Effects of short light-dark regimes on in vitro shoot rooting of some fruit tree rootstocks

Experiments were carried out to evaluate the effects of 4/2 light-dark cycles (4 h of light followed by 2 h of dark) on the rooting responses of shoots cultivated in vitro of the fruit tree rootstocks GF 677 (peach × almond hybrid), Mr.S. 2/5 (Prunus cerasifera), MM 106 (apple Nothern Spy × Paradise M1) and BA 29 (Cydonia oblonga). Under this light regime rooting percentage of GF 677, Mr.S. 2/5 and MM 106 shoots reached 100 % as in the control treatment (16/8), while in BA 29 shoots, short light-dark cycles increased rooting response by about 65 %. Moreover, the shoots of all rootstocks submitted to the 4/2 cycle showed an appreciable increase in root number and length, and an earlier root emergence of about 4 – 5 d compared to the 16/8 cycle. Finally, rooting percentage of BA 29 shoots submitted to the 4/2 light regime and treated with 0.2 mg dm⁻³ indolebutyric acid (IBA), was equal to that reported with 0.4 mg dm⁻³ IBA under the 16/8 regime, indicating that the former light regime also amplified the rhizogenic effect of auxin.     

In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants: influence of activated charcoal

The ability for adventitious rooting of micropropagated shoots from juvenile and mature Pinus pinaster Ait. explants was assessed in vitro on a rooting expression medium. The different rooting traits observed, namely the rooting rate, the number and the length of the adventitious roots, and the root score, were greatly influenced by the age of the donor plant: 98% of juvenile explants rooted, while only 49% of mature explants did. Addition of activated charcoal in the rooting expression medium improved the overall rooting capacity of the mature explants to an average of 78%. Whatever the plant material, the number and the length of the adventitious roots, as well as the root score, fluctuated according to the sampling date.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid - REM rooting expression medium - RIM rooting induction medium     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

Identification of QTLs for root characteristics in maize grown in hydroponics and analysis of their overlap with QTLs for grain yield in the field at two water regimes

We investigated the overlap among quantitative trait loci (QTLs) in maize for seminal root traits measured in hydroponics with QTLs for grain yield under well-watered (GY-WW) and water-stressed (GY-WS) field conditions as well as for a drought tolerance index (DTI) computed as GY-WS/GY-WW. In hydroponics, 11, 7, 9, and 10 QTLs were identified for primary root length (R1L), primary root diameter (R1D), primary root weight (R1W), and for the weight of the adventitious seminal roots (R2W), respectively. In the field, 7, 8, and 9 QTLs were identified for GY-WW, GY-WS, and DTI, respectively. Despite the weak correlation of root traits in hydroponics with GY-WW, GY-WS, and DTI, a noticeable overlap between the corresponding QTLs was observed. QTLs for R2W most frequently and consistently overlapped with QTLs for GY-WW, GY-WS, and/or DTI. At four QTL regions, an increase in R2W was positively associated with GY-WW, GY-WS, and/or DTI. A 10 cM interval on chromosome 1 between PGAMCTA205 and php20644 showed the strongest effect on R1L, R1D, R2W, GY-WW, GY-WS, and DTI. These results indicate the feasibility of using hydroponics in maize to identify QTL regions controlling root traits at an early growth stage and also influencing GY in the field. A comparative analysis of the QTL regions herein identified with those described in previous studies investigating root traits in different maize populations revealed a number of QTLs in common.     

Benzyladenine-induced adventitious shoot regeneration from hypocotyls of adzukibean (Vigna angularis {Willd.} Ohwi & Ohashi)

