The temporary immersion system bioreactor named GreenTray^® presents advantages over the existing ones. Firstly, there is no need to use forceps to move the shoots or plantlets in or out of the recipient, nor is the use of a scalpel necessary to divide them. Secondly, the basis of the shoot can remain in the GreenTray^® and can sprout again over several cycles of growth. These two characteristics reduce the costs in the process of micropropagation. In addition, shoot or plantlet development is enhanced by the aeration of the recipient, thus enriching the recipient’s atmospheric CO₂. This article describes and provides images and values of interest in (1) the micropropagation process of some commercial Prunus or Pyrus rootstocks; (2) the plantlet growth under autotrophic conditions; and (3) the inoculation of plants with pathogens that reproduce in vitro the symptoms observed in vivo, using the GreenTray^® bioreactor.

The major factors affecting the in vitro immature embryo rescue efficiencies from Prunus persica or P. armeniaca accessions have been identified, along with improving the feasibility. Variations in the woody plant medium (WPM) were used depending on the embryo size. Embryos less than 5 mm long were cultured in WPM supplemented with 1 μM BAP and 1 μM GA₃, while embryos bigger than 5 mm long were cultured in hormone-free medium, with or without vermiculite. The environmental in vitro culture conditions consisted of three phases: a (I) stratification at 4 °C during a 3- to 5-month-long period in the dark, followed by (II) growth of germinated embryos at 14 °C for a 4-week-long period, with 12 h light a day, which favors plantlet development, and finally, (III) growth at 24 °C, with 16 h light a day, until the plantlets were acclimatized in the greenhouse. The germination of smaller embryos, at the end of phase I, ranged from 82.2% to 22.1% for apricots and flat peaches, respectively, whereas for bigger embryos, the germination varied from 97.3% to 53.2% for the same species. The embryo germination for peaches and nectarines ranged from 40.1% to 30.3% for smaller embryos, and from 91.9% to 63.0% for bigger embryos. Endo- and epiphytic contamination, affecting from 7.4% to 52.9% of cultured embryos, depending on the fruit type and conservation conditions, and the capacity to acclimate to soil conditions, ranging from 50.4% to 93.2%, were the two most important factors influencing the protocol’s efficiency and feasibility. Considering the overall efficiencies, expressed as hardened plants transferred to field plots over clean uncontaminated embryo, the values ranged from 55.8% for nectarines, 54.0% for peaches, 45.6% for apricots, and 23.3% for flat fruits. The addition of vermiculite to the culture medium significantly improved the plantlet development, avoiding subculture to fresh medium when an extension of phase III was required before acclimatization. Compared to laboratory glassware, the use of food glass containers with air-permeable sealing film, along with vermiculite-containing medium, significantly reduced the costs when handling the large number of embryos required for breeding programs.

Rosa canina L. is a medicinal and nutritionally valuable species with increasing industrial demand, yet its conventional propagation is limited by low rooting capacity and high genetic heterogeneity. In this study, a complete and reproducible in vitro micropropagation protocol was established, from explant introduction to plantlet acclimatization. Axillary buds were disinfected and introduced into Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP). Shoot multiplication was achieved using sequential cytokinin treatments, and shoot elongation was promoted by adding liquid MS medium containing activated charcoal (AC). The highest and fastest root induction percentage (up to 75%) was obtained on WPM with 2 mg·L⁻¹ IBA and under a 16 h light/8 h dark photoperiod. Light promoted adventitious root formation depending on the nutrient formulation. Thereafter, shoots developed well-structured root systems in vitro, and plantlets fully survived to ex vitro acclimatization. This protocol provides an efficient platform for the large-scale propagation of R. canina and demonstrates that its auxin-driven adventitious rooting is strongly conditioned by the interaction between basal medium composition and photoperiod.

Rosa canina L. is a medicinal and nutritionally valuable species with increasing industrial demand, yet its conventional propagation is limited by low rooting capacity and high genetic heterogeneity. In this study, a complete and reproducible in vitro micropropagation protocol was established, from explant introduction to plantlet acclimatization. Axillary buds were disinfected and introduced into Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP). Shoot multiplication was achieved using sequential cytokinin treatments, and shoot elongation was promoted by adding liquid MS medium containing activated charcoal (AC). The highest and fastest root induction percentage (up to 75%) was obtained on WPM with 2 mg·L^-1 IBA and under 16 h light / 8 h dark photoperiod. Light promoted adventitious root formation depending on the nutrient formulation. Thereafter, shoots developed well-structured root systems in vitro, and plantlets fully survive to ex vitro acclimatization. This protocol provides an efficient platform for the large‑scale propagation of R. canina and offers new insights into the coordinated effects of mineral nutrition, hormones, and light conditions on adventitious rooting in woody species.

Major factors affecting in vitro immature embryo rescue efficiencies from Prunus persica or P. armeniaca accessions, have been identified along with improving its economic feasibility. Three culture media, based on Woody Plant Medium (WPM) have been used depending on the embryo size. Embryos less than 5-mm-long were cultured in WPM supplemented with 1 mM BAP and 1 mM GA3, while embryos bigger than 5-mm-long were cultured in hormone-free medium, with or without vermiculite. Environmental in vitro culture conditions consisted of three phases, a (I) stratification at 4 C during a 3 to 5-month-long period in the dark, followed by (II) growth of germinated embryos at 14 C for 4-week-long period, with 12h light a day, which favors plantlet development, and finally (III) growth at 24 C, with 16h light a day, until the plantlets were moved to the greenhouse for acclimatization. Germination of smaller embryos, at the end of the phase I, ranged from 82.2% to 22.1%, for apricot and flat peaches, respectively, whereas for bigger embryos the germination varied from 97.4% to 53.2% for the same type of fruits. Embryo germination for peaches and nectarines ranged from 40.1% to 30.3% for smaller embryos, and from 91.9% to 64.0% for bigger embryos. Endo- and exophytic contamination, affecting from 7.4% to 52.9% of cultured embryos depending on the fruit type and conservation conditions, and the capacity to acclimate to soil conditions, ranging from 50.4% to 93.2%, were the two most important factors influencing protocol’s efficiency and economic feasibility. Considering the overall efficiencies, expressed as hardened plants transferred to field plots over clean uncontaminated embryo, values ranged from 55.8% for nectarines, 54.0% for peaches, 45.6% for apricot, and 23.3% for flat fruits. Addition of vermiculite to the culture medium significantly improved plantlet development, avoiding subculture to fresh medium when an extension of the phase III was required before acclimatization. Compared to laboratory glassware, use of food glass containers with air-permeable sealing film, along with vermiculite-containing medium significantly reduced costs, when handling the large number of embryos required for breeding programs.

The temporary immersion system bioreactor named GreenTray®[2], [3]presents advantages over the existing ones. Firstly, there is no need to use forceps to move the shoots or plantlets in or out of the recipient, neither the use of scalpel to divide them. Secondly, the basis of the shoot can remain in the GreenTray® and can sprout again in several cycles of growth. These two characteristics reduce the costs in the process of micropropagation. Besides, shoots or plantlets development is enhanced by the aeration of the recipient, thus enriching with CO2 the recipient atmosphere. The chapter describes and provides images and values of interest in (1) the micropropagation process of some commercial Prunus or Pyrus rootstocks; (2) the plantlet growth under autotrophic conditions; and (2) the inoculation of plants with pathogens that reproduce in vitro the symptoms observed in vivo, using the GreenTray® bioreactor.