Benjamin Steinitz

Gene silencing of mannose 6‐phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant

Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%-80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.

Precocious gladiolus corm formation in liquid shake cultures

Conditions were defined for precocious differentiation and improved growth of corms at the base of gladiolus shoots. Shoots were derived from explants cultured on agar solidified media, and corm regeneration was obtained in subsequent liquid shake cultures. Benzyladenine (BA), at 10-7 M, was found to have a stimulating effect mainly when provided to the shoots prior to manifestation of corm growth. Paclobutrazol and sucrose promoted corm formation when supplemented to the liquid media. Paclobutrazol, at 10 mg l-1, shifted assimilate allocation towards the growing corm. A differential promotion of corm development by sucrose was not observed, and the concentration of sucrose at which the sugar demand for maximal shoot and corm growth is satisfied (60 g l-1) was unaltered by the presence of paclobutrazol. The rate of corm growth on shoots cultured in a liquid medium supplemented with paclobutrazol and a saturating sucrose concentration, was a function of the length of the shoots leaf blades, and was similar in light and in dark.

Adventitious regeneration in vitro occurs across a wide spectrum of squash (Cucurbita pepo) genotypes

Cucurbita pepo L. (squash, pumpkin) is a highly polymorphic vegetable species of major importance. Our study characterized a spectrum of C. pepo germplasm for the ability to regenerate in vitro by direct organogenesis from cotyledon explants. Cultivars tested included both cultivated subspecies, texana and pepo, and nearly all of their respective cultivar-groups. Direct shoot regeneration occurred in all accessions, and was generally high (56–94%), with a single exception of 22% (‘Bolognese’). There was no significant difference between the percentage regeneration of the two subspecies. Shoot regeneration per responding explant was uniform (1.2–1.6 shoots per explant). Only ‘True French’ produced statistically more shoots (3.9 per explant) than other accessions. The morphology of regeneration varied. Most cultivars produced long shoots, often fasciated, amid a few small buds. Some subspecies pepo cultivars (Beirut, Yugoslavia 7, Ma’yan and True French) produced short, massive, hollow shoots, sometimes accompanied by shoots that were more normal. Two subspecies texana cultivars (Creamy Straightneck and Small Bicolor) produced single (sometimes double) shoots without other buds. The production of chimeric (mixoploid) regenerants varied and there was a tendency to regenerate chimeric plants from the widest-fruited accessions (i.e. lowest length-to-width ratio) in each subspecies. Subspecies pepo Pumpkin Group ‘Tondo di Nizza’ showed significantly greater production of chimeric regenerants. In comparison with the great range of variation observed in fruit shape, the variation of in vitro responses (mostly less than 2-fold in regeneration and shoot production) was less than expected.

Pepper (capsicum annuum L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot

SummaryThree auxin-type herbicides, namely 2.4-dichlorophenoxyacetic acid (2,4-D), (4-chlorophenoxy)acetic acid 2-(dimethylamino)ethyl ester (centrophenoxine), and quinolinecarboxylic acid (quinclorac) induced direct somatic embryogenesis in seed-derived zygotic embryo explants of sweet pepper (Capsicum annuum L.) when added to Murashige and Skoog medium with 200 mM sucrose. Optimum concentrations for embryogenesis induction were 0.40–0.45 mM and 1.15–1.30 μM for 2.4-D and centrophenoxine, respectively (in the presence of 5.0 gl−1 activated charcoal), or 40 μM for quinclorac (in medium without activated charcoal). Somatic embryos emerged from the epidermal and subepidermal tissues and developed on the surface of the explant. Centrophenoxine- or 2.4-D-mediated embryogenesis was accomplished from 95% of the explants in about 3 wk and, on average, six embryos were formed per explant. Induction efficieney was lower for quinelorac. Centrophenoxine-mediated embryognesis was possible in 10 pepper cultivars, the extent of the reponse-being genotype-dependent. embryos detached from the explant and transplanted onto a growth regulator-free medium germinated; however, the recovered regenerants were without a shoot, and some of them bore a single deformed cotyledon while others had no cotyledons. Regenerants lacking a shoot were generated irrespective of the auxin type applied and across all responsive genotypes investigated. Absence of a shoot, resulting from a failure in the establishment of a normal functioning apical shoot meristem, was the principal developmental disorder that precluded regeneration of normal plants via direct somatic embryogenesis. Since stem cells of the shoot meristem become established in globular and heart-stage embryos, we deduce that the absence of a shoot in germinating embryos could orginate from deviant differentiation at these early stages of embryogeny.

