Recovery of fertile plants from isolated,cultured maize shoot apices

Maize shoot apices (1 to 2mm size) from two sources were used to recover normal plantlets. The first explant source included shoot apices from the embryonic axis of immature embryos, 12–14 days post pollination in the glasshouse (spring) or 15–20 days post pollination in the summer nursery. In most explants, the shoot apical meristem was surrounded by a coleoptile primordium which was removed before culture. The second explant source included shoot apices from the plumules of 72 h imbibed mature kernels. The coleoptile and all other leaf layers (leaf-1 to leaf-3 or 4) of the plumule were removed before culture to expose the apical meristem. Among the genotypes studied, a recovery of 43% (Mo17) to 100% (Oh43) of plantlets was achieved from shoot apices from immature embryo plumules cultured in MS medium. Recovery of 80% of Oh43 plantlets in MS medium and 40% of A188 plantlets from apices of plumules of imbibed (72 h) seeds in MS medium containing 2,4-dichlorophenoxyacetic acid was recorded. The plantlets derived from both explant sources grew normally and produced viable seeds upon pollination.     

Cell-lineage patterns in the shoot apical meristem of the germinating maize embryo

A fate map for the shoot apical meristem of Zea mays L. at the time of germination was constructed by examining somatic sectors (clones) induced by -rays. The shoot apical meristem produced stem, leaves, and reproductive structures above leaf 6 after germination and the analysis here concerns their formation. On 160 adult plants which had produced 17 or 18 leaves, 277 anthocyanin-deficient sectors were scored for size and position. Sectors found on the ear shoot or in the tassel most often extended into the vegetative part of the plant. Sectors ranged from one to six internodes in length and some sectors of more than one internode were observed at all positions on the plant. Single-internode sectors predominated in the basal internodes (7,8,9) while longer sectors were common in the middle and upper internodes. The apparent number of cells which gave rise to a particular internode was variable and sectors were not restricted to the lineage unit: a leaf, the internode below it, and the axillary bud and prophyll at the base of the internode. These observations established two major features of meristem activity: 1) at the time of germination the developmental fate of any cell or group of cells was not fixed, and 2) at the time of germination cells at the same location in a meristem could produce greatly different amounts of tissue in the adult plant. Consequently, the developmental fate of specific cells in the germinating meristem could only be assigned in a general way.Abbreviations ACN apparent cell number - LI, LII, LI-LII sectors restricted to the epidermis, the subepidermis, or encompassing epidermis and subepidermis - PCN progenitor cell     

Determination of floral organ type in cultured Silene shoot apices

Shoot apices of the long day plant, Silene coeli-rosa , were cultured on a basal medium (+3% sucrose) in non-inductive short days (SD) following their excision from plants which had been exposed to long day (LD) treatments in order to examine the period for determination of each floral whorl. In response to the inductive LD treatments, the pattern of whorl formation in vitro reflected their normal appearance in Silene : sepals, stamens 1–5, petals, stamens 6–10 and carpels, although the number of apices initiating each whorl was lower in vitro compared with apices in vivo. However, supplementing the medium with 7 instead of 3% sucrose corrected this deficiency and, for the first time, resulted in apices initiating floral whorls in SD. The interval between the shortest treatment to result in whorl initiation in vitro, 4 LD (which also resulted in 50% flowering in vivo), and the treatment which gave 50% initiation of the corresponding whorl in vitro, was taken to be the period for determination of that whorl. The determination times on the 3% medium were: sepals (2 days), stamens 1–5 (3 days), petals (3 days), stamens 6–10 (4 days) and carpels (4 days); all of these periods shortened to about 1 day on the 7% medium. Tissue culture did not perturb the pattern of initiation of each whorl since apices excised and cultured from plants which had received 7 LD + 2 SD, exhibited each whorl over the same time scale as those of intact plants which received the same treatment. The data are consistent with a sequential determination and initiation of each whorl in the order that they appear normally in Silene . Synchronisation of cell division, as represented by peaks of the mitotic index and G₂/G₁ ratios on day 8 (7 LD + 2 SD), did not occur in vitro but the mitotic index did not descend to zero, further emphasising that tissue culture did not perturb the Silene apex.     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices

This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium. Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm. Shoot apices of Oryza sativa L. Tropical Japonica, cv. Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter. Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT. After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT. Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation. Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests. A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern. Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation.     

