Meristematic Centers Research Articles

Induction of leafy galls in Acacia mearnsii De Wild seedlings infected by Rhodococcus fascians

Plantlets of blackwattle (Acacia mearnsii De Wild) were inoculated with the bacterium Rhodococcus fascians and cultured in vitro. Leafy galls appeared at the cotyledonary nodes in 75% of the infected plants. The galls were separated from the plants and cultured on a medium containing three-quarters-strength MS salts (Murashige and Skoog, 1962), MS vitamins, 2% sucrose and an antibiotic (cephalothin), supplemented with or without 0.2% activated charcoal. Histological studies conducted from the sixth to the twenty-second day after plant infection revealed the presence of newly formed meristematic centers, first in the axillary region, then on the petioles and lamina of the leaflets around the apical meristem. Approximately 37% of the galls developed one shoot with both concentrations of cephalothin.

Carbohydrate, metabolic, and osmotic changes in scaled-up liquid cultures of Narcissus leaves

The use of scaled-up liquid cultures could be an efficient system for mass propagation of Narcissus, as it can greatly reduce the costs involved with manual handling. Induction of hyperhydric meristematic leaf section clusters and proliferation were carried out in an ancymidol (ANC)-containing liquid medium in flasks and disposable presterilized plastic bioreactors. Non-hyperhydric bulblets started to develop from hyperhydric meristematic leaf section clusters after subculture on a 0.8% agar strength medium, and young bulbs formed after 10 mo. in vivo acclimatization with a 98% survival rate. The present study reveals that in Narcissus leaf sections cultured in liquid medium, morphogenetic changes in leaf sections were associated with metabolic changes. The changes in carbohydrate, protein, and water potential of the liquid media and leaf sections were found to be closely related to meristematic center initiation on Narcissus hyperhydric leaf sections. Starch, sucrose, and glucose were significantly higher in the hyperhydric leaf sections cultured in ANC medium. The water potential was signifieantly higher in ANC-treated leaf sections and significantly lower in the medium containing ANC, at the stage shortly before or after hyperhydricity and meristematic centers hegan forming on the leaf sections. A 30kDa protein was found to be present in the hyperhydric leaf sections. Based on the present study, a largescale micropropagation protocol of Narcissus in agar and liquid cultures is proposed.

Competence, determination, and meristemoid plasticity in tobacco organogenesis in vitro

Tobacco leaf explants can produce both shoots and roots depending on the phytohormones in the medium. These arise directly via meristemoids (meristematic centers), which form distinct primordia and then organs. In this study it was found that shoot primordia arose from the palisade mesophyll cells at the adaxial surface, while root primordia arose from the rib parenchyma cells, near the existing vascular bundles. In studies on competency and determination, it was found that the tobacco leaf explants required 4–6 days in culture on a shoot-inducing medium (SIM) to become determined for shoot formation, while the explants were competent for rooting at excision and needed only 1 day on the root-inducing medium (RIM) to become determined for root formation. Transfer of explants from SIM or RIM to basal medium (BM without phytohormones) and vice versa supported the above findings. Transfer of explants from SIM to RIM and vice versa, delayed the timing of root and shoot formation, but not the position in the explant from which the organs arose. On transfer from SIM to RIM or vice versa, meristemoids that were already determined for shoot or root formation continued to develop, while those not yet determined were inhibited and (or) reverted to parenchymatous tissue. Thus under our culture conditions meristemoids in tobacco leaf explants are not plastic.Key words: competence, determination, meristemoid plasticity, organogenesis, tobacco.

The effect of ancymidol on hyperhydricity, regeneration, starch and antioxidant enzymatic activities in liquid-cultured Narcissus.

Addition of the growth retardant ancymidol to Narcissus shoots and lower inner leaf sections isolated from shoots cultured in liquid medium induced hyperhydric malformations associated with morphogenetic changes. Meristematic centers initiated on the basal proximal ends appeared over the entire surface of the hyperhydric leaf sections after 6 weeks in culture. The meristematic centers which formed clusters on the leaf sections developed later into buds. In leaf sections grown in the liquid medium lacking ancymidol, hyperhydricity was not induced, and regeneration was not observed. Starch and protein levels and ascorbate peroxidase and catalase activities were examined in shoots and isolated leaf sections that were either hyperhydric or non-hyperhydric. In ancymidol-treated, hyperhydric leaf sections, ascorbate peroxidase and catalase activities were lower than in control, untreated leaf sections. The changes in starch and protein levels and in antioxidant enzymatic activities appeared to be related to the onset of meristematic-center initiation and further bud development on Narcissus hyperhydric leaf sections.

