Rhizogenesis and root growth ofCarica papaya L.in vitro in relation to auxin sensitive phases and use of riboflavin

High rooting percentages and high-quality adventitious root systems for papaya (Carica papaya L.) were obtainedin vitro by appropriate auxin source, duration of exposure to auxin and use of riboflavin. Root initiation of papaya shoots was higher using IBA than IAA, NAA or PCPA. Maximum rooting percentage (96%) was achieved by exposure of shoots to a medium containing 10 µM IBA for 3 days before transfer to a hormone-free medium. However, the resultant plants had small shoots and callused roots. Shoot and root growth were improved when shoots were transferred after 2 days from medium containing 10 µM IBA to hormone-free medium containing 10 µM riboflavin. Good root initiation, and root and shoot growth were also obtained when shoots were incubated for 2 days in darkness on a medium containing 10 µM IBA and 31 µM riboflavin before transfer to light. Alternatively, cultures could be placed in the light on medium containing 10 µM IBA, and after 1 day the medium overlaid with 300 µM riboflavin (1 ml over 10 ml of medium).     

Direct shoot formation in spontaneously occurring root pseudonodules of alfalfa (Medicago sativa L.)

Summary A simple and rapid procedure for direct organogenesis from root nodulelike structures of alfalfa (Medicago sativa L.) line SGg, spontaneously induced on growth regulator-free Gamborg (B₅) medium, was developed. Prolific adventitious shoot initiation was obtained using a combination of 1.0 mg/liter TIBA and 0.5 mg/liter 2iP. Transfer of shoots to a medium containing 0.5 mg/liter ABA and reduced concentration of TIBA (0.5 mg/liter) before rooting markedly stimulated shoot development. Regenerated shoots rooted easily and revealed the early appearance of nodulelike structures on basal medium (B₅) lacking growth regulators. Analysis of endogenous growth regulator levels of SGg roots maintained on growth regulators free media, showed that spontaneous shoot appearances was correlated with high cytokinin-to-auxin ratios.     

Micropropagation of four cultivars of Saskatoon berry (Amelanchier alnifolia NUTT.)

In vitro culture establishment, shoot proliferation, ex vitro rooting and dormancy breaking of the newly rooted plantlets were examined on Saskatoon berry (Amelanchier alnifolia NUTT.) cultivars Northline, Pembina, Smoky and Thiessen. Shoot-tip explants taken from actively growing plants were better for culture initiation than dormant buds. MS gave the most satisfactory results of the media formulations. Optimal shoot proliferation occurred at 8.8 and 13.3 M BA. Higher BA concentrations caused culture deterioration during long-term maintenance. Auxin treatments significantly stimulated ex vitro rooting of shoots in all cultivars. The best rooting was achieved with IAA/NAA (2.8/1.1 M) mixture. Satisfactory results were also obtained with commercial powder formulation, Rootone F, containing IBA/NAA mixture. Foliar application of BA and GA₄₊₇ was successful in breaking dormancy of newly rooted plantlets. Combinations of these two growth regulators caused formation of axillary shoots and vigorous plant growth. There were significant differences in the cultivar responses to culture conditions and treatments with growth regulators. The best culture establishment and the highest rate of shoot proliferation was observed in cv. Thiessen; the best rooting and the most vigorous post-dormancy growth was recorded in cv. Smoky. Cultivar Northland gave the most erratic responses.Abbreviations BA benzyladenine - cv(s) cultivar(s) - GA gibberellin - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - MS Murashige & Skoog's medium     

