Alvaro Standardi

Encapsulation of micropropagated buds of six woody species

Regrowth after encapsulation in a sodium alginate matrix of micropropagated buds from six different in vitro proliferated woody species was evaluated. Actinidia deliciosa Liang & Ferguson (kiwifruit), Betula pendula Roth (birch), Crataegus oxyacantha L. (hawthorn), Malus spp. (apple), Rubus spp. (blackberry) and Rubus idaeus L. (raspberry) propagated in vitro were used as bud sources. Encapsulation with sodium alginate and subsequent regrowth on nutrient rich medium was compared to encapsulation with nutrient-enriched alginate capsules followed by regrowth on nutrientless medium. Apical and sub-apical buds of Malus (rootstock ‘M. 27’ and cultivar ‘Starkspur Red’) were also compared for encapsulation and regrowth ability. All species showed a regrowth after encapsulation, but only if cultured on enriched media. ‘M.27’ apical and sub-apical buds showed different regrowth ability after encapsulation with sodium alginate. Applicability of encapsulation of single micropropagated tree buds is discussed.

Review: role of carbon sources for in vitro plant growth and development

In vitro plant cells, tissues and organ cultures are not fully autotrophic establishing a need for carbohydrates in culture media to maintain the osmotic potential, as well as to serve as energy and carbon sources for developmental processes including shoot proliferation, root induction as well as emission, embryogenesis and organogenesis, which are highly energy demanding developmental processes in plant biology. A variety of carbon sources (both reducing and non-reducing) are used in culture media depending upon genotypes and specific stages of growth. However, sucrose is most widely used as a major transport-sugar in the phloem sap of many plants. In micropropagation systems, morphogenetic potential of plant tissues can greatly be manipulated by varying type and concentration of carbon sources. The present article reviews the past and current findings on carbon sources and their sustainable utilization for in vitro plant tissue culture to achieve better growth rate and development.

Effect of different treatments on the conversion of 'Hayward' kiwifruit synthetic seeds to whole plants following encapsulation of in vitro-derived buds.

Abstract Encapsulated buds excised from in vitro proliferated shoots of the ‘Hayward’ kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson) can be used for non‐embryogenic synthetic seed production. Three experiments were carried out to evaluate the aptitude of apical and axillary buds (microcuttings) towards encapsulation and synthetic seed productionJuland to find the treatments able to induce conversion of the synthetic seeds to whole plantlets. ‘Hayward’ proliferating shoots are useful sources of microcuttings for encapsulation and synseed production, since a proliferation protocol is already available. Encapsulation, although considered necessary, depressed microcuttings’ vigour and vegetative activity. Cold treatments provided to the in vitro proliferating mother shoots boosted bud vigour and subsequent conversion. Increase of concentration of sucrose in some steps of the protocol also enhanced conversion, which in some conditions reached a rate of 57.5%. Potential applications of encapsulation and the synthetic seed technology in kiwifruit germplasm exchange and commerce are also discussed.

Micropropagation and synthetic seed in M.26 apple rootstock (II): A new protocol for production of encapsulated differentiating propagules

Synthetic seed technology is an alternative to traditional micropropagation for production and delivery of cloned plantlets. Several aspects of the technique are still underdeveloped and hinder its commercial application. One of these aspects is the high hand labor requirements and costly procedures for the production of encapsulated explants. Direct organogenesis is an efficient shoot regeneration method and uninodal explants can be used for encapsulated synseed production. In this paper, we compare the performance of encapsulated organogenetic explants of M.26 apple rootstock that were prepared by hand and mechanical manipulation. Conversion of synseed into plantlets was 25% from encapsulated hand-cut uninodal microcuttings and 11% from machine-ground explants. The results demonstrate that synthetic seeds of M.26 apple rootstock can be produced through organogenesis from machine processed explants followed by root induction and encapsulation of differentiating propagules.

Micropropagation and preparation of synthetic seed in M.26 apple rootstock I: Attempts towards saving labor in the production of adventitious shoot tips suitable for encapsulation

In order to achieve a time and hand-labor saving procedure for the use of direct organogenesis in the production of shoot tips suitable for encapsulation, a set of experiments aimed at the gradual reduction in the accuracy of selection, hand and machine excision of the explants (leaves and whole shoot clusters) was attempted. Vegetative performance of the regenerated shoots was evaluated and encapsulation and subsequent regrowth of adventitious shoot tips was performed. The research provided useful information to devise a mechanical protocol for the production of synthetic seed through encapsulation of differentiating propagules (tissue fragments with shoot primordia) in woody species.

