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In vitro shoot proliferation of Tectona grandis L. (teak) using temporary immersion system (TIS) and different concentrations of BA is presented. Shoots grown on semi-solid MS medium supplemented with 2% (w/v) sucrose and 4.44 µM of BA were used as starting materials. In the TIS, three BA concentrations (2.22, 4.44 and 6.66 µM) and a cytokinin-free medium (CK-free medium) were assayed. A high average number of shoot was reached, applying 4.44 and 6.66 µM of BA (7.7 and 10.3 shoots/explant), respectively. The high BA concentrations decreased the accumulation of phenolic compounds and the deposition of lignin in the vascular cells of the teak shoots. The morphometric analysis by scanning microscopy revealed that the leaves of the shoots cultured in the TIS in Ck-free medium and with 2.22 and 4.44 µM of BA showed elliptical stomata; however, in the leaves developed with 6.66 µM of BA, the stomata were majorly ring-shaped, raised and wide open. Deformed stomata with broken epidermis of the guard cells, typical of hyperhydric leaves, were also observed. The survival percentages after ex vitro rooting on the IBA (492.1 µM) solution were higher in plantlets from CK-free medium and with 2.22 µM of BA, (96.7 and 91.7%, respectively) than those cultured on semi-solid medium (73%). The shoots from both TIS treatments developed a good root system. Plantlets from 6.66 µM of BA showed the lowest survival percentages (60%). A survival percentage was 100% two weeks after transplanting, and three months after ex vitro transfer, the plants were ready for field plantation. Here we present the first report of the successful propagation of teak by TIS.

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The positive effect of ventilation of the culture container on in vitro shoot proliferation and quality was already proven for different species. Hereafter we report on the evolution of the headspace during in vitro culture of plantain in a Temporary Immersion Bioreactor (TIB) on the one hand, and culture on semi-solid medium on the other hand. The CO 2 and C 2 H 4 concentration reached a maximum of 12% and 0.45 ll l )1 , respectively in the control treatment on semi-solid medium, compared to 5.7% CO 2 and 0.06 ll l )1 C 2 H 4 in TIB. The minimal O 2 -concentration on semi-solid medium was 15.1%, compared to 19.3% in TIB. The multiplication rate was best in TIB, 6.4 compared to 4.3 in semi-solid conditions, and this was also the case for shoot height (4.3 cm compared to 3.3 cm), and leaf number (2.6 compared to 1.6). Moreover shoots produced on semi-solid medium showed distorted leaves. A typical day-night pattern in CO 2 and O 2 concentration was observed in TIB, as well as on semi-solid medium; this is illustrative for the photosynthetic capacity of the plant material produced in both systems.

In traditional in vitro culture, the low CO 2 concentration inside the vessels restricts photosynthesis and necessitates the addition of sucrose to the culture medium as the main energy source, thus bringing about changes in the absorption of mineral elements from the culture medium. In this study, we investigated macronutrient absorption and sugar consumption in Actinidia deliciosa Chevalier Liang and Ferguson cv. Hayward (kiwi), cultured on medium supplemented with varying amounts of sucrose (0, 10, and 20 g l −1 ) under both heterotrophy and autotrophy, flushed with different concentrations of CO 2 (non-ventilation, 300, 600, and 2,000 μl l −1 ). In ventilated systems with 20 g l −1 of sucrose, sucrose absorption was less than under non-ventilation. The lowest rate of sucrose absorption was recorded when the explants were cultured on medium supplemented with 20 g l −1 of sucrose and flushed with 600 μl l −1 CO 2 . Absorption of NO 3 − , PO 4 3− , and Mg 2+ were high (maximum) at the end of the culture period (40 d) in explants flushed with 600 μl l −1 CO 2 that have been cultured 20 d in the presence of sucrose and then transferred to a sucrose-free medium. These autotrophic conditions promoted maximum plant growth in terms of both fresh and dry mass as well as the length and number of shoots and leaves. The study shows that to maintain an optimum regime of mineral nutrition for prolonged culture of kiwi in vitro, an increased amount of these three ions should be supplemented in Murashige and Skoog's medium.

The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades. The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA3 and GA4, salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA4, the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction.

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