No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Histological and ecophysiological study of the changes occurring during the acclimatization of in vitro cultured roses
... A reduction of the inside container RH can therefore improve microplants' water retention, resulting in their better performance upon transplanting to ex vitro conditions. Through reducing the inside vessel RH by using a bottom cooling system, Capellades (1989) obtained more 'normally' structured stomates in cultured roses. Also, when RH is reduced by an increase in the vessel ventilation, leaf resistance to desiccation is increased and stomatal regulation improved (Smith et al., 1990, Ghashghaie et al., 1992. ...
... In most cases, this PPF already exceeds the plantlet light saturation point, however with increasing concentration of CO 2 inside the container the light saturation point of plantlets in vitro also rises (Niu et al., 1995;Nguyen et al., 1999b). The P-I curves of different plants in vitro obtained by Niu and Kozai (1997), Ohyama and Kozai (1998), and Kozai et al. (1998) as well as the Kautsky curves of tissue cultured roses recorded by Capellades (1989) indicate a similarity between the photosynthetic responses of plantlets in vitro and of those growing in the greenhouse or in the field. Once the light intensity exceeds a certain level, tissue cultured plants also suffer from photoinhibition (Nguyen et al., 1999b). ...
... Regarding the effects of sugar concentration, Tichá et al. (1998) revealed that 3% sucrose in the medium increased the photosynthetic potential of tobacco plantlets grown in vitro, while Capellades and co-workers (Capellades, 1989;Capellades et al., 1991) observed a decrease in the photosynthetic rate of in vitro cultured roses with increasing sucrose concentrations in the culture medium, and Vorackova et al. (1998) found that both too much or too little sucrose inhibit photosynthesis of wheat and rape plantlets in vitro and hindered their subsequent acclimatization in the greenhouse. The effects of sugars on the photosynthetic capacity of plantlets in vitro was also studied by Kozai and co-workers, who also noticed a discrepancy among the results obtained; ...
... Pigment synthesis and ribulosebisphosphate carboxylase (RubPcase) activity may be impaired in culture; photosynthetic pigment content was low-normal in cultured red raspberry [19] and cauliflower, which also had low RubPcase activity [33,35]. Some in vitro shoots and plant1ets had starch in their chloroplasts [13,51,54,72], while others had little or no starch, as in Leucaena leucocephala (Lam) De Wit. [13] and sweetgum [43,73]. ...
... [13] and sweetgum [43,73]. As sucrose concentration of the medium was augmented starch concentration in the chloroplasts increased [54]. Little starch was exported from the chloroplasts during the dark period and tended to accumulate. ...
... Little starch was exported from the chloroplasts during the dark period and tended to accumulate. In vitro leaves exhibited flattened, disorganized chloroplasts, in some cases with swollen thylakoids [43,54,73]. Accumulation of starch and disorganization of thylakoid structure was attributed to the relatively low light levels in culture [54] or altered light spectrum resulting from glass containers [ 43]. ...
The successful ex vitro acclimatization of micropropagated plants determines the quality of the end product and, in commercial production, the economic viability of the enterprise [6]. When shoots or plantlets are transplanted from culture to greenhouse conditions they may desiccate or wilt rapidly and can die as a result of the change in environment, unless substantial precautions are taken to accomodate them. In commercial micropropagation this step is often the limiting factor [53] and at best, is challenging, labour intensive and costly [6,7,10]. Methods which work for ex vitro establishment of one species are not necessarily satisfactory to ensure the survival of another [47].
... Some in vitro plants contain chloroplastic starch granules while others have little or none depending upon sucrose concentration (Dhawan and Bhojwani, 1987;Lee et al., 1985;Queralt, 1989). ...
... Low light levels during culture result in swollen thylakoids in chloroplasts of certain plant species (Queralt, 1989). Similarly, under the low light intensity characteristic of Stage II, swollen thylakoids appeared more frequently in sea oats genotypes than when they were cultured under higher light intensity during Stage III. ...
... Frequently, swollen thylakoids occur after starch granules have accumulated within the chloroplast (Queralt, 1989). When plants utilize their stored reserves, starch is used but the thylakoid membranes remain swollen. ...