A complete in vitro plant regeneration systemvia adventitious shoot-bud formation from seedlinghypocotyls of adzukibean was developed. Cotyledonarynode (CN) and root explants from 4-d-old invitro-germinated seedlings of 4 cultivars werecultured on agar-solidified R medium consisting of MSsalts, B5 vitamins, 3.0% (w/v) sucrose and 4.4 µM6-benzyladenine (BA). Shoot buds arose adventitiouslyat the basipetal cut of the hypocotyl in 40 to 85%of type 1 CN (8–10 mm) explants 6 weeks later with cvsBansei aki azuki and Tanbadainagon azuki exhibitingsignificantly higher response. Shorter type 2 (4–5 mm) CN explants responded poorly (0–6%) in culturewhile intact roots were regenerative but at lowerfrequencies (5 to 31%). In cv Bansei aki azuki,optimum shoot initiation (78.7%) was achieved whentype 1 CN explants from BA-preconditioned seedlingswere re-cultured in R medium (SC0). BA supplied at8.8 µM during SC0 significantly reduced the shootinitiation response to 46.7%. In the same manner,substituting equimolar concentration (4.4 µM) ofkinetin (KN), zeatin (Z) or thidiazuron (TDZ) to BAduring SC0 yielded no or significantly lowerresponses. Mean shoot production ranged from 6.2 to9.8 shoots per explant 6 weeks after subculture (SC1)and varied significantly among the 4 cultivars used. Efficient rooting (93–96%) was achieved on excisedshoots transferred to MS salts + B5 vitamins basalmedium alone. The tissue culture system covered only16 weeks to completion. Regenerated plantlets weretransferred to soil with 86–100% survival rate andall surviving plants were morphologically normal andseed-fertile.     

The influence of ethylene on in vitro rooting of GF 677 (Prunus persica × Prunus amygdalus) hybrid peach rootstock

Summary Treatments differing from each other for the type of tube closure (i.e., cotton plug for free gas exchange, airtight rubber cap, and rubber cap with ethysorb) and/or rooting culture medium (i.e., enriched or not by 25 to 100 μM acetylsalicylic acid) were compared for their effects on gaseous composition of the culture atmosphere and microcutting rooting of the GF 677 (Prunus persica × Prunus amygdalus) hybrid. Rubber capping, which leads to rapid ethylene accumulation inside tubes, strongly reduced rooting time and in some cases enhanced final rooting percentage over that of cotton plugs. Ethysorb almost completely absorbed ethylene produced by shoots, which showed lower rooting percentages within 9 d than microcuttings cultured in the absence of ethysorb. In contrast, no significant difference in rooting was found between the two treatments after 14 d. Carbon dioxide concentration was similar in all treatments within 5 to 9 d and seemed to be ineffective for rooting. The influence of acetylsalicylic acid on rooting was unclear. Root number and length were not significantly influenced by the treatments. These results demonstrate that the use of airtight closures, leading to rapid ethylene accumulation, can reduce time of rooting expression for GF 677 microcuttings. However, free gas exchange towards the end of the rooting period (from Day 9 to Day 14) is advisable to prevent leaf yellowing. No significant difference in plantlet survival and growth after transfer ex vitro was found among treatments.     

N,N′-bis-(2,3-Methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious rooting in Pinus radiata and affect expression of genes induced during adventitious rooting in the presence of exogenous auxin

We have analyzed the effect of N,N′-bis-(2,3-methylenedioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methylenedioxyphenyl)urea (3,4-MDPU), two symmetrically substituted diphenylurea derivatives with no auxin or cytokinin-like activity, on the rooting capacity of Pinus radiata stem cuttings. Results indicate that both diphenylurea derivatives enhance adventitious rooting in the presence of exogenous auxin (indole-3-butyric acid, IBA), even at low auxin concentration, in rooting-competent cuttings, but have no effect on the adventitious rooting of low or null competent-to-root cuttings. Histological analyses show that, in the simultaneous presence of MDPUs and low concentration of exogenous auxin, adventitious root formation is induced in the cell types that retain intrinsic competence to form adventitious roots in response to auxin. The time course of cellular events leading to root formation and the time of root emergence are closely similar to that observed in cuttings treated only with higher auxin concentration. In addition, the mRNA level of a P. radiata SCARECROW-LIKE gene, which is significantly induced in the presence of the optimal concentration (10 μM) of exogenous auxin needed for cuttings to root, is increased in the presence of MDPUs and low concentration of exogenous auxin (1 μM). The expression of a P. radiata SHORT-ROOT gene in rooting-competent cuttings during adventitious rooting is also affected by the presence of MDPUs when combined with auxin. As MDPUs do not affect the expression of either gene in the absence of exogenous auxin, but only in its presence, we suggest that MDPUs could interact, directly or indirectly, with the auxin-signalling pathways in rooting-competent cuttings during adventitious rooting.     

Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development

Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process induced by IAA in a NO-mediated but cGMP-independent pathway. The activation of MAPKs is discussed in relation to the cell responses modulating mitotic process.     

Nitric oxide and cyclic GMP are messengers in the indole acetic acid-induced adventitious rooting process

This report describes part of the signaling pathway and some of the molecules involved in the auxin-induced adventitious root formation in cucumber (Cucumis sativus). Previous results showed that nitric oxide (NO) mediates the auxin response during adventitious root formation (Pagnussat et al., 2002). To determine the order of action of indole acetic acid (IAA) and NO within the signal transduction pathway and to elucidate the target molecules that are downstream of NO action, cucumber hypocotyl cuttings were submitted to a pretreatment leading to endogenous auxin depletion. The auxin depletion treatment provoked a 3-fold reduction of the root number in comparison to the nondepleted explants. The NO-donor sodium nitroprusside was able to promote adventitious rooting in auxin-depleted explants, whereas the specific NO scavenger cPTIO prevented the effect of sodium nitroprusside. The endogenous NO level was monitored in both control and auxin-depleted explants using a NO-specific fluorescent probe. The NO level was 3.5-fold higher in control (nondepleted) explants than in auxin-depleted ones. The exogenous application of IAA restored the NO concentration to the level found in nondepleted explants. Because NO activates the enzyme guanylate cyclase (GC), we analyzed the involvement of the messenger cGMP in the adventitious root development mediated by IAA and NO. The GC inhibitor LY83583 reduced root development induced by IAA and NO, whereas the cell-permeable cGMP derivative 8-Br-cGMP reversed this effect. The endogenous level of cGMP is regulated by both the synthesis via GC and its degradation by the phosphodiesterase activity. When assayed, the phosphodiesterase inhibitor sildenafil citrate was able to induce adventitious rooting in both nondepleted and auxin-depleted explants. Results indicate that NO operates downstream of IAA promoting adventitious root development through the GC-catalyzed synthesis of cGMP.     

Architectural Tradeoffs between Adventitious and Basal Roots for Phosphorus Acquisition

Adventitious rooting contributes to efficient phosphorus acquisition by enhancing topsoil foraging. However, metabolic investment in adventitious roots may retard the development of other root classes such as basal roots, which are also important for phosphorus acquisition. In this study we quantitatively assessed the potential effects of adventitious rooting on basal root growth and whole plant phosphorus acquisition in young bean plants. The geometric simulation model SimRoot was used to dynamically model root systems with varying architecture and C availability growing for 21 days at 3 planting depths in 3 soil types with contrasting nutrient mobility. Simulated root architectures, tradeoffs between adventitious and basal root growth, and phosphorus acquisition were validated with empirical measurements. Phosphorus acquisition and phosphorus acquisition efficiency (defined as mol phosphorus acquired per mol C allocated to roots) were estimated for plants growing in soil in which phosphorus availability was uniform with depth or was greatest in the topsoil, as occurs in most natural soils. Phosphorus acquisition and acquisition efficiency increased with increasing allocation to adventitious roots in stratified soil, due to increased phosphorus depletion of surface soil. In uniform soil, increased adventitious rooting decreased phosphorus acquisition by reducing the growth of lateral roots arising from the tap root and basal roots. The benefit of adventitious roots for phosphorus acquisition was dependent on the specific respiration rate of adventitious roots as well as on whether overall C allocation to root growth was increased, as occurs in plants under phosphorus stress, or was lower, as observed in unstressed plants. In stratified soil, adventitious rooting reduced the growth of tap and basal lateral roots, yet phosphorus acquisition increased by up to 10% when total C allocation to roots was high and adventitious root respiration was similar to that in basal roots. With C allocation to roots decreased by 38%, adventitious roots still increased phosphorus acquisition by 5%. Allocation to adventitious roots enhanced phosphorus acquisition and efficiency as long as the specific respiration of adventitious roots was similar to that of basal roots and less than twice that of tap roots. When adventitious roots were assigned greater specific respiration rates, increased adventitious rooting reduced phosphorus acquisition and efficiency by diverting carbohydrate from other root types. Varying the phosphorus diffusion coefficient to reflect varying mobilities in different soil types had little effect on the value of adventitious rooting for phosphorus acquisition. Adventitious roots benefited plants regardless of basal root growth angle. Seed planting depth only affected phosphorus uptake and efficiency when seed was planted below the high phosphorus surface stratum. Our results confirm the importance of root respiration in nutrient foraging strategies, and demonstrate functional tradeoffs among distinct components of the root system. These results will be useful in developing ideotypes for more nutrient efficient crops.     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters

Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5±3.5%) as compared to stem (32.7±4.8%) or cotyledon (16.2±5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared. The frequency of adventitious shoot formation from transformed roots was much higher (88.5±9.8%) than that of non-transformed roots (31.7 ±9.5%) on hormone-free medium. Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots. The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants. Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. The differences observed may reflect the modification of morphological root characters by introduction of rol genes.Communicated by M.R. Davey     

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 μM sucrose, 0.8 % agar, 3.62 μM 2,4-dichlorophenoxy acetic acid and 2.22 μM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant⁻¹) was achieved on MS medium supplemented with 8.88 μM BA, 2.5 μM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 μM) favoured shoot elongation and indole 3-butyric acid (7.36 μM) induced rooting. Rooted plants were hardened and successfully established in soil.     

侧柏扦插不定根发生模式研究

该研究以侧柏一年生硬枝插穗为实验材料,利用连续组织切片技术观察插穗不定根发生发育过程中的组织结构变化,分析插穗外部形态变化、不定根原基起源和不定根的形成过程,探讨侧柏插穗不定根发生模式和不定根的组织学起源。结果显示:侧柏扦插后可由愈伤组织、皮部诱导产生不定根,出现皮部生根、愈伤组织生根、愈伤组织兼具皮部生根3种类型;侧柏插穗中存在少量潜伏根原基,但插穗生根类型以诱导生根为主;不定根原基诱导产生于愈伤组织、木质部、形成层及次生韧皮部等部位。研究认为侧柏扦插生根属于多位点发生模式,不定根原基的组织学起源是愈伤组织、髓射线、射线原始细胞、尚未分化成熟的木质部细胞,通过人工诱导同时激活这些不定根起源位点能够显著提高生根率和生根质量。     

Effects of explant types and media salt strength on growth and secondary metabolite accumulation in adventitious roots of <Emphasis Type="Italic">Periploca sepium</Emphasis> Bunge

In order to study the capabilities of Periploca sepium adventitious root induction in different types of explants, we selected leaves, roots and stems with or without buds. The growth of adventitious roots and periplocin content in these roots were determined. In order to investigate the suitable media salt strength, we cultured the adventitious roots in different salt strength (0.25, 0.50, 1.0, 1.5, 2.0) of Murashige and Skoog (MS) media supplemented with 1 mg/l indole butyric acid (IBA) and 30 g/l sucrose. The results showed that both leaf and root explants were proven suitable for the adventitious root induction; however, the stems could hardly induce adventitious roots no matter whether the stems had buds or not. Further studies reported that adventitious root proliferation and periplocin production derived from root explants were higher than those derived from leaf explants. So the root explants were the optimum explants for adventitious root induction, growth and periplocin production. The salt strength experiment showed that with the increasing salt strength (1.0–2.0 MS), adventitious root growth decreased significantly, as well as periplocin content in comparison with lower (0.25–0.5 MS) salt strength media.     

Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development

A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.     

