The incorporation of uridine-3H into the shoot apices of photoperiodically induced and non-induced plants ofChenopodium rubrum L.

The influence of photoperiodic induction on the incorporation of uridine-³H into the shoot apices ofChenopodium rubrum was studied using the technique of autoradiography. No increase in uridine incorporation was detected either during induction lasting three days or immediately after its termination. Pyroninophylia likewise did not rise. However, changes in uridine incorporation related to morphogenetic activity during leaf formation and later during differentiation of inflorescences were well marked. The distribution of label in the nucleus immediately after three inductive cycles shows the ratio of extranucleolar to nucleolar incorporation to be higher in non-induced control plants than in induced ones. Data from literature pointing to an activation of RNA synthesis during transition to flowering are discussed and compared with other systems where ontogenetic changes are accompanied by marked changes in RNA synthesis. It is assumed that the activation of RNA synthesis after induction is connected mainly with the activation of growth. However, inChenopodium rubrum photoperiodic induction proceeds together with limited growth and without activation of RNA synthesis.     

Autoradiography of tritiated uridine in shoot apices ofChenopodium rubrum

The incorporation of uridine-5-³H into shoot apices ofChenopodium rubrum, plants was studied using autoradiography. The evaluation of the rate of incorporation into the nucleolus and the extranucleolar part of the nucleus as a function of the total radioactivity in the apex yields quantitative data on the distribution of labeling in these parts of the nucleus. Incubation of intact germinating plants in uridine-³H makes it possible to carry out chase experiments. Curves of uridine incorporation into the nucleolus and the extranucleolar part of the nucleus were obtained which demonstrated a non-linear course of incorporation. When incubating with uridine from 30 to 120 min the nucleolar/extranucleolar ratio of labelling was found to increase from 2 to 3. In chase experiments this ratio changed within three days from 3 to 1. Interpretation of these results in view of the function of RNA localized in different parts of the nucleus is discussed.     

Gel electrophoresis studies of proteins from leaves of photoperiodically induced and vegetative cocklebur plants

Comparisons of proteins synthesized in photoperiodically-sensitiveleaves of induced and vegetative cocklebur plants were made.Fifteen or more protein bands could be separated by polyacrylamidegel electrophoresis when polyvinylpyrrolidone was used in theextracting buffer to remove phenolics. As contrasted to resultsof others with Pharbilis nil, no differences in stained bandingpatterns could be detected in vegetative and induced plants.Radioactive leucine, lysine and phenylalanine were incorporatedinto similar leaf proteins during the last half of an inductivedark period. These experiments and dual-labeling studies with³H- and ¹⁴C-phenylalanine indicated no consistent differencesin the types of extracted proteins synthesized by leaves ofvegetative and induced plants. ¹Present address: Plant Science Dept., Utah State University,Logan 84321, U.S.A. (Received October 26, 1970; )     

Development of maize plants from cultured shoot apices

Excised shoot apices of maize (Zea mays L.), comprising the apical meristem and one or two leaf primordia, have been cultured and can form rooted plantlets. The plantlets, derived from meristems that had previously formed 7–10 nodes, develop into mature, morphologically normal plants with as many nodes as seed-grown plants. These culture-derived plants exhibited the normal pattern of development, with regard to the progression of leaf lengths along the plant and position of axillary buds and aar shoots. Isolation of the meristem from previously formed nodes reinitiates the pattern and number of nodes formed in the new plant. Thus, cells of the meristem of a maize plant at the seedling stage are not determined to form a limited number of nodes.     

The changes in the growth pattern of organs ofChenopodium rubrum photoperiodically induced to flowering

The relationship between photoperiodically changed growth of leaves, cotyledons, hypocotyl, roots and flowering has been investigated inChenopodium rubrum. It was found that all the growth characteristics recorded in leaves and cotyledons,i.e. length, area, dry weight and chlorophyll content, were inhibited during three inductive photoperiods (16 h darkness, 8 h light-SD) as compared with control plants grown under continuous illumination. Similarly, the cessation of root elongation and a decrease in root dry weight were observed. On the contrary, the elongation and dry weight of hypocotyl are stimulated by SD. The degree of the effect exerted by SD on the growth of different organs depends both on actual growth stage and the number of SD photoperiods. Increased relative rate of growth of roots and cotyledons was recorded in plants transferred after SD treatment to continuous illumination. However, this rise possesses only transitional character and the relative growth rate of treated plants equals that of control ones afterwards. The above growth changes are discussed as a possible modifying factor of floral differentiation.     

