Black Currant Leaf Spot: II. Laboratory Tests of Fungicides for the Prevention of Sporing of Pseudopeziza Ribis on Overwintered Leaves

Summary

We have developed a two-step procedure for rooting of tea microshoots in vitro. The effectiveness of different auxin treatments for root formation was found to differ. Among the auxins tested, 25 μM -naphthalene acetic acid (NAA) gave the best results, with 100% rooting, compared to 25 μM indole-3-butyric acid (IBA) or 25 μM indole-3-acetic acid (IAA), which induced 17% and 58% rooting, respectively. Incubation of tea microshoots on 0.33 Murashige and Skoog (MS) medium, supplemented with 25.0 μM NAA or 175.0 μM IBA for 10 d, followed by transfer to auxin-free 0.33 MS medium resulted in 100% rooting, whereas 50.0 μM IAA induced 91.7% rooting. Besides the different auxin treatments, the strength of the MS medium, the duration of incubation of microshoots in auxin-containing medium, the sucrose concentration, the gelling agent, the pH of the medium, the incubation temperature, the light intensity, and the quality of the shoots also played a significant role during in vitro rooting of micropropagated tea shoots. Among the combinations tested, the most effective results were obtained when green microshoots were incubated on 0.33 MS medium supplemented with 25.0 μM NAA, 50.0 mM sucrose, pH 5.5, gelled with 0.2% (w/v) Phytagel^TM for 10 d at 25° – 30°C at a light intensity of 40 μmol m^–2 s^–1, followed by transfer of shoots to auxin-free 0.3 MS medium. This resulted in 100% rooting and, on average, 11 long roots were formed per shoot. Anatomical changes during adventitious rooting of micropropagated tea shoots in vitro were also studied to understand the process of rooting.     

Integration of the rolA gene into the genome of the vigorous apple rootstock A2 reduced plant height and shortened internodes

Summary

The aim of this work was to dwarf the vigorous apple rootstock A2 by insertion of the rolA gene. To optimize conditions for a successful transformation, regeneration tests were carried out. The use of sucrose in the regeneration medium gave higher regeneration frequency than sorbitol in some cases and the shoot number per regenerated leaf was higher at 10 |j.M TDZ compared with 2.5 |j.M TDZ on the sucrose medium. Two transgenic clones, verified by PCR and Southern analysis, have been obtained on the sucrose medium together with 2.5 |j.M TDZ and 1.0 or 2.5 |j.M NAA and wounding by forceps. The two clones, named LAI and LA2, contained both the rolA and nptll genes. The results of in vitro rooting showed that LAI had a lower rooting percentage and a reduced root number per rooted shoot than the untransformed control shoots and the clone LA2 on the rooting medium containing 5 |JLM IBA. Growth analysis revealed that both transgenic clones had a reduced plant height and a shortened internode length compared with the control plants. However, the node number and the stem diameter were significantly larger for clone LAI than clone LA2 and the control plants.     

The effect of light,darkness and temperature on micropropagation of the pear rootstock BP10030

There was no effect of irradiance level on surviving percentages of shoot tip explants of the pear rootstock BP10030, but low irradiance stimulated the initial growth of the explant. Irradiance had a strong effect on shoot multiplication. With an increase in photosynthetic photon flux (PPF) from 10 to 80 μmol m^?2s^?1, shoot number and length and shoot fresh and dry weights increased. The greatest number of shoots and the longest ones were obtained with a 16 h photoperiod, while the highest fresh and dry weight of shoots were produced with a 24 h photoperiod. Rooting percentage and the number of roots were markedly promoted under 80 μmolm^?2s^?1 PPF. Photoperiods of 8, 16 and 24 h produced similar effects on rooting percentages and the numbers of roots. Four to seven days of darkness were the optimum for rooting. Rooting percentage and the number of roots increased with increased temperature during darkness between 5 and 25°C. A further increase in dark temperature up to 30°C reduced rooting percentage and root number.     

Effects of media,carbon sources and cytokinins on shoot organogenesis in the Christmas tree Scots pine (Pinus sylvestris L.)

Summary

Significant effects of seven basal media and three carbon sources (sucrose, glucose and fructose) on the induction of adventitious buds from embryos of Pinus sylvestris L. were observed. Moreover, hyperhydricity of expiants and shoot regenerants was observed on basal media containing fructose, especially with half-strength Murashige and Skoog (MS), MS, and woody plant medium (WPM). Expiants grown on a Gresshoff and Doy (GD) medium with sucrose produced the highest frequency of regeneration (81%) and with no hyperhydricity observed of developing adventitious shoots. Among three cytokinins tested including BA, BPA, and TDZ (at four concentrations each), 5 μM BA resulted in the highest regeneration frequency and mean number of adventitious shoots per embryo. Shoot régénérants were elongated after transfer to a GD medium containing 2 g-l_–1 activated charcoal and no growth regulators. After one month, rooting was induced on 10% of expiants.     