Vegetative micro-cloning to sustain biodiversity of threatened Moringa species

Efficient vegetative cloning in vitro requires definition of plant growth regulator regimes for each genotype, and therefore formulation of a uniform culture protocol for a genetically heterogeneous wild or uncultivated plant population is often impossible. The likelihood of cloning a wide array of plant genotypes by avoiding the use of plant growth regulator(s) was explored with Moringa oleifera Lamk., Moringa stenopetala (Baker f.) Cufod, and Moringa peregrina Forssk. ex Fiori tree seedlings. Propagation was achieved by multiple shoot regeneration from the cotyledonary node of decapitated seedlings, followed by axillary shoot growth from single node shoot segments and rooting of excised shoots. All steps were accomplished on basal Murashige and Skoog medium without plant growth regulator supplements. The results revealed competence for generation of multiple shoots from cotyledonary node tissue, stimulated by repeated shoot harvest, in seedlings of all three tree species. Tens of plants per seedling were regenerated in about 4 mo from culture initiation. In a given species clone size was seedling-dependent, which presumably stems from genotypic variability among seedlings in regeneration ability in vitro. By this means the laborious search for a plant growth regulator regime suitable for organogenesis induction and adapted per genotype became redundant, and biodiversity of the seed germplasm could be maintained. The approach ideally suits establishment of clones of wild plants of endangered species, like those of the Moringaceae, species with high ability for producing supplementary shoots, and without the need to add plant growth regulators, including the rooting stage.     

Efficient in vitro regeneration of fireweed,a medicinal plant

Epilobium angustifolium L. (fireweed) is a medicinal plant that has been used to treat diarrhea, mucous colitis, irritable-bowel syndrome, skin problems, prostate problems, menstrual disorders, asthma, whooping cough, and hiccups. A highly efficient and rapid regeneration system via multiple shoot formation was developed for fireweed. Explants (leaf, petiole, root, and stem segments) excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. Explant browning, a major problem for regeneration, was overcome by adding 100 mg/l ascorbic acid to all prepared media containing growth regulator combinations. Root explants formed more shoots than other explants. Best shoot proliferation was obtained from root explants cultured on media with 0.1 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were transferred to rooting media containing different concentrations of IAA, IBA, NAA or 2,4-D. Most shoots developed roots on medium with 0.5 mg/l IAA. Rooted explants were transferred to vermiculate in Magenta containers for acclimatization and after 3 weeks they were planted in to plastic pots containing potting soil and maintained in the plant growth room.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators

The influence of various basal medium and plant growth regulators on the efficient micropropagation of nodal explants from mature trees of alemow, sour orange, and ??Cleopatra?? mandarin citrus rootstocks was studied. All three citrus rootstock shoot cultures showed a preference for high-salt media, like Murashige and Skoog or Driver and Kuniyuki Walnut medium. Several combinations of N ⁶-benzyladenine (BA) and adenine (AD), kinetin (KIN) or gibberellic acid (GA) were tested to optimize the shoot proliferation phase. BA/GA combinations improved the proliferation of all the rootstocks studied, especially alemow. The addition of BA and AD to the culture medium improved shoot proliferation in sour orange and ??Cleopatra?? mandarin in the same way as BA and GA. The addition of different combinations of BA/KIN did not result in further improvement of any of the studied variables. The transfer of in vitro shoots to rooting media, containing different concentrations of indolebutyric acid (IBA) and indoleacetic acid (IAA), resulted in regeneration of complete plantlets. Alemow and ??Cleopatra?? mandarin shoots rooted well using these plant growth regulators; however, all combinations of IBA and IAA tested resulted in very low rooting percentages in sour orange. To improve rooting in sour orange and ??Cleopatra?? mandarin, different combinations of naphthaleneacetic acid (NAA) and IBA were tested. All NAA/IBA combinations produced higher rooting percentages than did the IBA/IAA combinations, and in sour orange nearly 100 % of explants developed roots. An efficient and simple protocol for the micropropagation of three citrus rootstocks, alemow, ??Cleopatra?? mandarin, and sour orange, by culturing nodes from mature plants, has been established.     

