Shoot Primordia Research Articles

Clonal propagation ofPoncirus trifoliatethrough culture of shoot primordia

SummaryIn Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.

Plant regeneration via organogenesis and somatic embryogenesis in callus cultures derived from mature zygotic embryos of leek (Allium ampeloprasum L.)

A high frequency plant regeneration system via organogenesis and somatic embryogenesis was established with callus cultures derived from mature zygotic embryos of different leek genotypes (Allium ampeloprasum L.). Four different callus types with varying morphogenetic potential were obtained. Relatively high concentrations of the auxin 2,4-dichlorophenoxy-acetic acid reduced callus weight and subsequent shoot regeneration and primordia formation of the callus. Shoot regeneration and primordia formation of the callus decreased after prolonged subculture on media containing 2,4-dichlorophenoxy acetic acid. A callus growth period of six weeks on Murashige and Skoog medium with 0.25–0.5 mg l-1 2,4-dichlorophenoxy acetic acid showed the highest rate of shoot regeneration after transfer of callus to regeneration medium with 1 mg l-1 kinetin.

Long-term shoot regeneration from pampas grass (Cortaderia selloana Schult.) through manipulation of growth regulators in vitro

A regeneration system for 'Pumila' pampas grass (Cortaderia selloana Schult.) that yields plants over many months and allows control of morphogenesis was developed. Immature inflorescence explants cultured for three 4-week passages on MS basal medium supplemented with 4.5 μM 2,4-D and 8.9 μM BA yielded a dark green callus that organized into shoot primordia. Rate of shoot development was increased after transfer of shoot primordia to medium supplemented with 9.8 μM IBA. Subculture every 4-6 weeks onto medium containing IBA yielded a continuous production of shoots. Control of morphogenesis was achieved by transferring cultures to medium containing 4.5 μM 2,4-D and 8.9 μM BA for shoot bud proliferation and to medium containing IBA for shoot production.

Genetic variation and control of plant regeneration in leek (Allium ampeloprasum L.)

AbstractZygotic embryos of leek (Allium ampeloprasum L.) were isolated from mature seeds of different cultivars, selfings and full‐sib families. The embryos were cultured on callus induction and shoot regeneration medium and employed to study several parameters: percentage of embryos forming calluses, percentage of embryos forming compact calluses, callus weight, percentage of regenerating calluses, numbers of shoot primordia and numbers of regenerated shoots. Differences between cultivars and selfings were found for most parameters studied. For all cultivars all parameters, except callus weight, decreased after one generation of selfing. Compact callus types enhanced primordia formation and shoot regeneration. Genetic characteristics of callus development and plant regeneration were studied in a 4 × 4 diallel cross. Analysis of variance revealed significant differences between full‐sib families. The diallel analysis showed that additive gene effects were significant for all parameters. The predominance of additive gene effects indicated high narrow‐sense heritability. Breeding for an increased number of regenerated shoots was successful.

Indole butyric acid induces regeneration of phenotypically normal apricot (Prunus armeniaca L.) plants from immature embryos

A prerequisite for most transformation systems is an efficient and reliable method to regenerate phenotypically normal plants. Immature embryos or cotyledons were cultured at three developmental stages (stage 1, 2 and 3, PF=3, 30–60, and 100, respectively) from two unrelated apricot genotypes, ‘Zard’ and ‘NJA82’. Explants were cultured on MS media supplemented with either BA or TDZ at four concentrations (0, 0.5, 5.0 or 20 μM) and 2,4-D at 0 or 1 μM. Stage 1 embryos cultured on MS medium without growth regulators formed embryoid-like structures. Shoot primordia induction was greatest with stage 2 cotyledons on media containing 5–20 μM TDZ and 1 μM 2,4-D, although shoot morphology was abnormal, especially with the highest level of TDZ. In another factorial experiment, stage 2 cotyledons were cultured on media containing TDZ (0, 5, 7.5, 10, 15 or 20 μM) in combination with either no auxin, 1 μM 2,4-D, 1 μM IBA, or 5 μMIBA. Regeneration percentages of 80% or more were observed on media containing 1–5 μM IBA and 5–10 μM TDZ. The medium containing 5 μM IBA and no TDZ exhibited the highest frequency of phenotypically normal plantlet regeneration.

