Abscisic acid antagonizes the effect of cytokinin on DNA-replication origins

In previous studies (Houssa et al., 1990, 1994) we observedthat cytokinins stimulate the cell division process in vegetativeand reproductive shoot meristems of monocotyledonous and dicotyledonousspecies by activating latent DNA-replication origins. Here wereport that abscisic acid antagonizes this effect in the shootmeristem of Sinapis alba L. Abscisic acid reduces DNA synthesisby inactivating some DNA-replication origins resulting in alengthening of the replicon size. It is hypothesized that thebalance between abscisic acid and cytokinin levels is one ofthe major factors controlling the rate of DNA replication, andultimately the rate of cell division, in shoot meristems. Key words: Abscisic acid, cell division, cytokinin, DNA replication, replicon, shoot meristem, Sinapis alba     

In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass

Summary An efficient and reproduciblein vitro culture system has been developed for regeneration of multiple shoot clumps from intact seedlings of both lowland and upland cultivars of switchgrass (Panicum virgatum L.). The multiple shoots were induced on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-phenyl-3-(1,2,3-thiadiazol-5YL)-urea (thidiazuron or TDZ). Maximum response was obtained with 4.5 μM 2,4-D and 18.2 μM TDZ. These shoots proliferated and rooted efficiently on MS medium without growth regulators. The developmental pattern of the multiple shoots indicated their origin from the enlarged shoot apex via proliferation of axillary buds and subsequent reprogramming of shoot meristems followed by secondary differentiation of adventitious shoots The simplicity of the protocol and direct production of multiple shoots make this a potential system that is highly attractive and amenable for microprojectile-mediated gene transfer.     

Somatic embryogenesis from the axillary meristems of peanut (Arachis hypogaea L.)

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with 13.6 μM 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.     

Effect of mutations in the PINHEAD gene of Arabidopsis on the formation of shoot apical meristems

The primary shoot apical meristem of angiosperm plants is formed during embryogenesis. Lateral shoot apical meristems arise postembryonically in the axils of leaves. Recessive mutations at the PINHEAD locus of Arabidopsis interfere with the ability of both the primary shoot apical meristem as well as lateral shoot apical meristems to form. However, adventitious shoot apical meristems can form in pinhead mutant seedlings from the axils of the cotyledons and also from cultred root explants. In this report, the phenotype of pinhead mutants is described, and a hypothesis for the role of the wild-type PINHEAD gene product in shoot meristem initiation is presented. © 1995 Wiley-Liss, Inc.     

Nucleotide sequence and spatial expression pattern of a drought- and abscisic Acid-induced gene of tomato

The nucleotide sequence of le16, a tomato (Lycopersicon esculentum Mill.) gene induced by drought stress and regulated by abscisic acid specifically in aerial vegetative tissue, is presented. The single open reading frame contained within the gene has the capacity to encode a polypeptide of 12.7 kilodaltons and is interrupted by a small intron. The predicted polypeptide is rich in leucine, glycine, and alanine and has an isoelectric point of 8.7. The amino terminus is hydrophobic and characteristic of signal sequences that target polypeptides for export from the cytoplasm. There is homology (47.2% identity) between the amino terminus of the LE 16 polypeptide and the corresponding amino terminal domain of the maize phospholipid transfer protein. le16 was expressed in drought-stressed leaf, petiole, and stem tissue and to a much lower extent in the pericarp of mature green tomato fruit and developing seeds. No expression was detected in the pericarp of red fruit or in drought-stressed roots. Expression of le16 was also induced in leaf tissue by a variety of other abiotic stresses including polyethylene glycol-mediated water deficit, salinity, cold stress, and heat stress. None of these stresses or direct applications of abscisic acid induced the expression of le16 in the roots of the same plants. The unique expression characteristics of this gene indicates that novel regulatory mechanisms, in addition to endogenous abscisic acid, are involved in controlling gene expression.     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Gibberellic acid regulation of adventitious shoot formation from tuber discs of potato

Summary The formation of adventitious shoots from potato tuber discs explanted onto a modified Murashige and Skoog (MS) medium containingN ⁶-benzylaminopurine (BAP) (3.0 mg/l), and α-naphthaleneacetic, acid (NAA) (0.01 mg/l), was affected by gibberellic acid (GA). The presence of GA in the explant medium was required for shoot formation and 3×10⁻¹⁰ M GA appeared optimum. However, microscopic examination of the tissue protuberances on the surface of the tuber discs from which shoots arose revealed that GA inhibited the formation of shoot meristems. Tuber discs cultured for 6 wk on MS medium containing BAP and NAA without GA did not initiate adventitious shoots that could be determined visually, but microscopic examination of the tissue protuberances revealed the presence of numerous shoot meristems. Subsequent transfer of these tuber discs to medium with GA but without BAP or NAA resulted in the formation of shoots from 100% of the recultrued dises. Thus it appears that although GA inhibits shoot meristem initiation from potato tuber discs, it is required for shoot development once meristems are initiated. This is Journal Paper 8297 of the Purdue University Agricultural Experiment Station. The research was supported by Purdue University Agricultural Experiment Station Program Improvement Funds. Potato tubers were supplied by Wm. Gehring Farms, Inc., Rensselaer, Indiana.     