ABSTRACT: Sea oats (Uniola paniculata L.), a dune species native to the southeastern U.S., is commonly used for dune stabilization and restoration in Florida. A micropropagation protocol was developed for sea oats commercial production. However, significant variability in shoot production, rooting and ex vitro survival among sea oats genotypes was observed. Understanding the morphological, anatomical, and physiological basis for differences among genotypes would allow development of efficient micropropagation protocols to produce diverse sea oats genotypes for dune stabilization. Growth and development of two sea oats genotypes with differing acclimatization capacities were compared at morphological and anatomical levels as a function of the duration of multiplication and rooting stages using light and electron microscopy. During in vitro and ex vitro stages, changes in photosynthetic rates were monitored, and carbohydrate levels and photosynthetic enzymes activities were measured. Additionally, sea oats photosynthetic capacity in vitro and ex vitro and during acclimatization was evaluated using in vitro photoautotrophic and photomixotrophic culture conditions. During the rooting stage, the easy-to-acclimatize genotype (EK 16-3) developed short but numerous roots and grass-like leaves with fully expanded blades. Conversely, the difficult-to-acclimatize genotype (EK 11-1) developed few long roots and short and thick lance-like leaves without expanded blades. During in vitro development of grass-like leaves, EK 16-3 plantlets exhibited increases in activities of the two photosynthetic enzymes phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), and chlorophyll and total soluble protein content. These increases were correlated with higher net photosynthetic rates in EK 16-3 than EK 11-1 plantlets after ex vitro transfer. Both genotypes accumulated carbohydrate and starch reserves during in vitro rooting, which were depleted during the transition from photomixotrophic to photoautotrophic mode of nutrition. However, due to the lack of production of photosynthetically competent leaves on EK 11-1 plants coupled with rapid exhaustion of carbohydrate reserves, acclimatization and survival ex vitro for this genotype were low. Conversely, rapid production of photosynthetically competent leaves resulted in 100% survival ex vitro in EK 16-3 plantlets. In conclusion, the major cause for low acclimatization was the in vitro formation of leaves with abnormal anatomy correlated with limited photosynthetic capacity. Text (Electronic thesis) in PDF format. System requirements: World Wide Web browser and PDF reader. Mode of access: World Wide Web. Title from title page of source document. Document formatted into pages; contains 173 pages. Thesis (Ph.D.)--University of Florida, 2005. Includes vita. Includes bibliographical references.
... 'Comet' plantlet growth and photosynthesis were not greatly affected by reducing in vitro RH to 90%. The photosynthetic CO2 uptake of rose plantlets grown at 75 and 85% RH was 65 and 9l% that of control plantlets grown at lN% RH (Capellades 1989). In vitro RH reduction caused reduced water loss from detached leaves (Fig. 2) Culture closures are necessary in conventional micropropagation systems to prevent contamination. ...
Micropropagated 'Festival' red raspberry (Rubus idaeus L.) shoots were rooted in specially constructed plexiglass chambers in ambient (340 ± 20 ppm) or enriched (1500 ±50 ppm) CO 2 conditions on a medium containing 0, 10, 20, or 30 g sucrose/liter. Plantlet growth and leaf 14 CO 2 fixation rates were evaluated before and 4 weeks after ex vitro transplantation. In vitro CO 2 enrichment promoted in vitro hardening; it increased root count and length, plantlet fresh weight, and photosynthetic capacity but did not affect other variables such as plantlet height, dry weight, or leaf count and area. No residual effects of in vitro CO 2 enrichment were observed on 4-week-old transplants. Sucrose in the medium promoted plantlet growth but depressed photosynthesis and reduced in vitro hardening. Photoautotrophic plantlets were obtained on sucrose-free rooting medium under ambient and enriched CO 2 conditions and they performed better ex vitro than mixotrophi plantlets grown with sucrose. Root hairs were more abundant and longer on root tips of photoautotrophic plantlets than on mixotrophic plantlets. The maximum CO 2 uptake rate of plantlet leaves was 52% that of greenhouse control plant leaves. This did not change in the persistent leaves up to 4 weeks after ex vitro transplantation. The photosyn-thetic ability of persistent and new leaves of 4-week-old ex vitro transplants related neither to in vitro CO 2 nor medium sucrose concentration. Consecutive new leaves of transplants took up more CO 2 than persistent leaves. The third new leaf of transplants had photosyn-thetic rates up to 90% that of greenhouse control plant leaves. These results indicate that in vitro CO 2 enrichment was beneficial to in vitro hardening and that sucrose may be reduced substantially or eliminated from red raspberry rooting medium when CO 2 enrichment is used.
