Effect of growth regulators on ginsenoside production in the cell culture of two ginseng species

Plants belonging to the genus Panax produce ginsenosides that possess pharmacological properties. The ability to synthesize these compounds is preserved in some cultured cells of ginseng. In this work, we used suspension cell cultures of two species of ginseng: Panax japonicus var. repens C. A. Mey and P. ginseng C. A. Mey. The first culture was grown on MS medium supplemented with α-NAA. After one subculturing, cell biomass increased 5–6 times with the level of ginsenosides being equal to 2.5–3.0% of dry weight. The second culture was grown on the same medium supplemented with 2,4-D. In this case, biomass increment was 3–5-fold, and ginsenosides were produced in trace amounts. Substitution of 2,4-D for NAA in suspension cell culture of P. japonicus brought about deterioration of growth characteristics, but the content and composition of ginsenosides was not changed. In the suspension cell culture of P. ginseng, substitution of NAA for 2,4-D decreased the rate of biomass accumulation and increased the extent of cell aggregation, with total content of ginsenosides increasing 25 times and their assortment being complete.     

Influence of auxins on organogenesis and ginsenoside production in Panax ginseng calluses

Panax ginseng calluses were cultured for 5 weeks on solid MS medium supplemented with kinetin 0.46 mM (0.1 mg l^–1) and 2 mg l^–1 of 2,4-D (9.05 mM), IBA (9.98 mM) or NAA (10.74 mM). In the conditions studied, 2,4-D inhibited the organogenic capacity of the calluses, whereas IBA or NAA increased this capacity. IBA induced the formation of a high number of buds and roots, but the roots were thin and necrotized. Calluses grown with NAA produced fewer buds and roots than those grown in IBA medium, but the roots were thick and showed good growth. The highest ginsenoside content was found in root forming calluses grown in the presence of NAA.In calluses forming roots or buds, 2,4-D, NAA and especially IBA increased the Rb group of ginsenosides rather than that of the Rg group.     

Growth and biosynthetic characteristics of ginseng (Panax japonicus var. repens) deep-tank cell culture in bioreactors

The aim of the work was to study the growth characteristics of cultured cells of Panax japonicus var. repens, an endemic plant of the Primorski Krai of Russia, grown in laboratory bioreactors and to determine the content of basic ginsenosides under these conditions. An increase of the inoculum size of the culture produced higher biomass accumulation and economic coefficient but slightly reduced the specific growth rate. An increase in the auxin concentration in a medium by adding 2,4-D practically did not affect growth characteristics of the culture but significantly reduced the size of cell aggregates. In all treatments tested, all major ginsenosides (Rb₁, Rc, Rb₂, Rd, Rf, Rg₁, and Re) were found in the culture. The total ginsenoside content was 2–3% per biomass dry weight. Meantime, ginsenosides of the Rg-series with protopanaxatriol as aglycone prevailed (70% of the total ginsenoside content). The culture conditions considerably affected the ratio of individual ginsenosides. In 2,4-D-containing medium, the preferential synthesis of Re ginsenoside was observed while both Rg₁ and Re were synthesized in other treatments.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata)

Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium     

Regeneration of a metal tolerant grass Echinochloa colona via somatic embryogenesis from suspension culture

An efficient protocol was developed for in vitro plant regeneration via somatic embryogenesis from cell suspension cultures of metal tolerant grass Echinochloa colona (L.) Link. Callus was obtained by culturing leaf base on MS medium supplemented with 0.5 mg dm^-3 of 6-benzylaminopurine (BAP) and 2.0 mg dm^-3 of 1-naphthaleneacetic acid (NAA). Cell suspensions were initiated and established in MS liquid medium containing 0.5 mg dm^-3 BAP, 1.0 mg dm^-3 NAA and 2.0 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D). A reduction in the concentration of 2,4-D to 0.5 mg dm^-3 induced formation of somatic embryos. The embryos developed and grew into normal plants in the presence of half strength MS medium without growth regulators. The regenerated plants were hardened in the greenhouse and subsequently grown in the open. This system may be also used for isolation and culture of protoplasts as a first step in somatic hybridization. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence des régulateurs de croissance sur la prolifération,l'activité peroxydasique et les isoperoxydases de la suspension cellulaire deSilene alba