Transformation of plants via the shoot apex

Summary We have transformed petunia byAgrobacterium tumefaciens containing genes for kanamycin resistance and beta-glucuronidase using isolated shoot apices from seedling tissue. Regeneration of transformed plants in this model system was rapid. The technique of shoot apex transformation is an alternative for use inAgrobacterium-mediated transformation of dicotyledonous crop species for which a method of regeneration via protoplasts, leaf disks, or epidermal strips does not exist. This approach offers direct and rapid regeneration of plants and low risk of tissue-culture-induced genetic variation. Texas Agricultural Experiment Station Technical Article No. 23317.     

Cytokinin production by Asparagus shoot apex cultured in vitro

The cytokinin producing capacity of asparagus (Asparagus officinalis L.) shoot apex was examined by means of shoot apex culture in vitro, where adventitious roots were never formed. The cultured shoot apices continued to diffuse a small but constant amount of cytokinin into the medium throughout five passages of subculture. The cytokinin content in the apices at the end of the subculture was not different from that at the beginning of the subculture. These results indicate a production of cytokinin by the apices. However, the finding is not in conflict with the hypothesis that the root tip is the major source of cytokinin supply, because the root tip of asparagus produced more cytokinin than the shoot apex and the decline of shoot growth observed during the subculture was partially restored by an application of zeatin into the medium.     

Direct differentiation of ears and tassels from cultured shoot apices of maize

In vitro morphogenesis of inflorescences from the cultured corn seedling shoot tips was obtained on modified Murashige and Skoog (MS) medium in complete darkness. Some shoot tip meristems excised from seedlings of inbred line 515, inbred line 8112 and their filial generations would directly give rise to florets on modified MS medium supplemented with 2.0 mg/L N6-bezyladenine (6-BA) in five or six weeks. On the medium with 1.0 mg/L 6-BA and 0. 2 mg/L 2, 4-dichlorophenoxy acetic acid (2, 4-D), the explants swelled first, and produced multiple shoot clumps, then the culture of the shoot tips from all of the six inbred lines in experiment would ultimately initiate to develop ears and tassels accompanied by multiple shoot clumps developing on the medium with 1.0 mg/L 6-BA and 0. 2 mg/Lin-dole-3-butyric acid (IBA). The developmental patterns of the corn inflorescences were similar to the controls of normal plants in the field, but the number of the ears was much more than that of the tassels in vitro. It seem     

Clonal propagation ofBougainvillea glabra ‘Magnifica’ through shoot apex culture

Shoot apices ofBougainvillea glabra ‘Magnifica’ were induced to regenerate an average of ten shoots from their bases in response to BAP (0.5 mg/l) plus IAA (1.5 mg/l). All the isolated shoots from such cultures were rooted in a medium containing 0.1 mg/l each of IBA and 2,4,5-T and lacking BAP. Plantlets were then successfully grown in potted soil where they flowered normally. NBRI, research publication no. 32/80.     

