Possibilities and constraints in the regeneration of trees from cotyledonary needles of Picea abies in vitro

Although use of embryonic or seedling tissues for mass clonal micropropagation in vitro in conifer reforestation programmes is questionable, there is a potential application in the regeneration of plants from scarce and costly seed derived from controlled pollination. In addition, in vitro culture shortens considerably the lag phase in numbers during the initial stages of vegetative propagation via rooted cuttings. Successive steps of the present technique are described whereby cotyledonary needles (secondary explants) were subcultured on a hormone-free medium after administration of cytokinin or auxin to 14-day-old seedling (primary) explants of Picea abies. For bud induction, N⁶-benzyladenine (BA) was applied either as a short-duration (3 h), high-concentration (125 μM) pulse or by vacuum infiltration and incubation in a BA-containmg (5 μM) infusion medium. Induced adventitious shoots were elongated with the aid of far-red light and rooted in vivo after a long-duration (12 h), high-concentration (625 μM) application of indolebutyric acid. Pulse and infusion treatments resulted in the induction of greater numbers of adventitious buds (average of 12 per needle) over a three to four week shorter culture period than was the case with the conventional inclusion of growth regulators in the agarified medium. No exogenous auxin was required in the bud-induction programme; its inclusion even at nanomolar levels promoted histo- rather than morphogenesis. In cotyledonary needles, to the primary explants of which BA was applied as a pulse or by infusion, the cell divisions which gave rise to the meristemoids from which adventitious buds were produced, appeared to commence mainly in undifferentiated hypodermal layer cells but also in the mesophyll immediately below. By contrast, where BA was incorporated in the agarified medium the first divisions occurred mainly in cells of the epidermal layer. A number of factors affected plantlet regeneration, for instance seed variability, age of seedlings, and mode of application of growth substances. It should also be accepted that the xeromorphic nature of the conifer leaf might impose physiological and morphological constraints on its culture in vitro that could militate against easy morphogenic manipulation. It is deemed essential that the current mean ratio of regenerated plants to cotyledonary needles of 1:1 be increased 10 to 20 fold in order to approach commercial feasibility.     

Shoot regeneration from seedling explants of Acacia mangium Willd

Summary Regeneration of adventitious shoots was obtained in over 80% of explants, consisting of wounded cotyledonary nodes of Acacia mangium, by culturing germinated seedlings on DKW medium with combinations of N⁶-benzyladenine and either thidiazuron or N-(2-chloro-4-pyridyl)-N-phenylurea. Electron microscopy showed the presence of adventitious buds arising from wound tissue of the cotyledons and cotyledonary nodes. Shoot regeneration was also obtained at lower frequency in isolated cotyledon explants cultured with 6% sucrose alone (10%), or with 3% sucrose and 30.0 mg l⁻¹ (0.1 μM) 2–4-dichlorophenoxyacetic acid (2,4-D; 16%). With 2,4-D,>60% of explants produced organized structures but these did not develop into shoots or somatic embryos. Shoot formation was not induced in either hypocotyl or root explants.     

Structural and Functional Damage Caused by Boron Deficiency in Sunflower Leaves

Boron deficiency induced a dramatic inhibition in sunflower plant growth, shown by a reduction in dry mass of roots and shoots of plants grown for 10 d in nutrient solution supplied with 0.02 μM B. This low B supply facilitated the appearance of brown purple pigmentation on the plant leaves over the entire growth period. Compared to B-sufficient (BS) leaves, leakage from B-deficient (BD) leaves was 20 fold higher for potassium, 38 fold for sucrose, and 6 fold for phenolic compounds. High level of membrane peroxidation was detected by measuring peroxidase activities as well as peroxidative products in BD sunflower plants. Soluble and bound peroxidase activities measured in BD thylakoid membranes were accelerated two fold compared to those detected in BS-membranes. No detectable change in soluble peroxidase activity in roots whereas a 4 fold stimulation in bound peroxidase activity was detected. Thylakoid membranes subjected to low B supply showed enhancement in lipoxygenase activity and malondialdehyde (MDA) content in parallel with 40 and 30 % decrease of linoleic and linolenic acid contents (related to total unsaturated fatty acids). A slower rate of Hill reaction activity (40 %) and a suppressed flow of electron transfer of the whole chain (30 %) were detected in BD thylakoid membranes. This reduction was accompanied with a decline in the activity of photosystem 2 shown by a diminished rate of oxygen evolution (42 %) coupled with a quenching (27.5 %) in chlorophyll a fluorescence emission spectra at 685 nm (F₆₈₅). Thus B is an important element for membrane maintenance, protection, and function by minimizing or limiting production of free oxygen radicals in thylakoid membranes of sunflower leaves. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants

Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in natural habitat is low and the tree is overexploited. An in vitro propagation protocol has been developed from nodal explants obtained from in vitro raised 18-day-old axenic seedlings. The highest shoot regeneration frequency (85%), maximum number of multiple shoots (8.6) as well as length (4.8 cm) were induced from nodal explants on Murashige and Skoog (MS) medium amended with 4.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 20 μM adenine sulphate (AdS). The percentage of shoot multiplication as well as the number of shoots per node remained the same during the first two subculture, afterwards a decline was recorded. Rooting was best induced in microshoots excised from proliferated shoot cultures on semisolid hormone-free half-strength MS medium, after a pulse (dip) treatment for 7 days in half-strength MS liquid medium containing 100 μM indole-3-butyric acid (IBA) and 15.84 μM phloroglucinol (PG). The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 4 weeks before their transfer to a greenhouse, where the established plants showed 75% survival.     

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L^?1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.     

Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds

Cypripedium flavum, known as the rare lady’s slipper orchid, is one of the endemics with a yellow flower in China. Due to its conservation and commercial requirement, establishment of an efficient method for micropropogation is urgently needed. Multiple shoots were obtained by placing seedlings from seeds of C. flavum on Harvais media supplemented with two cytokinins (BAP or KIN) used alone or in addition to different concentration of potato homogenate. The effect of BAP was better than that of KIN on shoot multiplication. The Havais media supplemented with BAP (2.22 μM) and potato homogenate (20 g l⁻¹) was the most effective, providing high shoot multiplication frequencies (95%) associated with a high number of shoots per explant (2.55 shoots/plant). For root formation, high rooting and survival were achieved using 1/2 Harvais media supplemented with 0.6 g l⁻¹activated charcoals. High-level activated charcoal increased the number and the length of roots because the activated charcoal could absorb BAP in the media. This study demonstrated that C. flavum could be micropropagated by using multiple shoots of seedlings derived from mature seeds.     

Patterns of leaf size and morphology in relation to shoot length in Tsuga canadensis

Summary Pattern of change in leaf character was assessed along the length and around the circumference of Tsuga canadensis (L.) Carr. shoots of different length classes. Leaf size decreased, and number of leaves per unit length of shoot increased, with decrease in shoot length. Acropetally along the shoots, lamina length and width decreased, relative petiole length increased, apices became more pointed, and leaf margins bore more teeth. Around the shoot, from upper to side, and to lower surfaces, leaf size and number of marginal teeth in the proximal halves of leaves increased. These patterns were related both to production of preformed versus neoformed leaves, though their separation was indistinct, and to secondary orientation of leaves by twisting in their petiolar regions into two major bi-lateral ranks. An additional minor rank occurred along the upper surfaces of the shoots where secondary orientation of the leaves was minimal. Surface features of leaves did not differ in any obvious manner. Leaves on sylleptic shoots, which by definition were all neoformed, exhibited similar patterns, but were generally smaller than those on their parent shoots.     

Decomposition of litter of three mudflat annual species in a northern prairie marsh during drawdown*

The loss of dry mass, nitrogen and phosphorus from shoot and root litter of mudflat annuals was examined in a series of experimental marshes in the Delta Marsh, Manitoba, Canada. Litter bags containing shoot material of three mudflat annuals (Aster laurentianus Fern., Atriplex patula L., and Chenopodium rubrum L.) were placed on the sediment surface of the marshes under drawdown conditions. In addition, litter bags containing root material of these three species were shallowly buried. Approximately 70% and 50% of both shoot and root litter, respectively, was still present after one year in the field. During the second year when the marshes were flooded, shoot and root litter lost an additional 20% and 0% of their mass, respectively. Except for Chenopodium roots, which accumulated nitrogen and phosphorus during both years, shoot and root litter lost from 0 to 50% of their nitrogen and phosphorus early in the first year, with levels generally remaining constant through the remainder of the study period. Our results indicate that mudflat annual litter decomposed slowly and would provide abundant habitat for aquatic invertebrates when these marshes were reflooded. However, most nutrient loss took place in the first year when the litter was unflooded, with little loss occurring in the second year when flooded.