The effect of light and of explant orientation on the regeneration and subsequent growth of bulblets on Lilium longiflorum Thunb, bulb-scale sections cultured in vitro

Abstract Lilium longiflorum, cultivar ‘Osnat’, bulb-scale sections were cultured in the light and in the dark with explants placed with their dorsal (abaxial) side on the medium, and in the dark with their ventral (adaxial) side up or down, or in the upright or inverted position. Scale sections regenerated bulblets, roots and callus. In all cases, bulblets developed only on the ventral side of the scale. Light did not affect the regeneration capacity of the scale piece but did significantly affect bulblet growth. Bulblets developing in the light were small and bore many leaves. In the dark, a smaller number of bulblets, which were much larger but bore fewer leaves, developed. In the light, leaf dry weight was double that of bulb dry weight, whereas in the dark this proportion was reversed. In contrast to light, explant orientation greatly affected regeneration percentage. Scale sections planted with their ventral side down regenerated fewer and smaller bulbs, less root, but much more callus than those with their dorsal side down. The upright and inverted positions also reduced the number of bulblets regenerated and increased callus formation.

Sugar Alcohols Display Nonosmotic Roles in Regulating Morphogenesis and Metabolism in Plants that do not Produce Polyols as Primary Photosynthetic Products

Summary Sugar alcohols are frequent supplements to media of cultures from plant species that do not produce polyols as primary photosynthetic products. They comprise one of the major medium constituents and thereby contribute substantially to the medium osmotic potential. It is generally believed that plants that do not have a native pathway for sugar alcohol biosynthesis are also deficient in pathways to assimilate them. Therefore, polyol-media addenda are generally considered to be metabolically inert, and in vitro polyol-dependent phenomena are defined predominantly as mere osmotic (stress) effects. The article shows that this concept does not provide satisfactory explanations for experimental results described in the literature. Sugar alcohols commonly penetrate cells, accumulate, can be translocated and, whenever stringently tested, have always been found to be metabolized to various degrees. Heterotrophic cultures, such as those of tobacco, maize, rice, citrus and chicory, can adjust their metabolism from consumption of saccharides to that of polyols as carbon-energy source when the medium is sucrose-deficient or when exogenous saccharides are depleted to a level lower than a threshold. concentration. In these, as well as in other cultures (i.e. of barley, tobacco, or tomato), sugar alcohols stimulate specific molecular and physiological responses that do not belong to primary carbon metabolism. These responses are dependent on chemical stimuli rather than being mediated by nonspecific, physical, osmotic signals in the medium. One conclusion of this review is that it would be wise to assume that sugar alcohols may be metabolized until shown otherwise. Further, it is suggested that polyols are perceived by cells as chemical signals. This hypothesis is compatible with the emerging conception of sugars (hexoses) as signals in plants, distinctly separate from their possible metabolic and osmotic roles. At very high concentrations provided in vitro , sugar alcohols can probably in some cases be perceived as chemical stress agents, while in other instances they possibly act as protectants against unidentified detrimental medium constituents. In view of the multiple ways of polyol action, administration of these compounds as a means of water stress simulation can lead to misinterpretations of experimental results.

