Pierre Debergh

A scheme for commercial propagation of ornamental plants by tissue culture

Abstract The use of tissue culture for cloning ornamentals is expensive and presently limited to a certain number of species. However, the introduction of some additional new techniques may possibly reduce the cost and broaden the range of plants that can be propagated economically in vitro. In this report, a survey is given of the methodology followed in our laboratory and its adaptation to commercial practices. Stock plants are grown under controlled conditions prior to in vitro culture in order to obtain healthier explants and uniform response (stage 0). After the establishment of aseptic cultures (stage I), buds are propagated (stage II), and are then prepared to harvest uniform cuttings (stage IIIa). Those cuttings are rooted under in vivo conditions (stage IIIb).

Liquid medium additions to established tissue cultures to improve elongation and rooting in vivo

In order to save manual labour in plant tissue propagation, we tried adding liquid media to established, exhausted cultures, instead of transplanting the tissues to a fresh medium. This has been investigated for the elongation and for root induction stages with Cordyline, Philodendron, Magnolia and Spathiphyllum. The salt concentration and the application time are important factors, determining the success of this method. Charcoal added to the liquid stage IIIa medium eliminated the residual effects of BA applied during stage II.

Effects of sucrose on starch accumulation and rate of photosynthesis in Rosa cultured in vitro

Shootlets of Rosa multiflora L. cv. Montse were cultured in vitro with four different levels of sucrose (0, 1, 3 and 5%). Chloroplasts of shootlets grown in a medium without sucrose contained numerous, large plastoglobuli and were lacking in starch granules. The size and number of starch granules increased with the level of sucrose in the culture medium. Starch content in leaves of shootlets grown with 5% sucrose was higher (ca 1, 3%) than those grown with 3% (ca 0, 45%) and 1% sucrose (ca 0, 27%). Starch might be used by the in vitro shootlets during the acclimation period.

Photosynthesis and carbon metabolism in leaves formed prior and during ex vitro acclimatization of micropropagated plants

Abstract During the first days after transplanting micropropagated plants to greenhouse conditions, in vitro leaves are the only source to cover metabolic demands and to sustain plants’ adaptation and regrowth. However, the way these leaves act can differ depending on plant species and in vitro conditions. Here, we describe two main groups. In Calathea the in vitro leaves function as storage organs, from which the accumulated reserves (glucose, fructose) are consumed during the first days after transfer, until new leaves appear; these in vitro leaves never become fully autotrophic. On the contrary, in Spathiphyllum in vitro leaves are photosynthetically competent and normal source-sink relations are observed. Any surplus in photoassimilates at the end of the photoperiod is converted into starch. Three weeks after transfer, those leaves start to senesce and newly developed leaves become the main source of carbohydrates. In both plant species, higher photosynthetic activities are measured once new leaves are fully developed.

Evaluation of Analytical Techniques to Predict Viability after Cryopreservation

Preservation of plant germplasm is important to safeguard biodiversity and to store elite plants. Cryopreservation is one of the possible preservation techniques. Research for a cryopreservation protocol is often inefficient because of slow or poor regrowth of plant material. Therefore, at least one technique, that allows a quick and accurate prognosis of viability after cryopreservation, is required. We evaluated five techniques: electrolyte leakage, triphe-nyltetrazoliumchloride (TTC) staining (visual and spectrophotometrical analysis), malondialdehyde concentrations in plant tissue and a mathematical model that relates ‘water content’ to the weight of encapsulated plant material. Electrolyte leakage and TTC-staining (if visually analysed) are efficient to predict viability. Our mathematical model allows us to save time and plant material in order to develop an efficient encapsulation—dehydration protocol. All other techniques were rejected because of the high variability of the results. This is due to the variability of biochemical activity in plant tissue and the small amount of tissue used in the experiments.

The metabolism of benzyladenine in Spathiphyllum floribundum 'Schott Petite' in relation to acclimatisation problems.

In Spathiphyllum floribundum ‘Petite’, which was cultured on medium containing benzyladenine (BA), uptake of this cytokinin and its conversion to 9-ß-D-ribofuranosyl-benzyladenine (9R-BA) or 9-ß-glucopyranosyl-benzyladenine (9G-BA) was monitored. BA and extremely large quantities of 9G-BA were exclusively located in the basal part of the plant (callus and meristems). 9R-BA was found in the basal part, the petioles and the leaf blades. After an acclimatisation period of 9 weeks the plants still contained high levels of 9G-BA, but BA and 9R-BA could no longer be detected after one week. The possible role of BA and its derivatives on inhibition of root initiation or irreversible chloroplast deficiency is discussed.