Micropropagation of Prunus scoparia,a Suitable Rootstock for Almond under Drought Conditions

Prunus scoparia is a wild deciduous shrub, usually living on dry calcareous soils of the rocky mountains and has been used as a grafting rootstock for domesticated almonds to provide drought resistance. In the current study, micropropagation ability of P. scoparia was investigated using cytokinin and auxin. Uniform nodal shoot pieces (3–5 cm in length) of seedlings were used as explants. The explants were disinfected with 10% sodium hypochlorite solution. For adventitious shoot induction and proliferation, Murashige and Skoog (MS) media containing 7.00 g/l agar and 30.00 g/l sucrose containing five concentrations of benzyl adenine (BA) (0.00, 0.50, 1.00, 2.00, and 4.00 mg/1) and also containing six concentrations of Thidiazuron (TDZ) (0.00, 0.50, 1.00, 2.00, 5.00, and 7.00 mg/1) were compared. For rooting, in vitro shoots (2–3 cm) were transferred into ½ MS medium supplemented with 30 g/l sucrose, 7.50 g/l agar, and different concentrations of IBA (0.00, 0.25, 0.50, and 1.00 mg/l) and NAA (0.00, 0.25, 0.50, and 1.00 mg/l). Based on the results obtained for shoot proliferation, only 2.00 and 4.00 mg/l BA and 2.00 mg/l TDZ concentrations generated shoots, while other treatments did not show shoot proliferation. Among the three treatments that generated shoots, the best results for shoot number, leaf number, and leaf color quality were observed in media containing 2.00 mg/l TDZ. Based on the results obtained for rooting, the effect of IBA concentrations on the rooting percentage, root number, and root length was significant. Among IBA concentrations, only 0.50 mg/l IBA induced rooting, while there was no rooting in the media containing other IBA concentrations. None of the NAA concentrations showed rooting. In conclusion, MS culture medium supplemented with 2.00 mg/l TDZ and ½ MS culture medium supplemented with 0.50 mg/l IBA are suggested for in vitro shoot proliferation and rooting of P. scoparia, respectively. The results presented herein could be used for in vitro selection and micropropagation of P. scoparia.     

Progress towards clonal propagation of Camellia japonica cv. Alba Plena by tissue culture techniques

Summary

Optimal rooting conditions have been determined for shoot cultures of Camellia japonica cv. Alba Plena derived from a 50 year old tree: dipping the base of shoots in 1 g l^?l solution for 15 min, followed by 12 days’ darkness, induced 87% rooting in shoots cultured with Woody Plant Medium (WPM) macronutrients. Halving the auxin concentration or exposure time considerably reduced this rate, and root formation was severely inhibited if an initial dark period for the entire shoot was not used. The type of support (agar or paper bridges) did not significantly affect the rooting percentage or number of roots per rooted shoot, but liquid media induced greater root elongation. No significant differences were observed between the use of WPM and a modified Heller’s medium as regards the rooting percentages achieved, but the number of roots formed was considerably greater with WPM, as was the survival rate after transfer to soil (70% for transfer as against 35% with the modified Heller’s medium). Best survival rates were achieved when shoots were transferred to the acclimatization soil 12 days after auxin treatment, i.e. immediately after the dark period.     

Clonal propagation of Poncirus trifoliate through culture of shoot primordia

Summary

In Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.     

Partitioning sources of rooting potential in plum hardwood cuttings

SUMMARY

Rooting of leafless winter hardwood cuttings of the plum rootstock Prunus insititia ‘Pixy’ increased as the location from which the shoots were taken from within specially grown stockplants decreased in height above ground, associated with a parallel reduction in shoot thickness. However, the actual height of the least-ready-rooting crown cuttings had no effect on rooting, suggesting that relative rather than absolute position is important. Rooting was unaffected by bark-ringing and trunk incision distal to the shoot position, suggesting that such treatments did not interfere with a basipetally translocated root promotor which might have accounted for improved rooting of cuttings in the lower parts of the hedge. The rooting of crown cuttings above a bark ring was reduced considerably compared with that of cuttings from normal bushes, and this was associated with increased thickness of shoots distal to the ring. Delaying pruning in spring until after growth had started resulted in thinner crown shoots compared with those from plants pruned normally while dormant, and the rooting of these thinner crown shoots was much higher than that of the normal crown cuttings. It was shown by covariance analysis that shoot thickness accounted for part of the rooting response but could not account for the total effect due to shoot position within the bush, ringing, or time of pruning. Competence to root appears to develop independently in individual shoots, modified by a shoot thickness factor which favours the subordinate shoots induced in the shoot hierarchy of severely pruned hedges.     