In vitro regeneration of Acacia mangium via organogenesis

Plant regeneration of Acacia mangium was achieved through organogenesis in callus cultures. Calli were induced from five types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaflets, petioles and stems of seedlings) of A. mangium on MS (Murashige and Skoog, 1962) basal medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 13.95 μM kinetin (KT). Green or green purple compact nodules containing clusters of meristematic centers were induced in these calli after transfer to MS basal medium containing 1.14–22.75 μM thidiazuron (TDZ) and 1.43–2.86 μM indole-3-acetic acid (IAA). A combination of 4.55 μM TDZ and 1.43 μM IAA promoted the highest percentage of calli to form nodules, in 8–11% of calli derived from cotyledons, embryo axes, leaflets or petiole and in 4% of calli derived from stems. Twenty-two percent of the nodules formed adventitious shoots on MS basal medium containing 0.045 μM TDZ. Shoots were elongated on MS medium containing 0.045 μM TDZ supplemented with 7.22 μM gibberellic acid. The medium containing 10.75 μM NAA and 2.33 μM KT promoted rooting of 10% of the elongated shoots. Plantlets grew up well in the green house.

Somatic embryogenesis from leaf cultures of potato

An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and α-naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA3) and BA. Treatment with zeatin (22.8 μM) and BA (10.0 μM) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.

Biochemical and Histological Changes during In Vitro Organogenesis in Jatropha Integerrima

Biochemical changes associated with adventitious shoot regeneration during in vitro culture of hypocotyl explants of Jatropha integerrima were determined. Histological and biochemical studies were undertaken at 7-d intervals, up to four weeks on hypocotyl explants cultured on basal Murashige and Skoog's medium supplemented with 0.5 mg dm-3 N6-benzyladenine and 1.0 mg dm-3 indole-3-butyric acid. Initial cell proliferation occurred within one week of culture; meristemoid differentiation within two to three weeks and shoot development after four weeks. Peak activities of alkaline phosphatase, peroxidase and polyphenol oxidase was observed at day 14 indicating their involvement in the formation of meristematic centers. Protein accumulation and acid phosphatase activity were maximum at day 28.

Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm−3 casein hydrolysate and 5 mg·dm−3 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm−3 dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm−3 2,4-D or 1 mg·dm−3 dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm−3 2,4-D or 1 mg·dm−3 dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm−3 kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.

In vitro development from leaf explants of sugar beet ( Beta vulgaris L). Rhizogenesis and the effect of sequential exposure to auxin and cytokinin

Adventitious root development in lamina and midrib-petiole junction expiants of sugar beet cv. Primo was investigated using scanning electron microscopy and light microscopy. Primordia developed close to the vascular strands and areas of newly dividing cells (meristematic centres) were seen adjacent to the intrafascicular cambium after 2 d incubation on medium containing 30 mg 1(-1)1-naphthalene acetic acid. Clearly defined primordia were visible at 4 d and the first roots had emerged by 6 d. A minimum of 24 h exposure to NAA was necessary for root induction. Four days on NAA caused twice as many roots to be initiated but more prolonged exposure (5 and 10 d) inhibited root development. Root initiation continued after transfer to medium containing no plant growth regulators, new primordia appearing as the older ones extended as roots. Attempts were made to modify the development of primordia by sequential culture on cytokinin after induction by auxin. Incubation on N6-benzylaminopurine within 48 h of exposure to NAA disrupted the development of primordia and roots but did not induce shoot formation.

Somatic Embryogenesis and Plant Regeneration from Seedling Cultures of Tomato ( Lycopersicon esculentum Mill.)

Summary Somatic embryogenesis was induced in several cultivars of tomato (Lycopersicon esculentum Mill.) using a simple, one-step method. Seeds were germinated in the presence of 50 or 80 μmol · L-1 N6-benzylaminopurine, and the resultant seedlings were maintained for six weeks on the same medium. Somatic embryogenesis occurred directly without an intervening callus proliferation phase from the meristematic centres formed in the subepidermal region of the nodular tissues developed at the basal ends of induced seedlings. Regenerated somatic embryos were successfully grown into whole plants.

