 1.0 mg l^?1, and indol-3-yl butyric acid (IBA), 0.1 mg l^?1, was optimal for shoot proliferation. The presence of phloroglucinol (PG) at a concentration of 162 mg l^?1 significantly increased shoot number at all auxin: cytokinin concentrations. Removal of the cytokinin and increasing the concentration of IBA to 1.0 mg l^?1 resulted in adventitious root formation. PG synergistically promoted the number of roots per rooted culture but did not significantly increase the percentage rooting. Viable plants were produced from all genotypes when transplanted to soil.     

Regeneration from in-vitro leaves of ‘Conference’ and other pear cultivars (Pyrus communis L.)

Summary

Regeneration of shoots from in-vitro grown leaf tissues of the pear cv. Conference was achieved at rates of up to 40%. The highest regeneration percentage was achieved using a phytohormone combination of benzylamino purine (5 mg 1^?1) and 1-naphthalene acetic acid (1.0 mg 1^?1) on a basal medium of Murashige and Skoog, to which had been added 200 mg I^?1 of the antibiotic cefotaxime. The percentage regeneration was reduced when a higher concentration of cefotaxime (400 mg 1^?1) was used. Cefotaxime had little beneficial effect on regeneration at a lower concentration of 1-naphthalene acetic acid (0.5 mg l^?1). Regenerated shoots were easily micropropagated, rooted and transplanted to soil. To date, the plants have a true-to-type phenotypic appearance. Regeneration was also achieved with other cultivars—‘Abbé Fetel’, ‘Doyenne d’Hiver’, ‘Doyenne du Comice’ and ‘Passé Crassane’—but percentage regeneration was lower and, apart from ‘Passé Crassane’, the regenerated shoots were weak and could not be subcultured.     

The Influence of Some Genetic and Environmental Factors on the Concentration of L-Ascorbic Acid in the Tomato Fruit

Summary

An efficient, reproducible protocol has been developed for in vitro multiplication of Sida cordifolia L. High-frequency, multiple shoots (90%) were obtained indirectly from nodal explants. Callus was induced when nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg l^–1 Kinetin (Kn). These nodal calli were then cultured in order to differentiate multiple shoots on MS medium supplemented with 0.5 mg l^–1 Kn plus 0.5 mg l^–1 naphthaleneacetic acid (NAA). Roots were induced from these multiple shoots following culture on MS medium supplemented with 0.8 mg l^–1 NAA for 4 weeks. Finally, these in vitro plantlets were hardened, acclimatised, and successfully transferred to the field.     

Direct somatic embryogenesis from explants obtained from in vitro germinated embryonic axes of Camellia sinensis (L.) O. Kuntze

Summary

Studies on direct somatic embryogenesis in several types of explant from in vitro plantlets of tea cultivar TRI 2025 were undertaken to select those most suitable to induce cotyledonary-type somatic embryos. Mature zygotic embryonic axes were surface-sterilised and cultured on MS medium without growth regulators containing 0.6% (w/v) agar. Results showed that 65% of embryonic axes that converted into plantlets at the fifth week of culture had succulent leaves. Several types of explant (normal leaves, large and small succulent leaves, hypocotyl segments and root tips) were isolated from in vitro plantlets at the fifth week and cultured on half-strength MS medium containing 2 mg l^–1 6-benzylaminopurine (BAP) and 0.2 mg l^–1 naphthalene acetic acid (NAA). Morphological and histological observations on somatic embryogenesis were made. The results indicated that somatic embryos were produced at high frequency (25 – 50%) directly from the surface of hypocotyl segments (HS) and large succulent leaves (LSL) after 6 weeks of culture. Efficient somatic embryogenesis was induced in small succulent leaves (SSL) after 16 weeks. Most somatic embryos originated directly from the cortical tissues of HS or the upper epidermal layers of SSL or LSL. HS and SSL from in vitro plantlets gave the highest production of typical, firm somatic embryos for use in tea improvement programmes and for in vitro conservation of tea germplasm.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Longevity of Epidendrum ibaguense flowers as affected by pre-loading treatments and vase solution

Summary

Post-harvest longevity of Epidendrum ibaguense cut flowers was maximum when treated for 6 h with 1 g m^–3 Ethylbloc^® [0.14% 1-methylcyclopropene; 1-MCP] followed, or not, by pulsing with 200 g l^–1 sucrose for 12 h. This extended the vase-life from 5.5 d to at least 11 d. Cut inflorescences pulsed with 2.0 mM silver thiosulphate (STS) had reduced abscission of flowers, similar to the effect of 1-MCP. When inflorescences were pulsed with 200 g l^–1 sucrose alone for 12 h, no effect was observed on the vase-life of the flowers. Flowers kept in a vase solution containing 20 g l^–1 sucrose, 150 mg l^–1 citric acid, and 200 mg l^–1 8-hydroxyquinoline citrate did not influence the longevity of 1-MCP or STS pre-treated flowers, but the vase solution had a small influence on retarding abscission compared with flowers kept in distilled water. Adding 0.2 mM STS to the vase solution improved vase-life 1.74- and 1.45-fold compared with the longevity of flowers kept in distilled water or in vase solution alone, respectively. The presence of 0.3 mM AgNO₃ alone, or mixed into the vase solution, had no affect on the vase-life of the flowers.     

