Analysis of surface growth in shoot apices

A salient feature of shoot meristem growth is the maintenance of distinct anatomical and morphological features despite a continuous flux of cells. To investigate how meristem organization is self-perpetuated, we developed a protocol for the analysis of meristem growth in 3-D. Our protocol uses a non-destructive replica method to follow the pattern of cell expansion and cell divisions on the meristem surface over several days. Algorithms to reconstruct the meristem surface and compute its curvature and rate of extension were implemented. We applied this approach to the shoot apical meristem of Anagallis arvensis and showed that a subcellular resolution of extension rates can be achieved. This is the first detailed quantitative analysis of meristem geometry and surface expansion in 3-D. This new approach will be useful to connect cellular activities such as cell expansion, cell division, and differential gene expression with overall meristem morphogenesis.     

Improved shoot regeneration protocol for hairy roots of the legume Astragalus sinicus

An improved protocol for shoot regeneration from hairy roots transformed by Agrobacterium rhizogenes of the legume species Astragalus sinicus (Chinese milk vetch) has been developed. The A. rhizogenes strain DC-AR2 harboring the binary vector pBI121 which carries the uidAgene encoding -glucuronidase activity and the kanamycin resistance gene nptII, was used to transform cut ends of plantlet hypocotyls. Transformed hairy roots were selected on medium containing 75 g ml^–1 kanamycin, and transformation was monitored by detection of the opine mikimopine, histochemical -glucuronidase activity, the polymerase chain reaction, and Southern blot analysis. The cytokinins benzylaminopurine, kinetin, and thidiazuron suppressed the growth of 8-month and 3-year-old hairy roots, but were necessary for adventitious shoot formation that could occur with some lines. Putative somatic embryos developed from transformed roots on medium with 7.5-10.0 mg l^–1 2,4-dichlorophenoxyacetic acid. Light did not affect shoot regeneration from transformed hairy roots. This transformation and shoot regeneration system should be useful for testing gene expression quickly and be amenable to studies of shoot morphogenesis and interactions with rol gene expression.     

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 M 6-benzyladenine. Addition of -naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 M combined, respectively, with 2.5, 1.25–5.0 or 5.0 M -naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 M indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.     

Periods of organogenesis in shoots of Nothofagus dombeyi (Mirb.) oersted (Nothofagaceae)

The organogenetic cycle of main-branch shoots of Nothofagus dombeyi (Nothofagaceae) was studied. Twelve samples of 52-59 parent shoots were collected from a roadside population between September 1999 and October 2000. Variations over time in the number of nodes of terminal and axillary buds, and the length, diameter and number of leaves of shoots derived from these buds (sibling shoots) were analysed. The number of nodes of buds developed by parent shoots was compared with the number of nodes of buds developed, I year later, by sibling shoots. The length, diameter and number of leaves of sibling shoots increased from October 1999 to February 2000 in those shoots with a terminal bud. However, extension of most sibling shoots, including the first five most distal leaf primordia, ceased before February due to abscission of the shoot apex. Axillary buds located most distally on a shoot had more nodes than both terminal buds and more proximal axillary buds. The longest shoots included a preformed part and a neoformed part. The organogenetic event which initiated the neoformed organs continued until early autumn, giving rise to the following year's preformation. The absence of cataphylls in terminal buds could indicate a low intensity of shoot rest. The naked terminal bud of Nothofagus spp. could be interpreted as a structure less specialized than the scaled bud found in genera of Fagaceae and Betulaceae.     

Root starch storage and allocation patterns in seeder and resprouter seedlings of two Cape Erica (Ericaceae) species

Post-fire sprouting of dormant buds in resprouter plants is facilitated by stored carbohydrate reserves, with starch being the critical reserve. Starch is mainly stored in xylem parenchyma ray tissue of woody underground organs, such as burls, lignotubers, and roots. We carried out a comparative analysis of the pattern of starch storage and the proportion of parenchymatic ray tissue in the upper root or cotyledonary region of seedlings from seeder and resprouter forms within two Cape Erica (Ericaceae) species: E. coccinea L. and E. calycina L., which were raised in the greenhouse under controlled irrigation. We also explored the root-to-shoot allocation patterns of seeder and resprouter seedlings in these two species. Resprouter seedlings of both species showed higher relative amounts of upper-root starch and upper-root storage tissue as well as a higher root-to-shoot allocation than their seeder counterparts. Pronounced swelling of the upper root region suggests ontogenetic development of a lignotuber in the resprouter forms of the two Erica species. The distinct allocation of starch in roots seems to be genetically determined and would account for the apparent differences in the root-to-shoot allocation patterns between both regeneration forms from the early seedling stage.     

