Tissue culture of oil palm : finding the balance between mass propagation and somaclonal variation

The oil palm (Elaeis guineensis Jacq.) is typically propagated in vitro by indirect somatic embryogenesis, a process in which somatic cells of an explant of choice are, via an intermediate phase of callus growth, induced to differentiate into somatic embryos. The architecture of the oil palm, lacking axillary shoots, does not allow for vegetative propagation. Therefore, somatic embryogenesis is the only alternative to seed propagation, which is hampered by long germination times and low germination rates, for the production of planting material. The current oil palm somatic embryogenesis procedure is associated with several difficulties, which are described in this review. The limited availability of explants, combined with low somatic embryo initiation and regeneration rates, necessitate the proliferation of embryogenic structures, increasing the risk for somaclonal variants such as the mantled phenotype. Several ways to improve the efficiency of the tissue culture method and to reduce the risk of somaclonal variation are described. These include the use of alternative explants and propagation techniques, the introduction of specific embryo maturation treatments and the detection of the mantled abnormality in an early stage. These methods have not yet been fully explored and provide interesting research field for the future. The development of an efficient oil palm micropropagation protocol is needed to keep up with the increasing demand for palm oil in a sustainable way. Mass production of selected, high-yielding palms by tissue culture could raise yields on existing plantations, reducing the need for further expansion of the cultivated area, which is often associated with negative environmental impacts

In vitro plantlet production of the endangered Pinguicula vulgaris

This study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free A1/4MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development

Hydrogen peroxide in micropropagation of Lilium: A comparison with a traditional methodology

The micropropagation of Lilium longiflorum requires adequate equipment which may not be afforded by small laboratories or producers. In this work we compared traditional methodology with a protocol that included easily available elements to sterilize materials and culture media, together with addition of hydrogen peroxide (H2O2) into the nutrient media as chemical sterilizer. A series of H2O2 concentrations (0.005, 0.010, 0.015 and 0.020% p/v) was used to control contamination during in vitro establishment and subsequent cultivation; the explant organogenic response was also examined and compared to the traditional micropropagation technique. The level of culture contamination was within acceptable limits in all treatments, though it was higher in the H2O2 treatments (40%) compared to the traditional methodology (20%). There were not significant differences in the number of bulblets per explant, and at the end of the multiplication phase, bulblets from 0.02% H2O2 treatment had greater biomass than from other treatments, indicating a beneficial effect. These bulblets also had a higher relative growth ratio with respect to the traditional method when cultivated for an additional period and showed the highest average bulblet fresh weight. It is expected that this higher bulblet mass would result in better performance during ex vitro cultivation.Fil: Curvetto, Nestor Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Marinangeli, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Mockel, Gabriela Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentin

Micropropagation of a recalcitrant pine (Pinus pinea L.): An overview of the effects of ectomycorrhizal inoculation

Stone pine (Pinus pinea L.) is an economically important forest species in some regions of Iberian Peninsula. Portugal and Spain have nearly 500,000 ha of stone pine stands, representing 85% of worldwide distribution. The main use of this species is for the production of seeds (pinion) for food industry. In addition to its enormous profitability as a producer of seeds, it has beneficial impact on soil protection, dunes fixation and is a pioneer species particularly for cork and holm oaks degraded ecosystems. Stone pine plantations are today a major source of income for forestry holdings. Investments have targeted breeding, reforestation, forest management and harvesting. The maternal inheritance of desirable characteristics such as cone weight, number of seeds per cone and seed length is considerably high in this species thus encouraging the selection of seeds from “plus” trees. The selected trees have been propagated by grafting and micropropagation. However, grafting generates high variability due to scion-rootstock interaction that varies production levels. The production of clonal plants from selected seeds by micropropagation techniques has advanced very slowly due to the recalcitrance of this species in tissue culture and particularly to adventitious rooting of microshoots. Due to the tremendous importance of developing a reproducible tissue culture method for clonal propagation, a study has been carried out for over a decade to enhance rooting and acclimation. During this period of time, continuous increments in the multiplication rate and rooting frequency were achieved by introducing variations in culture media composition and conditions. Auxins, carbohydrates, light quality and duration, temperature at different concentrations and levels as well as compounds such as coumarin; salicylic acid, polyamines, etc. were tested for induction and expression phases of adventitious rooting. Despite these efforts, microshoots regenerated through organogenesis from mature embryo cotyledons failed to root or to have sustained root growth. At this point, an in vitro co-culture technique of stone pine microshoots with ectomycorrhizal-fungi was introduced to overcome the adventitious root growth cessation in vitro and improve root development during acclimation phase. An overview of the results showing the positive effect of fungal inoculation in promoting root growth in vitro and on plantlet survival during acclimation will be presented. Preliminary results of biochemical signals between Pinus pinea/Pisolithus arhizus during early steps of in vitro culture detected by liquid chromatography-mass spectrometry that might be responsible for the positive effect on root growth will be also presented

