Micropropagation of adult Stone Pine (Pinus pinea L.)

This paper describes a micropropagation protocol for in vitro propagation of mature Stone Pine trees. Axillary bud development was achieved by culturing bud explants in media containing various cytokinins. Experiments were conducted to test the effect of asepsis conditions, type and concentration of cytokinin and rooting protocol. Four cytokinins were tested, namely, benzyladenine, meta-topolin, N-benzyl-9-(2-tetrahydropyranyl)-adenine and thidiazuron (TDZ) of which TDZ gave the best results, as 59% shoot development was obtained following the application of 1 μM TDZ to the culture medium. The shoot development was significantly influenced by the genotype of the tree, but was effective in explants from all 20 genotypes used in the trial. In vitro rooting was, however, difficult to achieve and could only be induced at low rates. This protocol represents the first successful biotechnological approach to the micropropagation of adult Pinus pinea trees. Paloma Moncaleán and Ricardo Javier Ordás contributed equally.     

Changes of morphogenic competence in mature Pinus sylvestris L. buds in vitro

The effects of season and cold storage on morphogenic competence in mature Pinus sylvestris buds were investigated. Peroxidase and polyphenol oxidase activity were measured as markers of oxidative metabolism. No growth in vitro was observed on explants detached from the end of January until the beginning of March. Brachioblasts, each with a couple of needles, formed on 11% of the buds without macrostrobili that were detached in early April and introduced immediately into culture. Of the explants detached in late July, 15% formed shoots with brachioblasts and needles. The lowest activity of peroxidase and polyphenol oxidase in pine buds was observed from the end of April until the beginning of June when morphogenic competence of tissues started to increase. Development of bud explants detached in January was achieved by cold storage for 5 months. Low polyphenol oxidase and peroxidase activity coincided with increased morphogenic potential. Results suggest that reduced or stable activity of peroxidase and polyphenol oxidase is associated with an increased ability of tissues to start growth in vitro.     

Proliferating sieve elements present in bud phloem anastomoses connect sieve tubes of axillary bud traces to stelar vascular bundles in the aquatic monocotyledonPotamogeton natans L. (Potamogetonaceae)

Summary The stem ofPotamogeton natans is characterized by a central stelar vascular system with reduced xylem and abundant phloem. Wide sieve tubes composed of short sieve-tube members joined by simple sieve plates and associated with companion cells establish an effective conduit for assimilates. At each node the phloem forms a network of parallel sieve elements connecting the stem phloem to leaf and bud traces. InP. natans an axillary bud rarely develops into a side branch, its procambial vascular bundles are each connected to the nodal complex via separate anastomoses. Their most unusual components are the anastomosai sieve elements (ANSE), characterized by thin cell walls pitted all over by tiny callose-lined pores resembling plasmodesmata, which can be detected as bright areas by fluorescence microscopy after staining with aniline blue. Several layers of ANSE make up the centre of an anastomosis and link to both the nodal and bud stelar sieve tubes via mediating (MSE) and connecting sieve elements (CSE). The ultrastructural differentiation of ANSE, MSE, and CSE corresponds to that of normal sieve elements, i.e., in the mature stage they are enucleate, evacuolate, and have lost most of their cytoplasm. Their plastids are of form-P2c, containing many cuneate protein crystals, typical of monocotyledonous sieve elements. Quantitative aspects of the pore areas are discussed in relation to the functional significance of bud anastomoses.Abbreviations ANSE anastomosai sieve elements - CSE connecting sieve elements - FM fluorescence microscopy - LM light microscopy - MSE mediating sieve elements - TEM transmission electron microscopy Dedicated to Professor Dr. Rainer Kollmann on the occasion of his retirement     

The effect of the gaseous state on bud induction and shoot multiplication in vitro in eastern white cedar