糖和氮对山楂组织培养新梢生根的影响

采用敞口山楂组培新梢研究糖和无机氮源对不定根形成的影响,含有高水平蔗糖的培养基比低水平蔗糖的培养基新梢生根率高。新梢对糖的这种反应与糖代谢和物理渗透都有关,用甘露醇作为渗透代替剂在低浓度蔗糖存在下无作用,当蔗糖浓度达到一定程度时(86.64mmol·1~(-1))能影响新梢的生根率。在培养基中分别加入NO_3~(-)-N或NH_4~(+)-N作为唯一氮源,或是在MS培养基中加入NO_3~-/NH_4~+比率为标准的混合氮源,在测试范围(0～120mmol·1~(-1))内均降低新梢的生根率,三者对生根的抑制作用大小顺序依次为NH_4~+>NO_3~(-)-NH_4~(+)-N>NO_3~(-)-N.NO_3~-/NH_4~+的比率影响生根。NO_3~-/NH_4~+比率与生根之间呈直线正相关。糖/氮比值高有利于生根。插穗中碳、氮的绝对量及碳/氮比率可作为山楂新梢生根的指标。     

Improved shoot regeneration from nodules of Charybdis numidica in a temporary immersion system

Summary

A procedure for improved shoot regeneration from meristematic nodules of Charybdis numidica in a temporary immersion culture system was developed by optimizing immersion frequencies, volume of the nutrient medium, and alternating application of growth regulators. Modified liquid MS medium with 3% sucrose, 20 μM BAP and 5 μM NAA (shoot induction medium) was used to induce microshoot formation on 15 g of nodules in 1000 ml bottles. Volumes of medium (250 or 500 ml) and immersion frequency (5 min every 12 or 24 h) did not significantly influence shoot regeneration rates. Shoot induction medium additionally supplemented with 5 μM paclobutrazol in most cases led to less shoots but this effect was not significant, either. Microshoots formed under these conditions were severely hyperhydrated. Nearly complete elimination of hyperhydricity and enhanced formation of properly elongated shoots were achieved by running a shoot induction step with induction medium containing paclobutrazol followed by an elongation step with a medium supplemented only with 5 μM gibberellic acid GA₃. This two-step procedure yielded about 900 healthy shoots per bottle after a two-month cultivation period. Root induction was performed ex vitro during acclimatization and the plantlets could be established in the greenhouse with good success.     

Influence of FeEDDHA on in vitro rooting and acclimatisation of red raspberry (Rubus idaeus L.) in peat and vermiculite

Summary

We investigated the dependence of in vitro rooting and acclimatisation to greenhouse conditions on the source of iron used in the shoot multiplication and rooting media using five raspberry (Rubus idaeus L.) cultivars (‘Beskid’, ‘Canby’, ‘Malling Seedling’, ‘Norna’, and ‘Veten’). Ethylenediamine di-2-hydroxy-phenyl acetate ferric (FeEDDHA) in the rooting medium led to higher chlorophyll contents, earlier and more abundant rooting (8.7 vs. 5.3 roots per shoot), 30% higher fresh and dry weights, and thus higher quality microplants than ethylenediaminetetra-acetate ferric sodium (FeEDTA). Higher quality microshoots had a beneficial effect on acclimatisation (i.e., percentage survival and length of shoots) when the microplants were planted in a peat-based substrate; however, when planted in vermiculite, the initial differences disappeared during a 4 week-long growth period in a greenhouse.     

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.     

In vitro regeneration and conservation of wild species of Arachis

Summary

Wild species of Arachis are restricted to South America and generally occur in regions under intensive environmental disturbance. Both in situ and ex situ conservation strategies are required in order to maintain the availability of these genotypes. This work developed in vitro regeneration systems from seed explants of 17 wild species of Arachis from six Sections (Heteranthae, Caulorrhizae, Triseminatae, Erectoides, Procumbentes and Arachis). After seed disinfection, embryonic axes, leaflets and cotyledons were excised aseptically and cultured on Murashige and Skoog (MS) medium supplemented with 8.8 µM, 22 µM or 110 µM 6-benzylaminopurine (BAP). Cultures were maintained in a growth chamber at 28° ± 2°C with a 16 h photoperiod. Regeneration patterns from seed explants were similar among species from all Sections. Embryonic axes produced plants through meristematic amplification or multiple shoot formation, while cotyledons and embryonic leaflets produced shoots at significantly lower frequencies through direct and indirect organogenesis, respectively. Shoots obtained from all explants were transferred to MS medium without growth regulators to induce root formation.     

