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.     

Adventitious shoot formation from sections of sweet potato grown in vitro

Sweet potato (Ipomoea batatas Lam.) root sections were obtained from the cultivars ‘Centennial’, ‘Redmar’ and ‘Jewel’ with a No. 6 cork borer. These sections were cut into 2–3-mm discs and explanted on to modified Murashige and Skoog (MS) medium consisting of MS high mineral salts, myo-inositol (100 mg l^?1), Staba vitamins, 6-benzyl-aminopurine (2.0 mg l^?1), naphthaleneacetic acid (0.1 mg l^?1), sucrose (30 g l^?1) and agar (10 g l^?1). Root discs from internal regions of the tuberous roots gave rise to calli and meristematic bud-like centers (MBLC's). A small percentage of ‘Centennial’ MBLC's burst open to reveal plantlets which grew and rooted well on the medium. Some of the ‘Jewel’ MBLC's contained only roots, while those of ‘Redmar’ did not differentiate. MBLC-formation occurred most often on discs taken from fresh (unstored) roots of ‘Centennial’. Petiole sections taken from in vitro-cultured plants of all 3 cultivars developed plants quite readily on the medium. Shoots of all 3 cultivars grew rapidly, to yield whole rooted plants which could easily be moved to soil and grown in the greenhouse and field.     

Chemical composition of French and Polish cloudy apple juices

Summary

Cloudy juices from six apple cultivars from Poland (‘Ariwa’, ‘Gold Milenium’, ‘Florina’, ‘Melfree’, ‘Novamac’, and ‘Rajka’) and four French cultivars (‘Ariane’, ‘Chanteline’, ‘Judeline’, and ‘Judor’) were produced and chemically characterised. The analyses encompassed 23 chemical parameters and phenolics profiles. The most important parameter, differentiating cloudy juice from clear juice, was turbidity. Cloudy juices were characterised by having an average total turbidity of 1,210 Nephelometric Turbidity Units (NTU) and a stability of turbidity of 42%. Some of the results deviated from the accepted ranges given in reference values for apple juices in the Code of Practice of the European Fruit Juice Association. This occurred in the cases of simple sugars and saccharose contents of the Polish apple juices (e.g., up to 56.9 mg l^–1 of saccharose vs. a maximum value of 30 mg l^?1), and for some mineral compounds in the French apple juices (e.g., sodium values up to 10 mg l^?1). Large variations were found in case of important healthconferring components. Water-soluble pectin contents varied from 205 mg l^?1 for ‘Chanteline’, to 1,289 mg l^?1 for ‘Gold Milenium’; while, in the case of phenolic compounds, the range was from 85.7 mg l^?1 for ‘Novamac’, to 524.8 mg l^?1 for ‘Melfree’.     

Temporary immersion and stationary bioreactors for mass propagation of true-to-type highbush,half-high,and hybrid blueberries (Vaccinium spp.)

True-to-type propagules in half-high, highbush, and hybrid blueberries (Vaccinium L. spp.) were produced using stationary (SB) and temporary immersion bioreactor (TIB) systems containing a liquid medium. Multiple shoots were produced in vitro from nodal segments of blueberry cultivars ‘St. Cloud’ and ‘Polaris’, and of six blueberry hybrids obtained from crossing between half-high/highbush and lowbush blueberries. Shoot proliferation was best in a liquid medium containing 4.6 µM zeatin in both TIB and SB systems, but the performance was genotype dependent. Shoot proliferation was better in hybrids than in cultivars. Although SB produced longer shoots with more leaves per shoot in most of the genotypes, TIB-derived shoots were more vigorous and rooted better under ex vitro condition. Liquid culture-derived elongated shoots were rooted ex vitro by treating with indole-3-butyric acid (39.4 mM) before planting on a 3 peat:2 perlite (v/v) medium. Micropropagules were acclimatized and maintained in a greenhouse with 80?90% survival rate of rooted plantlets. Expressed sequence tag (EST)-polymerase chain reaction (PCR) and EST- and genomic-simple sequence repeat (SSR) marker assay formed a homogenous monomorphic banding pattern in the in vitro-derived and donor control plants proving the clonal fidelity of liquid-culture-derived micropropagated plants.     

