Aluminium-induced effects on growth,morphogenesis and oxidative stress reactions in in vitro cultures of quince

The effects of Al³⁺ [supplied as Al₂(SO₄)₃·18H₂O] addition to culture media (pH 4.0) on growth, morphogenesis (in leaf explants), and oxidative stress reactions in in vitro cultures of ‘BA 29’ quince were investigated. Aluminium (Al 0.5 mM) strongly inhibited shoot growth in the proliferation and rooting phases (Al 2.2 mM), reduced shoot proliferation (Al 1.1 mM), and induced tissue browning. Superoxide dismutase (SOD) activity was increased in shoot cultures supplemented with 2 mM Al. Malondialdehyde (MDA) content of shoots was strongly increased by Al during proliferation (starting from Al 1.7 mM) and rooting (already at Al 1.1 mM), thus serving as a good ‘marker’ for Al toxicity. Even a low concentration of Al (0.5 mM) in the shoot induction medium was found to inhibit shoot regeneration. When standard (Al 0) shoot induction medium was used, leaf explant growth was only reduced by 2.2 mM Al in the subsequent growth phases. Following a preliminary selection for their growth on Al-enriched media, 82 potentially Al-tolerant quince somaclones were selected for further trials.     

In vitro proliferation from axillary buds and ex vitro protocol for effective propagation of Syringa × hyacinthiflora ‘Luo Lan Zi’

As a precondition for lilac mass propagation, the optimal shoot-multiplication medium for Syringa × hyacinthiflora ‘Luo Lan Zi’ was ascertained mainly based on clustered microshoot inducement and large leaf area establishment in 6-benzyladenine (BAP) (1.00 mg L⁻¹) and zeatin (Z) (0.10 mg L⁻¹) combination. Medium supplied with lower level of BAP (0.50 mg L⁻¹) and auxin (IAA) (0.25 mg L⁻¹) was not suitable for lilac shoot proliferation, but it could be competent for long-term preservation of the un-rooted shoots so that subsequent proliferation culture could be carried out at anytime. In addition, excess height growth which resulted in low transplanting survival rate was effectively controlled by decrease in node number when paclobutrazol (PBZ) was applied in rooting medium at a concentration of 1.00 mg L⁻¹ after taking into account the effects on shoot height, rooting, persistent leaf area and PBZ carry-over. An important overwintering treatment was to use a plastic chamber covering for plants in the greenhouse prior to field planting to ensure adequate biomass of stem and underground parts not only in the current growing season but also in the subsequent years.     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

Clonal propagation of Rosa clinophylla Thory. through axillary bud culture

A protocol for clonal propagation of Rosa clinophylla, a rare and endangered species but very important for breeding purposes had been standardized through in vitro axillary bud culture. Although cytokinins alone were able to induce shoot buds, but their proper growth and number could be increased only when they were used in combination with GA₃. However, there was shoot tip necrosis and leaf fall in the proliferated shoots. AgNO₃ at 58.85 μM proved effective to avoid shoot necrosis and yellowing of leaves. Activated charcoal (AC) at 250 mg l⁻¹ was found necessary at all the stages of shoot multiplication as well as rooting. Ninety percent rooting could be achieved in 1/2 MS medium supplemented with 4.92 μM IBA and 250 mg l⁻¹ AC. Rooted plantlets were hardened and transferred to the field successfully with 80% survival rate.     

