In vitro propagation ofSalix tarraconensis Pau ex Font Quer,an endemic and threatened plant

Summary Salix tarraconensis Pau ex Font Quer, an endemic willow species from northeast Spain, was micropropagated with nodal segments. Shoot multiplication was obtained with different cytokinins, either on Murashige and Skoog medium or woody plant medium. Best results for shoot formation were obtained on Murashige and Skoog medium containing 4.9 μM of 6-γ-γ-dimethylallylaminopurine. Shoots showed strong apical dominance, and some cultures displayed apical necrosis. Benzyladenine gave the worst results; shoots displayed very slow growth, deformed leaves, and hyperhydrity. Good rooting of shoots was obtained with different auxins or without plant growth regulators on woody plant medium. The best results (90-100%) were obtained within 20 d. On rooting media with indole-3-butyric acid or indoleacetic acid, shoot elongation was good (35-40 mm length). Apical necrosis was observed in elongating shoots on rooting medium, but this disturbance favored axillary bud sprouting and formation of new shoots. Shoot length and quality of roots decreased gradually as the concentration of naphthaleneacetic acid increased. Plant survival was 90% 4 weeks after removal fromin vitro conditions.     

Distribution of assimilate during the flush cycle of growth in Theobroma cacao L.

The growth of the shoot and roots of seedling plants of cocoa (Theobroma cacao L.) under constant glasshouse conditions showed a rhythmic cycle, with the maximum growth stages of each alternating in a regular sequence. When the growth cycle of the shoot was upset by removing all new leaves immediately after unfolding, the roots showed a high constant growth rate during this period, suggesting that normally the rapidly expanding leaves exert an inhibitory influence on the roots. Conversely removal of portions of the root delayed the production of new leaves in the shoot. The level of soluble and starch carbohydrate in the mature leaves, roots and stem declined during the period of expansion of the flush leaves, but accumulated again at the end of the leaf expansion stage. It is likely that this reserve carbohydrate was remobilised and translocated to the flush leaves during their period of expansion. A large proportion of newly formed photoassimilate, as shown by the distribution of ¹⁴C radioactivity from different source leaves, was also translocated to the young leaves during expansion. The large sink created by these leaves may cause photoassimilate and reserve carbohydrate to be diverted from the roots, thereby inhibiting root growth during the stage of leaf expansion. It is suggested that the rhythmic leaf production at the apex may control the growth cycle of the roots.     

Abscisic acid: one of the factors affecting male sterility in Brassica napus

In Sinapis alba , a long-day plant (LDP) which can be induced by a single long day (LD), it has been suggested that cytokinins may be part of a multicomponent floral stimulus. In order to determine cytokinin fluxes during floral transition, we developed a technique to collect phloem sap reaching the apical part of the shoot, close to the target bud. Exudates collected from roots, leaves, and the apical part of the shoot were analysed by radioimmunoassay for cytokinins. Such analyses confirm previous observations, obtained using the Amaranthus bioassay. indicating thai cytokinin export from the roots and mature leaves is enhanced 2–5 fold during floral transition. The flux of cytokinins directed to the upper part of the shoot through the phloem is also rapidly increased (ca 1.5–2 fold) by the inductive treatment, between 9 and 25 h after start of the LD. We suggested that the shoot apical merislem of 2-month-old Sinapis plants probably has a low cytokinin level. Induced leaves rapidly produce a signal which is transported to the roots where it alters cytokinin production and/or export. In addition, or as a consequence, leaf-cytokinins are exported via the phloem to the apical meristem where they induce a mitotic peak and some other events normally associated with the floral transition.     

