Claude Teisson

Comparison of methods of liquid medium culture for banana micropropagation : effects of temporary immersion of explants

AbstractFive different liquid medium culture methods for meristem propagation of bananas were investigated and compared with solid medium culture. Treatments studied were: gelled culture medium (treatment 1); liquid medium with immersion of the plants (treatment 2); liquid medium with cellulose culture support (treatment 3); liquid medium with partial immersion of the plants (treatment 4); liquid medium aerated by bubbling (treatment 5); liquid medium with temporary immersion of the explants for 20 min every 2h (treatment 6). After 20 days of culture, three culture groups with statistically different multiplication rates were observed:-shoots in simple liquid medium and those on cellulose substrate proliferated little or not at all,-shoots on gelled medium, those subjected to partial immersion and those in aerated medium displayed multiplication rates of 2.2 to 3.1, and-the highest multiplication rate (>5) was observed in explants subjected to temporary immersion in the medium. Two groups of treatments differed in the accumulation of dry matter: the smallest weight (around 0.5 g) was observed in treatment 1, 2, 3 and 4, and accumulation was 2 to 5 times greater in the explants in aerated liquid medium and those subjected to temporary immersion. The highest multiplication rates and weight gains were observed in aerated treatments (treatments 4 and 5). Shoots in liquid medium continuously aerated by bubbling displayed hyperhydricity of the outer leaf sheaths. This was not observed with temporary immersion of explants.

Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa spp.)

SummarySomatic embryogenesis and plant regeneration of banana and plantain cultivars (Musa spp.) were obtained by culturing young male flowers. Multiplication and maintenance of embryogenic cultures were achieved by culturing somatic embryos in a temporary immersion system (SIT). A multiplication rate of 40 allowed us to obtain more than 6000 somatic embryos after 6 mo. of subculture. Plant recovery frequencies were 60 to 70%. This method was expanded to different banana and plantain genomic groups.

Sugarcane shoot formation in an improved temporary immersion system

A new protocol was established for sugarcane cv. C-1051-73 shoot formation in a temporary immersion system. The two-step protocol involves shoot formation in 50 ml of culture medium per explant and 1.0 mg l-1 paclobutrazol for 30 days followed by shoot elongation by exposure to 1.0 mg l-1 gibberellic acid for 15 days. The multiplication rate was doubled in comparison with the conventional micropropagation protocol (Jiménez et al., 1995) and the cost has been reduced by 46%. Three additional sugarcane varieties have been micropropagated according to this new protocol and results are comparable. Temporary immersion-derived plants have also been compared with conventionally propagated plants in sugarcane fields for more than 9 months and their agricultural indicators of performance are similar.

Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique

SummaryA culture procedure using temporary immersion in a liquid medium was tested for somatic embryogenesis of Hevea brasiliensis (Müll. Arg.). Embryogenic callus was placed under regeneration conditions, either on a gelled medium (Phytagel, Sigma, St. Louis, MO) or in a container designed for temporary immersion. The latter technique has some advantages over the use of a gelled medium during both the early steps of somatic embryogenesis, i.e., embryo development, and later on, i.e., during maturation, desiccation and germination. Somatic embryo production in a liquid medium was three to four times greater than on a semi-solid medium: 400 embryos/g fresh weight under the best embryogenesis induction conditions. Somatic embryogenesis had to be initiated on a gelled medium before the embryogenic callus was transferred to temporary immersion, and the amounts of 3,4- dichlorophenoxyacetic acid and N6-benzyladenine had to be reduced. Temporary immersion resulted in substantially more consistent, synchronized somatic embryo development, reducing the number of abnormal embryos by half and stimulating germination. All of the late events could be carried out in the temporary immersion container. Effective drying conditions were achieved after 12 wk without immersion and without selection of the embryos. Temporary immersion during germination greatly stimulated root development (+60%) and epicotyl emergency (+35%), combined with increased synchronization and a substantially reduced workload.

Somatic embryogenesis in plantain banana

SummaryA cell suspension of French Sombre plantain banana (Musa spp. AAB genome) was initiated from callus obtained from young male flowers. Histological examination enabled us to describe and follow the evolution of the suspension consisting of: embryogenic aggregates, proembryos, nodules, and isolated cells. It demonstrated the unicellular origin of somatic embryos, either during maintenance of the suspension or after plating on a semisolid medium. The cells from which the embryos originated had no starch but only protein reserves. Plating 1 ml of packed cells from the suspension led to the formation of 105 embryos of which 10 to 40% could be converted into plantlets.

Somatic embryogenesis and plants from immature zygotic embryos of the species Musa acuminata and Musa balbisiana.