Culture of triploid tissue from the endosperm of an endangered Chilean tree species Gomortega keule

The endangered Chilean tree species Gomortega keule (Mol.) Baillon produces an edible fruit, but is not cultivated at present. Recent advances in micropropagation may allow the further development of this species as a fruit crop. Triploid plants have been regenerated from the endosperm of seed of a number of species. This is the first report on in vitro culture of the seed endosperm of G. keule in order to obtain triploid plants. Callus was formed from endosperm after 1.5 months on 1.0× Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-(γ,γ-dimethylallylamino) purine. Shoot primordia developed and produced shoots that could be cultured on Rugini medium containing 0.1 mg l^–1 α-naphthaleneacetic acid and 1 mg l^–1 6-benzyladenine. Shoot primordia cultured on Rugini medium containing 2 g l^–1 activated charcoal produced longer shoots and longer leaves compared to diploid genotypes. Flow cytometry and chromosome observations indicated that the callus tissue and plantlets derived from the seed endosperm were triploid. Endosperm culture represents a feasible method to regenerate triploid plantlets of this tree species within 18 months. Such material may be of value for the genetic improvement and future development of G. keule as a commercial fruit tree species.     

An improved micropropagation protocol for the recalcitrant plant Capsicum – a study with ten cultivars of Capsicum spp. (C. annuum,C. chinense,and C. frutescens) collected from diverse geographical regions of India and Mexico

Capsicum spp. is a commercially important crop of the Solanaceae family, well-known for its multipurpose use as a vegetable, spice, medicinal and ornamental plants. The genus Capsicum is a recalcitrant species in terms of in vitro morphogenesis and plant regeneration. An efficient method was developed for multiple shoot regeneration in 10 cultivars of Capsicum collected from diverse geographical regions of India and Mexico. Seeds germinated in vitro on a half-strength Murashige and Skoog (MS) medium supplemented with 3.0 % sucrose. Nodes of the in vitro germinated seedlings were used as explant for micropropagation. The combination of the 6-benzylaminopurine, indole-3-acetic acid, and spermidine was found to be the best for multiple shoot induction. However, the optimum responcse varied accompanied by different cultivers with maximum 8.9 ± 0.52 (Capsi-10) to 15.3 ± 0.69 (Capsi-5) multiple shoot per explant. Depending on the cultivar, multiplied shoots were successfully rooted with maximum 18.4 ± 0.20 (highest for Capsi-9) to 36.8 ± 0.29 (highest for Capsi-5) roots per shoot on half-strength MS medium supplemented with 2.0 mg l^?1 indole-3-butyric acid, 1.0 mg l^?1 α-naphthalene acetic acid, and 1.5 mM spermidine. Finally, the micropropagated plantlets were acclimatized with 40.0–86.7 % survival rate, depending on different cultivars.     

Regeneration of various species of Crassulaceae,with special reference to Kalanchoë

Summary

Four species of the genus Kalanchoë (Crassulaceae), K. peltata, K. laxiflora, K. tubiflora and K. marmorata, were regenerated from leaf explants by direct organogenesis. Each species was tested on 19 media, all based on MS-medium. One medium was without growth regulators, the remaining 18 contained a combination of auxin and cytokinin. Auxin was indole-3-acetic acid (IAA): 1.1, 2.3 or 4.6 μM (0.2, 0.4 or 0.8 mg l^–1). Cytokinin was either 1-phenyl-3-(1,2,3-thidiazol-5-yl)urea(TDZ): 1.1, 2.3 or 4.5 μM (0.25, 0.5 or 1.0 mg l^–1), or 6-benzylaminopurine (BAP): 1.1, 2.2 or 4.4 μM (0.25, 0.5 or 1.0 mg l^–1). For each species an optimum level of growth regulators were obtained. One medium, called K22, containing 0.5 mg l^–1 TDZ and 0.04 mg l^–1 IAA, showed good shoot-generating capacity with all four species. Shoot elongation proved to be a problem only with K. marmorata. This could be bypassed by transferring shoots to a gibberellic acid (GA₃)-containing medium, or by ventilating the containers. Shoots were rooted on MS-medium and rooted shoots were transferred to soil. K. laxiflora failed to root, but plantlets produced on the leaves were easily used for vegetable proliferation of the regenerated shoots. Eight additional Kalanchoë species and four species from other genera of Crassulaceae: Crassula, Echeveria and Sedum, were tested for regenerative capacity on K22-medium. From four Kalanchoë species and three other species, regenerated plants were established in soil. These results suggest that this medium has a high regenerative capacity within the Crassulaceae. No close dependency was found between systematic position and ability to regenerate on this medium.     

Castanea sativa plantlets proliferated from axillary buds cultivated in vitro

Chestnut plants were proliferated in vitro from axillary buds of juvenile shoots. N6-Benzyl-aminopurine (BAP) at 0.1?0.5 mg l^?1 was optimal for shoot multiplication. The important role played by the macronutrient formula on shoot multiplication, and especially on the rooting-stage, is emphasized. The MS ($\text{1}\text{2}$ NO₃) macronutrients gave the best rooting percentage as well as the highest number of roots per rooted shoot. In these experiments, shoots remained in the 3 mg l^?1 indole-3-butyric acid (IBA) medium for 12 days, after which they were transferred to an auxin-free medium where roots developed fully. Optimum rooting was achieved by immersing the 1 cm basal end of shoots in concentrated IBA solutions (0.5?1 mg ml^?1) for periods ranging from 2 to 15 min.