Direct induction of protocorm-like bodies from shoot tips, plantlet formation, and clonal fidelity analysis in Anthurium andreanum cv. CanCan

Protocorm-like bodies (PLBs) were induced directly at high frequency from wounded surface of Anthurium andreanum cv. CanCan shoot tip-ends, used as explants. In order to obtain PLB directly, the influence of different types and concentrations of cytokinins were evaluated. Amid the cytokinins, N⁶-(?²-isopentenyl)-adenine (2-iP) at a concentration of 15?μM was most effective in inducing PLB whereby ~98 (97.8)?% of explants induced PLB with an average of 120 PLBs per shoot tip within 50?days of culture. Stereomicroscopic observation meticulously revealed the sequential changes from initiation to maturation of PLB gradually forming shoot apical meristem, shoot primordia and leaf primordia. Mature PLBs showed significant shoot proliferation (98.4?%) in media containing 10?μM 6-furfurylaminopurine forming 17 shoots per PLB within 30?days. The inclusion of activated charcoal (AC) in media containing auxin had promotive effect on rooting whereby 5?μM indole-3-butyric acid plus 500?μM AC resulted in highest number and length of roots. Successfully acclimatized plants, subjected to random amplified polymorphic DNA assessment for genetic fidelity, did not show any variation. Thus, this complete study has successfully outlined a rapid, high frequency direct induction of PLB of Anthurium from shoot tips inclusive of shoot proliferation, rooting and acclimatization.     

THE ROLE OF RESERVES IN LEAVES,BRANCHES, STEMS,AND ROOTS ON SHOOT GROWTH OF RED PINE

By defoliation, girdling, and various combinations of these before the growing season began, the role of foods in old needles, branches, main stems, and roots on shoot growth of 8-yr-old red pine (Pinus resinosa) trees was studied in northern Wisconsin. Defoliation and girdling, alone or in combination, reduced shoot growth but defoliation reduced shoot dry weight more than it reduced shoot elongation. Reductions in shoot growth due to treatment were in the following decreasing order: branch girdling + needle removal > girdling at stem base + needle removal > needle removal > branch girdling > base girdling. The old needles were the major source of food for shoot growth and accounted for four-fifths or more of all shoot growth. The combined reserves in the branches, main stem, and roots accounted for less than 15% of shoot growth. The contribution of reserves from tissues other than old leaves was in the following order: branches > main stem > roots. Defoliation weakened apical dominance relations. In defoliated trees, many secondary axes elongated more than the terminal leader, and secondary axes in lower whorls often grew more than those in upper ones. The data suggest an important role of nutritional factors in correlative growth inhibition.     

Microcutting leaf area,weight and position on the stock shoot influence root vigour,shoot growth and incidence of shoot tip necrosis in grape plantlets in vitro

The physiological parameters of microcuttings, namely leaf area, weight (wt) and position on the stock shoot, had significant effects on root vigour, microshoot growth and incidence of shoot tip necrosis (STN) in cultures of grape (Vitis vinifera L.) ‘Arka Neelamani’. Single-node leafy cuttings cultured in MS medium containing 1 μM IAA and 0.1 μM GA₃ generally rooted first and subsequently sprouted into a single shoot. Small leafed cuttings exhibited slow root emergence, poor quality roots, early sprouting and weak shoot growth. Large leafed cuttings on the other hand, showed early rooting, vigorous roots, delayed sprouting and healthier shoots. Significant correlations were observed between fresh root wt per plantlet at 1 month in culture and wt of lamina, total wt and leaf area of the cuttings in that order. A significant correlation also existed between wt of roots and height or wt of the sprout that developed. The study suggests that the weight or area of the leaf governed the root growth in a microcutting. STN was observed in some plantlets particularly those derived from large leafed cuttings. Such cuttings showed vigorous roots and delayed but fast sprout growth that ended in STN. Plantlets showing STN had less Ca⁺⁺ and Mg⁺⁺ in the shoot tissue than in the shoots of normal plantlets while the roots showed similar Ca⁺⁺ or higher Mg⁺⁺ contents. Both had comparable amounts of cytokinins in shoot and root tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of leaves and roots on meristem development inNicotiana tabacum L. cv. Wisconsin 38

The terminal, apical shoot meristem ofN. tabacum cv. Wisconsin 38 normally differentiates into a flower after producing 30 to 40 nodes. The influence of leaves and roots on the regulation of flowering was evaluated by counting the number of nodes produced after removal of leaves or the induction of adventitious roots. Leaf removal has no effect on the number of nodes produced before flower formation. Root induction significantly increases the number of nodes produced before flower formation. The plant behaves as if it were measuring the number of nodes between the meristem and the roots as a means of regulating meristem conversion from vegetative to floral differentiation.     

