Plants

Research

16 pages, 1813 KB

Open AccessArticle

Incubation Time and Size Effects of Biodegradable Mulch Microplastics on Lettuce Plantlets In Vitro

by Mathilde Henrion, Lluis Martin-Closas, Iseult Lynch and Ana M. Pelacho

Plants 2026, 15(5), 849; https://doi.org/10.3390/plants15050849 - 9 Mar 2026

Viewed by 456

The use of biodegradable mulch films (BDM) in agriculture has raised concerns about the potential impact of the microplastics (MPs) they release over time, after the BDM’s useful life. The effects of BDM MPs have been explored through a diversity of assays, with [...] Read more.

The use of biodegradable mulch films (BDM) in agriculture has raised concerns about the potential impact of the microplastics (MPs) they release over time, after the BDM’s useful life. The effects of BDM MPs have been explored through a diversity of assays, with still poorly understood and frequently contrasting results. Furthermore, the impact on plants as the MPs evolve in size and as a function of residence time in the soil remains largely unexplored. Through a controlled in vitro lettuce culture, this study explores the effect of BDM MPs size, using fractions 5 to <0.2 mm and pre-incubation times of 0 to 8 weeks, on plant development. Short incubation times, of 1 and 2 weeks, and freshly adding the BDM MPs inhibited plantlet growth, with smaller MPs inducing stronger effects. In contrast, longer MPs incubation, of 8 weeks, promoted plantlet development, enhancing leaf and particularly root elongation while reducing lateral root branching. The effects on roots were more pronounced, as the MPs size decreased. Germination and photosynthetic pigments were unaffected by any treatment. Overall, BDM MPs’ impact on plants was mainly driven by particle size and incubation time in the medium prior to seeding, with adverse effects on plant development observed at short incubation times that were no longer present when incubation was extended. These findings highlight the need to unravel the dynamic and temporal nature of the BDM MPs’ interaction with plants. Full article

(This article belongs to the Special Issue Development and Application of In Vitro Culture Techniques in Plants)

► Show Figures

Figure 1

12 pages, 755 KB

Open AccessArticle

Genotyping-by-Sequencing Reveals Marker-Based Genome Stability in Tetraploid Clementines for Triploid Breeding

by Pablo Aleza, Andres Garcia-Lor, Pierre Mournet, Luis Navarro and Patrick Ollitrault

Plants 2026, 15(2), 336; https://doi.org/10.3390/plants15020336 - 22 Jan 2026

Viewed by 354

Abstract

Tetraploid non-apomictic citrus genotypes are key female parents for 4x × 2x hybridizations aimed at producing seedless triploid hybrids. However, the extent to which different tetraploidization methods affect genome integrity remains insufficiently characterized at a genome-wide scale. In this study, genotyping-by-sequencing (GBS) was [...] Read more.

Tetraploid non-apomictic citrus genotypes are key female parents for 4x × 2x hybridizations aimed at producing seedless triploid hybrids. However, the extent to which different tetraploidization methods affect genome integrity remains insufficiently characterized at a genome-wide scale. In this study, genotyping-by-sequencing (GBS) was used to evaluate marker-based genomic stability in ten tetraploid plants of ‘Clemenules’, ‘Fina’, and ‘Marisol’ clementines obtained via colchicine treatment, in vitro adventitious organogenesis, or somatic cybridization. Diploid parental plants, two haploid plants of ‘Clemenules’ and ‘Fina’ clementines, and one doubled haploid plant of ‘Clemenules’ clementine were included, being the haploid and double haploid essential to resolve allelic phases. After quality filtering, 3333 SNP (Single Nucleotide Polymorphism) markers distributed across the nine citrus chromosomes were identified and used to compare allele dosage patterns along the genome. Across all GBS-covered regions, no major marker-based genomic gains or losses were detected in any tetraploid plant. These results indicate that, at the resolution provided by GBS, all three tetraploidization methods largely preserve chromosome structure, supporting their suitability for citrus triploid breeding programs based on 4x × 2x sexual hybridizations. Full article

(This article belongs to the Special Issue Development and Application of In Vitro Culture Techniques in Plants)

► Show Figures

Figure 1

20 pages, 4538 KB

Open AccessArticle

Doubled Haploid Production in Cucurbita pepo L. Through Ovary Culture

by Ana García-Pérez, Malen Escánez, Sandra Gil, Alejandro Miralles-Rodríguez, Santiago Vilanova, Francisco Bermúdez and Edgar García-Fortea

Plants 2025, 14(24), 3733; https://doi.org/10.3390/plants14243733 - 8 Dec 2025

Viewed by 953

Abstract

Gynogenesis offers a promising route for doubled haploid (DH) production in Cucurbita, yet efficient protocols remain scarce. This study established a reproducible ovary culture system for Cucurbita pepo and evaluated zeatin riboside (ZR) as an alternative cytokinin. Ovaries collected at anthesis and [...] Read more.