High-frequency Organogenesis from Direct Seed Culture in Lathyrus

Culture conditions were developed for inducing a high frequency of direct shoot morphogenesis and whole plant regeneration in cultures of intact seedlings of Lathyrus cicera L., L. ochrus (L.) DC., L. sativus L., and L. tingitanus L. The procedure of shoot regeneration involved culturing of whole, mature seeds on MS medium containing cytokinins, or thidiazuron (TDZ), a substituted phenylurea with cytokinin-like activity. Differentiation of shoots occurred without an intervening callus phase from the cotyledonary node and surrounding tissues of the intact seedlings developed from seeds germinated on media containing kinetin, BAP or TDZ. An average of 19·0, 15·8, 28·8 and 43·0 shoots were regenerated at optimal concentrations of 50·0, 50·0 and 80·0 μM BAP in L. cicera, L. tingitanus, L. ochrus and L. sativus, respectively. TDZ enhanced the shoot formation at the concentration of 10 μM to an average of 33·1 and 33·7 shoots per seedling in L. cicera and L. tingitanus and at 50 μM, to 57·4 shoots per seedling in L. sativus. Regenerated shoots developed roots on a modified MS medium containing 2·5 μM NAA and the surviving plantlets were transferred to soil. Histological studies revealed de novo formation of shoot buds from the epidermal or subepidermal cells of the basal and nodal region of multiple epicotyls. Several meristematic centres consisting of actively-dividing cells developed in the subepidermal cell layer of the nodal tissue and adjacent areas within 7 d of seed culture on a cytokinin- or TDZ-supplemented medium. The pattern of the development of these meristematic centres and shoot development was similar in all four species.

A Developmental Perspective of the Evolution of the Stele

The complexity of developmental processes constrains evolutionary changes. A rare change, an evolutionary innovation, may be inferred from the combined information of fossil steles and of the controls of vascular differentiation. This change was the gradual appearance of a new shoot apex, with determinate but dominant appendages and a weak, yet persistent, promeristem. The controls of vascular differentiation could be used to indicate additional evolutionary events. These could include the responses of the differentiating vascular tissues themselves and not only the formation of inductive signals by the meristematic centers. They could also involve the interactions between the phloem and the xylem, expressed by the development of vascular rays and perhaps by the ribbing of steles. Since the specification of orientation is an essential aspect of vascular differentiation, continued work requires information on the three-dimensional cellular structure of fossil tissues. Complementary studies would be experim...

Effect of cytokinins on plastid development and photosynthetic polypeptides during organogenesis ofPinus ponderosa Dougl. cotyledons culturedin vitro

InPinus ponderosa Dougl., application of the cytokinins, benzyladenine and 2-isopentenyl adenine, to excised cotyledons, promoted thein vitro formation of meristematic centers which led to bud and shoot production. Meristematic cells showed plastids with poorly developed thylakoid membranes and rudimentary grana, whereas cells in non-meristematic tissues and in growth regulator free medium, had chloroplasts with well developed inner membranes, and more thylakoid membranes and grana than plastids of meristematic cells. Chlorophyll and six polypeptides associated with photosynthesis were present in lower concentrations in cytokinin-treated cotyledons than in those cultured in growth regulator free medium. Both benzyladenine and 2-isopentenyl adenine are effective in inhibiting the accumulation of at least two photosynthetic polypeptides in the first 24 h in culture. The ability of cotyledons to respond in this way to cytokinins is lost after three days in culture in growth regulator free medium prior to treatment with cytokinin.

Genetic transformation by Agrobacterium tumefaciens in the interspecific hybrid Helianthus annuus × Helianthus tuberosus

A genetic transformation method has been developed in the interspecific hybrid Helianthus annuus × Helianthus tuberosus using Agrobacterium tumefaciens. Leaf explants of a clone with an efficient tissue culture regeneration capacity were inoculated with A. tumefaciens carrying a disarmed Ti plasmid containing the Cauliflower Mosaic Virus (CaMV) 35S- GUS fusion gene with the nopaline sinthase (NOS) neomycin phosphotransferase II ( NPT II) gene. On selection medium containing 25 mg/l of kanamycin, the inoculated leaf explants formed meristematic centers with buds and embryo-like structures that successively developed into putative transformed shoots, when transferred onto medium without growth regulators. Under suitable conditions, three days of cocultivation on medium containing BAP and NAA, the highest transformation frequency was 5.4%. Histochemical staining for the β-glucuronidase ( GUS) activity provided evidence for transformation in different tissues and organs of transgenic plants. Integration of foreign DNA into genomic H. annuus × H. tuberosus DNA was demonstrated by Southern analysis.

