Use of gentian violet to differentiate in vitro and ex vitro-formed roots during acclimatization of grapevine

The dye gentian violet was added to culture medium in order to distinguish in vitro and ex vitro-formed roots during acclimatization of micropropagated plantlets. Shoots of the grapevine rootstock Kober 5BB were rooted on media containing the dye (0.3 and 0.15 mg·l^–1) for 3 weeks. The dye coloured the roots. Root length was reduced by the presence of the dye, but root number and shoot growth were not affected. Most in vitro-formed roots continued to grow during acclimatization, and 3 weeks after the transfer to soil the root system was 60% composed of in vitro-formed roots. Our results suggest that in grapevine Kober 5BB, the in vitro-formed roots contribute to plantlet growth at least during acclimatization.     

Physiological changes and growth of micropropagated chile ancho pepper plantlets during acclimatization and post-acclimatization

Little is known about physiological changes that occur with micropropagated chile ancho pepper (Capsicum annuum L. cv. San Luis) plantlets during acclimatization. Plantlets were transferred to ex vitro conditions to study selected physiological changes and growth performance during acclimatization and post-acclimatization. The physiology of the plantlets was characterized by measuring leaf gas exchange and water status. Plant growth was determined by assessing plant height, leaf number, total leaf area, relative growth rate (RGR), and leaf, root, and stem dry matter (DM). Chile pepper plantlets became acclimatized within 6 days after transplantation. During this period, physiological adjustments occurred, which were critical for plantlet survival. After initial ex vitro transplanting, plantlets experienced water deficit [leaf wilting and reduced relative water content (RWC)], which corresponded with reduced stomatal conductance (g _s) and transpiration (E), and an increase in stomatal resistance (r _s). Thus, leaf stomata that developed in vitro were functional ex vitro. Because of this stomatal control, plantlets minimized transplant shock, recovered and survived. Prior to transplanting, plantlets were photomixotrophic, as indicated by low photosynthetic rates (A). During acclimatization, RWC, g _s, E, and A were significantly lower two days after transplanting. However, within 6 days after transplanting, plantlets recovered and became photoautotrophic – attaining high A, g _s, and E. Water use efficiency was initially low during the first days after transplanting, but increased dramatically at the end of the acclimatization period in part due to increased A. The stabilization and improvement of plantlet water status and gas exchange during acclimatization and post-acclimatization closely correlated with increased plantlet growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of a semi-dwarfing cherry rootstock

A successful in vitro propagation system for the semi-dwarfing cherry rootstock Maxma-14 (Prunus avium L.) has been developed. Shoot tips and axillary buds were successfully established in vitro. Multiplication rate of about 6 was achieved over a 4-week period using Murashige and Skoog medium with 4.44 μM benzyladenine and 0.49 μM indole-3-butyric acid (IBA). Rooting occurred within 4 weeks on liquid and agar-gelled media containing 0.49 μM NAA or 0.49, 2.45 μM IBA. On liquid media, a maximum rooting efficiency of up to 100% was obtained. However, high concentrations of auxins delayed the time of root initiation for 3–5 days. Acclimatization was affected directly by rooting conditions. Survival was best when plantlets were transferred to pots after a short period of root emergence on rooting media. Multiplication medium was also important for successful acclimatization. Shoots transferred to rooting media from that with kinetin resulted in better acclimatization and survival than that derived from media with benzyladenine. Further, plantlets rooted on liquid media had better survival than that rooted on agar-gelled media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction,growth and direct rooting of adventitious shoots of Begonia x hiemalis