Microcallus growth from maize protoplasts

Maize (Zea mays L.) protoplasts obtained from Type I and Type II calli from several genotypes were shown to be capable of synthesizing cell walls and forming small clusters of cells. The medium used also supported cluster formation from protoplasts obtained from root tips. The effects of various additions to the medium (such as casein hydrolysate, coconut water, amino acids, sugars, phytohormones, nitrate, calcium, and dimethylsulfoxide as well as pH variations on cellcluster formation were determined. The method of culture (protoplasts plated in agarose or supported in alginate beads in liquid medium) as well as several components of the medium were found to be critical for microcallus formation. Protoplasts obtained from embryogenic Type I callus and cultured in the medium of C. Nitsch and J.P. Nitsch (1967, Planta 72, 355–370) modified by various additions (NN 67-mod medium) were affected most by various sugars, casein hydrolysate, coconut water, and a combination of the auxins napthalene-1-acetic acid (2 mg/l) and 2,4-dichlorophenoxyacetic acid (0.1 mg/l), and the cytokinin N⁶-benzylaminopurine (0.5 mg/l). Cluster size in the agarose culture system was from 0.1 to 0.5 mm diameter and in the alginate culture system, up to 2.0 mm diameter.     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

The blockage in the G 1 phase of the cell cycle in the dormant shoot apex of ash

The shoot apex of the terminal bud was studied in four successive physiological states: during dormancy, when dormancy breaks, during the third week after the break of dormancy, and during a later typical period of active growth. DNA content was measured in Feulgen-stained nuclei of the axial zone, of the lateral zone, and of the rib meristem. The mitotic index was established for each zone of the meristem. During the period of dormancy, all the nuclei of the meristem are in phase G ₁ of the cell cycle and are blocked at the same point common to all nuclei. When dormancy breaks, this blockage is removed simultaneously and all nuclei in the shoot apex resume their cell cycles starting at the same point. The cycles remain synchronized for awhile. In the axial zone they remain synchronized until the third week after resumption of active growth.     

Factors affecting in vitro shoot formation from vegetative shoot apices of apple and relationship between organogenic response and cytokinin localisation

ABSTRACT

The effects of macro- and micro-elements, benzyladenine (BA) concentration, and the period of auxin application on adventitious shoot formation from callus originating from vegetative shoot apices were tested on apple (Malus domestica Borkh) rootstock Jork 9. The putative relationship between organogenic response and cytokinin localisation was also studied by an immunolocalisation technique for in situ determination of free cytokinins. The use of MS (Murashige & Skoog, 1962) salts in the medium instead of those of LP (Quoirin & Lepoivre, 1977) had a strong positive effect both on shoot formation rate and on the number of shoots produced. The highest organogenic response from callus was induced using 17.8 μM BA in the presence of 2.7 μM NAA and by maintaining the explants for 20 days in darkness, then transferring them to fresh auxin-free medium and to the light. The in situ localisation studies, performed using antibodies with a marked specificity against zeatin and isopentenyladenine, revealed changes in the localisation of free zeatin in the tissues during the shoot-forming process, in particular during the active cell division phase leading to callus formation, and in the initial phase of bud formation. Changes in zeatin distribution in the tissues of the vegetative shoot apex during shoot formation may indicate a role for this cytokinin free base in cell differentiation and organogenesis.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Efficient plant regeneration from shoot apices of sorghum

An efficient and rapid regeneration protocol was developed using shoot apices from germinating seedlings of two cultivars of sorghum, SPV-462 and M35-1, as explants. A vertical slit given from the base of each dissected apex enhanced the efficiency of callusing response by two fold. MS medium containing 0.5 mg dm⁻³ each of 2,4-D and kinetin was most effective in producing friable and embryogenic calli. Scanning electron microscopy of these calli detected somatic embryogenesis. Calli thus induced gave rise to approximately 42 green shoots per callus in both the genotypes when transferred to regeneration medium containing 1.5 mg dm⁻³ kinetin.     

In vitro shoot-tip grafting improves recovery of cotton plants from culture

A rapid in vitro shoot-tip grafting (STG) technique was adapted to increase recovery of intact cotton plants from shoots developed in culture. Induction of root organogenesis in cotton shoots is genotype dependent and unreliable. The resulting loss of regeneration potential due to failure to form roots can vary from 30 to 80% according to genotype and represents a significant bottleneck in the overall recovery of plants from culture. If the non-rooting shoots are transgenic, the loss in regenerated plant material can be substantial. In vitro grafting of cotton shoots to seedling rootstock proved to be a simple and reliable method allowing 90–100% recovery of non-rooting shoots from culture. Success of any given graft was directly related to scion size (0.8–1.0 cm) and age (14–35 days) of the seedling rootstock. The method appeared to be genotype independent, and varietal differences between rootstock and scion did not effect the rate of plant recovery from culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Appearance of peroxidase isozymes in floral‐initiated shoot apices of Pharbitis nil

