Regeneration of plantlets from embryogenic suspension cultures of pickling cucumber (Cucumis Sativus L. CV. Endeavor)

Summary Procedures have been developed for the initiation and long-term maintenance of embryogenic suspension cultures of pickling cucumber (Cucumis sativus) cultivar Endeavor and for the regeneration of normal plantlets. Embryogenic calluses from petiole explants plated on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), both at 5μM, were used to initiate the embryogenic suspension cultures. Among various growth regulator combinations evaluated for initiation and maintenance of these suspension cultures, only MS medium with 2,4-D and BA, both at 1μM, produced cultures that were yellow, friable, and still regenerable after repeated subculture (every two wk) over a 3- to 15-mo. period. The effects of various concentrations of auxin and cytokinin in the plating medium, the addition of AgNO₃, and various plating procedures were also evaluated. The highest frequency of regeneration of shoots and plantlets was achieved by plating aggregates onto filter paper overlaid on MS medium with naphthalene acetic acid (NAA)/BA at a concentration of 2:1 or 1:1μM. The addition of activated charcoal (0.5%) or AgNO₃ (30μM) in the plating medium did not enhance the frequency of plantlet regeneration. The highest frequency of normal-appearing plantlets recovered was 42 to 46% per petri dish. The procedures described in this study can be used to increase plantlet recovery from individual embryogenic calluses of pickling cucumber.     

Changes in DNA strand breaks in non parenchymal cells following hepatocyte regeneration in CCl4-induced rat liver injury

DNA strand breaks (nicks) in non-parenchymal cells (NPCs) in CCl₄-induced acute or chronic liver injury in rats were detected using an in situ nick translation method; their dynamic changes were analysed in relation to the proliferation pattern of hepatocytes and NPCs, as revealed by bromodeoxyuridine (BrdU)-up-take. In acute injury, hepatocyte proliferation started before centrilobular necrosis had occurred, whereas BrdU-labeled sinusoidal NPCs markedly increased only after centrilobular necrosis was apparent. DNA breakages in NPCs paralleled the proliferation pattern of these cells, suggesting that nicks are physiological, and reflect proliferation and activated gene expression. In chronic injury, liver cirrhosis developed after 9 weeks, but BrdU-labeling of hepatocytes was almost the same level as that in untreated liver. The number of BrdU-labeled NPCs showed only a slight increase, while those with DNA breakages were much more frequent in the cirrhotic stage, suggesting a significant role for NPCs in the fibrotic process. These results indicate that DNA strand breaks in NPCs act as a marker for activation states such as proliferation, differentiation and/or activated gene expression.     

Reproductive collapse in European beech results from declining pollination efficiency in large trees

Climate warming increases tree mortality which will require sufficient reproduction to ensure population viability. However, the response of tree reproduction to climate change remains poorly understood. Warming can reduce synchrony and interannual variability of seed production (“masting breakdown”) which can increase seed predation and decrease pollination efficiency in trees. Here, using 40 years of observations of individual seed production in European beech (Fagus sylvatica), we showed that masting breakdown results in declining viable seed production over time, in contrast to the positive trend apparent in raw seed count data. Furthermore, tree size modulates the consequences of masting breakdown on viable seed production. While seed predation increased over time mainly in small trees, pollination efficiency disproportionately decreased in larger individuals. Consequently, fecundity declined over time across all size classes, but the overall effect was greatest in large trees. Our study showed that a fundamental biological relationship—correlation between tree size and viable seed production—has been reversed as the climate has warmed. That reversal has diverse consequences for forest dynamics; including for stand- and biogeographical-level dynamics of forest regeneration. The tree size effects suggest management options to increase forest resilience under changing climates.     

Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.)