... One hypothesis to explain these findings could be that these structures are hydathodes, which occur in many plant families, including the Rosaceae (Fahn, 1979). The significant differences in the ion content observed with an energy-dispersive X-ray analyzer (EDAX) between the stomata and the supposed hydathodes, and the very high frequency of hydathodes when compared to the stomata in the Stage IIIa-100% RH leaves, would support this hypothesis (Capellades, 1989). Hydathodes are thought to be involved in the regulation of water uptake, maintaining an upward flow of water and minerals when the transpiration stream is suppressed because of the humid conditions of the environment (Devlin and Witham, 1983;Juniper, 1960). ...
The surface structure of rose (Rosa multiflora L. cv . Montse) leaves formed in vitro under several environmental conditions (light level, relative humidity) and with various growth regulator treatments was studied by light and scanning electron microscopy. The epidermis from leaves developed in cultures grown under a higher light level and a lower relative humidity (80 μmol·s ⁻¹ ·m ⁻² and 75% RH) than the conditions used in commercial laboratories (25 μmol·s ⁻¹ ·m ⁻² and 100% RH) showed anatomical modifications of the epicuticular wax, stomata, and epidermal cells similar to that of greenhouse-grown plant leaves. These results indicate that cultured plantlets can resemble greenhouse-grown plants under modified environmental conditions. In vitro pretreatment will reduce transplant losses and shorten the acclimatization period in the greenhouse.
... To some extent, this strategy has been discounted as apt to heighten evapotranspiration losses in transplants. However, it seems to hold promise for some plants 4,5 . Wainwright and Scrace 6 found that maximum values for shoot height, fresh and dry weight of Potentilla fruticosa and Ficus lyrata were obtained in vivo when previously conditioned with 2 or 4% sucrose. ...
A substantial number of micropropagated plants do not survive transfer from in vitro conditions to greenhouse or field environment. The greenhouse and field have substantially lower relative humidity, higher light level and septic environment that are stressful to micropropagated plants compared to in vitro conditions. The benefit of any micropropagation system can, however, only be fully realized by the successful transfer of plantlets from tissue-culture vessels to the ambient conditions found ex vitro. Most species grown in vitro require an acclimatization process in order to ensure that sufficient number of plants survive and grow vigorously when transferred to soil. This article reviews current and developing methods for the acclimatization of micropropagated plantlets.
... To some extent, this strategy has been discounted as apt to heighten evapotranspiration losses in transplants. However, it seems to hold promise for some plants 4,5 . Wainwright and Scrace 6 found that maximum values for shoot height, fresh and dry weight of Potentilla fruticosa and Ficus lyrata were obtained in vivo when previously conditioned with 2 or 4% sucrose. ...
Thesis (Ph. D.)--Oxford Polytechnic, 1990.
The reasons for high production costs of micropropagated plantlets are analyzed. Problems requiring solutions by proper control of the in vitro environment for improving shoot/plantlet quality and cost effectiveness are revealed. Features of the in vitro environment and responses of shoots/plantlets cultured in vitro to the environment in conventional micropropagation are discussed in detail. The features and responses are interpreted from an environmental control point of view. Similarity of growth patterns between plantlets and seedlings under different in vitro environmental conditions is shown. Strategies for environmental control in micropropagation are discussed. The environmental effect on growth and development of shoots/plantlets cultured in vitro under controlled environments is shown. Advantages and disadvantages of heterotrophic, photomixotrophic and photoautotrophic micropropagation are discussed. Some novel environmental control systems are introduced.