Role of auxin and its modulators in the adventitious rooting of <Emphasis Type="Italic">Eucalyptus</Emphasis> species differing in recalcitrance

It is well established that auxins play a central role in the determination of rooting capacity, which is essential for vegetative propagation. Recent studies with apple trees have pointed to significant effects of auxin stability, wound related phenolics and ethylene production in the control of adventitious rooting. In the present study, a comparative analysis of the adventitious rooting of microcuttings of Eucalyptus saligna (easy-to-root species) and Eucalyptus globulus (difficult-to-root species) was carried out with different types of auxins, light intensities, presence or absence of apical meristem, different concentrations of phenolic compounds and presence or absence of an ethylene action inhibitor. Parameters evaluated were the percent rooting, number of roots per rooted cutting, length of longest root and mean rooting time. Results showed that auxins of intermediate stability are more favorable to rooting (particularly for the recalcitrant species), higher light intensities in the presence of exogenous auxins promote the rooting response, the absence of meristematic apex or externally supplied phenolics are not limiting for the rooting induced by exogenous auxins, and ethylene appears to play a minor role in the development of adventitious roots in microcuttings of Eucalyptus, indicating that the rhizogenic response results from direct effect of auxins.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

Promotion of petunia (Petunia hybrida L.) regeneration in vitro by ethylene

The influence of ethylene on shoot and root formation from petunia leaf explants was studied in cultures in test tubes placed in 51 glass jars. Reduction of the endogenously produced ethylene by inclusion of ethysorb (KMnO₄), an ethylene absorbent, caused a decrease of the number of shoots. On the other hand, supplementing the cultures with ethylene (0.01–10 ppm) caused a marked increase of the number of shoots without, however, any effect on the length and fresh weight. Ethylene treatments (1 ppm) were found to be most effective when they were applied in the second week of culturing of petunia explants. Addition of Co⁺⁺ to the medium resulted in a reduction of the endogenously produced ethylene and concomitantly reduced shoot formation. Similarly, inclusion of Ag⁺, an inhibitor of ethylene action, resulted in poor shoot formation. Ethylene also appeared to play a role on rooting of petunia microshoots in vitro in an auxin-free medium. Ethylene at a concentration of 10 ppm induced adventitious root formation considerably, whereas at low levels (0.01–1 ppm) it had no influence on rooting.     

The dynamic distribution of NO and NADPH–diaphorase activity during IBA-induced adventitious root formation

Nitric oxide (NO) is a multifunctional molecule involved in numerous physiological processes in plants. In this study, we investigate the spatiotemporal changes in NO levels and endogenous NO‐generating system in auxin‐induced adventitious root formation. We demonstrate that NO mediates the auxin response, leading to adventitious root formation. Treatment of explants with the auxin indole‐3‐butyric acid (IBA) plus the NO donor sodium nitroprusside (SNP) together resulted in an increased number of adventitious roots compared with explants treated with SNP or IBA alone. The action of IBA was significantly reduced by the specific NO scavenger, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (c‐PTIO), and the nitric oxide synthase (NOS, enzyme commission 1.14.13.39) inhibitor, N^G‐nitro‐l ‐arg‐methyl ester (l ‐NAME). Detection of endogenous NO by the specific probe 4,5‐diaminofluorescein diacetate and survey of NADPH–diaphorase activity (commonly employed as a marker for NOS activity) by histochemical staining revealed that during adventitious root formation, NO and NADPH–diaphorase signals were specifically located in the adventitious root primordia in the basal 2‐mm region (as zone I) of both control and IBA‐treated explants. With the development of root primordia, NO and NADPH–diaphorase signals increased gradually and were mainly distributed in the root meristem. Endogenous NO and NADPH–diaphorase activity showed overall similarities in their tissue localization. Distribution of NO and NADPH–diaphorase activity similar to that in zone I were also observed in the basal 2–4‐mm region (zone II) of IBA‐treated explants, but neither NO nor NADPH–diaphorase signals were detected in this region of the control explants. l ‐NAME and c‐PTIO inhibited the formation of adventitious roots induced by IBA and reduced both NADPH–diaphorase staining and NO fluorescence. These results show the dynamic distribution of endogenous NO in the developing root primordia and demonstrate that NO plays a vital role in IBA‐induced adventitious rooting. Also, the production of NO in this process may be catalyzed by a NOS‐like enzyme.