Bud structure and resprouting in coppiced stools of Salix viminalis L., S. eriocephala Michx., and S. amygdaloides Anders

Fast-growing willows are cultivated as coppice in short rotation biomass plantations. The production and sustainability of the system is based on the ability of trees to resprout after repeated harvesting. The large variation in coppicing ability is due to plant genotypic differences in structure and physiology as well as environmental factors. Morphological and structural prerequisites for resprouting were compared in two shrubby willows with high coppicing ability, S. viminalis and S. eriocephala, and one tree-formed species, S. amygdaloides, with low coppicing ability. The initiation and development of buds and the resprouting pattern of coppiced stools were compared. All buds were axillary in origin and showed the same principal structure consisting of one main shoot primordium and two lateral primordia. In S. viminalis and S. eriocephala the lateral buds contained several leaf primordia and sprouted shortly after the main bud. In S. amygdaloides further development of lateral buds was inhibited after formation of two budscales, and leaf primordia were not formed until the buds were forced to sprout. The number of sprouts developing after coppicing were correlated to the structure and number of buds and their position on the stools. Self-thinning rate was high and many shoots originating from lateral buds died. Most buds were located above ground on the remaining basal portions of harvested stems. No adventitious buds were found on the stools. Significantly different bud differentiation pattern and frequent sylleptic sprouting resulted in lower coppice response in S. amygdaloides compared to S. viminalis and S. eriocephala.

Cells within the nodal region of carnation shoots exhibit a high potential for adventitious shoot formation

Adventitious shoot formation was studied with leaf, stem and axillary bud explants of carnation (Dianthus caryophyllus L.). The shoot regeneration procedures were applicable for a wide range of cultivars and shoot regeneration percentages were high for all explant types. Using axillary bud explants, shoot regeneration efficiency was independent of the size of the bud and of its original position in the plant. In contrast, shoot regeneration from stem and leaf explants was strongly dependent on their original position on the plant. The most distal explants (just below the apex) showed the highest level of shoot regeneration. The adventitious shoot primordia developed at the periphery of the stem segment and at the base of leaf explants. In axillary bud, stem and leaf explants, shoot regeneration originated from node cells, located at the transition area between leaf and stem tissue. Moreover, a gradient in shoot regeneration response was observed, increasing towards the apical meristem.

Micropropagation of Passion fruit from Subcultured Multiple Shoot Primordia

A technique for long-term micropropagation of Passiflora edulis from subcultured multiple shoot primordia is described. Excised tissues from leaves cultured on Murashige and Skoog medium (MS medium) supplemented with 3 % (w/v) sucrose, 1 μmol L -1 6-benzylaminopurine (BA) and 1 μmol L -1 indole-3-butyric acid (IBA) formed multiple shoot primordia. These shoot primordia were transferred to a medium containing 10 μM BA and subcultured repeatedly at 3-week intervals. When subcultured shoot primordia were transferred to a medium containing lower concentrations (0.1 or 1.0 μmol L -1 ) of BA, shoots were formed. Subcultured shoot primordia have continued to regenerate plants for more than 3 years. Shoots developed and rooted on MS medium lacking growth regulators. Rooted plants were transferred to pots and grown in a greenhouse. According to the morphology of leaves of regenerated plants, rejuvenation was obtained by this method of in vitro culture.

Plant Regeneration and Microprojectile-Mediated Gene Transfer in Embryonic Leaflets of Peanut (Arachis hypogaea L.)

Excised leaflets from mature embryos of peanut cultivars Robut, Gajah, McCubbin and NC-7 produced multiple shoot primordia via organogenesis on Murashige and Skoog medium supplemented with 3 mg L-1 BAP and 1 mg L-1 NAA. Plants were regenerated by elongation of shoot primordia on transfer to MS medium supplemented with 5 mg L-1 BAP. The regeneration frequency of five plants per embryonic leaflet was 10-40-fold higher than previously reported from seedling leaflets. Microprojectile bombardment parameters were optimised for high transient expression rates of the β-glucuronidase (uidA or gus) reporter gene in peanut embryos (> 2000 cells per bombardment) and in excised embryonic leaflets (> 1200 cells per bombardment). The firefly luciferase reporter gene (luc) allowed repeated non-toxic assays, revealing the transition from hundreds of transiently expressing cells soon after bombardment, to a few stably expressing regions in callus grown without selection for 8 weeks after bombardment of embryonic leaflets.