Cloning crops in a CELSS via tissue culture: Prospects and problems

Micropropagation is currently used to clone fruits, nuts, and vegetables and involves controlling the outgrowth in vitro of basal, axillary, or adventitious buds. Following clonal multiplication, shoots are divided and rooted. This process has greatly reduced space and energy requirements in greenhouses and field nurseries and has increased multiplication rates by greater than 20 fold for some vegetatively propagated crops and breeding lines. Cereal and legume crops can also be cloned by tissue culture through somatic embryogenesis. Somatic embryos can be used to produce 'synthetic seed', which can tolerate desiccation and germinate upon rehydration. Synthetic seed of hybrid wheat, rice, soybean and other crops could be produced in a controlled ecological life support system. Thus, yield advantages of hybreds over inbreds (10 to 20 percent) could be exploited without having to provide additional facilities and energy for parental-line and hybrid seed nurseries

The rootstock clones in rubber tree: a new varietal type toward the rejuvenated bi-clone

Cloning rubber rootstocks by microcutting will remove the genetic variability of the seedling rootstocks; Moreover, it will open the way to the breeding of rootstock clones regarding main challenges as the growth-vigor of the trees, the water stress tolerance or the root diseases tolerance. In other respects, using rejuvenated selected clones for budding will counteract the weakening effect of ageing. The conventional rubber clones are two-parts trees built up by budding mature budwood of selected clones [trunk and canopy] on unselected and uncloned seedling rootstocks [root system]. Outputs from biotechnology on Hevea brasiliensis, make it possible now to create the "rejuvenated biclone" associating a new rootstock clone A with a rejuvenated budded clone B. Such "bi-clone" will allow to optimize each part of the tree and their specific interaction; thus making the propagation more reliable and with higher quality regarding growth and rubber yield. This paper gathers laboratory and field data which grounded this project. The cloning of young seedlings by microcutting was previously defined by the CIRAD team in France since 1988 and more than 50.000 vitroplants were produced then. Field development of such self-rooted vitroplants was analysed at the CNRA research centre, in Ivory Coast, during ten years for growth and rubber yield. Results give evidence for the conformity of the root system and for the vigorous development of these trees which could have been used as clonal rootstocks. Work in progress with Michelin shows that rejuvenated budded clones, issued from primary somatic embryogenesis, display higher growth speed than the conventional mature clones. Currently, the IBRIEC and IRRI teams, in Indonesia, are successfully implementing the same microcutting process. The first "bi-clones" have been built and planted at Bogor early in 2008, showing the feasibility of it. (Résumé d'auteur

Hand pollination to increase seed-set of red helleborine Cephalanthera rubra in the Chiltern Hills, Buckinghamshire, England

In 2007 and in previous years, as part of ongoing attempts to improve red helleborine Cephalanthera rubra seed-set, hand pollination of florets has been undertaken at a small colony of this species in Buckinghamshire, southern England. Natural pollination rarely occurs (one mature pod recorded in 10 years) at this site. In 2007, hand pollination resulted in the production of four seed pods, of which one withered and died. Upon ripening, the three remaining pods were removed for attempted micropropagation of the seeds. Ongoing conservation management has probably benefited the solitary bee Chelostoma campanularum which now appears fairly plentiful at the site, but despite the presence of this red helleborine flower visitor, natural pollination remains virtually unrecorded at this locality; field observations suggest that C.campanularum is in fact probably not large enough to act as an effective red helleborine pollinator as it can slip in and out of the flowers without removing the pollinia, unlike it larger relative C.fuliginosum, absent from the UK but which is a known pollinator of red helleborine in continental Europe