Effects of Trichoderma harzianum strain T-22 on the growth of two Prunus rootstocks during the rooting phase

Summary

Trichoderma harzianum strain T-22 (T22) is one of the most effective strains of this fungus that is able to colonise the roots of most plant species across a wide range of soil types. This fungus is used as a biocontrol agent during crop production, and for the improvement of the rooting and acclimatisation phases in plant nurseries. In vitro-cultured shoots of GiSeLa6^® (Prunus cerasus P. canescens) and of GF677 (P. amygdalus P. persica), two important Prunus varieties used as commercial rootstocks, were inoculated with T22. The results showed that early inoculation of the fungus (at the stage of shoot transfer to root-inducing medium) seriously damaged both GiSeLa6^® and GF677 plants; whereas, following later inoculation (7 d after shoot transfer to root-inducing medium), the plants survived and showed significant increases in shoot growth and root development. In particular, root lengths in GiSeLa6^® and GF677 plants increased by 180% and 136%, respectively, compared to non-inoculated controls. Microscopic analysis revealed T22 hyphae spreading on the root surface in GiSeLa6^® (fungus colonisation frequency = 20%), but not in GF677 roots. Our results demonstrate that the application of T22 during the rooting phase resulted in greater shoot lengths, as well as increased numbers of leaves, roots, and stem diameters. These morphological characteristics could increase the quality and viability of nursery planting material and provide advantages during the plant acclimatisation phase.     

Root shoot interactions in passionfruit (Passiflora sp.) under the influence of changing root volumes and soil temperatures

Summary

Passionfruit are grown in the tropics and subtropics where mean monthly soil temperatures at 15 cm range from about 10° to 30°C. The choice of rootstock can also influence production with most industries exploiting either the purple (Passiflora edulis f. edulis) or golden passionfruit (P. edulis tflavicarpa). We examined the relationship between shoot and root growth in purple x golden hybrid E-23 grafted onto golden passionfruit seedlings. Growth was manipulated by varying the volume of the soil available to the roots or temperature of the root zone. Shoot and root growth increased as root zone volume increased from 0.3, 1.4, 4, 12 to 24 1. Shoot weight (W_s) was correlated with root weight (W_R):W_S = 12.697 + 5.272 W_R + 0.195 W_R² (r² = 91%, P<0.001), with the plants allocating a smaller proportion of dry matter to the roots as root weight increased. Differences in shoot growth with pot volume were not due to changes in water or nutrient status. In the temperature experiment, the two critical root zone temperatures at 90% of maximum growth were about 20° and 35° C for vine extension, leaf area, node and leaf production, and 20° and 30°C for flower production. Leaf and stem dry weight were optimal between about 18° and 34°C, while maximum root growth occurred at 38°C. There was a weak relationship between shoot (W_s) and root dry weight (W_R): W_s = ?19.346 + 24.500 W_R ?1.046 W_R² (r² = 53%, .P<0.001). Apparently, variations in shoot growth at different soil temperatures cannot be explained solely by differences in root growth. Reduced growth at 10°C was associated with lower chlorophyll concentration, stomatal conductance and net CO₂ assimilation, but not lower leaf water potential. The concentration of most nutrients were lower at 10°C than at higher temperatures, but none was outside the range which would be expected to restrict growth. There appears to be a co-ordination of shoot and root growth as the soil volume available for root growth increases, whereas root temperature affects the roots and tops differently. The results of the pot volume experiment demonstrate the importance of rootstock vigour in passionfruit breeding. Productivity would be affected in cool subtropical areas with soil <20°C and in tropical areas with soil >30°C.     

Effect of agar,galactomannan and indole-butyric acid on in vitro rooting of the pear cultivar ‘Durondeau’ and apple rootstock cultivar ‘Marubakaido’

Summary

The influence of partial substitution of agar by galactomannans in culture media supplemented with different concentrations of indole-3-butyric acid (IBA) was studied on in vitro rooting of pear (Pyrus communis L.) cultivar ‘Durondeau’ and apple rootstock (Malus prunifolia Borkh.) cultivar ‘Marubakaido’. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum) seeds. The results obtained with mixtures of agar and galactomannan (3 g l^–1 each) were compared with those from media solidified with a standard concentration of agar (6 g l^–1). The rooting of pear shoots was enhanced significantly in the presence of a mixture of agar plus cassia galactomannan compared to medium solidified with agar only. The modified media promoted a higher number of roots than the control, and increased the percentage of rooted shoots. A maximum of 84.8% rooting was obtained on half-strength MS medium (¹?2MS) supplemented with 0.49 µM IBA and solidified with a blend of agar plus cassia galactomannan. For the apple rootstock, only the number of roots per shoot was influenced significantly by the addition of galactomannan to the rooting medium. The highest number of roots per shoot was 16.67 on ¹?2MS medium gelled with a mixture of agar plus guar galactomannan supplemented with 4.90 µM IBA. The behaviour of the agar-galactomannan gel and the possibility of reduced costs when compared with systems containing only agar, suggest new biological and commercial applications for galactomannans.     

