Jose Javier Regalado

Polyploidization is a powerful tool in plant breeding that can induce desirable morphological and physiological modifications. This study aimed to establish an efficient in vitro protocol for inducing autotetraploid plants in cherimoya (Annona cherimola Mill. cv. Fino de Jete) using colchicine. Hypocotyl explants from seedlings germinated in vitro were treated with different colchicine concentrations (0.01–0.2%) for 24 and 48 h, and the effects on shoot regeneration and ploidy level were evaluated by flow cytometry and chromosome counting. Regeneration and survival rates decreased with increasing colchicine concentration and exposure time. The most effective treatment for autotetraploid induction was 0.1% colchicine for 24 h, yielding a 10.5% polyploidization rate with 5.8% autotetraploids. Tetraploid shoots were successfully rooted (80%) and acclimatized (100%) under greenhouse conditions. Autotetraploid plants exhibited significantly larger and more rounded leaves, higher chlorophyll contents and an increased Chl a/Chl b ratio compared with diploids, indicating enhanced photosynthetic efficiency. The induction of stable autotetraploid lines in A. cherimola provides a reliable approach for generating novel genotypes with improved physiological traits and potential tolerance to abiotic stress. These results offer valuable material for future breeding programs aimed at developing new cherimoya rootstocks and cultivars with enhanced vigor and adaptability.

Ri plants characteristically exhibit a compact architectural growth habit, enhancing the economic value of ornamental varieties. These plants are obtained through the natural transformation of plant cells using wild strains of Rhizobium rhizogenes, which means they are not classified as genetically modified organisms (GMOs) in some countries, as USA or Japan. In the standard transformation protocol, rol genes are transferred to plant cells, resulting in the formation of hairy roots from which complete Ri plants can be regenerated. However, there are plant species, such as petunias, in which the regeneration of complete plantlets from hairy roots is not possible. In our research, we have developed a natural transformation protocol that enables the production of Ri petunias through direct organogenesis from leaves, bypassing the hairy root stage. The presence of rolB and rolC genes in these petunias was confirmed by PCR. The phenotypic characterization, both in vitro (laboratory-based experiments) and ex vitro (outside artificial tissue culture), showed that all Ri petunias had a more compact growth, with lower height due to reduced elongation of the internodes. Moreover, two of nine Ri petunia lines exhibited enhanced branching, resulting in an increased number of internodes, leaves, and flowers. These phenotypic changes, especially the increase in the number of flowers, represent an additional increase in their commercial value. However, there were no observable changes in the specific morphology of leaves or flowers of Ri petunias. The transformation protocol developed in this work could be adapted to other species or genotypes in which shoot regeneration from hairy roots is not possible.

The development of somatic embryogenesis in avocado (Persea americana Mill.) has been hampered by different chronic problems. One such problem is the low level of induction of white-opaque somatic embryos (WOSEs) during the process of obtaining full avocado plants. We detected the induction of multiple WOSEs promoted after the placement of three or four small WOSEs over the embryogenic callus of Duke-7. Among the other possible chemical inductors of the Arabinogalactans (AGPs), we identified a family of extracellular plant proteoglycans implicated in many aspects of the in vitro induction of somatic embryos (SE). We extracted AGPs directly from embryogenic cultures of avocado. When the induction/proliferation medium of embryogenic avocado calli (MS-0.1 mg L⁻¹ Picloram) was supplemented with 1–2 mg L⁻¹ AGP, the induction rate of good-quality WOSEs from the embryogenic callus increased significantly (more than ten times that of the control without AGP) and this effect persisted for at least five subcultures after the initial treatment with AGP. AGP also modified the texture and quality of the callus. The effect of AGP extends to other cultivars and proliferation media. Our objectives were to improve the induction of WOSEs and study the effect of AGP in the somatic embryogenesis of avocado.

In this article, we present proliferation data from 10 years of the continuous in vitro incubation of cv. Solo papaya shoots and propose a reliable method for the long-term micropropagation of papaya, using microshoots developed from the axillary buds of papaya shoots as primary explants. Three different media were assayed. The proliferation medium (PPRM) allowed us to maintain papaya shoots under continuous proliferation for 20 years, maintaining consistent behavior. Most of the shoots developed in the PPRM rooted during the incubation and then acclimated easily, maintaining the ploidy and morphological characteristics of the parental plants, and flowering and setting fruits normally. The PPRM medium consisted of MS medium supplemented with naphthalene acetic acid (NAA) (0.1 mg L⁻¹), benzyladenine (BA) (0.5 mg L⁻¹), gibberellic acid (GA₃) (0.5 mg L⁻¹), and adenine hemisulphate (40 mg L⁻¹). The average multiplication rate was higher than 20 shoots per explant during the long-term assay. The elongation medium (PELM) was designed to recover shoots with poor growth and allowed the development of high-quality shoots ready for rooting. It consisted of an MS basal medium supplemented with NAA (0.1 mg L⁻¹), kinetin (KIN) (0.5 mg L⁻¹), and GA₃ (1 mg L⁻¹). The rooting medium (PROM) was designed to induce high-quality roots from nonrooted shoots and consisted of a half-strength MS medium and indole-3-butiryc acid (IBA) (1 mg L⁻¹). On PROM, agar can be exchanged for expanded vermiculite. Acclimation took place inside an acclimatization tunnel under progressive hydric stress. After 4 weeks, the plant recovery rate was 90% for plants maintained under continuous proliferation for ten years. The main objective of this work was to provide a micropropagation method which would maintain healthy elite genotypes of papaya for long periods of time and produce a high number of good quality plants.