The inhibition and stimulation of DNA synthesis in shoot apices ofChenopodium rubrum L. during photoperiodic induction of flowering

Three short-day inductive cycles bring about inhibition followed by transitional enhancement of growth, not only in roots and leaves but also in different zones of shoot apical meristem, as shown by measurement of DNA synthesis using³H-thymidine autoradiography. The first inductive cycle resulted in marked inhibition of the cells of the central zone (CZ), rib meristem (RM), and peripheral zone (PZ). Subsequent enhancement of DNA synthesis occurs in RM during the second inductive cycle, but in CZ only in the third cycle. The growth activation in PZ is counteracted by decrease in apical dominance which results in further inhibition of leaf primordia and increases in bud primordia. In plants induced only by one cycle, which later reverse the vegetative pattern of growth and differentiation, increased DNA synthesis in RM and CZ was not observed. The significance of inhibitory and stimulatory processes in particular zones of the shoot apex is discussed considering flower morphogenesis.     

Adventitious shoot regeneration from vegetative shoot apices in pear and putative role of cytokinin accumulation in the morphogenetic process

Adventitious shoot regeneration was obtained from callus produced from main vegetative apices of pear of in vitrogrown shoots of Italian cultivars Spadona and Precoce di Fiorano and wild pear genotypes ISF54 and ISF61. The highest morphogenetic response was obtained on a medium containing 8.8 M 6-benzyladenine, 1.0 M -naphthaleneacetic acid and 250 mg l^–1cefotaxime. The explants were maintained for 30 days in darkness and then transferred to an auxin-free medium and to the light. Histological studies revealed that the new vegetative buds originated from callus that completely altered the morphology of the explant tissues by the 30th day of culture. The in situ localisation of cytokinins, performed using antibodies with marked specificity against zeatin (Z) and isopentenyladenine, revealed an accumulation of Z in the cambiform cells of the leaf primordia and in the shell zone of the new forming buds showing a primary role of this cytokinin in cell differentiation of in vitro pear organogenesis.     

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.     

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.     

Efficient shoot and root formation from cotton shoot apices

Successful shoot and root induction were obtained from shoot apices of two cotton (Gossypium hirsutum L.) genotypes, Nazilli 84S and Çukurova 1518, which are widely planted in Turkey. Plant tissue culture systems were established on Murashige and Skoog (MS) medium supplemented with various plant growth regulators using seven-day-old shoot apices as explants. The shoot apex size was of 2–3 mm; it contained the meristem and unexpanded leaves. Shoot apices were placed on MS plus vitamins and combinations of various plant hormones. The best regeneration responses were obtained for cv. Nazilli 84S (98%) on MS + 0.1 mg/l kinetin (KIN) + 1 g/l polyvinylpyrrolidone (PVP) and for Çukurova 1518 (94%) on MS + 0.1 mg/l KIN + 2 mg/l NAA + 1 g/l PVP. Including germination, all regeneration and rooting processes lasted only 5 weeks. The shoot apices of both genotypes developed successfully without intervening callus formation, and no significant differences between cultivars were found. All regenerated plants of both genotypes were phenotypically normal and set seeds. This shoot meristem-based rapid regeneration method can also be used in the cases of biolistic and Agrobacterium-mediated transformation.     

Histochemical investigation of dehydrogenases in shoot apices of wheat plants at different ontogenetic stages

The activity and localization of alcohol (AD), lactic (LD), malic (MD), isocitric (ICD) and succinic (SD) dehydrogenase, resp. was histochemically determined in shoot apices of plants in the vegetative condition during transition to flowering and at the reproductive state. The enzymes were determined in freehand sections, as well as in shoot apices incubatedin toto according toNachlas et al. (1958) using Nitro-BT. The characteristic localization of the enzymes depending on the ontogenic stage of the shoot apex is described. Different parts, layers and cell groups of the apex showed differences in enzyme activity. SD could not be detected in shoot apices. The histochemical findings are discussed in relation to former results obtained from a biochemical investigation of respiration in the same material.     