Effect of nitrogen form and radiation on growth and mineral concentration of two Brassica species

Three greenhouse experiments were carried out to determine the growth, yield, nitrate, total N and S concentration in shoots, and water uptake of hydroponically grown Brassica rapa L. subsp. nipposinica var. chinoleifera and Brassica juncea L. In each experiment, daily photosynthetically active radiation (PAR) level was 5.0 mol m⁻² (low), 6.8 mol m⁻² (medium) or 9.0 mol m⁻² (high). Plants were supplied with nutrient solutions having equal N concentrations of 11 mM in different forms: 100% NH₄, 50% NH₄ + 50% NO₃, and 100% NO₃. Nitrogen supplied as 100% NH₄ reduced fresh and dry shoot biomass, leaf area, and leaf number in both Brassica species, especially at low and medium PAR levels. In both Brassica species, S concentrations were highest, while nitrate concentrations were lowest in leaves of plants grown at N supplied as 100% NH₄. No differences in leaf nitrate concentrations were observed between 50% NH₄ + 50% NO₃ and 100% NO₃ treatments. Low and high PAR levels increased the nitrate concentrations and decreased the N/S ratio in leaves of both crops compared to medium PAR level. Fresh shoot biomass was maximized in Brassica rapa when PAR level was above the medium value and nitrate was supplied in the nutrient solution as NO₃ or as a mixture of 50% NO₃ and 50% NH₄. The highest fresh shoot biomass of Brassica juncea was observed in all nutrient solution treatments at high PAR level.     

Callus induction and plant regeneration from embryonic axes of Kosteletzkya virginica

Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L⁻¹ indole-3-acetic acid (IAA), 0.3 mg L⁻¹ kinetin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L⁻¹ IAA, 0.5 mg L⁻¹ zeatin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (3) 96.67% rooting on MS medium containing 30 g L⁻¹ sucrose and 8 g L⁻¹ agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.     

Regeneration and transformation system in Mirabilis jalapa

Protocols for in vitro regeneration and production of in vitro-propagated plants and a transformation system were developed for Mirabilis jalapa (Nyctaginaceae). Among the types of explants and the different media tested, consistent shoot regeneration was obtained only from nodal segments grown in a regeneration medium consisting of Murshashige and Skoog medium supplemented with 2 mg l⁻¹ 6-benzyladenine, 2 mg l⁻¹ zeatin and 1 mg l⁻¹ indole acetic acid. Regeneration efficiency was dependent on the type of plant – white or pink flowers – used as the source of explants. Stable transformation was obtained following inoculation of nodal segments with Agrobacterium tumefasciens strain EHA105, which harbours the binary plasmid pAD1339 containing both nptII and gus genes under the control of the 35S promoter. Transformation was confirmed by PCR and Southern blot analysis of genomic DNA from mature regenerated plants. β-Glucuronidase (GUS) activity was observed only in tissues regenerated from in vitro-grown plants and not in tissues originating from greenhouse-grown plants. GUS expression was not uniform in regenerated leaves and showed a chimera pattern.     

Nitrogen content determines adventitious rooting in Euphorbia pulcherrima under adequate light independently of pre-rooting carbohydrate depletion of cuttings

Root regeneration in shoot tip cuttings responds to graduated nitrogen (N) fertilization of stock plants. In pelargonium cuttings, reduced carbohydrate reserves caused by high N absorption by the donor plants and post-harvest storage of cuttings limit adventitious root formation, especially in low-light environments. In contrast, in chrysanthemum, similar carbohydrate reserves do not have this dominant effect on rooting capacity. The positive correlation between rooting capacity and internal N status is stable across a wide range of environments and is genotypically consistent for this species. However, the influence of N and carbohydrates on adventitious rooting of Euphorbia pulcherrima is unknown. We investigated the consequences of different N fertilization regimens applied to E. pulcherrima stock plants and cold and dark storage of the cuttings on N absorption, carbohydrate distribution, and rooting capacity of cuttings. Increasing time of stock plant cultivation with graduated N nutrition produced cuttings with N contents, ranging from 19 to 51 mg N g⁻¹ dry mass (DM). High N absorption resulted in low carbohydrate concentrations in cuttings, and subsequent storage decreased carbohydrate concentrations further, particularly in stems. Lower sucrose contents in leaves were correlated with reduced rooting of stored cuttings at a particular harvest date. However, despite the lower carbohydrate levels, root numbers and lengths correlated positively with internal N concentrations. These relationships remained stable in unstored and stored cuttings, even when overall rooting intensity was reduced under lower natural light during autumn. Multivariate regressions accounting for nitrogen content, sucrose content and daily light integral during rooting highlighted these relationships and explained up to 79% of rooting variances. We conclude N nutrition of stock plants and N absorption by cuttings are the dominant factors determining the rooting capacity of poinsettia when rooting occurs under sufficient light, as is commonly available during propagation. To maximize rooting capacity of poinsettia cuttings their nitrogen content should exceed a threshold of 40 mg N g⁻¹ DM.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