In vitro plant tissue culture and biotechnology used to assist and support the development of plant breeding when classical methods of propagation must be accelerated or it was necessary to overcome barriers inaccessible by classical approaches. In asparagus, to improve multiple breeding tasks, a high number of in vitro methods have been used, such as plant regeneration methods through organogenesis, embryogenesis, manipulation of ploidy, protoplast isolation, genetic manipulation (protoplast fusion, genetic transformation), embryo rescue and germplasm preservation (in vitro, in vitro slow growth, cryopreservation). Plant tissue culture methods can overcome multiple problems in asparagus breeding such as, barriers of self and cross-incompatibility between asparagus species through embryo rescue of interspecific hybrids and protoplast fusion or genetic transformation, introgression of new genes, clonal propagation of elite genotypes of asparagus, mass screening, and the generation of haploid and polyploid genotypes, among others, becoming the tool of choice for asparagus breeding programs. Some of these in vitro methods are still under development.

A mass micropropagation method was developed for Mammillaria vetula ssp. gracilis cv. Arizonica Snowcap, a cactus species with high ornamental value. A culture media based in MS medium (Murashige and Skoog, 1962) supplemented with 0.1 mg l^− 1 NAA + 0.3 mg l^− 1 KIN + 5.0 mg l^− 1 GA₃ + 30 g l^− 1 sucrose (MSM-1), was developed specifically for the establishment and multiplication of Mammillaria vetula cv. Snowcap offshoots, let us to produce a proliferation rate of 34.9 ± 5.9 new offshoots per explant. The non-rooted offshoots were rooted in MSM-2, consisting in MS salts supplemented with 0.1 mg l^− 1 NAA + 0.3 mg l^− 1 KIN plus 5 mg l^− 1 ancymidol and a lower dose (10 g l^− 1) of sucrose, with a rooting rate of 100%. The rate of acclimatization of the rooted offshoots was 100%. The full process of micropropagation from aseptic establishment in vitro to the end of plant acclimation takes approximately between 5 and 6 months and the plant production can be maintained continuously during all year.

We present the data on proliferation corresponding to 10 years of continuous incubation in vitro of papaya shoots, and propose a reliable method for long-term micropropagation for papaya, using two types of explants: Microshoots from somatic embryos, and from axillary buds of papaya. Three different media were assayed. The proliferation medium (PPRM) allowed to maintain papaya shoots under continuous proliferation during 20 years, maintaining a consistent behaviour. Most of the shoots developed in PPRM rooted during the incubation, and after acclimated easily, maintaining the morphological characteristics of the parental plants, flowering and setting fruits normally. The PPRM medium consist in MS medium supplemented with NAA (0.1 mg l^-1), BA (0.5 mg l^-1), GA₃ (0.5 mg l^-1) and Adenine sulphate (40 mg l^-1). The average multiplication rate was higher than 20 shoots per explant along the long-term assay. The elongation medium (PELM), was designed to recover shoots with a poor growth, and allowed the development of high quality shoots ready for rooting, and consist in a MS basal medium supplemented with NAA (0.1 mg l^-1), Kin (0.5 mg l^-1) and GA₃ (1 mg l^-1). The rooting medium (PROM) was designed to induce high quality roots from non-rooted shoots and consist in a half strength MS medium plus IBA (1mg l^-1). On PROM, agar can be exchanged for expanded vermiculite. Acclimation took place inside an acclimatization tunnel under progressive hydric stress. After 4 weeks, the plant recovery rate was 90% for plants maintained under continuous proliferation during ten years.

Aim of the study: We developed a faster micropropagation protocol specifically designed for Eucalyptus grandis. Eucalyptus breeding programs use micropropagation protocols to obtain high quality cloned seedlings, but current protocols are excessively time consuming.Area of the study: The protocol has been developed in Argentina, but it can be applied in anywhere.Materials and methods: We used nodal segments as initial explants to obtain micropropagated shoots, which were then simultaneously rooted ex vitro and acclimated in a hydroponic system. Nodal segments were cultured in a MS medium supplemented with 1 mg l-1 6-benzylaminopurine, 30 g l-1 sucrose, 1 g l-1 active charcoal and 8 g l-1 agar and incubated for four weeks at 25 ± 2°C under 16 h day photoperiod. Then, micropropagated shoots were exposed 15 seconds to 5000 ppm indol-butyric acid prior to being transferred to a hydroponic system, allowing simultaneous ex vitro rooting and acclimatization.Main results: 73 ± 9% of nodal segments grew to generate 1.73 ± 1.03 shoots per explant (length: 0.76 ± 0.44 cm). After four weeks in hydroponic system, 46 ± 4 % of micropropagated shoots developed roots, which represents an acceptable and intermediate rate of success, compared to the reported in vitro rooting rates.Research highlights: Our protocol allowed to obtain micropropagated seedlings in a total timespan of 8 weeks. Our results show that, by utilizing a hydroponic system, traditional protocols to micropropagate Eucalyptus can be substantially enhanced, allowing for improved production dynamics and potentially resulting in better organized seedling manufacturing facilities.Keywords: Woody plants; silviculture; nursery seedlings; rooting methods; hydroponics; acclimatization.