Plantlet regeneration from subculturable nodular callus of Pinus radiata

An efficient planlet regeneration system via nodular callus formation is described for Pinus radiata. Subculturable nodular callus was induced at its highest frequency (93%) on embryonic explants excised from seeds at an early stage of germination (radicle length 2–5 mm). The optimal medium for nodular callus tissue proliferation was LP basal medium that was modified by reducing the concentration of potassium nitrate to 500 mg l^–1 and supplemented with 22.2 M 6-benzyladenine (BAP) and 2.85 M indole-3-butyric acid (IBA). Bud differentiation from the nodules was achieved by reducing BAP and sucrose concentrations in the culture medium. The maximum frequency of adventitious bud formation occurred on LP basal medium containing 2% sucrose and 0.44 M BAP on which about 61% of the transferred nodules formed buds. During the next 6 weeks of culture on the same cytokinin-free medium multiple shoots elongated from the buds. These shoots were excised and transferred to root initiation medium (RIM2.1), consisting of full-stregth SH macro- and micro-salts, 1000 mg l^–1 myo-inositol, 0.4 mg l^–1 thiamine-HCl, 2% sucrose and a combination of naphthaleneacetic acid (NAA), IBA and BAP at concentrations of 2.69, 4.93 and 0.11 M, respectively. After 5–15 days, root meristems were initiated on the stem bases. The highest rooting frequency was achieved when shoots were treated for 10 days on RIM2.1 medium, before being transferred to half-strength Schenk and Hildebrandt medium with 1% sucrose and without growth regulators for root growth.     

Plant regeneration through shoot formation from callus of Areca catechu L.

In order to establish and optimize an in vitro micropropagation protocol of Venus fly trap (Dionaea muscipula Ellis), a carnivorous plant, the effects of medium type, MS medium concentration, pH, and cytokinin and auxin types on shoot proliferation and root formation were investigated using 3-month-old shoots. The shoot proliferation was most effective in 2.3 M kinetin-supplemented 1/3MS medium at pH 5.5. The best conditions for rooting were 1/3MS medium supplemented with 0.5 M IBA. All subcultured shoots produced extensive root systems after 5–6 weeks culture. When plantlets after rooting were planted in plastic pots filled with 1:1 peat moss and sand, the survival rate of plantlets was almost 100%, exhibiting normal development. With subculture every 8 weeks, hundreds of the plants were propagated from a single plant within a year.     

Production,storability, and regeneration of shoot tips of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) encapsulated in calcium alginate hollow beads

Summary Shoot tips, of four potato cultivars (Désirée, Genet, Tigoni, and Tomensa), 3–4 mm in size, were precultured for 2 d on Murashige and Skoog (MS) solid medium, then encapsulated in calcium alginate to produce hollow bead synthetic seed capsules averaging 0.78 cm in diameter. Regeneration and ‘regrowth’ were tested on MS solid medium and on soil in the greenhouse, respectively. The encapsulated shoot tips were stored at 4 and 10°C for up to 390 d. For all cultivars, the encapsulated shoot tips stored at both temperatures for 180 d and at 4°C,for 270 d, 100% regeneration on MS solid medium was recorded. After 360 d in storage at 4°C, 70.8% (Tigoni), 66.7% (Genet), 58.3% (Désirée), and 51.5% (Tomensa) regeneration was recorded on MS medium, reducing to 15% (Tigoni), 25% (Genet), 10% (Désirée), and 0% (Tomensa) regeneration after 390 d in storage. ‘Regrowth’ of 93–100% was recorded for non-stored encapsulated shoot tips, directly transferred on soil in the greenhouse after a 2 wk preculture on MS solid medium with an added fungicide (carbendazim) in the encapsulating gel. The ‘regrown’ shoot tips produced plants showing normal development. The results presented here demonstrate that hollow bead synthetic seed capsules are an alternative propagating method for potato seed production.     

Shoot population dynamics of persisting clones of<Emphasis Type="Italic"> Miscanthus sinensis</Emphasis> in the warm-temperate region of Japan

The population dynamics of annual shoots in four clonal patches of Miscanthus sinensis was surveyed in terms of clone persistence. Over a study period of 3 years, a stable net shoot number was attained through a balanced replacement of old shoots by tillering 2–3 times a year. The birth rate was significantly correlated with the average monthly temperature. This suggests that a warm climate advances the date of tillerings, and shoots become taller as they emerge earlier. Five cohorts were identified, corresponding to shoots that were born in spring, early summer, summer, autumn and early winter. The autumn cohorts were more numerous and had a greater longevity than the other cohorts; they were thus were best suited for overwintering and, consequently, maintaining the population from one generation to the next. The shoots of earlier cohorts grew too tall to survive the cold of winter, which might cause freezing injury, while later cohorts shoots excelled in overwintering because of their shortness. However, if the delayed shoots are very short, they will also have difficulty in surviving the early season of growth. Therefore, M. sinensis shoots have an optimum size for survival.