Optimizing the shoot proliferation protocol of Penthorum chinense by axillary buds

Four parameters, three hormones and sucrose, at seven concentrations, were designed for shoot proliferation of Penthorum chinense by uniform design. The obtained data were used for building two quadratic polynomial equations by partial least square to determine optimum concentrations of four factors. Experiments for verification confirmed that no significant difference existed between the predicted and the validated values in shoot number and length based on all inoculated explants.     

An improved micropropagation protocol for teak

An improved micropropagation protocol has been developed for teak (Tectona grandis). Nodal explants placed on MS medium supplemented with 22.2 M benzylaminopurine and then serially transferred to fresh medium after 12, 24, 48 and 72 h gave maximum culture establishment (76.8%). Establishment was reduced when explants were retained in the initial culture medium longer than 12 h. Explants collected in May showed maximum (76.8%) response. Placement of the explants on MS medium supplemented with 22.2 M benzylaminopurine and 0.57 M indole-3-acetic acid resulted in the maximum average number of shoots. In vitro raised micro shoots were rooted ex vitro by dipping in indole-3-butyric acid (9.8 mM) for 2 min followed by planting in polyethylene pots containing a soil:vermiculite (1:1 v/v) mixture. This treatment resulted in 77.9% survival of the plantlets. They were weaned in a glasshouse and finally moved to an agro-net shade house.     

A protocol for micropropagation of Alpinia galanga

Emerging buds of rhizome of Alpinia galanga Willd produced shoots and roots simultaneously when cultured in MS medium supplemented with kinetin 3.0 mg l^-1. Each explanted shoot bud produced 8 shoots in average and roots simultaneously within 8 weeks. Shoot proliferation could be continued even after a year by transferring each divided shoot explant to the same medium. Regenerated plantlets could be sucessfully transferred to soil where they grew well within 10–12 weeks with 80% survivality. This revised version was published online in June 2006 with corrections to the Cover Date.     

A protocol for rapid micropropagation of endangered Isoplexis

Summary An efficient protocol for large-scale micropropagation of Isoplexis canariensis (L.) Loud., I. chalcantha Svent, and O'Shan., and I. isabelliana (Webb and Berth.) Masf. (Scrophulariaceae) is reported. Multiple shoots were obtained from shoot tips and nodal segments isolated from seedlings when cultured under varying conditions. Factors such as nutrient media, concentration of growth regulators, and type of induction medium (liquid or solid) strongly influenced shoot proliferation and development. Multiple well-developed shoots were obtained after induction on solid media containing Murashige and Skoog full-strength salts and low cytokinin concentrations. By changing the major salt formulation to half strength and employing liquid media the morphogenic parameters evaluated were affected negatively. Many shoots rooted spontaneously in hormone-free media and plants grew successfully in the greenhouse. Flowering was observed 6 mo. after ex vitro cultures were established.     

Development of shoot inHydrangea macrophylla I. Terminal and axillary buds

The structure of shoots, in particular of winter buds, ofHydrangea macrophylla was examined. The non-flower-bearing shoot is usually composed of a lower and an upper part, between which a boundary is discernible by means of a distinctly short internode. This internode is the lowermost of the upper part, and it is usually shorter than the internodes immediately above and below, although the internodes tend to shorten successively from the proximal to the distal part of the shoot. Variations exist in the following characters among the terminal bud, the axillary bud on the lower part of the shoot and the axillary bud on the upper part: (1) length of bud; (2) character of the outermost pair of leaf primordia; (3) degree of development of secondary buds in the winter bud; and (4) the number of leaf primordia. Usually, the terminal bud contains several pairs of foliage leaf primordia with a primordial inflorescence at the terminal of the bud, but the axiallary bud contains only the primordia of foliage leaves in addition to a pair of bud scales.     