When micropropagating plants it is not always required that the final product in vitro is autotrophic. On the contrary, micropropagated roses with the lowest net photosynthesis yielded the best survival rate in the greenhouse, provided the culture conditions in the last stage of micropropagation were adequate. Sugar content, light intensity and controlled water retention capacity in the head space of the container are the most important factors. They can contribute to yield plants with a normal anatomy and physiology.
Micropropagation is the true-to-type propagation of a selected genotype using in vitro culture techniques. Most often micropropagation is also associated with mass production at a competitive price.
Micropropagation has many advantages over conventional vegetative propagation and its commercial use in horticulture, agriculture and forestry is currently expanding worldwide. However, its widespread commercial use for major crops is still restricted as a result of its relatively high production costs. The high production costs in conventional micropropagation are mainly due to high labor costs, limited rates of growth during multiplication, poor rooting, and low survival rates of the plantlets during acclimatization.
Growth of plantlets of asparagus (Asparagus officinalis L.), raspberry (Rubus idaeus L.) and strawberry (Fragaria × ananassa Duch.), treated during the in vitro rooting stage under three photosynthetic photon flux densities (PPFD) (80, 125 and 250 μmol s−1 m−2) (17.5, 26.9 and 53.8 W m−2 (PAR), respectively) and three CO2 enrichment levels (CDE) (330, 1650 and 3000 μmol mol−1), was monitored during the acclimatization stage. For the three species, generic differences were observed in the plant response to treatments. A significant residual growth enhancement was caused by CDE. High PPFD in vitro increased the dry weight of strawberry and fresh weight of asparagus in acclimatization. Raspberry leaf dry weight was increased by 262% in acclimatization after in vitro treatment with high CDE. This enhanced the performance of micropropagated plantlets in acclimatization and reduced by 2 weeks the acclimatization period with raspberry. Our results suggest that in vitro leaves may be a source of nutritional reserves for leaves initiated ex vitro, but do not exclude a morphogenetic effect of CO2 during the in vitro rooting stage.
Introduction Environmental Factors In Vitro Challenges with Conventional Micropropagation Environmental Influences on Growth and Development In Vitro Environmental Control and Photoautotrophic Micropropagation Concluding Remarks Literature Cited
The photosynthetic characteristics of the Cymbidium plantlet in vitro cultured on Hyponex-agar medium with 2% sucrose were determined based on the measurements of CO2 concentration inside and outside of the culture vessels. The CO2 measurements were made with a gas chromatograph at a PPF (photosynthetic photon flux) of 35, 102 and 226 mol m-2 s-1, a chamber air temperature of 15, 25 and 35C and a CO2 concentration outside the vessel of approximately 350, 1100 and 3000 ppm. The net photosynthetic rates were determined on individual plantlets and were expressed on a dry weight basis. The steady-state CO2 concentration during the photoperiod was lower inside the vessel than outside the vessel at any PPF greater than 35 mol m-2s-1 and at any chamber air temperature. The photosynthetic response curves relating the net photosynthetic rate, PPF, and CO2 concentration in the vessel and chamber air temperature were similar to those for Cymbidium plants grown outside and other C3 plants grown outside under shade. The results indicate that CO2 enrichment for the plantlets in vitro at a relatively high PPF would promote photosynthesis and hence the growth of chlorophyllous shoots/plantlets in vitro and that the plantlets in vitro would make photoautotrophic growth under environmental conditions favorable for photosynthesis.
The type of gelling agent can influence to a large extent clonal propagation of Ranunculus asiaticus L. through axillary bud stimulation. In preliminary experiments we identified three agar brands (Oxoid=OX, Merck=MK, and
Roth=RT) which affect the availability of water and minerals to tissues in different ways. In the present study we investigate
the influence of these agars on the in vitro performance of Ranunculus. On OX and MK gels, growth was satisfactory, although the former had a more promotive effect on fresh and dry weight production
and on multiplication rate. Growth and development of shoots were poor on RT; shoot clumps showed symptoms of hyperhydricity,
with shoots having large dark-green malformed leaves and very elongated petioles. Epidermal strips of leaves from shoots grown
on the different gels and collected at the end of the culture period revealed differences according to the agar brand on which
the plantlets were cultured. Severe structural deformations of stomata could be detected on RT-grown shoots. The analyses
of the sugar content of the gel at the end of the culture period demonstrated that the explants grown on RT gels are strictly
dependent on the carbohydrates in the medium. On OX and MK gels the heterotrophic metabolism was lowered compared to RT-grown
explants. The agar brand on which plantlets were grown also influenced water retention capacity and water content of the shoots.