同源四倍体青花菜离体再生及其倍性鉴定

以同源四倍体青花菜带柄子叶为外植体,研究其离体再生体系。结果表明： 10 d苗龄外植体在MS + 0.05 mg·L^-1 NAA + 3.0 mg·L^-1 6-BA+0.8% 琼脂 + 3% 蔗糖培养基上不定芽的再生频率最高为63.3％;继代培养(MS + 0.04 mg·L^-1 NAA + 4.0 mg·L^-1 6-BA + 0.8% 琼脂 + 3% 蔗糖)增殖系数为6,诱导(1/2MS + 0.1 mg·L^-1 NAA + 0.7% 琼脂 + 3% 蔗糖)生根率100％,移栽成活率90％。流式细胞技术分析及染色体计数鉴定结果表明,再生植株的DNA相对含量为二倍体的2 倍,染色体数为2n=4x=36。     

车轮梅茎段高效再生体系的建立

 以车轮梅 (Rhaphiolepis indica L.)茎段为外植体。探讨基本培养基种类、植物生长调节剂组合、蔗糖浓度和AgNO3等因素对茎段器官发生和植株再生的影响。结果表明：MS+6-BA 2.0 mg·L^-1 +NAA 0.5 mg·L^-1 + AgNO3 1.0 mg·L^-1+蔗糖40 g·L^-1培养基最适合不定芽的分化和增殖,不定芽分化率达90%以上,平均每外植体分化不定芽数达5.38个。不定芽可在1/2MS培养基中有效伸长,适宜生根培养基为1/2MS+NAA 1.0 mg·L^-1,生根率达到100%。     

Cryopreservation of in vitro almond shoot tips by vitrification

Summary

Shoot tips of two almond scion cultivars, ‘Ne Plus Ultra’ and ‘Nonpareil 15-1’, and one almond/peach hybrid rootstock were successfully cryopreserved using a one-step vitrification technique. Three week old in vitro cultures were cold-hardened at 4°C on the multiplication medium (Murashige and Skoog for ‘Ne Plus Ultra’ and the hybrid rootstock; Almehdi and Parfitt for ‘Nonpareil 15-1’) for three weeks. Shoot tips, 2–2.5 mm long, were excised and precultured for 1 d at 4°C on the same basal medium, without plant growth regulators, supplemented with 0.7 M sucrose. After the preculture, the shoot tips were incubated in vitrification solution at 25°C for 45 min for the almond scion cultivars and 60 min for the hybrid rootstock, and then stored under liquid nitrogen (LN) for at least 3 d. After rapid thawing at 30°C, the shoot tips were washed with the appropriate liquid basal medium containing 1.0 M sucrose and then cultured on the same basal medium, solidified with agar, but excluding NH₄NO₃ or (NH₄)₂SO₄. Shoot regeneration was usually observed within 2–3 weeks. Survival after LN, recorded as the percentage of shoot tips that produced at least one new shoot four weeks after thawing, was 87.5, 60.0 and 72.5% for ‘Ne Plus Ultra’, ‘Nonpareil 15–1’ and the hybrid rootstock respectively. The one-step vitrification method is a promising simple technique for cryopreserving almond scion and rootstock shoot tips from in vitro cultures.     

Adventitious shoot regeneration from leaf segments of in vitro cultured shoots of the apple rootstock Jork 9

SUMMARY

Adventitious shoot formation was investigated using leaf segments of in vitro cultured shoots of the apple rootstock Jork 9. Regeneration capacity was influenced by the pretreatment of the mother shoots, macroelements, hormone concentrations, the gelling agent and the carbohydrate source. The highest regeneration rate and most shoots per leaf explant resulted from young leaves on a medium based on MS macroelements supplemented with 22 µM BAP and 0.1 µM_NAA together with sorbitol, at concentrations of 165 mM or 220 mM. Sorbitol was more effective than sucrose, glucose, fructose or a combination of these sugars. A cold and dark pretreatment of the shoots enhanced the formation of adventitious shoots.