The efficiency of commercial micropropagation programs for Begonia x hiemalis depends on the production of large adventitious shoots for easy handling and on effective rooting and acclimatization procedures. Maximum induction of adventitious buds on petiole segments occurred in response to NAA (0.1 mg, l^-1) and BA (0.5 mg l^-1), but continued shoot growth was limited. With a lower concentration of BA (0.1 mg l^-1) fewer shoots were produced but shoot growth was enhanced. With a combined agar/liquid culture program the low BA (0.1 mg l^-1) medium produced 50 percent more shoots larger than 1 cm than did the high BA (0.5 mg l^-1) medium. In vitro rooted explants developed weak root systems and acclimatization losses occurred during adaptation to greenhouse conditions. Adventitious shoots treated with commercial rooting powder and placed directly in mist frames produced much stronger root systems and could be adapted to greenhouse conditions without loss. The elimination of the in vitro rooting stage also simplifies the micropropagation program.Contribution No. 743     

Anatomy of normal and hyperhydric leaves and shoots of in vitro grown Simmondsia chinesis (link) schn

Summary The anatomy of normal and hyperhydric in vitro shoots and leaves from micropropagated simmondsia chinensis (Link.) Schn. (jojoba) was compared with that of seedlings (control plants). In vitro normal plantlets displayed good development and survived during the acclimatization stage. In vitro hyperhydric plantlets presented numerous anatomical defects, such as hypertrophy of the mesophyll and of the stem cortex, malformed non-functional stomata, epidermal discontinuity, and xylem hypolignification; they did not survice acclimatization. The study of the anatomical features of in vitro jojoba shoots and leaves allowed determination of the structural condition of the plantlets and prediction of which plantlet would survive the critical acclimatization stage.     

Role of carbohydrates in micropropagation of cork oak

The influences of carbon sources, fructose, glucose, sorbitol and sucrose on shoot proliferation and in vitro rooting of cork oak (Quercus suber L.) were compared at a wide range of concentrations (1–6%, w/v). The highest number of shoots occurred on glucose-containing medium. Nevertheless, we have chosen 3% sucrose which induced a similar rate of proliferation but favoured shoot elongation, permitting an effectively higher number of shoots during transfers. Sorbitol and autoclaved fructose did not stimulate shoot proliferation. Adventitious root formation was strongly dependent on carbohydrate supply. Sorbitol and autoclaved fructose were completely ineffectively on rooting induction. Glucose was the most effective carbon source on rooting promotion followed by sucrose and filter-sterilized fructose. The rooting response induced by fructose was dependent on the sterilizing procedure. The number of adventitious roots produced per shoot increased with increasing glucose and sucrose concentration. The content of reducing sugars in leaves of proliferation cultures and in leaves and roots of rooted plantlets was more dependent on carbon concentration than on glucose or sucrose supplement. The results presented here show that carbohydrate requirements during cork oak micropropagation depend upon the phase of culture. Sucrose (3%) and glucose (4%) were the best carbon sources respectively during proliferation and rooting phases.     

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.     

Stimulation of adventitious root formation in non-woody stem cuttings by uridine

Uridine strongly stimulated adventitious root formation in stem cuttings of sunflower (Helianthus annuus L.), mung bean (Vigna radiata L.) and common bean (Phaseolus vulgaris L.). A dose response curve of uridine induced rooting showed that the optimum concentration of uridine was 0.1 µM. At all concentrations employed, uridine had no significant effect on root elongation. The rooting response of stem cuttings to the optimal concentration of indole-3-butyric acid (10 µM) in combination with 0.1 µM uridine did not significantly differ from their response to either of these compounds when applied alone. However, the rooting response of the cuttings to sub-optimal IBA (0.01 µM) was significantly stimulated by uridine. These findings suggested that uridine may have stimulated rooting by increasing the sensitivity of the rooting tissue to auxin.     

In vitro plantlet regeneration from nodal explants of field-grown culms in Bambusa glaucescens Willd.

Nodal segments from field-grown culms were used as explants to develop a method of in vitro plantlet regeneration in Bambusa glaucescens Willd. through axillary bud proliferation. Shoot multiplication experiments were carried out with different concentrations of benzyl adenine (BA) and kinetin (Kn), either singly or in combination. A synergistic effect of the two cytokinins was observed and the best interaction giving the highest rate of shoot multiplication (4.00-fold) was obtained for a combination of 5 μM BA and 15 μM Kn. The MS medium supplemented with 25 μM indole butyric acid (IBA) was most suitable for rooting of shoots. Hardening and acclimatization was successful and plantlets are growing normally in soil.     