In vitro shoot proliferation and enhancement of rooting for the large-scale propagation of yellow bamboo (Bambusa vulgaris ‘Striata’)

Continuous and rapidly proliferating axillary shoots were raised from axillary buds in secondary branches of adult field culms and nursery grown 1-year-old tissue culture-raised plants of Bambusa vulgaris ‘Striata’. Shoots continuously proliferated in a MS medium containing 4 mg L⁻¹ 6-benzyladenine (BA). The effects of indole butyric acid (IBA) levels, a pretreatment with thidiazuron (TDZ) (1-phenyl-1-([1,2,3-thidiazol-5-yl])urea) and illumination on rooting, were investigated after 6 months of shoot proliferation. A rooting medium with IBA at 3 mg L⁻¹ was optimum for root induction. Shoots of adult field culms that were proliferated in the presence of BA when induced to root in this medium resulted in 40% rooting in 27 days. In vitro shoots raised from 1-year-old tissue cultured plants showed 92% rooting under the same conditions. Rooting was enhanced when the relatively difficult-to-root in vitro shoots from adult field culms were pretreated with 0.5 mg L⁻¹ TDZ for two to three subcultures before placing in the root induction medium. Continuously illuminated shoots pretreated with TDZ for three subcultures showed 100% rooting compared to 83% rooting of shoots that were exposed to a 12 h photoperiod. These findings have been applied in the large-scale propagation of this species.     

Improvements in somatic embryogenesis protocol in Feijoa (Acca sellowiana (Berg) Burret): Induction,conversion and synthetic seeds

Pineapple guava (Acca sellowiana) syn. Feijoa sellowiana, a Brazilian indigenous Myrtaceae is under domestication in South Brazil. Previous works showed that this species is responsive to somatic embryogenesis and recalcitrant to conventional methods of clonal propagation. In the present work it was evaluated the role of components of culture medium in the induction and development of somatic embryos. The technology of synthetic seeds was also evaluated. Zygotic embryos were inoculated in LPm medium supplemented with 8 mM glutamic acid and 8 mM l-glutamine, 2,4-dichlophenoxiacetic acid (20 μM) and myo-inositol. For conversion of somatic embryos and synthetic seeds it was tested the effect of 6-benzylaminopurine and gibberellic acid combined or not with activated charcoal. The highest values for embryogenetic induction (100%) and number of somatic embryos/explant (113) were observed in the LPm medium supplemented with Glu (8 mM), and 2,4-D. The culture medium supplemented with BA (0.5 μM) and GA₃ (1 μM) and activated charcoal (1.5 g L⁻¹) enhanced the conversion of somatic embryos to plantlets. Pre-germinated somatic embryos encapsulated in sodium alginate with BA (0.5 μM) and GA₃ (1 μM) developed radicles. The use of synthetic seed was a requisite for the survival of plantlets.     

Restoration of rooting competence in a mature plant of Garcinia indica through serial shoot tip grafting in vitro

Apical and axillary buds from a high yielding, early fruiting elite tree (more than 20 years old) were cultured in woody plant medium (WPM) supplemented with 0.9 μM N⁶-benzyl adenine (BA). Multiple shoots were obtained on WPM basal medium containing 8.9 μM BA and 0.5 μM thidiazuron (TDZ). Elongation of axillary shoots was obtained in half-strength WPM medium supplemented with 0.4 μM BA. For root initiation, the elongated shoots were transferred to half strength WPM basal medium containing 2.5–245 μM indole-3-butyric acid (IBA) or 2.7–268.5 μM α-naphthaleneacetic acid (NAA) or the shoots were subjected to 2.5–53.9 mM IBA, 2.7–59.1 mM NAA dip for (30 s–30 min) and then transferred to half strength WPM basal medium. However, rooting was never achieved even after 2 months of culture.     