Development of suitable protocol to overcome hyperhydricity in carnation during micropropagation

Hyperhydricity during micropropagation of carnation (Dianthus caryophyllusL.) was reduced by media modifications. Three commercial varieties (White sim, Exquisite and Scania) tested, varied in optimal growth without hyperhydricity. Increased concentration of iron and/or magnesium reduced hyperhydricity with 0.7–0.8% agar. At some concentrations, hyperhydricity was reduced to 0% and shoot multiplication was increased. All nonhyperhydrified micropropagated plantlets survived in a glasshouse during acclimatization.     

Development of a new culture medium and efficient protocol for in vitro micropropagation of Ceratonia siliqua L.

A new basal culture medium was developed and tested using a rapid and efficient protocol of in vitro axillary shoot bud proliferation of Ceratonia siliqua L., an important Mediterranean Fabaceae plant species. In a first experiment, the new formulated ‘LA’ mineral composition significantly improved shoot growth and proliferation as compared with Murashige and Skoog medium (MS, 1962) in both solid and liquid culture media. However, the liquid culture system proved to be the most suitable for shoot induction, shoot length (about fourfold higher), and multiplication rate (about two-fold higher), the difference being significant. The measured growth and proliferation parameters were further improved when LA mineral composition was optimized, in a second experiment. The highest multiplication rate (6.3) was achieved during the second subculture using the optimized ‘LAC’ medium. Noticeably, hyperhydricity and shoot-tip necrosis symptoms were absent in both formulated LA and LAC compositions when using the liquid culture system. In vitro rooting in solid medium showed 41.7 to 46.3% response on a solid medium which was more suitable than the liquid culture system, the difference being significant. In contrast, pretreated microcuttings with 3 μM IBA (indole-3-butyric acid) were successfully rooted ex vitro, showing significantly higher response (91.7%), average root number (8.3), and root length (31.5 mm). The plantlets were successfully acclimatized showing more than 90% survivability and normal morphology. The present study is a first cost-effective protocol for carob micropropagation combining the use of the newly formulated LAC basal medium, a liquid culture system, and ex vitro rooting.

     

A protocol for Ulmus minor Mill. micropropagation and acclimatization

Here we report the establishment of a simple protocol for the micropropagation and acclimatization of U. minor. Branches with dormant buds were collected from mature elms and sprouted in a greenhouse. Tip and node segments were used as starting material for in vitro proliferation in a medium (designated here as DKW1) already used for the micropropagation of a clone of the English Elm (U. procera SR4). In the first assay, in which explants from nine different trees were used, 88.5% of the tip segments produced new axillary shoots thus proving to be the best explant type. Afterwards, material from four different trees (F4, F7, F13, F14), that had the highest sprouting rate in the greenhouse, was used to test for genotype influence. F14 proved to be the best genotype in culture and it was used for all the subsequent experiments. Shoots from F14 were used to assay in vitro rooting using five DKW based media. Rooting percentages were high for all media and varied between 80% and 100%. For acclimatization two approaches were assayed: the use of previously rooted in vitro plants and the direct acclimatization of shoots from cultures in DKW1. After 6 weeks, 86.4% of the in vitro rooted plants were successfully acclimatized and a slightly higher value, 88.6%, was attained by direct acclimatization of shoots with thick stems and hard leaves. These results proved that there is no need for a previous in vitro rooting step and that direct acclimatization can effectively reduce time and costs. Thus U. minor micropropagation and acclimatization can be divided into only two steps: proliferation of shoots in DKW1 and direct acclimatization of these shoots in a sterile soil mixture.     