A novel biochemical assay system for detecting the early stage of flowering is reported. Peroxidase isozymes in shoot apices of Pharbitis nil plants that had been exposed to flower‐inducing or non‐inducing conditions were analyzed by isoelectric focusing in polyacrylamide gels and activity staining for peroxidase. Several isozymes with pH 8.5–8.8 appeared for the first time 7 days after the beginning of short‐day treatment, but not after nightbreak (non‐inducing) treatment. When shoot tips were cultured in vitro, these same isozymes also appeared after short‐day treatment but not after night‐break treatment. The extent of the appearance of these isozymes was reduced by exposure to high or low temperature during the inductive dark period and removal of cotyledons after the inductive dark period. Such treatments also reduced the extent of flowering. The appearance of an isozyme with pH 8.5 was more closely correlated with flowering than that of the other isozymes. From these results, the appearance of this peroxidase isozyme in shoot apices is discussed as a biochemical marker of flowering in intact plants and in cultured shoot tips.     

Mitotic activity in the maize root apex after freeze-decapping

Mitosis and nuclear DNA synthesis have been examined in root apices of maize whose caps were removed by a freezing technique. These processes are not impaired by this technique even though cells at the surface of the decapped apex experience a temperature close to 0°±1.5° C for a brief period. We conclude that freeze-decapping is without significant deleterious effects to the apex and therefore the technique is a useful adjunct in studies of the role of the cap on root growth.     

Embryogenic callus formation from maize protoplasts

Maize (Zea mays L.) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos. The somatic embryos can be induced to form roots and small leaf-like structures. The genotype was the hybrid A188xBlack Mexican Sweet. Protoplasts were prepared from an embryogenic suspension culture derived from a Type II callus which had been selected from Type I callus produced by immature zygotic embryos. The basal medium for the suspension culture was N6 (C.C. Chu et al., 1975, Scientia Sinica 18, 659–668). The 2,4-dichlorophenoxyacetic acid concentration of the suspension culture was critical for subsequent protoplast growth and was optimal at 4.0 mg.l. Protoplasts had to be cultured in a low-osmoticum medium (0.3 M mannitol) for subsequent cell divisions to occur. The protoplasts have been transformed transiently with the gene chloramphenicol acetyltransferase (CAT) containing the 35S promoter obtained from cauliflower mosaic virus (CaMV-35S).Abbreviations FDA fluorescein diacetate - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Development of shoot inHydrangea macrophylla II. sequence and timing

The development of axillary buds, terminal buds, and the shoots extended from them was studied inHydrangea macrophylla. The upper and lower parts in a nonflower-bearing shoot are discernible; the preformed part of a shoot develops into the lower part and the neoformed part into the upper part (Zhou and Hare, 1988). These two part are formed by the different degrees of internode elongation at early and late phases during a growth season, respectively. Leaf pairs in the neoformed part of the shoot are initiated successively with a plastochron of 5–20 days after the bud burst in spring. The upper axillary buds are initiated at approximately the same intervals as those of leaf pairs, but 10–30 days later than their subtending leaves. Changes in numbers of leaf pairs and in lengths of successive axillary buds show a pattern similar to the changes in internode lengths of the shoot at the mature stage. The uppermost axillary buds of the flower-bearing shoot often begin extending into new lateral shoots when the flowering phase has ended. The secondary buds in terminal and lower axillary buds are initiated and developed in succession during the late phase of the growth season. Internode elongation seems to be important in determining the degrees of development of the axillary buds. Pattern of shoot elongation is suggested to be relatively primitive. Significances of apical dominance and environmental conditions to shoot development are discussed.