Morphogenesis and tissue cultures of three citrus species

Studies were performed to define tissue culture techniques and culture conditions for morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis (L.) Osb.), citron (C. medica L.) and lime (C. aurantifolia) (Christm. Swing). The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l^-1 for sweet orange and lime, and 3 mg l^-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l^-1 for sweet orange and citron, and 1 mg l^-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mg l^-1 NAA and 0.25 mg l^-1 BA. Callus was maintained by periodical subculture into the same medium supplemented with 10% (v:v) organge juice. In vitro plantlets of the three species were obtained by rooting of shoots developed from bud cultures, and of citron and lime by development of shoots from root cultures. The plants were successfully established on soil.     

Influence of beneficial microorganisms during in vivo acclimatization of in vitro-derived tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>) plants

The effects of native isolates of Pseudomonas fluorescens, Azospirillum brasilense, and Trichoderma harzianum on rooting and acclimatization of in vitro-grown shoots and plantlets of tea were evaluated. In vitro bacterization of P. fluorescens failed to establish, while both T. harzianum and A. brasilense retarded shoot growth, eventually overtaking shoot cultures in in vitro rooting. Acclimatization of rooted plantlets in soil amended with bioinoculants, either individually or in various combinations, promoted plantlet survival. Moreover, efficiency of nutrient uptake of plantlets was higher in the presence of microorganisms. Root rot or wilting of tissue culture-derived plants was not observed in bioinoculant-treated plants, as they possessed relatively higher activities of defense enzymes, including peroxidase and phenylalanine ammonia lyase.     

IBA浓度、扦插时间对江西杜鹃和百合花杜鹃扦插生根的影响

为探明有鳞大花亚组杜鹃扦插生根的最佳IBA浓度和扦插时间,该研究以江西杜鹃、百合花杜鹃为材料,分别采用腐叶土+河沙(1:1)、泥炭+珍珠岩+蛭石(3:1:1)基质,开展了4个IBA浓度和4个扦插时间的生根试验.结果表明:IBA浓度对除老叶留存数外的所有指标有显著影响,其中100 mg·L-1 IBA处理生根率、新梢长最大,腐烂率最低,其它指标也表现良好,为最佳生根浓度;50 mg·L-1 IBA处理根幅、新梢率最大,但不定根数最少,效果其次;200 mg·L-1 IBA处理促进根系生长,但生根率较低、特别是显著抑制新梢发育;对照处理生根效果最差.扦插时间对所有生根指标均有显著影响,早春(04-18)木质化硬枝扦插除老叶留存数较差外,其它指标均表现极佳,为最适扦插时间;秋季(10-19)半木质-木质化过渡枝扦插效果其次;夏季(06-21)嫩枝及(08-16)半木质化枝生根效果极差,不宜进行扦插育苗.物种、基质对生根指标也有显著影响,百合花杜鹃扦插生根能力强于江西杜鹃,泥炭+珍珠岩+蛭石(3:1:1)基质生根效果优于腐叶土+河沙(1:1).该研究结果首次发现早春新梢萌发前采用木质化硬枝扦插可以显著提高两种杜鹃的生根效果,为该亚组杜鹃的扦插育苗提供了科学依据.     

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.     