Sources of Free IAA in the Mesocotyl of Etiolated Maize Seedlings

Sources of free indole-3-acetic acid (IAA) for the mesocotyl of intact etiolized maize ((Zea mays L.) seedlings are evaluated. The coleoptile unit, which includes the primary leaves and the coleoptilar node, is the main source of free IAA for the mesocotyl. The seed and the roots are not immediate sources of IAA supply. Dependence of the apical growing region of the mesocotyl on the coleoptile unit as a source of free IAA is almost total. One-half or more of the supply of IAA comes from the coleoptile tip, the rest mainly from the primary leaves. Removal of the coleoptile tip results in inhibition of mesocotyl elongation. The hypothesis that growth of the mesocotyl is regulated by auxin supplied by the coleoptile is supported. Conjugated forms of IAA appear to play little part in regulating the levels of free IAA in the shoot.     

Adventitious rooting of Jatropha curcas L. is stimulated by phloroglucinol and by red LED light

An efficient root induction system has been established for in vitro-regenerated Jatropha curcas L. shoots. Callus formation on shoots transferred to auxin containing medium was found to be a prominent and recurrent problem for rooting of in vitro-cultivated J. curcas. In particular, the type of auxins and cytokinins applied in the culture media were shown to strongly influence the severity of callus formation. Shoots cultivated on meta-methoxytopolin riboside (MemTR) were free of callus and produced elongated stems and well-developed leaves in comparison to the cytokinins benzyl adenine, zeatin, and thidiazuron. Subsequent root induction experiments were performed with shoots precultured on MemTR-containing medium. Shoots were excised and transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphtaleneacetic acid (NAA). The induction of excessive callus formation was avoided only on IBA-containing medium. The optimum rooting medium with good root induction (35%) and 1.2 roots per shoot contained half-strength MS salts supplemented with 2.5 μM IBA. The same medium supplemented with 0.25% (w/v) activated charcoal produced 46% rooted shoots. Further improvement of rooting was obtained by transferring in vitro grown shoots to woody plant medium containing phloroglucinol (PG). In the presence of 2.5 μM IBA and 238 μM PG, 83% of the shoots rooted with on average 3.1 roots per shoot. We also analyzed the impact of light quality on the rooting capacity of Jatropha in vitro grown shoots. In general, light-emitting diodes (LEDs) light sources were less efficient for root induction. Red LED light provided the most favorable growth conditions, inducing a rooting response in 65% of the shoots, which produced on average 5.5 roots per shoot. These results indicate that adventitious rooting in J. curcas is under control of photoreceptors and that optimal rooting requires fine-tuning of the salt concentration, auxin, and cytokinin balance and application of synergistic compounds.     

In vitro rooting of almond (Prunus dulcis Mill.)

Summary Shoot cultures of the paper shell almond (Prunus dulcis Mill.) cultivars ‘Ne Plus Ultra’ and ‘Nonpareil’ were subcultured for 4 wk at 4°C on growth regulator-free basal medium under low light conditions. Elongated shoots were excised and their response to a range of rooting treatments determined. Various concentrations of indole-3-butyric acid (IBA) and α-naphthaleneacetic acid were compared over a range of incubation periods to determine the optimum auxin for root formation. In addition, the effect of shoot base shading, phloroglucinol (PG), and basal salt composition were examined. The treatment resulting in the best rooting of both cultivars was shoot insertion for 12 h into water-agar (0.6% w/v) with 1.0 mM IBA, followed by 2 wk in basal medium without auxin but with 100.0 μM PG. Explants were maintained under dark conditions for 3 d at the start of the treatment period, then exposed to light. Extending the darkening period did not improve rooting ability. Whilst half-strength Murashige and Skoog basal medium was suitable for rooting “Ne Plus Ultra’ shoots, full-strength Almehdi and Parfitt medium resulted in the best rooting of ‘Nonpareil’. Under these conditions, 60.0% of explants developed adventitious roots.     

Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro

Efficacy of phloroglucinol in promoting growth and development of in vitro-derived shoot tips was studied in six potato (Solanum tuberosum L.) genotypes. Different concentrations of phloroglucinol (0, 0.08, 0.4, 0.8, 1.2 and 1.6 mM) were tested in combination with either 0.1 or 0.2 M sucrose in shoot tip proliferation medium based on MS (Murashige and Skoog, 1962) medium supplemented with 5.8 μM GA₃ (gibberellic acid), 1.1 μM BA (N⁶-benzyladenine) and 8.39 μM D-calcium pantothenate. Phloroglucinol fostered multiple shoot formation, promoted axillary shoot proliferation in terms of shoot tip fresh weight and shoot length, and stimulated root formation on the shoot tips. There was significant phloroglucinol × sucrose interaction for number of shoots developed per shoot tip, shoot tip fresh weight and number of roots induced per shoot tip. The beneficial effect of phloroglucinol on shoot tip survival was conspicuous only in genotypes that showed poor survival in the control proliferation medium. There were significant differences in response between the two sucrose levels with regard to shoot tip fresh weight and number of roots per shoot tip. Phloroglucinol in combination with 0.2 M sucrose induced maximum number of roots per shoot tip. Optimum shoot tip growth was fostered in medium containing 0.8 mM phloroglucinol and 0.2 M sucrose. High frequency multiple shoot formation in this medium ensures a faster rate of potato shoot tip multiplication within a limited time and space. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Zeatin-induced one-step in vitro cloning affects the vegetative growth of cranberry (<Emphasis Type="Italic">Vaccinium macrocarpon</Emphasis> Ait.) micropropagules over stem cuttings

Shoot proliferation and rooting of three cranberry (Vaccinium macrocarpon Ait.) cultivars Bergman, Pilgrim, and Stevens were obtained in vitro on a modified nutrient medium containing zeatin following a one-step procedure. Bergman and Stevens differed in terms of shoot height, leaf number per shoot, rooting frequency, root number per explant, and root length; this was manifested with various concentrations of zeatin. Shoots proliferated and roots developed best when nodal segments were cultured in the medium supplemented with very low concentration of zeatin (2–4 μM). Such zeatin-induced tissue culture (TC) shoots of Bergman, Pilgrim, and Stevens were rooted ex vitro and compared with those propagated by conventional softwood cuttings (SC) for growth and morphology over four growth seasons. Significant interactions for leaf number per upright were observed among the treatments. The cultivars differed in terms of runner number per plant, upright length, number of leaves per upright, and shoot vigor. The propagation method had an effect on morphology of cranberry plants. The TC plants produced more runners and uprights with more leaves per upright than the conventional cuttings. This increase in vegetative growth of in vitro-derived plants over stem cuttings varied among genotypes. In vitro culture on zeatin-containing nutrient medium apparently induces the juvenile branching characteristics that favored enhanced vegetative growth with more shoots and leaf production.     

Developmental Morphology of Roots and root-borne shoots ofPodostemum subulatum as compared withZeylanidium olivaceum (Podostemaceae—Podostemoideae) Part VII of the series ‘Morphology of Podostemaceae’

The root structure ofPodostemum subulatum is investigated and compared with that ofZeylanidium olivaceum. Podostemum has thread- or ribbon-like roots. The root tip consists of an inner apical meristem and a single-layered root cap. From roots arise numerous shoots of endogeneous origin. Their vascular bundle isab initio connected with the root bundle.By the simple (reduced) apical zonation, the roots ofPodostemum subulatum appear more advanced than the crustose roots ofZeylanidium olivaceum, which bear an ordinary (though asymmetrical) root cap. With regard to the endogeneous root-borne shoots, however,Zeylanidium appears more advanced because of the shoot dimorphism. The floriferous shoots have a short axis that grows plagiotropously above the crust surface, whereas the axes of the vegetative shoots are extremely short and remain, together with the apical meristem, within the crust. Only the leaves protrude from the crust surface.     