Callus was obtained from immature zygotic embryos of semminiferous species (diploids) of Musa sp. using a medium derived from that of Murashige and Skoog. Picloram (7.5 μM) was added and the medium was solidified with gelrite (2 gl−1). Differentiation of the first somatic embryos occurred after transfer of the callus in the presence of 7.5 μM picloram or 5.3 μM NAA. Somatic embryos germinated on the medium supplemented with 5.3 μM NAA. Serial sections of zygotic and somatic embryos showed perfect homology in their structure (epidermis, cotyledonary slit, shoot apex and 3 root primordia). Embryonic callus was characterised by a large quantity of protein storage in the cytoplasm.

Improvement of Citrus somatic embryo development by temporary immersion

Liquid medium improves and facilitates somatic embryo development from Citrus deliciosa Ten. suspension cultures. Three different culture conditions were compared to determine a means of overcoming poor somatic embryo development. Somatic embryos derived from suspension cultures were plated on solid medium, maintained in suspension culture or temporarily immersed. About 60% of somatic embryos plated on solid medium developed to the cotyledonary stage, but were hyperhydric. Continuous growth in suspension culture at 100 rpm hindered cotyledon and protoderm formation, and somatic embryos were unable to develop beyond the globular stage. Temporary immersion promoted somatic embryo development, i.e. 66% of the somatic embryos produced were cotyledonary, and were morphologically similar to nucellar embryos. This latter culture system also improved regeneration synchronization by hampering secondary embryogenesis at the onset of germination. Irrespective of the culture system used, most cotyledonary somatic embryos studied had no caulinary meristem or starch and protein reserves, thus explaining the low germination rates obtained.

Somatic embryogenesis and plant regeneration through cell suspensions inMusa acuminata

SummaryCell suspensions ofMusa acuminata sspburmannicoides andMusa acuminata sspmalaccensis were obtained by culturing embryogenic callus initiated from immature zygotic embryos in liquid medium. Plant regeneration was then achieved through somatic embryogenesis. Germination of these embryos occurred in a modified MS medium containing auxin and cytokinin. Plant recovery frequencies were 20 to 36%. This method may allow a better utilization of biotechnologies in genetic improvement of theMusa diploid species, essential for banana and plantain breeding.

Physiological effects of temporary immersion on Hevea brasiliensis callus

In vitro culture by temporary immersion generates potentially stressful conditions for explants that may differ from those associated with classic methods. In order to evaluate the effects of these conditions on physiological changes in explants, different parameters of metabolic activity were investigated for a friable embryogenic callus of Hevea brasiliensis (Müll. Arg.), in response to 1 min, and 1, 12 and 24 h per day of immersion, using semi-solid and agitated liquid media as controls. The relative growth rate of the callus was not significantly different for the 1 min immersion treatment and the controls, but it decreased by about 60% for the 1-, 12- and 24-h immersion treatments. During the immersed stage, the rate of respiration of the callus was comparable for all the treatments. However, during the emersed stage, the respiration rate increased by 140 and 164% for the 12- and 24-h immersion treatments, respectively. Meanwhile, the total adenylate nucleotide concentration and the ratio of ATP/ADP remained almost constant, or even decreased. The adenylate energy charge was comparable for all the treatments, averaging 0.88. The superoxide dismutase activity and the lipid peroxidation increased with the immersion duration, and were significantly higher for the 12- and 24-h immersion treatments than for controls. However, after 24 h in emersed stage, there was no lipid peroxidation, regardless of previous immersion duration. It appears from these results that the immersed stage induced a substantial oxidative stress, which was not associated with the callus immersion per se.

Somaclonal variation rate evolution in plant tissue culture: Contribution to understanding through a statistical approach

SummaryIn order to better understand somaclonal variant rate evolution in plant tissue culture, a statistical approach has been adopted. According to this approach, the variant percentage could be calculated by: %V=[1−(1−p)n]×100, where %V is the percentage of variant, p the probability of variation and n the number of multiplication cycles. A numerical estimation was performed to characterize the variance of this function. It has been demonstrated that a wide scale of variance is associated with ‘%V’, due to the occurrence of variations after a variable number of multiplication cycles in the different lines of culture. Two main conclusions can be drawn from this model: (1) a variant rate increase can be expected as an exponential function of the number of multiplication cycles; (2) after a given number of multiplication cycles, variable off-types percentages can be expected. Due to the complexity of biological systems, this statistical approach could obviously not be applied directly for the calculation and forecasting of variant rates in tissue culture. However, this approach results in a better understanding of two apparently confusing experimental features often reported in tissue culture: the increase of the variant rate as a function of the length of the culture period on the one hand, and, on the other hand, the observations of different variant rates among lines cultured for the same lengths of time under strietly identical culture conditions. This approach also underlined that the comparison of somaclonal variant percentage between batches of plants from different in vitro treatments could be, in some cases, insufficient for ascertaining a difference of variability generated by tissue culture.