Floral determination in axillary buds of Nicotiana silvestris

At anthesis of the terminal flower the developmental fates of axillary buds of the long-day plant Nicotiana silvestris were assessed in situ and in isolation. The in situ developmental fate was assessed by decapitating the plant above the bud in question and letting the bud mature. The developmental fate of isolated buds was assessed by removing the bud from the main axis, rooting it, and letting it mature. The number of nodes below the terminal flower of the mature shoot was indicative of the developmental fate of the bud. Terminal meristems of rooted axillary buds exhibited two patterns of development: (1) Their developmental fate was the same as that of in situ buds at the same node or (2) their developmental fate was the same as that of seed-derived plants. For example, terminal meristems of rooted buds from the fourth node below the inflorescence produced either 15 to 19 nodes or 36 to 40 nodes. In situ fourth buds produced 12 to 14 nodes while seed-derived plants produced 33 to 39 nodes. Terminal meristems of rooted axillary buds that exhibited the same developmental fate as that of in situ buds were determined for floral development. Although determined buds produced a terminal flower, all but one had abnormal inflorescences. That is, in the place of floral branches determined buds produced vegetative branches. Four buds that were not determined for floral development had their shoot tips rooted each time the plant bolted. Only when the plants were allowed to grow without being rerooted did they flower. These results indicate that roots may prevent and/or destabilize floral determination in N. silvestris.     

Expression of a ribosomal protein gene in axillary buds of pea seedlings

Axillary buds of intact pea seedlings (Pisum sativum L. cv Alaska) do not grow and are said to be dormant. Decapitation of the terminal bud promotes the growth of these axillary buds, which then develop in the same manner as terminal buds. We previously showed that unique sets of proteins are expressed in dormant and growing buds. Here we describe the cloning, sequencing, and expression of a cDNA clone (pGB8) that is homologous to ribosomal protein L27 from rat. RNA corresponding to this clone increases 13-fold 3 h after decapitation, reaches a maximum enhancement of about 35-fold after 12 h, and persists at slightly reduced levels at later times. Terminal buds, root apices, and elongating internodes also contain pGB8 mRNA but fully expanded leaflets and fully elongated internodes do not. In situ hybridization analysis demonstrates that pGB8 mRNA increases in all parts of the bud within 1 h of decapitation. Under appropriate conditions, growing buds can be made to stop growing and become dormant; these buds subsequently can grow again. Therefore, buds have the capacity to undergo multiple cycles of growth and dormancy. RNA gel blots show that pGB8 expression is reduced to dormancy levels as soon as buds stop growing. However, in situ hybridization experiments show that pGB8 expression continues at growing-bud levels in the apical meristem for 2 d after it is reduced in the rest of the bud. When cultured stems containing buds are treated with indoleacetic acid at concentrations ≥10 μm, bud growth and expression of pGB8 in the buds are inhibited.     

Plant regeneration from axillary buds of a mature tree of Ficus religiosa

Dormant axillary buds of nodal explants collected from a mature (35-year-old) tree of Ficus religiosa L. sprouted on MS medium supplemented with 6-benzyladenine (BA, 5 mg l^–1) and indole-3-butyric acid (IBA, 0.2 mg l^–1 ) within 4 days. Multiple shoots were obtained when these explants were transferred to MS medium containing 1.5 mg l^–1 BA and 1.5 mg l^–1 adenine sulphate (ADS). These multiple shoots (1 cm) were allowed to elongate to a height of 4–5 cm by maintaining them on MS medium containing 2 mg l^–1 BA, 0.5 mg l^–1 1-naphthalene acetic acid (NAA), and activated charcoal (0.3% w/v). Nodal segments taken from these in vitro-proliferated shoots yielded multiple shoots when cultured on the multiple shoot-inducing MS medium mentioned above. Root induction in these shoots (4–5 cm in height) was achieved by transferring them onto MS medium supplemented with 2 mg l^–1 IBA and 0.1 mg l^–1 NAA for 1 week; upon transfer to half-strength MS basal medium these shoots exhibited root proliferation. These rooted plantlets were successfully established in soil after a short period of acclimatization. Received: 17 April 1997 / Revision received: 2 September 1997 / Accepted: 20 September 1997     

Standardization of a protocol for micropropagation of Eupatorium glandulosum L. an important medicinal plant