Growth ofSilene alba (MILLER) E. H. L. KRAUSE cells, as well as their peroxidase pattern and activity are studied. Cells were grown in the presence and absence either of IAA, or NAA or 2,4-D. The subculture is dependent upon the growth regulator used to sustain the growth of cells. For 14 days' passages, subculture is possible with 2,4-D (5 x 10^-7M) or NAA (10^-5M) but impossible with IAA or without any growth regulators. Cells grown using 2,4-D or NAA in the medium contain a smaller number of isoperoxidases and have lower activities than those grown using IAA or no growth regulator. The nature of growth substances does not affect the compartimontation of the peroxidase; in fact the bulk of the peroxidaso activity is always liable to the ionic wall bound fractions. Tho electrophorotic mobilities of peroxidase isoenzymes detected in the modium are not the same as those of tho eytoplasmic isoenzymes. Cell cultures grown with and without growth regulators show different patterns of modium peroxidase activities. Some forms are present both in cells and media and some other only in the media; this may indicate that there is some selection made in tho cells for retention of particulars forms; the others could be secreted as exoenzymes shortly after they are synthesized in the cells. The nature of the growth regulator used could act on the release of certain isoperoxidases. These results are discussed from the viewpoint of the correlation of isoperoxidase patterns with the possibility of subculture.     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.     

Somatic embryogenesis and regeneration of plantlets in protoplast cultures from somatic embryos of coffee (Coffea canephora P. ex Fr.)

Somatic embryogenesis was achieved in protoplast cultures of coffee. Protoplasts were isolated from cell suspension-derived somatic embryos of Coffea canephora. After repeated subculture in a medium supplemented with 0.5 mg/l of each of kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthaleneacetic acid (NAA), microcalli developed. Transfer of these microcalli to a medium lacking growth regulators resulted in the formation of globular embryos. Upon subculture without growth regulators they grew to well-differentiated embryos, Eventually some of them developed to plantlets which were transferred to the greenhouse for further observation.     

Achillea millefolium (yarrow) cell suspension cultures: Establishment and growth conditions

Summary Friable calli were obtained fromAchillea millefolium L. hypocotyls, in Gamborg B5 medium, supplemented with 1.5mg.1^–1 2,4-D / 0.1mg.1^–1 Kin, and used for the production of cell suspension cultures in the same liquid medium. The growth pattern of the cultures was determined in permanent light or dark conditions and with different inoculum densities, basal media, growth regulators and sucrose concentrations. Different sources and nitrogen amounts were assayed to study the effect on yarrow cell growth. The conditions found to be optimal for growth of yarrow cell suspension cultures were: 70g (f.w.).1^–1 of initial inoculum in Gamborg B5 medium, supplemented with 1.5mg. 1^–1 2,4-D / 0.1mg.1^–1 Kin, NO₃ ^–/NH₄ ⁺ (30/lmM), and 2% sucrose, in darkness. In these culture conditions the cell suspensions showed a doubling time of 35–40h.Abbreviations 2,4-D dichlorophenoxyacetic acid - NAA naphtalenacetic acid - BA benzyladenine - Kin Kinetin     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.     

Plant regeneration of Chorispora bungeana via somatic embryogenesis

Summary A method was developed for in vitro regeneration of plants via somatic embryogenesis in Chorispora bungeana, an alpine plant with freeze-tolerance, using cell suspensions initiated from leaf-derived callus. Primary calli were induced from leaves of C. bungeana grown on Murashige and Skoog (MS) media supplemented with 4.0 mg l⁻¹ gibberellic acid (GA₃), 0.2 mgl⁻¹ α-naphthaleneacetic acid (NAA) and 0.2 mgl⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension culture was initiated by incubating the callus particulates in liquid MS medium supplemented with 1.0 mgl⁻¹ kinetin (KT) and 0.2 mgl⁻¹ NAA. Individual early cotyledonary-stage somatic embryos isolated from cell suspension developed into whole plants on medium containing high levels of sucrose (60 and 90 gl⁻¹), whereas lower sucrose concentrations (0 and 30 gl⁻¹) were inhibitory to main root development. On the MS medium with 90 gl⁻¹ sucrose, one regenerated plant exhibited hetero-morphologic leaves, while other plants grown on different media showed a transformation from stem to root.     