核桃多倍体诱导技术研究

以‘绿岭’核桃种子、1a生‘绿岭’核桃实生苗的新梢、5a生采穗圃的‘绿岭’核桃新梢为试材,通过不同浓度的秋水仙素和不同时间的处理对核桃进行多倍体诱导,并用流式细胞仪进行倍性鉴定。结果表明:幼苗茎尖浸润法、新梢掐茎尖浸润法、种仁浸泡法、胚浸泡法、刺激隐芽法、刺激腋芽法处理后变异率均为0%,幼苗掐茎尖浸润法处理后,1.0%的秋水仙素溶液处理8d的30株幼苗中有4株变异,变异率为13.3%。对叶片明显变大、变厚,叶色变深,叶片变皱的植株进行倍性鉴定表明,1株是四倍体,3株是混倍体。     

Effects of grafting height of MM106 rootstock on growth,lateral shoot formation and yield in apple trees

Summary

Between 2004 and 2008, the effects of different grafting heights on sylleptic shoots were tested in the apple (Malus domestica Borkh.) cultivars ‘Granny Smith’ and ‘Gloster’, and cumulative fruit yields were evaluated. MM106 apple rootstocks were grafted 10, 20, 40, or 60 cm above soil level in August 2004. The results showed that an increased grafting height significantly decreased tree height in both cultivars. The tallest and shortest trees were observed at grafting heights of 10 cm (153.0 and 170.0 cm) and 60 cm (141.3 and 143.5 cm) in ‘Granny Smith’ and ‘Gloster’, respectively.Among the various grafting heights tested, 60 cm in ‘Granny Smith’ and 20 cm in ‘Gloster’ gave the largest stem diameters (17.6 mm and 16.8 mm, respectively). The number of lateral shoots increased significantly with increased grafting height in both cultivars. The largest numbers of lateral shoots in ‘Granny Smith’ (10.75) and ‘Gloster’ (2.00) were obtained from a grafting height of 60 cm, while 2.55 and zero lateral shoots occurred at 10 cm grafting height in ‘Granny Smith’ and ‘Gloster’, respectively. Shoot lengths decreased significantly by increasing the grafting height. Grafting heights of 10 cm and 60 cm resulted in the tallest and shortest shoots in both cultivars. Cumulative fruit yields were significantly affected by grafting height in both cultivars. The highest yield was found for a 60 cm grafting height in both ‘Granny Smith’ (11.295 kg tree^–1) and ‘Gloster’ (4.818 kg tree^–1).The results of this study suggest that grafting heights of 40 cm and 60 cm have the potential to promote branching and early bearing for apple fruit production in sustainable and organic agricultural systems.     

In vitro propagation of garlic by shoot proliferation

Shoot buds (5–8 mm long), excised from dormant cloves of the New Zealand commercial garlic (Allium sativum L.) and a virus-free French cultivar ‘Rose-de-Kakylis’, proliferated both axillary and adventitious shoots on B-5 basal medium supplemented with 0.5 mg l^?1 isopentenyladenine (2-ip) and 0.1 mg l^?1 naphthaleneacetic acid (NAA). An 8-fold increase in shoot number occurred every 6 weeks. Shoots were readily rooted in B-5 + 0.01 mg l^?1 2-ip + 0.2 mg l^?1 NAA and transferred to pots, where about 70% of the shoots formed established plants. The plants raised by this shoot-proliferation method retained the diploid condition of the parents.     