The effect of vessel type and the gaseous phase on the morphogenic response of Thuja occidentalis L. explants in vitro was studied. Explants were cultured in container types that varied in their degree of gas exchange. Traps for ethylene and CO₂ were employed. During shoot bud induction from embryonic explants, the number and elongation of shoot buds improved significantly when gastight, serum-capped flasks were used compared to the foam bung-capped flasks or the regularly used Petri dishes. Elimination of the two gases from the headspace of the flasks either singly or together reduced shoot bud induction and especially elongation of shoots. A similar response was seen during axillary bud development from cultured shoots. Ethylene and CO₂ accumulation promoted development and elongation of axillary shoots. An increase in the zeatin concentration in the medium produced a greater number of axillary shoots and higher levels of ethylene in the culture vessels. Removal of CO₂ caused gradual death of the shoots, while removal of ethylene alone reduced axillary shoot lengths significantly. Inclusion of aminoethoxyvinylglycine in the medium combined with ethylene traps produced an effect similar to the use of ethylene traps alone.     

Effect of light quality (red: far-red ratio) and defoliation treatments applied at a single phytomer on axillary bud outgrowth in Trifolium repens L.

We studied the effects of light quality and defoliation on the rate of phytomer appearance and axillary bud outgrowth in white clover. The treatments were applied to one phytomer, a phytomer being defined as the structural unit comprising a node, internode, axillary bud, subtending leaf and two nodal root primordia. Light of a low red:far-red (R:FR) ratio (0.27) was applied to a target phytomer either (i) within the apical bud and then to the axillary bud after emergence of the phytomer from the apical bud, or (ii) to the axillary bud only after emergence. The light conditions were directed to these specific parts of the plant by collimating light from small FR light-emitting diodes; with this technique we were able to change the light quality without any change in the level of photosynthetically active radiation. The subtending leaf of the target phytomer was retained or defoliated when it had emerged from the apical bud. FR light applied from the time the phytomer was within the apical bud caused a delay in branch appearance at the target phytomer. In contrast, direct treatment of the axillary bud with FR light after it had emerged from the apical bud did not result in any delay in branch appearance. As the light treatment of the apical bud may have changed the light environment of any of the organs contained in the bud we were unable to ascribe the delay in branch appearance to light perception by any particular organ. However, indirect evidence leads to the conclusion that the likely site of light perception was the developing leaf subtending the axillary bud while it was the outermost phytomer within the apical bud. These results do not support the hypothesis that the R:FR ratio of light incident at an axillary bud site is the environmental factor that controls bud development. Defoliation of the unfolding leaf reduced the rate of phytomer appearance on the main stolon but had no immediate effect on branch appearance. As a consequence there was a reduction in the number of phytomers between the stolon apical meristem and the first phytomer with a branch. This is frequently taken to indicate a relaxation of apical dominance, but in this case was found not to involve a direct effect on bud activity. A current model of white clover growth suggests that there is integration of activity between apical meristems but independence of activity and response to the local micro-environment by axillary buds. In contrast, we found that (i) defoliation reduced phytomer appearance only at the main stolon apical meristem and not at all the meristems in the plant and (ii) that a change in the local light environment of an axillary bud had no discernible effect on bud activity once the bud had emerged from the apical bud but could delay branching if applied before emergence. These results are at variance with the predictions of the model.     

茭白冬芽的发育及抗寒性的形态学研究

节间横向维管组织是冬芽发育过程中所形成的特殊越冬维管结构,它们约起源于冬芽苗端倒数第五节间,其原始细胞由紧贴周缘维管束环外侧的一层薄壁细胞壁反分化而成。节间横向维管组织具有发达的木质部,但不呈V形,它们对加强冬芽发育时期及来年春天萌发期的水份和无机盐的运输起重要作用。冬芽发育过程中,其生长锥及幼叶的细胞发生了一系列细胞学变化,可归纳如下:①细胞分裂活动逐渐减弱,最终停止;②核仁周围的亮环越来越小,直至基本消失;③细胞所积累的淀粉颗粒逐渐增多增大;④在休眠期,细胞普遍产生质壁分离现象。这些细胞学变化可作为确定茭白冬芽抗寒性的有力依据。     

Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

Long-term effects of thidiazuron are intermediate between benzyladenine,kinetin or isopentenyladenine in Miscanthus sinensis

The thidiazolylurea derivative thidiazuron has been reported to be considerably more effective than benzyladenine in promotion of in vitro shoot formation in a number of dicotyledonous species. In the present study, axillary shoots of Miscanthus sinensis (Thunb.) Anderss. Giganteus that had been subcultured four times on modified Murashige & Skoog medium with 20M benzyladenine were transferred to media with benzyladenine, kinetin, isopentenyladenine or thidiazuron at concentrations of 0.01, 0.1, 1, 10, 30 or 100M and grown over four subcultures. Shoot and root formation stabilized after the first subculture and results from the three subsequent subcultures are presented. The common effects of cytokinins, i.e., promotion of axillary bud growth, inhibition of root formation, reduced stem growth and delay of senescence, were observed for all four cytokinins. In a descending order regarding shoot formation, the four cytokinins at the optimum concentration could be ranked as follows: benzyladenine, thidiazuron, kinetin and isopentenyladenine. Benzyladenine and thidiazuron had optimum effects at the same concentration with regard to axillary shoot formation but thidiazuron induced a significantly lower number of shoots than benzyladenine. The number of roots, shoot size and percentage of chlorotic shoots were also the same for benzyladenine and thidiazuron. When transferring shoots from benzyladenine or thidiazuron medium to rooting medium, shoots previously grown on thidiazuron became taller and formed fewer roots than shoots previously grown on benzyladenine.Abbreviations BA benzyladenine - 2iP isopentenyladenine - KIN 6-(furfurylamino)-purine (kinetin) - MS Murashige & Skoog medium - NAA naphthaleneacetic acid - THI N-phenyl-N(1,2,3-thidiazol-5-yl)-urea (thidiazuron)     

Plate-shaped nuclear pole bodies in the monothalamous foraminiferKibisidytes sp.

Summary Cultures capable of continuous plantlet production have been established from excised, immature embryos ofSorghum bicolor and the course of development of the plantlets has been followed by light and scanning electron microscopy. Such analyzes revealed that there are two distinct methods of plantlet production. Shoot primordia and embryo-like structures arisede novo from cells of the scutellum. However, when these cultures are transferred to fresh medium a further proliferation of shoot buds occurs by the formation of axillary shoot primordia. The cultures are therefore more comparable to shoot cultures than to callus cultures. Over 300 plantlets producedin vitro have been transferred to potting compost and grown to maturity. Most plants flowered and set seed. Fifteen plants were sterile but were of normal chromosome number (2 n=20) and it is presumed that the sterility was due to segregation of restorer genes in the immature embryos used to initiate some of the cultures.Abbreviations used in the text 2,4-D 2,4 dichlorophenoxyacetic acid - MS Murashige and Skoog - NAA -Naphthalene acetic acid - 6-BAP 6-Benzylaminopurine     

Nitrogen and phosphorus fertilization of aquatic vascular plants and algae in replicated ponds I. Initial response to fertilization

Populations of common submerged vascular plants were established in a series of 18 experimental ponds in 1967 and subjected to a replicated inorganic N-P fertilization program. The 18 ponds were fertilized as follows in 1968: 6 unfertilized controls, 6 low fertility (.75 mg. P/1) and 6 high fertility (75 mg. N/1., 7·5 mg. P/1.). The high fertility levels tended to eliminate the benthic plant populations and increase the phytoplankton standing crops. Elodea canadensis grew in the highest nutrient levels but Myriophyllum spicatum var. exalbescens and Ceratophyllum demersum appeared to be eliminated. Potamogeton crispus produced an abundance of winter buds under conditions of high fertility. There were no obvious differences in the benthic plant and phytoplankton populations among the control and low fertility ponds.Supported by funds from OWRR Title II Matching Grant and the College of Agriculture at Cornell University.