Development of a Polygonum minus cell suspension culture system and analysis of secondary metabolites enhanced by elicitation

Polygonum minus has been reported to contain valuable metabolites and to date, there is no report on using cell culture technique for metabolite production in P. minus. Naphthalene acetic acid (NAA) concentrations in the range of 2–6 mg L^?1 were used in a matrix of combinations with dichlorophenoxyacetic acid (2,4-D) concentrations in the range of 2–10 mg L^?1 as plant growth regulators (PGRs) to induce callus cultures. Media that were supplemented with 2 mg L^?1 2,4-D + 4 mg L^?1 NAA, 2 mg L^?1 2,4-D + 6 mg L^?1 NAA and 6 mg L^?1 2,4-D + 8 mg L^?1 NAA were effective for callus induction (93.3 % of the explants produced callus). To establish cell culture, the best growth was obtained from medium that was supplemented with 1 mg L^?1 2,4-D + 2 mg L^?1 NAA. From a 1-g inoculum size, the fresh weight increases exponentially after 5–10 days of culture, and a 26.71 g maximum fresh weight was obtained after 25 days of culture. The cell culture medium was then analyzed using gas chromatography–mass spectrometry (GC–MS). Jasmonic acid (100, 50, 25 and 5 μM), salicylic acid (100, 50, 25 and 5 μM), yeast extract (500, 250 and 100 mg L^?1) and glass beads were used in this research as elicitors. The cell cultures were then incubated with the different elicitors for 1, 2, 3 and 4 days. Several compounds with high peak area percentages were detected, including 2-furancarboxaldehyde, 5-hydroxymethyl, furfural, and 2-cyclopenten-1-one, 2-hydroxy. These results show the diversity of metabolites released by P. minus cell into the culture medium under control conditions.     

Nodule culture and regeneration of Eucalyptus grandis hybrids

Callus of three superior Eucalyptus grandis hybrids was induced from immature inflorescences, floral parts, shoot tips, zygotic embryos, and hypocotyl explants on various auxin (2,4-D or NAA) and cytokinin (kinetin) supplemented media. Hypocotyl callus initiated on 4 mg/l NAA and 1 mg/l kinetin formed massive nodular structures, and shoots and roots after four weeks on hormone free-medium. Callus from all other expiants turned brown and died upon transfer to hormone free or reduced hormone media. The nodular structures originating from hypocotyl-callus were maintained by subculture for over three years and retained the ability to form thousands of shoots. Shoots were successfully rooted (98% rooting) and plantlets developed were transferred to mist-greenhouse and then to greenhouse conditions with 95% survival. Plantlets were grown for six months in the greenhouse without sign of abnormal growth.Abbreviations NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - MS Murashige and Skoog Medium (1962) - IBA indolebutyric acid     

The establishment of cell suspension culture of sabah snake grass (<Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Burm.F.) Lindau)

Clinacanthus nutans (Burm.F.) Lindau is an herbaceous plant that has long been used for traditional medicinal purposes in Asia. It has recently gained popularity as an alternative treatment for cancer. The aim of this study was to establish cell suspension cultures of C. nutans and to identify targeted bioactive compounds in the cultures. Young leaf explants were cultured on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin to identify a suitable medium for callus induction and proliferation. Proliferated, friable calluses were cultured in different combinations of plant growth regulators (2,4-D, naphthaleneacetic acid [NAA], picloram, kinetin, and 6-benzylaminopurine) in liquid medium to establish cell suspension cultures. Three cell lines of suspension culture, callus, and intact plant parts were subjected to ethyl acetate extraction followed by thin layer chromatography for identification of selected bioactive compounds. Medium supplemented with 0.25 mg L^?1 2,4-D and 0.75 mg L^?1 kinetin was found to be optimal for callus induction, whereas supplementation with 0.50 mg L^?1 2,4-D was efficient for callus proliferation. Liquid medium supplemented with 0.25 mg L^?1 2,4-D and 0.50 mg L^?1 NAA produced the highest growth index (2.52). Quercetin, catechin, and luteolin were present together in the callus and cell suspension cultures of C. nutans, but all three compounds were detected separately in young leaves, mature leaves, and stems. This study is the first to report the establishment of cell suspension culture of C. nutans with both cell and callus cultures producing quercetin, catechin, and luteolin.     