Evaluation,tissue culture propagation,and dissemination of ‘Saba’ and ‘Pelipita’ plantains in Costa Rica

Fruit characteristics of the disease-resistant bluggoe-type (ABB) cooking bananas (Musa × paradisiaca L.) ‘Saba’ and ‘Pelipita’ were compared with those of the susceptible ‘Currare’ (‘Horn’). Yields of ‘Saba’ and ‘Pelipita’ were similar to those of ‘Currare’; however, ‘Saba’ and ‘Pelipita’ yielded fewer hands with a greater number of small fruit when compared with ‘Currare’. Shoot-tip cultures of both clones were readily initiated on a modified Murashige and Skoog (MS) basal medium supplemented with N⁶-benzyladenine (BA) in combination with indole-3-acetic acid (IAA). Propagation cultures were initiated by splitting shoot tips along their longitudinal axis and re-culturing the individual pieces to basal medium supplemented with 5 mg l^?1 BA. Transfer of axillary shoots to hormone-free medium resulted in rapid and extensive root formation. Plantlet survival after transfer to methyl bromide-treated soil exceeded 90%. Establishment in the field was achieved following procedures normally used for vegetative propagation of this crop. Trial plantings of in vitro propagated ‘Saba’ and ‘Pelipita’ were established in various provinces of Costa Rica for grower evaluation and for future comparison of growth and reproductive development with plants of these and other cultivars propagated from corms.     

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.     

Cost-effective tissue culture media for large-scale propagation of three commercial banana (Musa spp.) varieties

Cost-effective tissue culture protocols have been established for the commercial multiplication of three banana varieties, ‘Rasthali’ (AAB – Silk), ‘Grand Naine’ (AAA – Cavendish), and ‘Udhayam’ (ABB – Pisang Awak). Reverse osmosis water and 3% (w/v) table sugar were used as the low-cost water and carbon source, respectively. Six different gelling agent treatments were tested: sago alone (T1), Isabgol alone (T2), sago + agar (T3), Isabgol + agar (T4), sago + Isabgol (T5), and agar alone as a control (T6). Full-strength Murashige and Skoog (MS) medium supplemented with 3 mg l^–1 6-benzylaminopurine (BAP) and 1 mg l^–1 indole-3-acetic acid (IAA) were used for culture initiation and subculturing. Rooting was accomplished on low-cost MS medium containing 1.0 mg l^–1 α-napthaleneacetic acid (NAA), 1.0 mg l^–1 indole-3-butyric acid (IBA), and 250 mg l^–1 activated charcoal. Statistical analysis indicated that sago + Isabgol (T5) produced the maximum number of shoots (10 per explant) in ‘Udhayam’ and ‘Rasthali’, while sago alone (T1) produced the maximum number of shoots (6 per explant) in ‘Grand Naine’. The genetic stability of tissue-cultured banana plantlets produced using these low-cost substitutes was assessed using inter-simple sequence repeat (ISSR) markers. The results indicated that the ISSR profiles of the five treatments and the control (T6) were similar, indicating genetic stability using these cost-effective tissue culture protocols. Reductions in cost over the control (l^–1 of MS medium) ranged from 65% to 86%, while the per plant production cost was reduced by 12.5%–20.0%. Adoption of these treatments (T1–T5) as low-cost tissue culture protocols for in vitro propagation would reduce production costs significantly, leading to an expansion of the area planted with tissue-cultured banana, thereby increasing productivity.     

Growth of Vitis vinifera L. and Vitis aestivalis Michx. as Affected by Temperature