Experiments with tritiated water were undertaken to better understand the water fluxes inside the vessel and to investigate
the difference in “pump function” exerted by shoots cultured on the three gels. Shoots grown on OX media showed the best “pump
function,” which would account for the better results obtained on this gel. On the basis of the relationship between gel properties
and the growth of Ranunculus shoots, we conclude that the different physiological responses on the three gels are a reflection of different water and
nutrient availability in the different media.
Trials were carried out to test if the higher growth response shown by shoot clusters of Mr. S. 2/5, a clonal selection of Prunus cerasifera, submitted to short and frequent light-dark regimes could be related to the amount of sucrose added to growth medium.
The reduction of sucrose from 30 gl-1 (control) to 22.5 gl-1, 15 gl-1 and 7.5 gl-1 caused a progressive and remarkable inhibition of shoot tip growth. With 15 gl-1 the value of some growth parameters was reduced by more than half. Under 16-h daylength, the best sucrose concentration was 30 gl-1, while with 4-h light-2-h dark no statistical differences appeared between 30 gl-1 and 22.5 gl-1 sucrose. Compared to 16-h light-8-h dark, the 4-h light-2-h dark cycle at the three highest sucrose concentrations gave rise to higher values of fresh and dry weight as well as increasing the number of axillary shoots produced.
The increment in growth response induced by the shorter light-dark regime decreased with diminishing growth capacity in the cultures when sucrose concentration was lowered, but it was still appreciable even with 7.5 gl-1. Since the 4-h light-2-h dark cycle induced a favourable effect in culture growth with all sucrose concentrations, we conclude that the greater growth response observed with this light regime was not triggered by carbohydrate availability but by some other unknown factors.
To quantify the C-metabolism in micropropagated roses, two labeled substrates were tested:14C-sucrose and14CO2. In the14C-sucrose experiment four parameters were followed during the culture period: the decrease of the radioactivity of the culture
medium (by liquid scintillation counting), the dry matter accumulation, the radioactivity of the dry matter, and the radioactivity
of the captured CO2. These values were related to the total sucrose uptake, the total biomass increase, the biomass increase by sucrose incorporation,
and the sucrose respired as CO2. C-incorporation during photosynthesis was calculated as the difference between total biomass increase and biomass increase
by sucrose incorporation. The14CO2 experiment did not allow measurement of the net CO2 fixation in a direct way because the ratio of14CO2 over total CO2 in the headspace of the container could not be kept constant over the culture period. Respiration caused a build up of the
CO2 concentration in the headspace. Indirect calculation established a comparable photosynthetic portion of CO2 uptake for the tested variety.
Stomatal malfunctioning is one of the main reasons why plants desiccate when transferred from in vitro to greenhouse conditions. In order to overcome this problem in Rosa hybrida cv. Madame G. Delbard (R) Deladel, two techniques, bottom cooling and water vapour permeable lid, were used. Both methods aimed to increase the vapour pressure gradient between leaf and atmosphere and consequently to improve plant transpiration.
The results showed that these techniques increased leaf resistance to dehydration and improved stomatal regulation. Water relations of treated plantlets were similar to those generally observed in hardened plants: lower leaf water and osmotic potentials, and lower leaf water content than in the control ones. Osmotic adjustment occurred in treated plantlets maintaining turgor pressure. Each technique also induced some effects on growth during the rooting phase: with bottom cooling, roots were shorter, with permeable lids, apices were necrosed.
These results are discussed in terms of physiological causes and in terms of effect during the following acclimatization.