In vitro propagation of three commercial cut flower cultivars of Anthurium andraeanum Hort

In vitro propagation of Anthurium andraeanum Hort. cut flower cultivars viz. Lima White, Tropical White and Tropical Red through organogenesis using mature plant derived leaf explants was established on Murashige and Skoog (MS) medium fortified with different growth regulators. Cultivar, stage and different regions of the source leaf, and type of growth regulators significantly influenced callus induction. Explants from folded brown leaves were superior in induction of callus. Half strength MS medium fortified with 0.88 microM of benzyiadenine (BA), 0.9 microM of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.46 microM of kinetin (Kn) at pH 5.5 was most effective for callus induction. Transfer of callus to medium with 0.54 microM of NAA in place of 2,4-D induced higher number of shoots. Subsequent cultures displayed enhanced rate of shoot initiation and multiplication. Transfer of shoots onto half strength MS medium supplemented with 0.54 microM of NAA favoured rooting of shoots. Cultivar Tropical White was superior in callus, shoot and root induction compared to Lima White and Tropical Red. Plantlets after acclimation in greenhouse were transferred to net-house, that exhibited ninety seven per cent survival. Plants flowered normally between 12 and 15 months and were morphologically similar to that of the mother plants.     

Propagation of rose speciesin vitro

Summary Several rose species (Rosa rugosa, R. wichuraiana, R. setigera, R. laevigata, R. banksiae, R. roxburghii, R. odorata) and interspecific hybrids were cultured to determine the appropriate concentrations of nutrients and growth regulators for shoot proliferation and root initiation. Cultured shoot tips and lateral buds from different genotypes proliferated multiple shoots on a basal medium [Murashige and Skoog (MS) salts, vitamins, glycine, sucrose, and agar] supplemented with 0 μM to 17.8 μM (4 mg·l⁻¹) 6-benzyladenine (BA) and 0 μM to 0.54 μM (0.1 mg·l⁻¹) naphthalene, acetic acid (NAA). The ability of the explants to proliferate shoots and initiate roots was affected by the genotype, the nodal position of the explant, the strength of the MS basal salts, and the growth regulators used. The buds nearest the apex exhibited the slowest rate of development. Most species had the highest shoot proliferation when cultured on basal MS medium supplemented with 8.9 μM (2 mg·l⁻¹) BA, but the degree varied by species. Root development was enhanced by lowering the concentration of MS salts. With difficult-to-root species, rooting was improved by supplementing the media with 11.4 μM (2 mg·l⁻¹) indole-3-acetic acid (IAA) or by giving them a 7-d dark treatment at 10°C.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

Different inhibitors of the gibberellin biosynthesis pathway elicit varied responses during in vitro culture of aspen (<Emphasis Type="Italic">Populus tremula</Emphasis> L.)

Several synthetic plant growth regulators (PGRs), including prohexadione-calcium (ProCa), paclobutrazol (PBZ), and chlormequat chloride (CCC), known for their ability to inhibit gibberellin (GA) biosynthesis, were investigated for their influence on Populus tremula L. (aspen) shoots grown in vitro. Changes in plant growth induced by these inhibitors were compared to the effects of exogenous gibberellins (GA₃ and GA_4/7). All PGRs were added to the nutrient medium at concentrations of either 1 or 5 μM. Stem segments with and without apical buds were excised from in vitro-grown shoot culture, and these explants were incubated either in test tubes or Petri dishes. In the presence of 5 μM ProCa, shoot growth and rooting were inhibited when grown in test tubes, while shoots grown in Petri dishes exhibited strongly enhanced shoot and root growth. PBZ suppressed shoot development both in test tubes and Petri dishes, although 1 μM PBZ promoted adventitious root formation when shoots were grown in test tubes. Five micromolars CCC suppressed shoot and root development in test tubes, but promoted shoot growth in Petri dishes.     

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.     