Conservation of Gene-resources in Epiphytic Vanda pumila (Orchidaceae) by Tissue-cultured Shoot Primordia.

Shoot apices of 60-day-old protocorms of Vanda pumila Hook. f., were transplanted and cultured in B5 liquid media supplemented with 2% sucrose and NAA and BAP at various concentrations at pH 5.8 by shaking at 2 cycles/minute on a rotary culture apparatus under 2, 000-10, 000lux illumination for 24 hours daily. Tissuecultured shoot primordia were primarily induced from some of those cultures and were subcultured in B5 liquid medium supplemented with 2% sucrose and 0.02mg/l BAP. Those tissue-cultured shoot primordia showed a high ability to regenerate plantlets approximately 120 days after they were placed onto 1/2 B5 agar medium supplemented with 1% sucrose and no plant growth substance at pH 5.8. The subculture line has been retaining the diploid chromosome number of 2n=38, typical of this species, for three years. This method may have promise and be applied to certain important clones of Vanda for micropropagation and conservation of genetic resources.

Ultrastructural Changes Leading to Plantlet Differentiation in Callus Cultures of African Rice.

Semithin and ultrathin sections of callus tissues of African rice (Oryza glaberrima Steud) after 7 and 14 days of initiation were compared with similar cultures after 3, 5, 7 and 10 days of incubation on a differentiation medium. Histological examinations indicated that at the early stages of development, both types of callus tissue appear structurally similar with small, almost isodiametric and meristematic cells located toward the callus periphery. In addition to this cell type, cultures on a differentiation medium also contained large parenchymatous and highly vacuolate non-differentiating cells. Ultrastructurally, while the former were characterized by a relatively large number of organelles, the non-differentiating cells contained only a thin peripheral layer of cytoplasm. Later in culture, the differentiating cells undergo periclinal divisions resulting in the formation of radial files of tussue which correspond to the meristematic regions where shoot primordia differentiation is generally initiated. There is also evidence to show that the differentiation of shoot and/or root primordia in callus tissues is a high energy demanding process. This energy requirement is fulfilled by the accumulation, mobilization and utilization of starch and other reserve substances prior to the onset of plantlet regeneration.

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10(4) protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.

Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.

Influence of the Herbicide Oryzalin on the Sporophytic Morphogenesis of Equisetum arvense

Sporophytic shoot primordia are initiated on gametophyte tissue of Equisetum arvense when cultured in a medium supplemented with cytokinin. The initiated shoots can develop on a cytokinin-free medium. Oryzalin, a dinitroaniline herbicide, inhibited the initiation and development of sporophytic shoots. But gametophytic growth with normal morphogenesis was not inhibited by oryzalin. These results suggest that some alteration induced by oryzalin is fatal to sporophytic, but not to gametophytic morphogenesis. Histological features in relation to the inhibition of sporophytic morphogenesis by oryzalin are discussed.

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 μM) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 μM) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.

Bud and cambial zone phenology of lateral branches from Douglas-fir (Pseudotsugamenziesii) seedlings