Improvement of the maturation and germination of horse chestnut somatic embryos

Embryogenic tissues obtained from stamen filament cultures of horse chestnut (Aesculum hippocastanum L.) were cultured on maturation media supplemented with different combinations of abscisic acid, polyethylene glycol 4000, mannitol or activated charcoal. Somatic embryos were subjected to different desiccation procedures after a culture period on maturation media. After a slow desiccation, obtained by placing the somatic embryos in empty and non-sealed Petri dishes under the laminar air flow for 48 h, an increase in viability, shoot elongation and conversion was observed for the embryos previously cultured on medium enriched with ABA (80 μM) alone or plus PEG (50 g l−1).

Somatic embryogenesis and plant regeneration in Anthurium scherzerianum

Several types of explants, growth regulators, sugars and gelling agents were tested to induce somatic embryogenesis in Anthurium scherzerianum Schott. For induction, leaf pieces from micropropagated plants were found to be the best explant on a medium with 18 μM 2,4-dichlorophenoxyacetic acid and a high sucrose concentration (6), solidified with Gelrite. Somatic embryos converted to entire plants on a medium with 0.46 μM kinetin. Plantlets were transferred to greenhouse conditions for evaluation of plant uniformity.

The effect of light quality on the morphogenesis of in vitro cultures of Azorina vidalii (Wats.) Feer

In the present study, the possibility to improve the multiplication rate and quality of in vitro cultured plants of Azorina vidalii (Wats.) Feer was investigated. For these purposes the influence of high and low ratios of blue/red light (2.3 and 0.9, respectively) and red/far red light (1.1 and 0.6, respectively) on the development of shoot cultures of Azorina vidalii (Wats.) Feer was evaluated. Maximum plant length (87.9 ± 5.3 mm) (p ≤ 0.05) and internode length (6.6 ± 0.5 mm) (p ≤ 0.05) were obtained for the plants cultured under the low ratio of red/far red light. In contrast, when blue light was supplemented, plant height was reduced, due to shorter internodes. Highest number of axillary shoots per plant (2.4 ± 0.5) was obtained in the plants growing under a high level of red/far red light, while the lowest number (0.7 ± 0.2) was obtained in the plants cultured under the low level of red/far red light. Leaf area was increased in plants cultured under a low level of red/far red (1,522.5 ± 129.5 mm2) while blue/red light treatments inhibited leaf expansion. For chlorophyll a and total carotenoid contents, statistical differences (p ≤ 0.05) were observed between the plants exposed to the red/far red light treatments and the plants from the control.

PHOTOINHIBITION DURING ACCLIMATIZATION OF MICROPROPAGATED SPATHIPHYLLUM "PETITE" PLANTLETS

SummaryMicropropagatedSpathiphyllum “Petite” plantlets were acclimatized at low- or high-light intensities [photosynthetic photon flux density (PPFD) 100 or 300 µmol·m−2·s−1]. During the first days chlorophyll fluorescence measurements show a partial photoinhibition of the photosynthetic apparatus, expressed by a decrease of the variable over maximal fluorescence ratio (Fv/Fm). This inhibition of Fv/Fm was significantly higher for plants grown at high-light intensity, leading to a photooxidation of chlorophyll. Newly formed leaves were better adapted to the ex vitro climatic condition (as shown by the increase of the Fv/Fm ratio) and had a higher net photosynthesis compared with in vitro formed leaves. Nevertheless, plants grown at 300 µmol·m−2·s−1 were photoinhibited, compared with those at 100 µmol·m−2·s−1. A sudden exposure to high-light intensity of 1-, 10- or 25-d-old transplanted plants (shift in PPFD from 100 to 300 µmol·m−2·s−1) gave a linear decrease of Fv/Fm over a 12-h period, which was reflected in a 50% reduction of net photosynthesis. No significant interaction between day and hour was found, indicating high-light exposure causes the same photoinhibitory effect on in vitro and ex vitro formed leaves.