Abstract

Four European (Vitis vinifera L.) winegrape cvs., ‘Semillon’, ‘Pinot Noir,’ ‘Chardonnay’, and ‘Cabernet Sauvignon’, and one American (Vitis aestivalis Michx.) winegrape cv. ‘Cynthiana’, were subjected to three temperature regimes in growth chambers set at 20/15°C, 30/ 25°C, or 40/35°C, for 16/8 hr day/night to determine the influence of temperatures on vine growth and development. In general, the best temperature for shoot and root growth 28 days after temperature treatments was 20/15°C for ‘Semillon’, ‘Cabernet Sauvignon’, and ‘Cynthiana’, and 30/25°C for ‘Pinot Noir’ and ‘Chardonnay’. Although 40/35°C reduced number of leaves, shoots, tendrils, and internodes, total leaf area (LA), and total shoot biomass of all the cultivars, the reduction was more pronounced in ‘Cynthiana’ than in the European cultivars. The average reduction in number of leaves at 40/35°C for the European cultivars was 47%, compared with 92% for ‘Cynthiana’. The two types of grapes adapted differently to high temperature. Shoot growth in the European cultivars continued under high temperature, whereas growth ceased in ‘Cynthiana’. Roots of ‘Cynthiana’, however, were less susceptible to the adverse effect of high temperatures than were the shoots. This study shows that the European cultivars were relatively more tolerant to high temperature than the American cultivar and they have a potential for production of wine in the climate of south central Kansas.     

Rooting stem cuttings of Chinese chestnut

Rooting of Chinese chestnut (Castanea mollissima Blume.) stem cuttings following treatment with indole-3-butyric acid (IBA) was investigated. Tip and basal cutting from vigorous epicormic shoots and terminal shoots with 2–3 cm of the previous year's wood were taken from mature regions of trees approximately 40 years of age. Cuttings were dipped for 5 s in an aqueous solution of either 3 or 6 g l^?1 IBA. Rooting occurred only in the basal softwood cuttings. Average rooting of 33.5, 5.0 and 1.6% for ‘AU-Leader’, ‘AU-Homestead’ and ‘AU-Cropper’, respectively, was obtained using the 3 g l^?1 IBA solution, and 35.0, 6.7 and 3.3%, respectively, using the 6 g l^?1 IBA solution.     

The use of CPPU for efficient propagation of pineapple

The use of forchlorfenuron (N-(2-chloro-4-pyridyl)-N′-phenylurea) (CPPU) for efficient propagation of pineapples was investigated. About 85% of axillary buds can be forced to sprout by soaking defoliated stem pieces (12 cm in length) in a 2.5 or 5.0 mg l⁻¹ CPPU solution for more than 3 h. The use of old stems taken from the third or fourth ratoon plants had the advantage of less liability to fungal decay, as compared to young stems from the first crop plants. The CPPU treatment combined with the removal of shoots from stems at monthly intervals significantly increased the number of shoots per stem piece (about 15 shoots per piece at 5.0 mg l⁻¹ CPPU), and resulted in a more uniform shoot size (the percentage of shoots within a range of 5–15 cm in length was about 90% at 5.0 mg l⁻¹ CPPU). The rooting of shoots was easily promoted within 1 month by treating the basal portion of shoots with 20 mg l⁻¹ indole-3-butyric acid (IBA) for 15 min. The CPPU method required about 5 months for plantlet propagation. From these results, we found that pineapple plantlets could be efficiently propagated by the following method: (1) soaking defoliated stems in a 2.5–5.0 mg l⁻¹ CPPU solution for more than 3 h; (2) harvest of developed shoots from the stems at regular intervals; and (3) promotion of rooting on the shoots at 20 mg l⁻¹ IBA for 15 min.     

In vitro colchicine-induced polyploid plantlet production and regeneration from leaf explants of the diploid pear (Pyrus communis L.) cultivar, ‘Fertility’

Summary

Polyploid plantlets, including triploid, tetraploid, and mixoploid, were induced from the European pear (Pyrus communis L.) cultivar ‘Fertility’ by in vitro colchicine treatment of leaf explants. The leaf explants were incubated in 0.4% (w/v) colchicine for 24, 48, or 72 h, then transferred to adventitious shoot-induction medium. Regenerated shoots were pre-selected according to their morphological characteristics when compared to control shoots from untreated shoot proliferation cultures. Shoots with putative polyploid morphological characteristics were maintained and proliferated. The ploidy levels of all putative polyploid individuals were analysed by flow cytometry and identified by chromosome counts of shoot tip tissue squashes. Polyploid shoots were rooted, and the resulting plantlets were transferred to the field. Polyploid plantlets had a higher specific leaf mass and larger stomata than those of diploid plantlets.     