Endogenous phenolic contents,peroxidase and polyphenoloxidase activities in date palm (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) offshoots related to rooting ability

Offshoots from three cultivars of date palm showed different rooting ability, in response to the same root inducing treatment (25 mM indole butyric acid), under misting system. Offshoots from Mejhoul rooted easily and produced numerous roots, while those from Khalt rooted poorly and produced fewer roots. No relationship was found between soluble and ionically bound peroxidase and polyphenoloxidase activities and phenolics level, determined spectrophotometrically in the offshoot leaves prior to the auxin application, and the rooting ability of these cultivars. However, changes of these parameters during the first month of culture were correlated with the rooting ability. In the well rooting cultivar, the soluble peroxidase activity showed a minimum on the 4^th day and a maximum on the 10^th day, while phenolics level and polyphenoloxidase activity showed an inverse trend. In the poorly rooting cultivar, these parameters changed weakly.     

RML1 and RML2, Arabidopsis genes required for cell proliferation at the root tip.

New cells are produced from the meristematic tissues located at the shoot and root tip throughout the life of higher plants. To investigate the genetic mechanism regulating meristematic activity, we isolated and characterized four single-gene, recessive mutants in Arabidopsis thaliana called root meristemless (rml). Complementation tests identified two RML loci; RML1 maps to chromosome IV and RML2 maps to chromosome III. These mutants produce normal embryonic roots that either did not undergo or experienced limited cell division following germination, resulting in primary roots of less than 2.0 mm in length. Mutants can produce lateral and adventitious roots, which can grow to a length comparable to the embryonic root and arrest, indicating that the growth arrest is unrelated to the embryonic dormancy process. Neither the addition of growth regulators to the media nor the removal of shoots can rescue mutant roots from growth arrest, indicating that the mutant phenotype is not caused by a shortage of known growth regulators or by a transmissible shoot inhibitor. Normal cell division ability in mutant embryo, shoot, and callus cells indicates that the RML gene functions are not part of the general cell division processes; rather, they are involved specifically in activating the cell division cycle in the root apical cells.     

Factors influencing direct shoot regeneration from mature leaves of Jatropha curcas, an important biofuel plant

In order to establish a highly efficient and sustainable regeneration system, we systematically researched the key factors affecting direct shoot regeneration from Jatropha curcas leaves that were collected from Hainan (HN1-1), Lijiang (LJ3-1), and Yuxi (YX2-12) provinces in China. The L₉(3⁴) orthogonal test of thidiazuron (TDZ), kinetin (Kn), and gibberellic acid (GA₃) were studied, and the explant type, growth age, and cultivar of leaves were subsequently investigated. Simultaneously, the combinations of plant growth regulators (PGRs) promoting shoot bud proliferation, elongation, and root establishment were examined. The results showed that the best medium for shoot bud induction was Murashige and Skoog (MS) medium supplemented with 1.0 mg/L TDZ, 0.5 mg/L Kn, and 0.5 mg/L GA₃. TDZ was the key PGR, while Kn and GA₃ played an important role in shoot bud elongation and the number of shoots per leaf disk, respectively. The induced shoot buds proliferated and readily elongated in MS medium with 0.3 mg/L 6-benzylaminopurine and 0.01 mg/L indole-3-butyric acid (IBA) and established roots in half-strength MS medium supplemented with 2.0 mg/L IBA. Using the previously described methods, the third to fifth leaves were found to be the best explant source for shoot bud induction, with a high induction rate, large shoot numbers per disk, excellent proliferation, and consistent rooting. With the use of this regeneration system, the shoot bud induction rate increased from the reported rate of 53.5% to more than 90% using different explants and cultivars, and the shoot number per leaf disk (shoot length?≥?0.5 cm) increased from 1.6 to 3.5. Thus, this optimized regeneration system will effectively promote the propagation and genetic transformation of J. curcas.     