Expression of knotted1 marks shoot meristem formation during maize embryogenesis

The formation of shoot and root meristems that ultimately give rise to all tissues of the plant body occurs for the first time during embryogenesis. Meristem formation has traditionally been defined in terms of the appearance of histological features of meristems; this approach has led to varying interpretations of the timing of meristem formation relative to other events in embryogenesis. Markers that would provide more objective criteria for the analysis of meristem formation have not been widely available. The maize homeobox gene, knotted1 (kn1), is expressed in shoot meristems throughout postembryonic stages of shoot development. In order to determine whether this gene is expressed in the shoot meristem from its earliest inception, we examined the expression of kn1 in embryos at a series of stages by in situ hybridization to kn1 mRNA and immunolocalization of KN1 protein. Our results show that the onset of kn1 expression is temporally and spatially coincident with the earliest histologically recognizable signs of shoot meristem formation in the embryo, and thus provides a valuable marker for this process. © 1995 Wiley-Liss, Inc.     

In vitro morphogenesis of pearl millet [Pennisetum glaucum (L.) R.Br.]: efficient production of multiple shoots and inflorescences from shoot apices

 This report presents a procedure for high-frequency multiple shoot production from cultured shoot apical meristems of pearl millet [Pennisetum glaucum (L.) R. Br.]. Shoot apices from 1-week-old aseptically germinated seedlings were cultured in vitro on MS medium containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) with biweekly subculture. A low concentration of 2,4-D coupled with four different concentrations of BA induced the production of adventitious shoots from the enlarged shoot apical meristems. Somatic embryogenesis was also observed at higher concentrations of BA. The use of higher levels of 2,4-D resulted in callusing of shoot apical meristems, while the shoot tips produced many leaves and in vitro flowering in 2,4-D-free media containing BA. All four pearl millet genotypes produced similar results. Fertile pearl millet plants were produced from in vitro-produced multiple shoots. Received: 1 April 1999 / Revision received: 8 July 1999 / Accepted: 17 August 1999     

Fruit-set of unpollinated ovaries of Pisum sativum L.

The influence of removing the apical shoot and different leaves above and below the flower on the fruit-set of unpollinated pea ovaries (Pisum sativum L. cv. Alaska) has been studied. Unpollinated ovaries were induced to set and develop either by topping or by removing certain developing leaves of the shoot. Topping had a maximum effect when carried out before or on the day of anthesis, and up to four consecutive ovaries were induced to set in the same plant. The inhibition of fruit-set was due to the developing leaves and not to the apex. The third leaf above the first flower, which had a simultaneous development to the ovary, had the stronger inhibitory effect on parthenocarpic fruit-set. The application of different plant-growth regulators (indoleacetic acid, naphthylacetic acid, 2,4-dichlorophenoxyacetic acid, gibberellic acid, benzyladenine and abscisic acid) did not mimic the negative effect of the shoot.Abbreviations CCC (2-chloroethyl)trimethylammonium chloride - MH maleic hydrazide - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - 6-BAP benzyladenine - ABA abscisic acid     

Plant regeneration of the legume Lens culinaris Medik. (lentil) in vitro

Until recently, grain legumes in general have proven recalcitrant at de novo regeneration in vitro. By culturing portions of lentil (Lens culinaris) shoot meristems and epicotyls on a medium containing kinetin and gibberellic acid, callus tissue was produced which could be induced to regenerate shoots in relatively large numbers, even after several subcultures. The shoots could be rooted in a mist chamber to yield whole, fertile plants.     

The SHOOT MERISTEMLESS gene is required for maintenance of undifferentiated cells in Arabidopsis shoot and floral meristems and acts at a different regulatory level than the meristem genes WUSCHEL and ZWILLE

The function of the SHOOT MERISTEMLESS (STM) gene in shoot and floral meristems throughout Arabidopsis development has been analyzed. The results show that STM plays a major role in maintaining shoot and floral meristems. In an allelic series of stm mutants the shoot meristem was either reduced or completely absent in mature embryos and mutant seedling cotyledons showed partial fusion, indicating that the STM gene affects embryonic shoot meristem development and spacing of cotyledons. Postembryonically, stm mutants initiated adventitious shoot development at a position corresponding to the shoot meristem in wild-type. Repetitively initiated defective mutant shoot and floral meristems were consumed during primordia formation and typically terminated prematurely in fused ectopic primordia, indicating that STM is required for continuous shoot and floral meristem function. Analogous defects were observed in stm embryonic and postembryonic development suggesting that similar mechanisms are employed in embryonic and postembryonic organ primordia initiation. Allelic combinations suggest different thresholds for STM requirement during plant development. STM requirement could not be bypassed by standard growth factor regimes or by shoot regeneration from calli. The results suggest that STM functions by preventing incorporation of cells in the meristem center into differentiating organ primordia and that this role can completely account for all defects observed in stm mutants. Mutations in the WUSCHEL (WUS) and ZWILLE (ZLL) genes result in defective organization and premature termination of shoot meristems. Genetic interactions between STM, WUS and ZLL were analyzed and the results indicate that STM acts upstream of WUS and ZLL. Therefore, while STM appears to function in keeping central meristem cells undifferentiated, WUS and ZLL seem to be subsequently required for proper function of these cells.     