Video evaluation of ethylene sensitivity after anthesis in carnation (Dianthus caryophyllus L.) flowers

Differences in ethylene sensitivity among carnation (Dianthus caryophyllus L.) cultivars were evaluated using a time-lapse video recording system. Measurement of time to petal inrolling of ‘White Sim’, ‘Nora’, ‘Chinera’, and line 64-54 flowers subjected to a range of 1–20 μl l⁻¹ ethylene showed that 10 μl l⁻¹ was the optimum concentration for sensitivity evaluation with our video system. With this system we found clear differences in ethylene sensitivity among 10 cultivars and one line. ‘Candy’, ‘Pallas’, ‘Chinera’, and line 64-54 had lower ethylene sensitivities than the other seven cultivars. Line 64-54 had the longest ethylene response time (20.6 h to start of petal inrolling). Video monitoring is a simple and accurate way of evaluating ethylene sensitivity. We have also used the system to study changes in the ethylene sensitivity of carnation flowers after anthesis. We were able to detect a shift in responsiveness to ethylene that was impossible to detect by previous methods. In the Sim-type carnation cultivars tested (‘White Sim’, ‘Scania’, ‘U Conn Sim’, and ‘Nora’), ethylene sensitivity after anthesis decreased significantly with age in both early-cut and late-cut flowers. These results clearly showed that decline of ethylene sensitivity is caused by the increasing physiological age of flowers. Ethylene sensitivity after anthesis decreased with age in normal Sim-type carnations in the same way as in long-vase-life variants such as ‘Sandrosa’.     

Influence of growth regulators on setting,retention and weight of fruits in two cultivars of litchi

Investigations were undertaken to explore the possibility of improving setting, retention and weight of fruits in ‘Early Seedless’ and ‘Calcuttia’ cultivars of lichi (Litchi chinensis) by means of growth regulators. Indole acetic acid (IAA) at 20, 40 and 80 mg l^?1, 2,4-dichlorophenoxy acetic acid (2,4-D) at 2,4 and 8 mg l^?1 and gibberellic acid (GA₃) at 50, 100 and 150 mg l^?1 were sprayed on panicles in the first fortnight of April, when 50–100% flowers had opened. All 3 growth regulators caused a favourable effect on fruit setting, fruit retention and weight of individual fruits, but IAA at 20 mg l^?1 proved the best for enhancing setting, GA₃ at 50 mg l^?1 for increasing retention and GA₃ at 100 mg l^?1 for improving fruit weight. IAA and GA₃ should, therefore, be used in combination. Between the 2 cultivars tested, ‘Calcuttia’ proved superior to ‘Early Seedless’ in fruit setting, fruit retention and weight of individual fruits.     

Factors affecting the quality and in vitro germination capacity of strawberry pollen

Summary

Poor pollen quality and germination capacity curtails early yield in strawberry. The aim of this study was to establish a reliable method for in vitro assessment of strawberry pollen germination ability and to investigate further the effects of photoperiod and gibberellin on pollen germination and quality. In the first part of the study, pollen from seven strawberry cultivars (Chandler, Selva, Tudla, Camarosa, Eris, Pajaro and Irvine) was collected and its germination capacity and incidence of deformed pollen grains assessed in vitro using the hanging-drop technique. Highest germination rates, in ‘Selva’, were observed in a nutrient medium of 10% sucrose. Addition of calcium nitrate to the medium decreased the germination percentages of all cultivars. There was no significant difference, on average, between the germination rate at 20° and 25°C. Genetic factors affected the incidence of deformed pollen grains significantly, with ‘Pajaro’ showing the highest percentage (76%). In the second part, groups of young strawberry plants, cultivar Seascape, grown either under natural early spring conditions or under long-day or short-day conditions were sprayed once with GA₃ at 0, 50, or 200 mg l^–1. Pollen germination and deformation and stamen length were assessed three months later. In plants of the first group, GA₃ at 50 mg l^–1 increased pollen germination and decreased the incidence of deformed pollen grains, while GA₃ at 200 mg l^–1 decreased pollen germination without affecting the formation of deformed pollen grains. Plants of the second group showed a higher rate of pollen germination under long than under short days. GA₃ at 200 mg l^–1 decreased pollen germination under either short- or long-day conditions compared with the controls but doubled the percentage of deformed pollen only under short days. Stamens in control plants grew four times as long under long- than short-day conditions. GA₃ did not affect stamen length under long days but significantly enhanced their growth under short days.     