Bud and cambial zone phenologies of outdoor-grown, containerized seedlings of Pseudotsugamenziesii (Mirb.) Franco (Douglas-fir) were determined and compared. Morphological characteristics of the live primordial shoots were related to known stages of bud development, while cambial zone phenology was based on anatomical characteristics. Secondary growth in branches of P. menziesii seedlings was separated into six stages: (i) early wood formation; (ii) latewood formation; (iii) cessation of cambial cell division; (iv) dormancy; (v) cambial cell expansion; and (vi) resumption of cell division. Early wood formation by the cambial zone, and primary leaf initiation in the shoot tips occurred until July. During this time of maximal activity, differences in wall thickness and shape distinguished cambial cells from vascular cells in early differentiation stages. By late July, earlywood formation was changing to latewood formation and bud-scale initiation had begun. The transition to preformed leaf initiation in the buds occurred over a period of 1 month in August and September. Cambial cell division ceased in early September but tracheid differentiation continued until about the end of October. Preformed leaf initiation lasted until the buds became dormant in mid-November. The cambial zone was dormant from the end of November until the beginning of March, and in transverse section was characterized by a distinct boundary at the border of the xylem and regular, cigar-shaped cambial cells with thick radial walls. Resumption of cambial activity in the 1st week of April began with radial expansion, followed about 2 weeks later by cell division. Differentiation of earlywood tracheids and bud swelling began in early April. The majority of buds flushed in the 3rd week of April. Rays in the lateral shoots were composed of monoseriate files of radially elongated cells. Ray initials were not observed. Ultrastructural features indicated that the dictyosomes of the cambial cells were active during the winter period.

多孔質担体への苗条原基の捕捉と種苗大量増殖への応用

Development of an efficient system for shoot mass propagation through the entrapment of shoot primordia was investigated on Stevia rebaudiana. Shoot primordia of Stevia rebaudiana could be trapped into cubes of reticulate form matrix of loofah, sponge of Luffa aegyptica, when the primordia were cultured in a rotary culture system (flask scale) or in the drum type jar fermentor system in the presence of the cubic matrices. The shoot primordia grew to form uniform clumps staying in loofah matrices and the growth was stimulated by the entrapment compared to the control culture without the matrices. Shoots were efficiently induced from the entrapped clumps of primordia under subsequent static culture condition and were acclimatized successfully without removing the matrices.

大型培養槽を用いた多芽体の大量増殖に関する研究

The shoot primordium is a suitable inoculant for large scale culture of shoots comparing with the shoot. itself. A culture method of multiple shoots starting from the shoot primordium was established. The influence of scale up conditions on the growth of multiple shoots originating from the primordia was also investigated.Shoot primordium of Stevia rebaudiana has been induced and maintained in modified MS liquid medium containing NAA and BA. The primordia grew constantly by about 7 times in 1 month in the liquid shake culture. The multiple shoots were then induced efficiently from the primordia by eliminating the growth hormones from the MS medium. They grew constantly by about 20 times in 1 month in the liquid shake culture. The growth of shoots in culture vessels of different sizes, 0.3 l (flask), 6 l (jar fermentor) and 500 l (bioreactor), were compared by inoculating the primordia at a fixed density of about 10 gl-1. The shoots grew actively to fill up culture vessels within a month, and the final weight of shoots per unit volume of medium was similar in each culture vessel. This result strongly suggests that multiple shoots can be mass propagated in a large scale without affecting the culture efficiency.

Mass propagation of shoots of Stevia rebaudiana using a large scale bioreactor

A procedure for the mass propagation of multiple shoots of Stevia rebaudiana is described. Isolated shoot primordia were used as the inoculum to obtain clusters of shoot primordia. Such clusters were grown in a 500 liter bioreactor to obtain shoots. A total of 64.6 Kg of shoots were propagated from 460 g of the inoculated shoot primordia. These shoots were easily acclimatized in soil.

Improvement of protoplast regeneration from a recalcitrant inbred line of Brassica oleracea: a morphogenic analysis

The regeneration potency of protoplast-derived calli from a regenerative and a recalcitrant inbred line of Brassica oleracea was studied. Microcallus of the regenerative line consisted of meristematic structures that generated multiple meristematic nodules on callus proliferation medium K3. After transfer to regeneration medium K3R multiple shoot primordia developed. Microcallus of the recalcitrant line also contained meristematic regions during early culture, but most of them disappeared after 3 weeks on proliferation medium. Accordingly, a low regeneration rate was found for this genotype. However, when the calli were transferred to regeneration medium after a culture period of 2 weeks on K3, a fourfold increase in regeneration rate was observed. It is concluded that the recalcitrance of the non-regenerative line is due to the inability to develop shoot primordia after prolonged culture on K3. We further noticed that the regeneration rate of both lines is strongly affected by the hormonal composition of the callus proliferation medium, in particular by the presence of 2,4-D. The period of callus proliferation is therefore considered as a critical step in shoot differentiation.