Analysis of surface growth in shoot apices

A salient feature of shoot meristem growth is the maintenance of distinct anatomical and morphological features despite a continuous flux of cells. To investigate how meristem organization is self-perpetuated, we developed a protocol for the analysis of meristem growth in 3-D. Our protocol uses a non-destructive replica method to follow the pattern of cell expansion and cell divisions on the meristem surface over several days. Algorithms to reconstruct the meristem surface and compute its curvature and rate of extension were implemented. We applied this approach to the shoot apical meristem of Anagallis arvensis and showed that a subcellular resolution of extension rates can be achieved. This is the first detailed quantitative analysis of meristem geometry and surface expansion in 3-D. This new approach will be useful to connect cellular activities such as cell expansion, cell division, and differential gene expression with overall meristem morphogenesis.     

Diffusible Cytokinins in shoot apices of Dahlia variabilis

Cytokinin activity (soyabean callus assay) has been determinedin excised apical buds of Dahlia variabilis before and aftera period of 3 h with cut surfaces in contact with agar gel,in the agar gel and in xylem exudates from cut shoot stumps. Buds before diffusion gave three peaks of activity in the butanolfraction, one in the aqueous fraction, following paper chromatography.Two of the former diffused into agar gel, the third (in whichmost activity was recorded) decreased in level during the 3-hperiod but was absent from the agar diffusate. The water-solublecytokinin remained at its original level and was absent fromthe agar diffusate. The three peaks of activity in the butanolfraction were also present in xylem exudates. Ether and ethylacetate fractions contained callus-growth inhibitors which diffusedinto agar gel.     

Effect of different conditions of light on the nucleic acid fractions in cotyledons ofChenopodium rubrutn L.

The nucleic acid fractions in cotyledons of young Chenopodium rubrum plants exposed to continuous light, continuous darkness and short (8 h) day, respectively and labelled with³²P 24 h prior to harvesting were studied by means of chromatography on MAK columns. Some parameters of cotyledon growth (dry weight, cotyledon area, occurrence of mitoses) were also investigated. The changes in the nucleic acid fractions agreed with the dynamics of cotyledon growth. In continuous light the content of all fractions increased. The radioactivity of DNA and s-RNA did not undergo any great changes and only r-RNA increased. The specific activity of r-RNA increased slightly, that of soluble RNA and DNA was reduced. In continuous darkness the content of all the fractions did not undergo any great changes. The radioactivity as well as the specific activity of all fractions decreased. In short day the content of the nucleic acid fractions did not change conspicuously. Only the specific activity of s-RNA increased in a noticeable way while the radioactivity of r-RNA and the specific activity of DNA decreased and this of r-RNA did not change. The changes in nucleic acid metabolism were partially connected with changes in³²P uptake which depended upon light conditions but they were not merely a consequence of this fact. Obviously, there also exists a more direct relationship between nucleic acid synthesis and growth.     

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     

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     

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.     

Growth correlations and rna synthesis in different parts of the shoot apical meristem ofChenopodium rubrunt L. induced to flowering

Uridine-³H incorporation and RNA concentration were investigated in different parts of the shoot apical meristem ofChenopodium rubrum using autoradiography and cytophotometry. A single inductive cycle was sufficient to bring about postinductive first events in the shoot apex but not for complete flower differentiation. The initial activation of RNA synthesis manifested itself in all zones of the apex. The first increase was more conspicuous in the peripheral than in the central zone. The indications of the first events in the apices after a single inductive cycle disappear prior to morphological reversal to the vegetative state. Induction by three short days led to rapid flower differentiation. The increase in RNA synthesis and concentration was most conspicuous in the central zone in this case. The ratio of RNA synthesis and content between bud and leaf primordia (B/L) also change in relation to photoperiodic induction. In vegetative plants the B/L ratio was low while after induction it increased. The shifts in activity of RNA synthesis observed in the shoot apical meristem are related to the changes in growth activity of the different parts of the apex. The growth ratios in the apices bear the character of growth correlations. The change in the growth correlations following photoperiodic induction together with the total activation of RNA synthesis are considered to represent one of the first events of the transition to the reproductive state.     

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.