Transitions in the functioning of the shoot apical meristem in birch (Betula pendula) involve ethylene

In many trees, a short photoperiod (SD) triggers substantial physiological adjustments necessary for over-wintering. We have used transgenic ethylene-insensitive birches (Betula pendula), which express the Arabidopsis ethylene receptor gene ETR1 carrying the dominant mutation etr1-1, to investigate the role of ethylene in SD-induced responses in the shoot apical meristem (SAM). Under SD, the ethylene-insensitive trees ceased elongation growth comparably to the wild-type. In contrast, the formation of terminal buds, which in trees is typically induced by SD, was abolished. However, although delayed, endo-dormancy did eventually develop in the ethylene-insensitive trees. This, together with the rapid resumption of growth in the ethylene-insensitive trees after transfer from non-permissive to permissive conditions suggests that ethylene facilitates the SD-induced terminal bud formation, as well as growth arrest. In addition, apical buds of the ethylene-insensitive birch did not accumulate abscisic acid (ABA) under SD, suggesting interaction between ethylene and ABA signalling in the bud. Alterations in SAM functioning were further exemplified by reduced apical dominance and early flowering in ethylene-insensitive birches. Gene expression analysis of shoot apices revealed that the ethylene-insensitive birch lacked the marked increase in expression of a beta-xylosidase gene typical to the SD-exposed wild-type. The ethylene-dependent beta-xylosidase gene expression is hypothesized to relate to modification of cell walls in terminal buds during SD-induced growth cessation. Our results suggest that ethylene is involved in terminal bud formation and in the timely suppression of SAM activity, not only in the shoot apex, but also in axillary and reproductive meristems.     

Localization and expression of serine racemase in Arabidopsis thaliana

Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with β-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization showed that expression of the serine racemase gene was not induced by l- and d-serine, light irradiation, biotic and abiotic stresses.     

CHARACTERIZATION OF AN EMBRYOGENIC CELL SUSPENSION CULTURE DERIVED FROM CULTURED INFLORESCENCES OF PENNISETUM AMERICANUM (PEARL MILLET,GRAMINEAE)

An embryogenic suspension culture was established from cultured inflorescence segments of Pennisetum americanum in Murashige and Skoog's medium supplemented with 2.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 5% coconut milk. The suspension was composed of two major cell types: 1) small, richly cytoplasmic and starch-containing cells, generally found in small, compact clumps, here termed embryogenic cells; and 2) elongated, thick-walled cells with large vacuoles. By manipulating the duration of culture and dilution ratios (cell suspension: fresh medium) at the time of subculture, suspensions consisting predominantly of embryogenic cells were obtained. Suspensions grown for 2-3 wks were transferred to agar media with reduced amounts of 2,4-D. This resulted in the production of hundreds of globular and early cotyledonary embryoids. Further development of the embryoids was promoted by their transfer to a medium containing abscisic acid. Many of the embryoids germinated and produced normal green plants. Atypical embryoids, some containing many shoot meristems and a leafy scutellum, were also observed. The relevance of such atypical embryoids in the interpretation of organogenesis and embryogenesis reported in tissue cultures of cereal species is discussed. It is also suggested that somatic embryogenesis occurs in tissue cultures of most, if not all, species of cereals and grasses.     

Effect of exogenous gibberellic acid,abscisic acid,and benzylaminopurine on epicotyl dormancy of cultured herbaceous peony embryos

Epicotyl dormancy was broken in cultured peony (Paeonia lactiflora Pall.) embryos after topical application of agarose gels containing gibberellic acid, with optimum growth at 1.5 mM gibberellic acid. Addition of 100 M abscisic acid to the medium resulted in complete inhibition of gibberellic acid-stimulated promotion of dormant epicotyls. Epicotyl dormancy was also broken in embryos by culture on media containing 1 or 10 M benzylaminopurine. A highly significant increase in leaf number occurred when embryos were both cultured on medium containing benzylaminopurine and treated topically with gibberellic acid. Anatomical and morphological studies indicated that the increase in shoot growth was due to the development and growth of 1) buds formed at the cotyledonary node, 2) axillary buds, and 3) adventitious meristems originating from subepidermal parenchymatous tissue.Abbreviations ABA abscisic acid - BA N⁶-benzylaminopurine - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - LS Linsmaier and Skoog