Plant Growth Regulators III : Gibberellins, Ethylene, Abscisic Acid, their Analogues and Inhibitors; Miscellaneous Compounds.

Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 102–105 colony forming units g−1 fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 105 – 4.0 × 107 colony forming units g−1 tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.

Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures

This study establishes the widespread prevalence of fastidious or viable but non-culturable endophytic bacteria in field shoots and in unsuspicious shoot-tip cultures of papaya (Carica papaya L.) against the norm of asepsis in vitro. A total of 150 shoot-tips (approximately 10 mm) were inoculated on MS-based culture medium after surface sterilization of field-derived axillary shoots of cv. Surya during November or January (100 and 50, respectively) when 35–50% cultures showed endophytic microbial growth on culture medium. Indexing of apparently clean cultures using bacteriological media helped in detecting and removing additional 14–17% stocks with covert bacteria during the first two passages. The rest of the stocks stayed consistently index-negative during the first eight subculture cycles, but appeared positive in PCR-screening undertaken thereafter employing universal bacterial 16S rRNA gene primers indicating the association of non-cultivable bacteria. Direct sequencing of the PCR product yielded overlapping nucleotide data signifying mixed template or the presence of diverse endophytic microorganisms. This was confirmed by light microscopy of tissue sap revealing viable bacteria in considerable numbers, which were detected under phase contrast or with negative staining. Planting tissue segments or applying homogenate from these stocks on diverse bacteriological media did not induce the organisms to grow in vitro. The shoot cultures displayed variation in growth and rooting potential, the onus of such variation was solely attributable to the associated microorganisms. The findings were confirmed with additional field shoots and fresh in vitro stocks established subsequently. The observations have implications in micropropagation and all other applications involving plant cell, tissue, organ, and protoplast culture.

Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya

The hyperhydricity, formerly called vitrification, is considered a physiological, biochemistry and morfologic disorder due to abnormal accumulation of water inside the cells and tissues. Plants grown in vitro are undoubtedly under continuous stress condition which results in metabolic changes characteristic of oxidative stress. Anatomically plants or shoots affected often become swollen, with pale green, translucent sheets, glass-like, low relative number of cells / cell area and hipolignification. Physiological changes occur in major metabolic pathways including photosynthesis, respiration and transpiration resulting in reduced efficiency of these metabolic pathways. Morphological, physiological and biochemical disorders are triggered by physical factors related to the environment of cultivation vessels and consistency of the culture medium or by chemical factors such as culture medium components, especially the growth regulators in high concentrations. The hyperhydricity occurs at various levels of severity, reaching result in irreversible loss of morphogenic capacity and the establishment of a state of neoplastic cells, however, in most cases hyperhydricity is considered reversible. This review focuses on the current knowledge about the phenomenon of hyperhydricity addressing morphological, physiological, biochemical, and reversibility of the process.

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Hiperidricidade: uma desordem metabólica

Cultured leaf cells of Nyoho strawberry (Fragaria ananassa Duch.) produced 4 times more anthocyanins than fruit, with additions of 2.0 mg/l of 1-naphthalenacetic acid (NAA) and 0.2 mg/l of benzyladenine (BA) for 2 weeks. They were habituated after 6 passages at NAA 1.25 and BA 0.125 μg/l in 107 cultures with stepwise decrease of the additions at 2-week intervals, and supervention of a gradual decrease in anthocyanin contents of 80%. They fully recovered through the following 43 passages without the additions. Finally, 3 types of cell line rich in cyanidin 3-glucoside (62%), peonidin 3-glucoside (55%) or both (49 and 36%, respectively) were obtained. They also produced pelargonidin 3-glucoside (0.6 to 1.8%), which was the main component in the fruit. The fruit produced peonidin 3-glucoside (0.4%), as well as pelargonidin 3-O-(6-O-malonyl-β-D-glucoside) and cyanidin 3-glucoside. The habituation seems to have caused some activation or inhibition of the cell’s anthocyanin formation, which originated from the mother plant.

Production of Anthocyanins by Habituated Cultured Cells of Nyoho Strawberry (Fragaria ananassa Duch.)

Fully habituated organogenic and nonorganogenic sugarbeet calluses reacted to application of the synthetic auxin [3-benzo(b) selenienyl] acetic acid by changes in growth and ethylene production. Treatment of fully habituated cells of periwinkle with 2,4-dichlorophenoxyacetic acid led to the decrease of free cytokinin contents (isopentenyl adenine, zeatin riboside, and zeatin) during the late exponential phase of growth. The polyamine contents were also modified and the capacity to biotransform secologanin into ajmalicine was decreased. Treatment of the habituated periwinkle cells with zeatin greatly increased the amount of a polypeptide of 16 kDa; this response was more marked than that displayed by the auxin-dependent line. These data show that hormone-independent calluses and cell suspensions can retain some sensitivity to growth hormones. However, differences of responses were observed between the auxin-dependent lines and the habituated lines.

https://www.jstor.org/stable/info/20064906

M. Filali

Papers

Habituation of plant cells does not mean insensitivity to plant growth regulators