The presence of residual female fertility in most of the parthenocarpic banana accessions encourages the banana breeder to develop new hybrids through conventional breeding. Desirable trait can be fixed in the first generation of hybrid progenies, but the evaluation of these hybrids in field is the time-consuming process owing to non-availability of uniform suckers/planting material. This can be overcome by developing multiple shoots from single embryo in a short period of time through embryo culture. A protocol for in vitro multiplication of plantlets from zygotic embryos was standardized in seeded accessions. Multiple shoots from zygotic embryos were achieved up to 55.2% and 64.1% in seeded accessions of Musa acuminata and M. velutina respectively in medium supplemented with 17.76 µM of BAP. The Single shoot derived (only germination) from zygotic embryos was decapitated and the apical meristem were disturbed for further multiple shoot formation in media supplemented with 17.76 µM of BAP. Present studies revealed that in total 75% and 91% of the M. acuminata and M.velutina embryos were able to produce multiple shoot from single embryo by manipulating the media composition and decortications technique. The above protocol was applied for zygotic embryos obtained from controlled pollination (18 cross combinations) and open pollination (nine accessions) of various genomic groups (ABB, AAB, AA). The multiple shoots derived from zygotic embryos and plantlet germinated from zygotic embryos was examined for genetic fidelity analysis by SSR markers.

     

A simplified protocol for micropropagation of guayule (Parthenium argentatum gray)

Summary A simple, efficient protocol for in vitro micropropagation of guayule is reported. Shoot cultures were maintained on MS (Murashige and Skoog, 1962) medium supplemented with 1.0 mg l⁻¹ (4.4 μM) 6-benzylaminopurine and 0.025 mg l⁻¹ (0.3 μM) α-naphthaleneacetic acid. Excised shoots were treated for 14–18 h with 100 mg l⁻¹ (492.1 μM) indole-3-butyric acid in 0.5 x MS salts to induce rooting. The shoots were subsequently inserted into cellulose plugs which were packed in sterile, ventilated plastic culture vessels and moistened with 0.5 x MS medium without growth regulators. Use of cellulose plugs, liquid medium and ventilated culture vessels facilitated acclimation. Rooted shoots were transplanted into potting medium and acclimated to greenhouse conditions by covering with a cloche for 2 d, followed by daily watering for the first week. Any mention of trade names or commercial products in this report is for informational purposes only and does not imply endorsement by the U.S. Department of Agriculture or the Agricultural Research Service.     

Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl₂, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds. Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.     

Elimination of five viruses from sugarcane using in vitro culture of axillary buds and apical meristems

Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus elimination through meristem tissue culture originating from both apical and axillary shoots were compared. The average rates of regeneration and elongation from apical meristem tissues were 91 and 66%, respectively, with the virus-free rate among elongated shoots ranging from 61–92%. Mature axillary buds were cultivated in vitro to produce axillary shoots, from which meristem tissues were excised and cultured. These meristem tissues regenerated (77–100%) and elongated (55–88%) in culture medium at approximately the same rate as the apical meristems. The average virus elimination rate was 90% among elongated shoots derived from mature axillary buds. All five viruses can be eliminated by meristem tissue culture from both apical and axillary shoots using a standardized procedure. The overall average efficiency of virus-free plant production was 45 and 58% from apical and axillary shoots, respectively. There were no significant differences for shoot induction or virus elimination when the meristems were harvested from either the apical or the axillary shoots. This is the first report of SrMV or SCSMV elimination from sugarcane, as well as elimination of any mixed virus infections. This new method of harvesting meristems from axillary buds greatly expands the amount of material available for therapeutic treatments and thereby increases the probability of eliminating viruses from infected sugarcane.     

Back to the roots: protocol for the photoautotrophic micropropagation of medicinal Cannabis

Plant Cell, Tissue and Organ Culture (PCTOC) - The aim of this protocol was to develop an alternative in vitro propagation system for Cannabis sativa L. by mimicking nursery-based vegetative...     

Callus formation and plantlet development from axillary buds of taro

Excised lateral buds of taro [Colocasia esculenta var. esculenta (L.) A.F. Hill] developed into plantlets and formed callus if cultured on media containing taro extract. α-Naphthaleneacetic acid enhanced the process but only if taro extract was also present. The tissue requirements for this variety of taro are different from those of Colocasia esculenta var. antiquorum (L.) A.F. Hill.