Leaf number and position effects on the survival and performance of grape microcuttings in vitro,and the sensitivity of the cut nodal region to the medium

The presence of leaf in microcuttings of grape cvs. Arka Neelamani and Thompson Seedless promoted rooting in vitro (MS, 1 μM IAA, 0.1 μM GA₃, 3% sucrose) but the effect varied depending on the number of leaves and position of the leaf on the cutting. Single node cuttings with a full-length lower internode and a lamina at top (LAT) showed earlier rooting and more root and shoot growth than cuttings with lamina positioned at the middle (LAM), while cuttings with a leaf at the base (LAB) of the cutting and full-length upper internode exhibited a lower percent rooting and sprouting, poor root and shoot growth, and low survival. Partial or complete removal of the upper internodal segment in LAB cuttings improved rooting and sprouting suggesting the possible operation of an inhibitory effect by the upper internode. Retaining an upper leaf in LAB cuttings (LAB+UL) resulted in necrosis of the upper leaf often followed by the lower one. The extent of necrotic damage was influenced by the leaf area and position or age of the cutting on the stock shoot. Retaining the lower internode in LAB and LAB+UL cuttings which held the node–leaf junction away from the medium, or reducing the concentration of MS medium helped significantly in improving the survival and performance of these cuttings. The difference in reaction between LAB and LAT cuttings was attributable mainly to the difference in the sensitivity of the stem part that came in contact with the medium. Removal of the leaf in LAB cuttings reduced this sensitivity. The majority of the LAB and LAB+UL cuttings, as well as non-rooting or delayed rooting LAT and LAM cuttings, exhibited high purple pigmentation of leaf, petiole and stem. Two-leafed cuttings in vitro showed poor survival, less rooting and low plantlet output compared to single-leafed cuttings. This revised version was published online in June 2006 with corrections to the Cover Date.     

Stimulation of adventitious rooting of Taxus species by thiamine

Summary Results obtained from using root inducing compounds on Taxus species cuttings suggested that rooting could be significantly enhanced by the presence of thiamine. This observation was verified using a root inducing solution containing a set concentration of IBA (0.2%), NAA (0.1%), and supplemented with various concentrations of thiamine. The best rooting response for Taxus cuspidata stem cuttings was found using this solution supplemented with 0.08% thiamine. Rooted cuttings were easily established and developed into vigorous plants. In addition, Taxus brevifolia shoots obtained from tissue cultures via in vitro organogenesis also responded favorably to this 0.08% thiamine supplemented rooting solution.     

Exogenous trehalose affects morphogenesis in vitro of jojoba

Trehalose is a stress protecting and reserve carbohydrate in a variety of organisms. The effect of trehalose on micropropagation of jojoba [Simmondsia chinensis (Link) Schneider] was investigated using a modified Murashige–Skoog as the basal medium (BM). Proliferation rate was significantly higher in explants grown on BM + 1 mM trehalose compared to that in the phytohormone control medium. In multiplication stage, shoot proliferation rate of 4.8 was achieved in BM with 4.44 μM BA and 1 mM trehalose. The rooting induction with 14.7 μM IBA had a significant effect on root regeneration; the highest rooting percentage (46.6%) was obtained with 7 days of induction. The incorporation of trehalose to the IBA-rhizogenesis media decreased basal callus but with trehalose alone, root development was scant. In vitro plants showed anatomical features typical for the acclimatization stage.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

In vitro sucrose concentration affects growth and acclimatization of <Emphasis Type="Italic">Alocasia amazonica</Emphasis> plantlets

Plantlets of Alocasia amazonica were regenerated on the MS medium supplemented with different concentrations (0–9%) of sucrose. An absence of sucrose in the growth medium induced generation of leaves, however, it decreased multiplication. On contrary, sucrose supply of 6% or 9% enhanced multiplication but hampered photoautotrophic growth (generation of leaves). Increasing sucrose supply also increased sugars and starch content and number of stomata and decreased water potential and size of stomata during in vitro growth period. During ex vitro acclimatization, shoot length, root length, leaf number and root number of Alocasia plantlets grown with 3% sucrose, were found to be better among the other studied sucrose concentrations. Under ex vitro acclimatization, number of stomata, contents of various carbohydrates in the leaves were increased but size of stomata decreased with increasing sucrose supply during in vitro growth period. Moreover, water potential of leaves of plantlets, which have been grown with a sucrose concentration other than 3%, was decreased. During in vitro growth, net CO₂ assimilation rate (P_N), transpiration (E), stomatal conductance (g_s) and variable fluorescence to maximum fluorescence ratio (Fv/Fm) were unaffected, however, during acclimatization these were changed and maximum P_N, E, and g_s were observed in the plantlets micropropagated with 3% sucrose. Fv/Fm was decreased severely in the plantlets micropropagated with 6% sucrose during acclimatization. Thus a sucrose concentration of 3% in the medium is appeared to be better among studied concentrations for both in vitro growth and ex vitro acclimatization of A. amazonica plantlets.     