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.     

The Incorporation of Growth Hormones in Seed Dressings

Summary

The consequences of using ex vitro, single-node explants from different topophysical positions in chrysanthemum (Chrysanthemum grandiflorum /Ramat./ Kitam) were determined. In particular, how explant topophysis affected the rate of propagation, which is important for the successful micropropagation of chrysanthemum. Uniform shoots of five cultivars of chrysanthemum, cultured in vitro, were each divided into three equal zones: distal, central, and proximal. Two single-node explants were isolated from each zone and cultured on MS medium without any added growth regulators. After 10 weeks of culture, 50% of the shoots that had developed from axillary buds on each single-node explant were excised and measurements were taken in order to compare those shoots that had developed from explants from the different topophysical zones. The remaining shoots were sub-cultured on rooting medium. After 4 weeks, the numbers of roots per plantlet, and the total fresh weight (FW) of roots were recorded. The cultivars fell into two groups. ‘Lady Amber’, ‘Lady Orange’, and ‘Lady Vitroflora’ explants were topophysis-dependent, while ‘Lady Bronze’ and ‘Lady Rosy’ explants were topophysis-independent. For the three topophysis-dependent cultivars, the propagation rate, growth rate, shoot length, internode length, single leaf weight, and total plantlet FW values were highest for those shoots derived from the central and proximal zones. Topophysis failed to affect the number of leaves per shoot or the number of days between the appearance of two successive leaves. The effects of topophysis on the number of roots per plantlet and on root FW were inconsistent. The unequal growth of chrysanthemum plantlets during in vitro micropropagation can be an effect of topophysis, and this phenomenon is cultivar-specific in chrysanthemum.     

Effects of IAA and BA on development of globular,heart- and torpedo-stage embryos from cell suspensions of three grape genotypes

In order to initiate cell and embryo suspensions, embryogenic calluses derived on NN solid medium with 2,4-D and BA from petioles of in vitro grown plants of three interspecific grapevine hybrids were cultured in three versions of liquid NN medium: (1) without growth regulators, (2) 0.1 mg l⁻¹ IAA and (3) 0.5 mg l⁻¹ BA. Cell and embryo suspensions were incubated two and three times in these versions of liquid media in various combinations. Incubating suspensions two times in hormone-free media and/or with 0.1 mg l⁻¹ IAA led to formation of globular embryos in the three cultivars studied and small numbers of heart-stage embryos in ‘Podarok Magaracha’ and ‘Intervitis Magaracha’. Numerous heart-stage embryos developed in ‘Intervitis Magaracha’ and ‘Podarok Magaracha’ when the suspensions had been initiated in medium with 0.5 mg l⁻¹ BA, and in ‘Bianca’ this was achieved after two incubations in the above medium. Torpedo-stage embryos were formed after subculturing heart-stage embryo suspensions in medium with 0.1 mg l⁻¹ IAA in all study cultivars. If only small numbers of embryos of a certain developmental heart- or torpedo- stage were formed, such cell and embryo suspensions need to be repeatedly subcultured in liquid medium with specific growth regulators to enable this process.