Sugar in the medium is considered to be an essential cause for the high production costs of plantlets in conventional, heterotrophic
micropropagation. Chlorophyllous explants, shoots, and plantlets in vitro have high photosynthetic ability to develop photoautotrophy,
but their photosynthetic activity is restricted largely by the low CO2 concentration in the vessel during the photoperiod and in part by the presence of sugar in the medium. The growth of plantlets
in vitro is often greater under photoautotrophic conditions than under heterotrophic conditions, provided that the in vitro
environment is properly controlled for promoting photosynthesis. The advantages and disadvantages of photoautotrophic micropropagation
are discussed.
Photosynthetic parameters have been investigated using complementary approaches throughout the in vitro development of coconut zygotic embryos into plantlets. Patterns of chlorophyll fluorescence were comparable in in vitro grown coconut plantlets (θMAXP=0.72 and θP=0.45) and in autotrophic adult palms (θMAXP=0.76 and θP=0.50). Chlorophyll content was lower in in vitro-cultured plantlets (0.92 mg/g fresh weight (FW)) than in autotrophic plants (2.43 mg/g FW). The photosynthetic rate (1.14 μmol CO2/m2 per s) of autotrophic palms was half that of in vitro grown plantlets, while transpiration rates were similar in both. Changes in the PEPC:RubisCO ratio during the development of in vitro grown plantlets (from 89.17 to 0.04 μmol CO2/h per mg total soluble protein (TSP)) reflected a transition from a heterotrophic towards a RubisCO-mediated mode of CO2 fixation. The RubisCO enzyme capacity (2.83 μmol CO2/h per mg TSP) and content (172.8 mg/g TSP) measured in in vitro-cultured plantlets were lower than those measured in autotrophic palms (6.60 μmol CO2/h per mg TSP and 217.6 mg/g TSP respectively). Transmission electronic microscopy (TEM) observations showed a complete ultrastructural organisation of chloroplasts in plantlets at the end of the in vitro culture process (6 weeks under light). All the studied parameters have shown that plantlets at the end of the in vitro culture process exhibit photosynthetic characteristics (θMAXP, θP, PEPC:RubisCO ratio and transpiration rates) similar to those of acclimatized plants. These results suggest an early establishment of a photosynthetic metabolism during the in vitro development of coconut plantlets. Nevertheless, RubisCO content and capacity together with chlorophyll content were found to remain lower in in vitro grown plantlets, which might explain the lower photosynthetic rates recorded, as compared to the autotrophic coconut palm.
Dans le cadre du programme de micropropagation du palmier à huile par embryogenèse somatique développé par l'ORSTOM et l'IRHO/CIRAD, nous avons étudié le passage en milieu liquide de différents stades de culture. Des suspensions embryogènes, appartenant à 10 clones différents, ont été établies à partir d'amas friables de nodules embryogènes localisés sur certains cals entretenus en milieu gélosé. Sur le plan histocytologique, l'origine unicellulaire des nodules qui génèrent la suspension est discutée ; le mode de prolifération de la suspension par fragmentation des massifs méristématiques qui la composent est décrit. Les conditions assurant une croissance pondérale optimale des cultures ont été recherchées. L'obtention d'embryons isolés et relativement synchrones est effective. PLus de cent plantes obtenues à partir de suspension ont déjà été transférées #ex vitro$ en Côte d'Ivoire. En vue de réaliser à terme l'ensemble des étapes d'expression de l'embryogenèse somatique en milieu liquide, une étude comparative du développement caulinaire des massifs d'embryons issus de la collection du laboratoire a été effectuée. L'établissement de suspensions embryogènes et la production d'embryons isolés, au développement synchrone, ouvrent de nouvelles perspectives pour l'étude des différentes phases du phénomène d'embryogenèse somatique chez les palmacées, monocotylédones pérennes dont la culture #in vitro$ est réputée difficile. Des recherches sur la compréhension des phénomènes mis en jeu aux niveaux moléculaire et biochimique sont proposées. Sur le plan pratique, les résultats obtenus conduiront à un changement d'échelle, par passage en bioréacteur. (D'après résumé d'auteur)
ResearchGate has not been able to resolve any references for this publication.