Effect of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of arbuscular mycorrhiza-forming Prunus avium and Prunus cerasus

 The effect of different genotypes of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of Prunus avium and P. cerasus was studied in an attempt to determine whether ectomycorrhizal fungi could enhance in vitro adventitious root formation in plants which form arbuscular endomycorrhizas. The rooting percentage of P. avium cuttings was approximately 16% in the absence of hormonal treatment; it increased up to 30% in the presence of 5.7 μM IAA which was the most favourable auxin concentration. The rooting percentage of cuttings cultivated in the absence of IAA was enhanced by all the studied strains of H. cylindrosporum. It ranged from 50 to 60% with the IAA-overproducing mutant D 111 or the wild-type dikaryon D1, to 100% in the presence of the mutants 331 or D 117. The cuttings of P. cerasus showed a higher rooting ability than those of P. avium since approximately 40% of them were able to root in the absence of hormonal treatment. Except for the mutant D117, their rooting percentage was not significantly improved by H. cylindrosporum. Fungal inoculation also affected the survival of cuttings at acclimatization: 50% of the uninoculated P. avium cuttings survived whereas the survival percentage of inoculated cuttings ranged from 30 to 100% depending on the fungal genotype. With P. cerasus, the percentage of survival of uninoculated cuttings ranged from 85 to 100% and fungi either did not significantly improve it or lowered it. At acclimatization fungal hyphae could be observed in close contact with adventitious roots, but they did not establish mycorrhizal association. The shoot height of P. avium plantlets obtained from inoculated cuttings was not significantly different from that of plantlets originating from uninoculated ones. By contrast, fungal inoculation generally depressed the growth of acclimatized P. cerasus plantlets. The possibility of using ectomycorrhizal fungi as a tool to enhance rooting of micropropagated cuttings of plants which do not form ectomycorrhizas is discussed. Received: 25 November 1996 / Accepted: 2 June 1997     

The effect of paclobutrazol on in vitro rooting,transplant establishment and growth of fruit plants

The effect of paclobutrazol on in vitro rooting and growth of sour cherry (Prunus cerasus) rootstock CAB 11E clone, of S 749 × S 1490 (Prunus persica × Prunus kansuensis) hybrid rootstock, and of pear (Pyrus communis), cv. Abbé Fetel is reported.PP333 increased rooting of S 749 × S 1490 and of Abbé Fetel, particularly at a concentration of 0.5 mg/l (a.i.); moreover, it induced a rooting percentage as high as auxin in the former and hastened rooting of the latter. By contrast, paclobutrazol did not affect root production of 11 E.PP333-treated plants had shorter and thicker roots than controls but similar survival rates during acclimatization. Otherwise they grew less than controls during the first part of the acclimatization phase.Abbreviations used in text and tables BA = 6-benzyladenine - IBA = indole-3-butyric acid - PP333 = paclobutrazol = (2RS,3RS)-1-(-4-chlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pentan-3-ol Part of the results referring to S 749 × S 1490 (P. persica × P. kansuensis) rootstock were presented at the meeting on Controllo della fruttificazione delle piante da frutto, Bologna, Italy, June 1986, and were published in the Riv. Ortoflorofrutt. It. 70 (6)(1986). This research was funded in part by the Italian Ministry of Education (M.P.I. 60%).