Restart of Lignification in Micropropagated Walnut Shoots Coincides with Rooting Induction

The lignin content of walnut shoots did not change during in vitro shoot multiplication. Lignin content started to increase as soon as shoots were passed to a rooting medium with auxin. Exogenous auxin (applied for rooting) caused a transient elevation of the endogenous free indoleacetic acid (IAA) content with a simultaneous decrease of peroxidase activity. These events typically marked the completion of the rooting inductive phase (before any visible histological event, that is before the cell divisions beginning the rooting initiation phase). This meant that either the given exogenous auxin or the endogenous IAA has served as signal for the stimulation of lignification. Continued increase of lignification in the shoots required completion of root formation; this increase indeed was slown down when root emergence did not occur. It was further shown that lignification varied conversely to the content of the soluble phenol content, itself apparently being related to the activity of phenylalanine ammonia-lyase activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of Chokecherry and Pincherry cultivars

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of chokecherry (Prunus virginiana L.), `Garrington', and pincherry (P. pensylvanica L.f), `Mary Liss' and `Jumping Pound', were examined using various combinations of growth regulators. Dormant winter buds were used as explants. MSMO medium supplemented with 0.49 μM IBA and either 4.44 or 8.87 μM BA was found to be optimal for culture initiation of both species and cultivars. GA₃ (28.89 μM) significantly reduced (p=0.0001) the number of successfully established cultures. BA concentrations 8.87–12.82 μM gave optimal shoot proliferation in chokecherry and 4.44 μM BA in both cultivars of pincherry. Auxin treatments were required for ex vitro rooting of approximately 10 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (84%) was obtained with IBA/NAA (9.80/2.69 μM). A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%) mixture, was also effective (75%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Organogenesis from shoot segments and via callus of endangered <Emphasis Type="Italic">Kosteletzkya pentacarpos</Emphasis> (L.) Ledeb.

Efficient plant regeneration systems both from shoot segments and via callus organogenesis were developed for Kosteletzkya pentacarpos (L.) Ledeb., a rare and endangered Eurasian species. In the experiments with existing meristems, factors affecting shoot proliferation, including explant type, i.e. decapitated and intact shoots, and plant growth regulators, indole-3-acetic acid or kinetin, were investigated. Shoot proliferation was significantly affected by the type of explant, the hormones and their interaction. The highest shoot multiplication rate was obtained from decapitated shoots. Increasing kinetin concentration promoted shoot elongation regardless of explant type. In intact shoots, shoot length was also affected by increasing auxin concentration, although this effect tends to decrease with higher concentration. Decapitated shoots were not responsive to the addition of auxin. Micropropagation through organogenesis from callus was also investigated. Calli were obtained from leaf, stem internode and root explants. Only the leaf-derived calli produced shoots and indole-3-acetic acid favoured increased numbers of shoots. A number of experiments were conducted for rooting of in vitro produced shoots. All of them induced high rooting frequency, the number and the length of roots being dependent on the strength of the basal medium. The use of 1–2 mg l⁻¹ indole-3-butyric acid resulted in refining the optimal concentration for root elongation. The regenerated plants (70%) survived and flowered in their first vegetative period.     

Influence of ventilation closure, gelling agent and explant type on shoot bud proliferation and hyperhydricity in Scrophularia yoshimurae—A medicinal plant

Summary We report an improved procedure of in vitro propagation of Scrophularia yoshimurae&#x2014;a medicinally important plant species indigenous to Taiwan. Induction of maximum shoot buds (22.75 per explant) was obtained with shoot tip explant cultured on Murashige and Skoog medium supplemented with 1.0mgl^&#x2212;1 benzyladenine (BA) and 0.2mgl^&#x2212;1 &#x03B1;-naphthaleneacetic acid and gelrite using dispense paper (DP) for ventilation closure of culture vessels. The type of gelling agents (agar and Gelrite) affected both quantity and quality of the shoots induced. Using aluminum foil for ventilation closure resulted in a higher number of hyperhydric shoots. Hyperhydricity was reduced by culturing shoots on a medium devoid of plant growth regulators in conjunction with the use of DP. Plantlet growth in vessels using DP was healthier and all plantlets survived after being transplanted to soil.     