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.     

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%).     

Application method and rate of Trichoderma species as a biological control against Pythium aphanidermatum (Edson) Fitzp. in the production of microgreen table beets (Beta vulgaris L.)

Seed balls of ‘Early Wonder Tall Top’ table beet (Beta vulgaris L.) were incubated for 2 days at 21 °C in moist exfoliated grade 3 vermiculite (3 g seed balls [168 seed balls] and 1.1 g vermiculite) containing equal weights of Trichoderma harzianum Rifai strain KRL-AG2 G41 and T. virens strain G-41 (ThTv) at 0, 0.25, 0.50, 0.75 or 1.00 mg ThTv per seed ball. ThTv also was applied to the peat-lite growth medium 14 days before planting, at the same rates per seed ball as the seed ball treatments. Four days before planting, the peat-lite was inoculated with Pythium aphanidermatum (Edson) Fitzp. (Pa) at 0, 0.5 and 1.0× the rate that resulted in 96% damping-off when non-ThTv-treated dry seed balls were sown in peat-lite containing 1.0 Pa. Increasing ThTv level per seed ball decreased percentage damping-off, with 1.0 Pa giving greater percentage damping-off than 0.5 Pa. At 1 mg ThTv per seed ball, damping-off was 5% and 19% at 0.5 and 1.0 Pa, respectively. Including Agro-Lig UF (mostly humic acids) in the incubating seed-ball-ThTv mixture further decreased damping-off by an average 13 and 10 percentage points in 0.5 and 1.0 Pa, respectively. Increasing ThTv per seed ball in growth media decreased percentage damping-off, but not to the extent achieved with seed ball treatment, with 1 mg ThTv per seed ball giving 20% and 55% damping-off in 0.5 and 1.0 Pa, respectively. Decreasing incidence of damping-off with increasing ThTv application to seed balls or growth media was associated with increasing shoot fresh weight m⁻² at 14 days after planting, a response attributable to increased percentage plant survival and not to a ThTv growth-promoting effect.     

Production protocol development for greenhouse cut Linaria, Lupinus, and Papaver flowers

Linaria maroccana Hook. f. Ann., ‘Lace Violet’, Lupinus hartwegii ssp. cruikshankii Lindl. ‘Sunrise’ and Papaver nudicaule L. ‘Meadow Pastels’ seeds were directly sown into 105 cell plug trays and received either ambient light or supplemental high intensity discharge (HID) lighting. For each species, a 2 × 3 × 3 factorial was used with two light intensities during propagation, three transplant stages, and three night temperatures. Seedlings were transplanted at the appearance of 2–3, 5–6, or 8–9 true leaves. Transplanted Linaria and Papaver seedlings were placed at 5/11, 10/16, or 15/21 ± 1 °C night/day temperatures and Lupinus seedlings were placed at 15/24, 18/25, or 20/26 ± 2 °C night/day temperatures. For this study, the optimum production temperature for Linaria was 10/16 °C as the cut stems produced at 15/21 °C were unmarketable and production time was excessively long at 5/11 °C. At 10/16 °C, Linaria seedlings should be transplanted at the 2–3 leaf stage to maximize stem number, stem length and profitability. For Lupinus the optimum temperature was 15/24 °C due to long stems and high profitability per plant. Lupinus seedlings should be transplanted at the 2–3 leaf stage when grown at 15/24 °C to obtain the longest and thickest stems; however, $/m² week was higher for plants transplanted at the 8–9 leaf stage due to less time in finishing production space. For Papaver, the 15/21 °C temperature was optimal as that temperature produced the longest stems in the shortest duration, resulting in the highest $/m² week. At 15/21 °C Papaver plants should be transplanted at the 2–3 leaf stage. Supplemental HID lighting had no effect on any of the species.     