Influence of phosphorus level on VA Mycorrhizal colonization and growth of cowpea cultivars

Vesicular-arbuscular mycorrhizal (VAM) associations often vary according to the abundance of available soil phosphorus (P). Therefore, understanding the response of crop plants to colonization by VAM fungi necessitates the study of the response of colonized and noncolonized plants, from a range of cultivars, to differing levels of P. Cowpea is grown throughout the world, often on impoverished soils in which it can benefit from formation of mycorrhizae. The present study was conducted to determine the response of four cultivars of cowpea (Vigna unguiculata (L.) Walp.), varying in nitrogen fixation capacity, to inoculation withGlomus fasciculatum at four levels of added P in the rooting medium. In a greenhouse experiment, four cowpea cultivars, Mississippi Silver, Brown Crowder, Six Week Browneye and MI 35, were grown with and without the mycorrhizal fungus at four levels of added P, 0, 10, 20 and 30 ppm. Root colonization (%) was negatively correlated with P content of the growth medium and shoot P concentration. Intraspecific variability was shown for shoot dry weight and leaf area in response to inoculation withG. fasciculatum at different P levels. The range of P required in the growth medium which allowed benefit fromG. fasciculatum was identified for individual cultivars using shoot dry weight and leaf area, and collectively across cultivars for other parameters.     

Accumulation and transport of abscisic Acid and its metabolites in ricinus and xanthium

When intact plants of Xanthium strumarium L. were water stressed, the youngest leaves accumulated the highest levels of abscisic acid (ABA). On the other hand, when leaves of different ages were detached and then stressed, the capacity to produce ABA was highest in the mature leaves. Radioactive ABA was transported from mature leaves to the shoot tips and young leaves, as well as to the roots, as evidenced by the presence of radioactive ABA and phaseic acid in the xylem exudate coming from the roots. Thus, ABA was recirculated in the plant, moving down the stem in the phloem and back up in the transpiration stream to the mature leaves. Phloem exudate collected by the use of the EDTA technique had a high concentration of ABA and phaseic acid which increased several-fold after water stress. The high ABA levels in immature leaves and apical buds are, therefore, mainly due to import from older leaves, rather than to in situ synthesis.     

Comparative effects of NaCl and polyethylene glycol on root distribution,growth, and stomatal conductance of sour orange seedlings

An experiment was conducted to study sour orange (Citrus aurantium L.) seedling root density, distribution, and morphological development under NaCl and polyethylene glycol (PEG) stresses in relation to shoot growth and stomatal conductance. Plants were treated with 2 stress levels (– 0.12 and – 0.24 MPa) of NaCl and PEG 4000 for 7 months. Root observation chambers were used to monitor root growth and distribution under stressed and non-stressed conditions. Seedlings receiving NaCl or PEG treatments produced fewer roots and shallower root systems with 46 to 65% of the roots occurring in the top portion of the soil. Fibrous root weight per unit length was increased by 24 to 30% by PEG but was not significantly increased by NaCl.Root growth rate usually alternated with shoot growth in a 2-month cycle. This alternating pattern was not shifted by NaCl and PEG stresses. In all NaCl and PEG treatments, growth was depressed and stomatal conductance was reduced. Compared to controls, plants that received NaCl or PEG had smaller shoot and root dry weights, fewer leaves, shorter height, and fewer roots. Sodium chloride usually caused less damage than PEG to sour orange seedlings suggesting that NaCl and PEG acted through different mechanisms.Florida Agricultural Experiment Station Journal Series No. 9941.