Control of Precocious Gladiolus Corm and Cormel Formation in Tissue Culture

Summary Gladiolus propagates vegetatively by virtue of corm formation at the base of the shoot and by cormel differentiation on tips of stolons. The precocious generation of both organs was accomplished in vitro on plantlets incubated for one month in a liquid shake culture supplemented with 1.0–10.0 mg/1 paclobutrazol. Paclobutrazol-dependent corm and cormel development could be prevented by addition of GA 3 to a paclobutrazol-containing medium, thereby indicating that paclobutrazol mediates its influence by reducing the endogenous GA level. BA was also found to inhibit corm formation. The dose-response relations for the influence of paclobutrazol, BA and GA 3 on corm development are presented. Paclobutrazol-induced cormels, when excised from the culture-derived plant, germinated and started to regenerate adventitious shoots upon transfer to an agar-solidified medium with 0.3 μM BA and 0.1 μM NAA.

Factors controlling the retardation of chlorophyll degradation during senescence of detached statice (Limonium sinuatum) flower stalks.

Summary Detachment of green, flowering Limonium sinuatum flower stalks stimulated chlorophyll degradation. The loss of chlorophyll, which is regarded as a marker process of senescence, was accelerated when flower stalks were kept in the dark. Exposure of sections of the stalk to continuous white light or to brief red irradiations inhibited chlorophyll degradation. The effectiveness of the brief red i rradiations was similar to that of continuous white light, and could be completely reversed if each red irradiation was followed by a brief far-red irradiation. It is concluded that phytochrome is present in the flower stalks and mediates the retardation of flower stem senescence by light. Treatment of excised flower stalks with cytokinin (BAP) or gibberellin (GA3) significantly retarded the destruction of chlorophyll which occurred in the dark.

The effect of various iron-containing fertilizers on growth and propagation of Gladiolus grandiflorus

Abstract Plants of Gladiolus grandiflorus ‘Friendship’ were grown on a CaCO 3 -containing soil treated with the following soil-applied Fe fertilizers: (1) self-produced Fe-enriched peat (Bar-Teva 55); (2) Fe-enriched ammonium polyphosphate (APP); (3) FeEDDHA (sequestrene 138). Foliar application of FeSO 4 solution in the presence of a wetting-agent (L-77) was also tested. Bar-Teva 55, FeEDDHA and FeSO 4 +L-77 were found to be effective in the prevention of chlorosis, while APP was not. About 95% of the Bar-Teva 55-treated plants flowered, compared to 79–89% for the other treatments. Flowering in Bar-Teva 55-treated plants started a few days earlier. None of the fertilizers affected inflorescence stem length or the post-harvest keeping-quality of the flowers. With the exception of APP, the fertilizer treatments improved daughter corm growth. The number of cormels per corm in the Bar-Teva 55-treated plants was markedly higher than in other treatments. FeEDDHA and FeSO 4 +L-77 treatments also increased cormel number per corm when compared to APP-treated plants and control. Effective Fe-containing fertilizers seem to be of great importance to flowering, corm growth and cormel formation. Fe-enriched peat was found to be the most efficient fertilizer among the ones tested.

Thigmomorphogenesis and its interaction with gravity in climbing plants of Epipremnum aureum

Summary The touch-mediated increase in the leaf lamina area of climbing Epipremnum aureum (Linden and Andre) Bunt. plants was characterized. The touch stimulation was provided by coupling plant internodes to a support, whereas hanging plants were regarded as mechanically unstimulated controls. We show that the thigmosensitive regions is located in the youngest internodes and is restricted to the stem flank facing the supports surface, i.e., the flank which forms adventitious aerial clasping roots. Further, two different interactions between the gravity- and the mechanical-sensing response systems were found: ( i ) The touch-mediated promotion of leaf size was best expressed in upwards-oriented stems and was completely absent in horizontally oriented stems. Thus, the gravitational vector dictates permissive or non-permissive conditions for expression of the response to the touch stimulus. ( ii ) In down-hanging stems, the negative gravitropic curvature response of the shoot apex was too weak to prevent downward growth. However, touch stimulation of the apex induced a negative gravitropic tip curvature response sufficiently strong to invert the stem growth to an opposite direction of the gravity vector. Hence, the touch signal alters the expression of the apexs response to gravity.