Summary Embryogenic callus was formed from several cultivars of cotton (Gossypium hirsutum L.) when sections of hypocotyl and cotyledon were cultured on medium supplemented with 5 mg/liter 6-(γ, γ-dimethylallyl-amino)-purine (2iP) and 0.1 mg/liter α-naphthaleneacetic acid (NAA) for callus initiation and proliferation, and subcultured on medium supplemented with 5 mg/liter NAA and 0.1 to 1 mg/liter 2iP for embryogenic callus induction. It seems that a high 2iP:auxin ratio is preferred for callus initiation and proliferation, but should be exchanged with a higher NAA:cytokinin ratio before differentiation will occur. Embryogenic calluses were recovered at a frequency of 2 to 85% depending on the cultivar used. Coker cultivars produced embryogenic callus faster and at higher frequencies than other cultivars. Embryogenic callus produced somatic embryos on phytohormone-free medium. This medium was used to maintain and proliferate embryogenic callus for a perid of 18 to 24 mo. Somatic embryos were converted to plants on a lower ionic strength medium supplemented with 0.1 mg/liter gibberellic acid (GA₃) and 0.01 mg/liter NAA. Glucose was the only carbohydrate used through all phases of tissue culture and was much better than sucrose, on which phenolic production was very high. High temperature (30° C) and low light intensity (9 μE · m⁻² · s⁻¹) were optimal conditions for callus initiation, embryogenic callus induction, and maintenance, whereas lower temperature (25° C) and high light intensity (90 μE · m⁻² s⁻¹) were the optimal conditions for somatic embryo maturation, germination, and plantlet development. Plants could be regenerated within 10 to 12 wk in Cokers or 7 to 8 mo. in others.     

Differences in growth of neurons from normal and regenerated teleost spinal cord in vitro

Summary Explants and dissociated cells from normal adult spinal cord and regenerating cord of the teleostApteronotus albifrons were grown in vitro for periods of 8 to 12 wk. During this time the neurons showed extensive neurite outgrowth. Neurite outgrowth from tissue explants and dissociated cells of regenerated spinal cord starts sooner and is more profuse than that from normal (unregenerated) cord. Neurite outgrowth is maximized by using adhesive substrata and a high density of explants or dissociated cells. Inasmuch asApteronotus does regenerate its spinal cord naturally after injury, whereas mammals do not, this culture system will be useful to study factors that control (permit) regeneration of spinal neurons in this adult vertebrate.     

Cellular expansion of MSCs: Shifting the regenerative potential

Mesenchymal-derived stromal or progenitor cells, commonly called “MSCs,” have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.     

Fertile plant regeneration from protoplasts of meadow fescue (Festuca pratensis Huds.)

Suspension cultures from mature embryo-derived compact callus were initiated in seven meadow fescue (Festuca pratensis Huds.) cultivars. Four to six months after initiation, embryogenic suspension cultures with a moderate growth rate were established from three of them (cvs. Barmondo, Belimo and Leopard). These suspension cultures showed the capacity, maintained over six months, to regenerate green plants which could be grown to maturity under greenhouse conditions.Morphogenic suspension cultures from single genotypes of three F. pratensis cultivars (cvs. Barmondo, Belimo and Leopard) yielded large numbers of protoplasts, which upon culture in agarose beads using nurse cells formed microcalli with an overall plating efficiency in the range of 10^-3 to 10^-4. Mature plants were reproducibly regenerated and established in soil, from such protoplasts during a period of six months. The regeneration of fertile plants from protoplasts derived from suspension cultures of meadow fescue and its implications on gene transfer technology for this species are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid.     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

Embryogenesis and plant regeneration of spinach (Spinacia oleracea L.) from hypocotyl segments

A system for somatic embryogenesis and plant regeneration of spinach from hypocotyl segments has been established. Callus was induced on solid media supplemented with 8.5–15.0 mg.l⁻¹ of indole-3-acetic acid and 3.46–34.64 mg.l⁻¹ gibberellic acid. Callus was then subcultured on different media (solid or liquid) with or without IAA, or continuously maintained on the initiating media. Somatic embryos were obtained in subcultures on IAA-containing media as well as in long-term cultures on initiating media. The best results were achieved in liquid subcultures. About 60% of plantlets survived after transplanting in pots.     

Total DNA 5-methylcytosine level of rice calli derived from protoplasts is independent of regeneration ability

To study the roles of m⁵C in the differentiation of rice calli derived from protoplasts (protoclones), the m⁵C level of the total DNA was analyzed using the³²P post-labeling method. The level of m⁵C in regenerable and nonregenerable protoclones was similar, as was in calli and leaves of plants regenerated from the same protoclones. Treatment with 0.5 mM 5-azacytidine caused significant reduction of the level of m⁵C and of the regeneration frequency of callus. Significantly increased m⁵C levels were observed during prolonged culture.