M.I. Trillas

Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation.

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our knowledge, this study is the first to evaluate the effect of TDZ on endogenous phytohormones in an organogenic process.

Development of photoautotrophy and photoinhibition of Gardenia jasminoides plantlets during micropropagation

This paper reports on the fast fluorescence responses of Gardenia jasminoides Ellis plantlets, at two successive stages (shoot multiplication and root induction) of culture in vitro. We test whether plantlets in vitro suffer photoinhibition during culture and whether the degree of photoautotrophy of these mixotrophic plantlets has any effect on the extent of photoinhibitory impairment. In this regard the effects of different sucrose levels in the medium and PPFD during growth on the development of photoautotrophy and the extent of photoinhibition were evaluated. Plantlets were grown under low, intermediate, and high (50, 100, and 300 μmol m-2 s-1) PPFD, and at 3 different sucrose concentrations (0.5, 1.5, and 3.0%, w/v) in the medium, during shoot multiplication. During root induction the same growth conditions were assayed except for the high PPFD. The development of photoautotrophy was assessed via the difference between the stable carbon isotope composition of sucrose used as heterotrophic carbon source and that of leaflets grown in vitro. Plantlets from root induction showed more developed photoautotrophy than those from shoot multiplication. For both stages the low-sucrose medium stimulated the photoautotrophy of plantlets in vitro. In addition, intermediate PPFD induced photoautotrophy during shoot multiplication. For plantlets of both culture stages at the lowest PPFD no photoinhibition occurred irrespective of the sucrose concentration in media. However, during the shoot multiplication stage chlorophyll fluorescence measurements showed a decrease in Fv/Fm and in t1/2 as growing PPFD increased, indicating photoinhibitory damage. The decline of Fv/Fm was caused mostly by an increase in Fo, indicating the inactivation of PSII reaction centers. However plantlets growing under low sucrose showed reduced susceptibility to photoinhibition. During root induction, only plantlets cultured with high sucrose showed a decrease in Fv/Fm as PPFD increased, although t1/2 remained unchanged. In this case, the decline of Fv/Fm was mostly due to a decrease in Fm, which indicates increased photoprotection rather than occurrence of photodamage. Therefore, growth in low-sucrose media had a protective effect on the resistance of PSII to light stress. In addition, plantlets were more resistant to photoinhibition during root induction than during shoot multiplication. Results suggest that increased photoautotrophy of plantlets reduces susceptibility to photoinhibition during gardenia culture in vitro.

The effect of different closure types, light, and sucrose concentrations on carbon isotope composition and growth ofGardenia jasminoides plantlets during micropropagation and subsequent acclimationex vitro

The growth ofGardenia jasminoides Ellis plantlets and the development of photoautotrophy during two successive culture stages (shoot multiplication and root induction)in vitro was analyzed. We examined the effects of changes in growth conditions (type of tube closure, light, and sugar levels) on the development of photoautotrophy and growth during micropropagation and sought to establish whether they affected later acclimation to conditionsex vitro. During the two stagesin vitro, plantlets were grown in tubes under two different PPFD (50 and 110 µmol m−2 s−1), in media with three different sucrose concentrations (0, 1.5, and 3.0%, w/v) and with two different CO2 levels inside the tubes (controlled by either tightly closed caps or loosely sealed caps, and with an external CO2 concentration of 750 µmol mol−1). The development of photoautotrophy was assessed by determining the difference between the stable carbon isotope composition (δ13C) of sugar cane sucrose used as a heterotrophic carbon source and that of leaflets grownin vitro. Plantlets from the root-induction stage showed a more highly developed photoautotrophy than those from the shoot- multiplication stage. At both stages, utilization of closed caps was the treatment which most stimulated development of photoautotrophy in plantlets. Also, lowering PPFD or sucrose concentration induced a greater degree of photoautotrophic development, the strongest effect being observed in plantlets cultured inside loosely sealed tubes. During acclimationex vitro, plantlets taken from loosely sealed tubesin vitro performed better than those cultured inside tightly sealed tubes. The former, as well as recording a larger increase in fresh weight during this stage, also showed more negative δ13C in the newly developed leaves, which would seem to indicate a better water status during acclimation. Present results validate the usefulness of δ13C analysis of leaflets as a simple technique in assessing the development of photoautotrophy during culturein vitro. In addition, δ13C analysis can be extended to evaluate growth conditions during acclimation toex vitro conditions.

Effects of agar concentration and vessel closure on the organogenesis and hyperhydricity of adventitious carnation shoots

Carnation plantlets (Dianthus caryophyllus L.) cultured in vitro often develop morphological and physiological anomalies, a phenomenon called hyperhydricity, which impairs their survival ex vitro. When the agar concentration of the growth medium was increased (from 0 to 12 g dm−3), thereby reducing water availability, the hyperhydricity of those adventitious shoots regenerated from carnation petals decreased. This was accompanied by a progressive fall in the water content of shoots (94.9 to 91.4 %), fresh mass (from 57.2 to 1.8 mg), number of leaf parenchyma cell layers (from 9.3 to 7.7), and the size of these cells (from 968 to 254 µm2). However, the number of regenerated shoots also decreased (17.7 in 2 g dm−3 agar to 4.3 in 12 g dm−3). Similarly, in ventilated tubes, which exhibit a lower relative humidity than tightly closed tubes, shoot organogenesis diminished up to 28 %, in tandem with shoot water content. Thus, relative humidity and water availability in culture vessels do not only influence shoot hyperhydricity in carnations, but also greatly affect adventitious shoot organogenesis.