Cotyledons: an Explant for Routine Regeneration of Sunflower Plants

The potential of plant regeneration from sunflower cotyledon explants was studied under conditions either to allow direct regeneration (organogenesis) or callus proliferation with high regeneration capacity. Analysis of histological modifications caused by tissue culture conditions showed that the regeneration protocol described here permitted the development of abundant meristematic centers in discrete regions of the basal part of the cotyledon. The protocol presented here proved to be suitable for the regeneration of 10 genotypes, among 20 assayed, including commercial inbred lines and hybrids of common use for production in Argentina. Furthermore, Agrobacterium infection experiments showed that basal halves of cotyledons were transformed, evidencing tumour proliferation.

When plant teratomas turn into cancers in the absence of pathogens

Habituated calli have long been classified as neoplasms together with tumors from different origins. The general opinion is that habituation is a reversible process with an epigenetic basis. This is probably true in most cases examined. However, we show here that there might be several degrees of habituation, which can be considered as steps of a neoplastic progression leading to cancerisation in the absence of an introduced oncogenic pathogen. Cell rejuvenation, loss of the capacity to organize meristematic centers, and loss of totipotency are proposed to define plant cancer through this neoplastic progression of a callus.Habituated tissues share many morphological and biochemical similarities with so‐called vitreous shoots from micropropagation. Vitrification and hyperhydric malformations of shoots raised in vitro may be considered as steps of another neoplastic progression, which leads to cancerisation also in the absence of introduced oncogenic pathogens. In this case death of the whole organism occurs either through direct apex necrosis or indirectly, from the loss of the capacity for the primary meristems to function normally, which gives rise to completely anarchic structures. As in the animal kingdom, carcinogenesis in plants is the final result of a multistep process involving the irreversible conversion of a stem cell to a terminal‐differentiation‐resistant cell.

Shoot, root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 μM concentrations. Direct shoot formation occurred on media with 10 μM kinetin and 0.001 μM IAA or NAA. Root formation was observed on media with 0.1–10 μM IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 μM IAA and 0.1 μM kinetin. Shoot organogenesis was increased by substituting 10 μM zeatin or N6-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (‘Red Alert’) to 5.3 (‘Large Red Cherry’). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.

Histological-anatomical studies of the structure of the organogenic callus inPapaver somniferum L.

Organogenic callus cultures ofPapaver somniferum L. were studied with the aim of describing the morphology of the callus and observing the changes occurring during differentiation and induction of organogenesis. The morphology of the cells and the formation of meristemoid areas were studied on a light microscopic level. Histological evidence showed that from the inner meristematic centres, protracheal elements are differentiated, from the surface meristematic centres meristemoids are formed and therefrom green leaf-like organoids.

Formation and growth of callus tissue of Arabidopsis under changed gravity

The changed gravity conditions do not prevent the process of cell dedifferentiation and formation of callus. Yet, callus grown on the clinostat and in space differs from the control one: its consistence is denser, occurence of meristematic centres is less frequent and it shows a reduced proliferative activity of cells. Average size of cell and nuclei area in the test variants is lower than in the control.

Effects of inhibitors of polyamine biosynthesis on growth and organization of meristematic centers in Petunia protoplast cultures

We examined the effects of inhibitors of the polyamine biosynthetic enzymes arginine decarboxylase (ADC), ornithine decarboxylase (ODC) and spermidine synthase (Spd synthase) on growth and organogenesis in cultures derived from mesophyll protoplasts of Petunia hybrida Vilm cv. Fire Chief. Both processes were dramatically inhibited when protoplasts were cultured in a mixture of dl-α-difluoromethylarginine (DFMA) and dl-ga-difluoromethylornithine (DFMO), specific inhibitors of ADC and ODC respectively, and cyclohexylamine sulfate (CHA) an inhibitor of Spd synthase. The inhibition was partially reversed by addition of micromolar amounts of both putrescine and spermidine. Treatment with DFMA markedly inhibited organization of meristematic centers and reduced levels of free and bound putrescine, while treatment with CHA was less effective. In contrast, treatment with DFMO promoted growth and organization with concomitant increases in bound putrescine and spermidine. The results suggest that putrescine synthesized via ADC is involved in growth and organogenesis.