Modulation of the morphogenic potential of the embryonic axis of Juglans regia by cultural conditions

Embryonic axes of Juglans regia werecultured in vitro on Murashige and Skoog mediumsupplemented with different auxin/cytokinin ratios.2,4D, NAA or IBA at 1, 2, 4, 8 mgl^-1, alone orcombined with Kn 0.3 mgl^-1 induced callus whichshowed browning. NAA and IBA allowed organ developmentwhich was inhibited by 2,4-D. Cell suspensions fromNAA-or IBA induced callus had embryogenic capacitywhen cultured with NAA or IBA, showing a heterogeneouscell population composed of giant, elongated, andmeristematic cells. Those cultured with IBA dividedfollowing embryogenic patterns and cell aggregateswhich were associated with the earliest steps ofembryoid formation. Cell suspension from 2,4-D inducedcallus, revealed homogeneous cell populations whichwere very vacuolated with no apparent differentiation.Axillary bud proliferation induced by BA at 0.1, 1 or5 mgl^-1 was affected by both the physical stateof the culture medium and the time period in contactwith the growth regulator. The culture of embryonicaxes for one week in MS liquid medium with BA5 mgl^-1 favoured proliferation as well as buddevelopment.     

Suspension and protoplast culture of U.S. rice cultivars

Efficient protoplast culture and plant regeneration of five U.S. rice cultivars (Oryza sativa L.) - Mercury, Lacassine, Maybelle, Cypress, and Lemont - were obtained from suspension cells maintained in modified General Medium. Embryogenic suspension cells were developed from calli grown on the original callus induction medium for 10–20 weeks without subculture. Weekly subculture of the suspensions for five to eight weeks yielded cells suitable for protoplast isolation. After 2 weeks, rate of colony formation from protoplasts varied among the cultivars and ranged from 2.5 to 6.8%. Improvement of plating efficiencies to as high as 13.7% was obtained by conducting a second cycle of protoplast culture. A total of 525 plants were regenerated from the cultivars studied.Abbreviations BAP 6-benzylaminopurine - CH casein acid hydrolysate - MGM modified General Medium - Kin kinetin - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Comparison of NAA and 2,4-D induced somatic embryogenesis in Cassava

NAA and 2,4-D were compared for their ability to induce somatic embryogenesis in cassava (Manihot esculenta Crantz). In all seven cultivars tested, only 2,4-D had the capacity to induce primary somatic embryos from leaf explants, however, both NAA and 2,4-D were capable of inducing secondary somatic embryos. More secondary somatic embryos were formed in NAA than in 2,4-D medium. Furthermore, the maturation period for secondary somatic embryos was shorter in NAA medium than in 2,4-D medium. In some cultivars, repeated subculture of secondary somatic embryos in NAA medium resulted in a gradual shift from somatic embryogenesis to adventitious root formation. This shift could be stopped and reversed by subculture of the material in 2,4-D medium. In NAA medium the most secondary somatic embryos were formed when they were subcultured every 15 days whereas in 2,4-D a 20 day subculture interval was optimal. Subculture of secondary somatic embryos at a high inoculum density (>1.5 g jar⁻¹) in NAA medium did not result in the formation of secondary somatic embryos, whereas in 2,4-D it lead to the formation of globular secondary somatic embryos. With 2,4-D the newly induced secondary somatic embryos were connected vertically to the explant and with NAA medium horizontally. For all cultivars tested, desiccation stimulated normal germination of NAA-induced somatic embryos. However, the desiccated, secondary somatic embryos required a medium supplemented with BA for high frequency germination. The concentration of BA needed for high frequency germination was higher when the desiccated secondary somatic embryos were cultured in light instead of dark. In only one cultivar desiccation enhanced germination of 2,4-D induced secondary somatic embryos and in three other cultivars it stimulated only root formation. This revised version was published online in June 2006 with corrections to the Cover Date.