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv. Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110 mM, BAP 10 μM and NAA 0.5 μM. A low light regime of 17 μmol m⁻² s⁻¹ improved regeneration frequency up to 86%. For transformation, leaf discs were inoculated and co-cultivated with two disarmed A. tumefaciens strains, EHA 101 and LBA 4404, both carrying the binary vector pFAJ3000 which contained the nptII selectable gene and the GUS reporter gene. A pre-culture period of 3 days and a short co-cultivation duration (1 day) improved the efficiency of transformation. Inoculation of only 10 min with agitation including (or not) vacuum infiltration was sufficient. If selection on kanamycin containing medium was applied after a 2 weeks culture period on shoot regeneration medium, the percentage of explants forming kanamycin-resistant shoots increased from 3.3 to 13.3%. Integration and expression of the introduced transgene were confirmed by histochemical GUS assay, PCR and Southern blot analysis. Flowering of transgenic plants in glasshouse occurred 10 months after acclimatization.     

High frequency multiplication of Phalaenopsis gigantea using trimmed bases protocorms technique

This study was conducted to determine the effects of coconut water (CW) and activated charcoal (AC) on multiplication of Phalaenopsis gigantea protocorms. The protocorms used for this study were obtained by germinating seeds in vitro. Protocorms with trimmed and untrimmed bases were cultured on XER basal medium containing 0, 10, 15 or 20% (v/v) CW; and 0, 1, 2 or 2.5 g AC l⁻¹. Trimmed protocorms exhibited the highest percentage of proliferation on a medium containing 15% (v/v) CW and 2.5 g AC l⁻¹ (56.82 ± 38.86%) with an average of 4.24 ± 2.89 protocorms formed per protocorm. Untrimmed protocorms cultured on a medium containing 20% (v/v) CW without AC produced the highest percentage of new protocorms (6.93 ± 6.28%) with an average of 0.72 ± 0.57 per protocorm. When CW was added to a medium singly, 10% (v/v) CW induced a higher degree of proliferation on trimmed protocorms (5.68 ± 10.14%) with an average 0.50 ± 0.84 new protocorms per protocorm. Untrimmed protocorms proliferate to a much lower extent (2.57 ± 2.74%) with an average of 0.72 ± 0.57 protocorms per protocorm when cultured on a similar medium. A high concentration of CW enhanced proliferation on untrimmed protocorms, but increased mortality of trimmed protocorms. The addition of CW with AC to media increased protocorm proliferation and survival of both trimmed and untrimmed protocorms. When cultured on all media, trimmed protocorms produced a higher number of new protocorms (an average 0.5–7.0) as compared to untrimmed protocorms (0.3–1.9). Comparative studies showed that trimmed protocorms produced up to 10 times more new protocorms than untrimmed ones. Altogether this study showed that trimmed protocorms cultured on a medium containing CW and AC can be used for high-frequency multiplication of P. gigantea seedlings.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

High frequency plant regeneration from microspore-derived embryos of ornamental kale (Brassica oleracea L. var. acephala)

The aim of this work was to study the effect of solid medium, developmental stage, embryonic age, cold treatment and additives to the medium on plant regeneration from microspore-derived embryos in four F₁ hybrids of ornamental kale (Brassica oleracea L. var. acephala). The results showed that all of the cultivars responded best when the embryos were cultured in solidified B5 medium with 1% agar. Optimal regeneration was gained when cotyledonary embryos were cultured for 25 days. Cold treatment significantly improved plant regeneration with a frequency of up to 79.0% under 4 °C for 2 d or 5 d. The addition of 3.0 or 5.0 mg/L silver nitrate (AgNO₃) increased the frequency of plant regeneration. In the Zhouyehongxin cultivar, the frequency of plantlet development reached 84.4%. The addition of activated charcoal reduced embryo hyperhydricity.