Effect of various growth regulators on growth in vitro of cherry shoot tips

An investigation was undertaken to examine the effects of nine different growth regulators on growth in vitro of shoot cultures of the semi-dwarfing cherry rootstock Colt (Prunus avium × P. pseudocerasus). The effects of each supplement on shoot extension and proliferation and also leaf and callus production were noted, and it was found that BAP has the ability to proliferate shoots, IBA nullifies this effect and that kinetin, ABA, GA₃ and ethylene inhibit the growth of colt cultures. Conditions were established which resulted in a) optimum shoot growth prior to subsequent rooting or grafting; b) maximum shoot proliferation for rapid clonal multiplication and c) minimum shoot growth. This study will form the basis of an investigation into germplasm conservation of temperate fruit trees by both cryogenic storage and minimal growth techniques.     

Regeneration of <Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl (Japanese plum) from hypocotyls of mature seeds

Plant regeneration of Prunus salicina (Japanese plum) using mature seeds was studied and evaluated. Shoots were effectively induced from hypocotyl slices of mature seeds on media containing cytokinins. Among three plant growth regulators evaluated, thidiazuron (TDZ) was the most effective for shoot induction overall. Shoots were also induced using 6-benzylaminopurine (BA), but the effectiveness was reduced at low concentrations. Low regeneration was induced using kinetin. Three plum varieties were evaluated and the regeneration appeared to be genotype dependent. Induced shoots elongated, roots formed, and plantlets developed upon transfer of the shoots to the rooting medium. Primary shoots, when sub-cultured on fresh induction medium, produced multiple shoots, and such multiplication could continue for more cycles. The plantlets were transferred to soil, and the full plants were readily recovered in a greenhouse. The regeneration process was relatively fast as plants could be recovered in 4 to 5 mo. after the culture initiation.     

High frequency shoot regeneration and plantlet formation from root tip of garlic

An efficient and novel method of direct shoot regeneration from root tips in garlic was developed. The influence of growth regulators, basal media and age of root explant on shoot initiation and proliferation was examined. The best growth regulator combination was 1-naphthaleneacetic acid and 6-benzyladenine at 1 and 10 μM, respectively, inducing shoot initiation from 75% of the explants. The frequency of shoot initiation on different basal media was similar. Explant root tips from plantlets taken 15 to 18 days after sprouting showed the highest shoot initiation (95%). In contrast to Murashige and Skoog medium, which produced more than 10 shoots per explant, B5 medium produced smaller shoots, although the number was higher. Rooting of individual shoots was induced after transfer to medium without growth regulators. Plantlets, after acclimatization in a growth cabinet, were successfully transplanted to the field, and no phenotypic variation was observed among them. The technique has potential applicability for rapid propagation of garlic. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of the Damask rose (Rosa damascena Mill.)

Roses are an important commercial crop available in a wide range of varieties in international markets. Due to its economic value, this study aimed to establish a new and reproducible protocol for the in vitro propagation of Rosa damascena Mill. We developed an efficient and cost-effective method for rapid and high-quality shoot multiplication and in vitro rooting of Damask rose using nodal explants. For each stage of the micropropagation procedure (i.e., explant establishment, shoot multiplication and growth, and rooting), different media and combinations of plant growth regulators were utilized. A new culture medium, termed A₁₉, resulted in significant improvements to shoot proliferation and root induction for this rose cultivar. For optimal explant establishment, shoot growth, and proliferation, a modified Murashige and Skoog medium with higher levels of nitrates, calcium, and iron plus supplementation with 4?mg/l 6-benzylaminopurine and 0.25?mg/l indole-3-acetic acid was utilized. To increase shoot length, 75?d after culture initiation (including two subcultures), shoots were transferred to the same medium additionally supplemented with 0.2?mg/l gibberellic acid. This resulted in vigorous shoot growth, with longer shoots and a greater number of shoots per explant. Shoots were then separated and transferred to various root induction medium for 30?d. The results clearly showed that a liquid ?A₁₉ medium-A (i.e., with half-strength macroelements) supplemented with 0.1?mg/l indole-3-butyric-acid was the most successful medium for in vitro rooting in this cultivar. Shoots were cultured in this medium for 7?d in the dark, before transfer to liquid ?A₁₉ medium-A without hormone supplementation under a 16-h photoperiod. This modified protocol resulted in significant improvement in shoot regeneration and proliferation and obtained stronger shoots over a period of about 20?wk.