Effects of double encapsulation and coating on synthetic seed conversion in M.26 apple rootstock

Encapsulated vitro -derived apical buds of M.26 apple rootstock (Malus pumila Mill) can be employed for the formation of the synthetic seed. Satisfactory levels of conversion (plantlets from synthetic seed) can be achieved if there are adequate (i) rooting induction treatment, (ii) protocol of encapsulation, and (iii) nutritive and environmental conditions. For capsule manufacturing, sodium alginate is largely used; however, this is excessively permeable with loss of the nutritive substances (artificial endosperm) and/or dehydration risks during conservation and transport causing detrimental effects on the synthetic seed conversion and on the plantlets growth. In order to overcome these problems, two experiments were carried out comparing simple encapsulation in alginate with double encapsulation, and with encapsulation-coating procedures. The presence of a second layer of alginate (double encapsulation) and of a thin external coating layer over the alginate (encapsulation-coating) did not show any detrimental effects on viability, sprouting and regrowth of the encapsulated microcuttings. Satisfactory conversion (70%) was reached with the encapsulation-coating procedure, whereas the double and simple encapsulation converted less than 40% of the synthetic seed. The effect of the addition to the capsule of an anti-microbial substance (Plant Preservative Mixture - PPM#174;) was examined: it did not compromise the conversion of the encapsulated microcuttings sown in ex-vitro non-aseptic conditions.

Rooting Induction in Encapsulated Buds of M.26 Apple Rootstock for Synthetic Seed

In 1993, Redenbaugh reported that the term “synthetic seed” (synseed) should be referred only to an encapsulated somatic embryo. In more recent years, synthetic seeds were defined as artificially encapsulated somatic embryos, shoots or other vitro-derived tissues that can be used for sowing under in vitro or ex vitro conditions (Aitken-Christie et al., 1995). It seems logical, therefore, that the terminology that was initially set up for somatic embryo synthetic seeds be now used for any kind of encapsulated expiant. The term “conversion” was explained as growth and development of both shoot and root systems, with minimal swellings, callus production, etc. In one word, “conversion” expresses the production of a green plant with a normal phenotype from a synthetic seed (Redenbaugh et al., 1988; Redenbaugh, 1993). When the above described criteria are met, it seems justified that the term “conversion” can be reasonably used to describe the development of a full plantlet from any synseed, either made from a bipolar (somatic embryo) or an unipolar explant. Talking about synseeds, there are other terms that should instead be avoided, such as “germination”, which is proper of the real seed, or “plantlet formation”, which can generally be related to any way of producing a small rooted plant from micropropagated units (Debergh and Read, 1991). Furthermore, the term “regrowth” should be used to indicate any other vegetative act different from conversion that is performed by an encapsulated propagule after sowing, such as rooting or sprouting or emission of a leaf, etc.

Encapsulation of In Vitro-Derived Explants: An Innovative Tool for Nurseries

The encapsulation technology consists of the inclusion of some millimeter-long plant portions in a nutritive and protective matrix. This technology represents a further and promising tool for exchange of plant material between private and public plant tissue culture laboratories, for short- and medium-term storage of valuable plant material and for use of in vitro-derived or micropropagated propagules directly in farm or in nurseries. After encapsulation, transport, storage and sowing in aseptic conditions, the enclosed explants (capsules) may evolve in shoots (regrowth) and be employed for subsequent micropropagation or culture in vitro. When the encapsulated explant evolves in plantlet (conversion) in in vitro or in vivo conditions, the product of the encapsulation is defined as synthetic seed or artificial seed or synseed. The different evolution of the encapsulated plant material depends on tissue or plant material, genotype, nutritive and culture conditions, and treatments before or after encapsulation. In order to make economical the application of the encapsulation technology in the commercial nursery, research is looking for efficient automation or mechanization of the procedure and for preparation of the encapsulable explants.

Effects of tree shelters on young olive (Olea europaea) tree growth and physiology

Abstract A 3‐year study was conducted to evaluate the effects of tree shelters on young olive (Olea europaea) tree growth and physiology. The trial was carried out in the Apulia region in southern Italy using 1‐year‐old plants of the cultivar ‘Coratina’. Four different types of polypropylene shelters were tested: 75‐cm‐high brown; 90‐cm‐high brown; 75‐cm‐high green; 120‐cm‐high light‐green vented, with holes in their basal part. Both green shelters significantly increased the vertical growth of the trees, especially the 120‐cm light‐green vented ones. Shelters did not increase the amount of dry matter produced, but they modified its allocation within the tree, favouring vertical growth and the development of shoots in the apical parts of the trees.

Micropropagation of mother plants of lucerne (Medicago sativa L.) for somatic embryogenesis

SummaryA reliable and standard method was established for micropropagation of the A70-34 selected genotype of lecerne (Medicago sativa L., genotype A70-34), aimed at reducing contamination problems, seasonal and phenological influences on regeneration and phytosanitary problems of the mother plants, while maintaining the regenerative potential for somatic embryogenesis of the plant tissues. Mother plants were routinely maintained for several subcultures and somatic embryogenesis was regularly obtained from the subcultured explants. Proliferation, rooting and embryogenetic ability of plants cultured for 30 days was greater than those cultured for 20 days. The regenerative potential of tissues from different organs and of triturated and intact whole plants was also tested. Petioles were confirmed as the best source for embryogenesis as far as efficiency and repeatability were concerned, even though regeneration from other explant types was also achieved. Production of somatic embryos through mechanical trituration of the in vitro cultured plants was obtained; the regeneration ability of the triturated plants was greater than that observed in the intact plants.