The rolC gene in carnation exhibits cytokinin- and auxin-like activities

The overexpression of the rolC gene of the Ri plasmid of Agrobacterium rhizogenes in transgenic plants alters their development. Few studies have been performed on adventitious shoot or root formation in rolC-transformed plants. In this study we evaluated a possible cytokinin-like and auxin-like effect on shoot and root regeneration from petals and leaves of four lines of rolC-transgenic carnation plants (Dianthus caryophyllus L. cv. White Sim). rolC was found to enhance shoot regeneration, by increasing either the number of shoots per regenerative explant, or the number of shoot-forming explants. Remarkable root regeneration, in a medium with only auxin, was obtained from rolC explants, due to the increased percentage of root-forming explants. Our results show that rolC exhibits both cytokinin-like and auxin-like activities in rolC-transgenic carnation tissues.

The Effect of In Vitro Culture Conditions on the Pattern of Photoinhibition during Acclimation of Gardenia Plantlets to Ex Vitro Conditions

We tested the effect of growing conditions during micropropagation on the fast kinetics of chlorophyll (Chl) fluorescence of Gardenia jasminoides Ellis plantlets during a 4-week acclimation to ex vitro. We studied whether photoautotrophic growing in vitro produced plantlets with less photoinhibition impairment during acclimation. Of the growing conditions stimulating photoautotrophy in vitro, only loose tube caps had a positive effect, whereas low sucrose or sucrose-free content in the medium and high PPFD showed a negative effect. Thus, plantlets cultured with 3 % (m/v) of sucrose were subsequently less photoinhibited throughout acclimation than those cultured with low sucrose (0.5 %) or sucrose-free media. Moreover, at the end of acclimation the former plantlets showed Fv/Fm and Fv/F0 ratios typical of unstressed ex vitro plants as well as a higher Chl content and ratio of Chls to carotenoids. Plantlets cultured at a photosynthetic photon fluence density (PPFD) of 50 µmol m−2 s−1 also showed a better performance at the end of acclimation than those cultured at a higher (110 µmol m−2 s−1) PPFD. Thus except in the case of loose-tube closure, gardenia plantlets cultured in vitro under conventional sucrose concentration and PPFD are the least photoinhibited during acclimation. Nevertheless, significant interactions between the in vitro growing factors were observed at the end of acclimation.

Selection of biological control agents against tomato Fusarium wilt and evaluation in greenhouse conditions of two selected agents in three growing media

Two biological control practices are the use of suppressive growing media and the application of biological control agents (BCAs). The goals of this study were: (i) to screen 584 potential BCAs obtained from Fusarium wilt (FW) suppressive growing media; (ii) to evaluate in greenhouse conditions selected BCAs in three growing media with different degrees of suppressiveness of tomato FW. Two isolates selected after screening were identified as Fusarium solani (305) and Streptomyces sp. (A19). Results showed that tomato FW was reduced and total production was improved when both BCAs were applied to a conducive medium (coir fiber). In highly suppressive growing medium (grape marc compost), A19 and 305 inoculations did not improve suppressiveness. In moderately suppressive growing medium (cork compost), only A19 improved this compost to natural grape marc compost suppressiveness level. Therefore, compost suppressiveness of tomato FW depended on the nature of the compost and on the isolates applied.

The Effect of Photoautotrophy on Photosynthesis and Photoinhibition of Gardenia Plantlets during Micropropagation

We studied the relationships between the degree of photoautotrophy, photosynthetic capacity, and extent of photoinhibition of Gardenia jasminoides Ellis plantlets in vitro. Two successive micropropagation stages (shoot multiplication and root induction), and three culture conditions [tube cap closure, photosynthetic photon flux density (PPFD), and sucrose concentration] which may influence the development of photoautotrophy in vitro were assayed. The ratios of variable chlorophyll fluorescence to either maximal (Fv/Fm) or ground (Fv/F0) values were low, irrespective of the culture stage or growing conditions. Incomplete development of the photosynthetic apparatus and permanent photoinhibition may be involved. However, Fv/Fm and Fv/F0 increased from shoot multiplication to root induction owing to a decrease in F0 and an increase in Fm. This suggests that photoinhibition decreases later during micropropagation, when the photoautotrophy of plantlets is more advanced. The low sucrose content and high PPFD increased the photoinhibition of plantlets, whereas growth in tubes with permeable caps showed the opposite effect. The only culture factor with a significant (positive) effect on maximum photosynthetic rate (Pmax) was PPFD. At shoot multiplication net photosynthetic rate (PN) was positively correlated with the half time of the increase from F0 to Fm (t1/2). Such association may be mainly due to a common response of both traits to higher PPFD in culture. Within each culture stage, no relationship was observed between PN and the degree of photoautotrophy, which was positively correlated with Fv/Fm and Fv/F0 during root induction. During shoot multiplication, these correlations were not significant, or were even negative. Hence during the last stage of micropropagation, plantlets with a higher degree of photoautotrophy are less photoinhibited, whereas they do not follow this pattern at the earlier stage.