Gynogenesis offers a promising route for doubled haploid (DH) production in Cucurbita, yet efficient protocols remain scarce. This study established a reproducible ovary culture system for Cucurbita pepo and evaluated zeatin riboside (ZR) as an alternative cytokinin. Ovaries collected at anthesis and one day before were cultured to screen nine media with different cytokinin–auxin combinations. Subsequently, four optimized ZR-based formulations were evaluated. Both floral stages showed morphogenic activity, but embryo formation occurred almost exclusively in pre-anthesis ovaries. Among ZR treatments, E6.1 (1 mg·L⁻¹ ZR + 3 mg·L⁻¹ NAA, 30 g·L⁻¹ sucrose) achieved the highest embryogenic output (approximately 97 embryos per 100 explants), while high-sucrose media (120 g·L⁻¹) induced abundant swollen ovules but poor conversion, suggesting that excessive osmotic pressure promotes morphogenesis but hampers embryogenic transition. In total, 415 embryos were obtained, and 52 regenerants were analyzed by flow cytometry, confirming haploid, diploid, and mixoploid plants and evidencing spontaneous chromosome doubling during in vitro development. A categorical A–D scoring system enabled early prediction of embryogenic potential. This represents the first successful application of ZR in cucurbit gynogenesis and highlights its value as a biologically compatible cytokinin for DH production. The findings open new avenues for testing ZR-based formulations in other Cucurbita species under different auxin and sucrose regimes. Full article

(This article belongs to the Special Issue Development and Application of In Vitro Culture Techniques in Plants)

► Show Figures

Figure 1

11 pages, 1796 KB

Open AccessArticle

Silicon Nanoparticles (SiNPs) Enhance Elongation and Rooting of In Vitro Shoots of Vanilla (Vanilla planifolia Andrews) During Micropropagation in RITA^® Bioreactors

by Marco A. Ramírez-Mosqueda

Plants 2025, 14(24), 3732; https://doi.org/10.3390/plants14243732 - 7 Dec 2025

Cited by 1 | Viewed by 775

Abstract

Vanilla (Vanilla planifolia Andrews) cultivation is globally relevant due to the extraction of vanillin from its cured fruits. However, the high demand for propagules for commercial plantations requires new propagation methodologies, including in vitro propagation. Currently, the use of biostimulants in plant [...] Read more.

Vanilla (Vanilla planifolia Andrews) cultivation is globally relevant due to the extraction of vanillin from its cured fruits. However, the high demand for propagules for commercial plantations requires new propagation methodologies, including in vitro propagation. Currently, the use of biostimulants in plant micropropagation protocols is being explored to increase the number of plants obtained and their vigor. Nanomaterials such as silicon dioxide nanoparticles (SiNPs) have shown a positive effect on plant growth and development. The objective of this study was to evaluate the effect of SiNPs on the micropropagation of V. planifolia in RITA^® bioreactors. In vitro plants were transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of SiNPs < 50 nm (0, 50, 100, and 150 mg L⁻¹) in RITA^® bioreactors. The obtained plants were then acclimatized in a greenhouse. The results indicated that 150 mg L⁻¹ of SiNPs produced the highest average shoot number, with 5.12 shoots per explant (5.48 cm in length), 9.50 leaves, and 5.00 roots per explant. The formation of an optimal root system in plants with SiNPs allowed for 98% survival. Results will enable more efficient in vitro propagation protocols through the obtainment of plants with greater length and a developed root system that facilitates ex vitro adaptation. Full article

(This article belongs to the Special Issue Development and Application of In Vitro Culture Techniques in Plants)

► Show Figures

Figure 1

22 pages, 5314 KB

Open AccessArticle

Micropropagation of Apple Cultivars ‘Golden Delicious’ and ‘Royal Gala’ in Bioreactors

by Simón Miranda, Mickael Malnoy, Anxela Aldrey, María José Cernadas, Conchi Sánchez, Bruce Christie and Nieves Vidal

Plants 2025, 14(17), 2740; https://doi.org/10.3390/plants14172740 - 2 Sep 2025

Cited by 3 | Viewed by 1512

Abstract

This study aimed to investigate culture conditions for the efficient micropropagation of apple cultivars ‘Golden Delicious’ and ‘Royal Gala’ in liquid medium by temporary immersion. RITA^® bioreactors were used for the multiplication stage whereas RITA^® or Plantform™ were used for the [...] Read more.

This study aimed to investigate culture conditions for the efficient micropropagation of apple cultivars ‘Golden Delicious’ and ‘Royal Gala’ in liquid medium by temporary immersion. RITA^® bioreactors were used for the multiplication stage whereas RITA^® or Plantform™ were used for the rooting stage. Murashige and Skoog media (MS) with N⁶-benzyladenine (BA) was used for shoot multiplication and indole-3-butyric acid (IBA) for root induction. During the multiplication phase, we evaluated the mineral medium, BA concentration, immersion frequency, silver nitrate and activated charcoal supplementation and the use of physical supports to hold explants in an upright position. The results demonstrated that longer incubation periods (10 weeks) were better than shorter periods (6 weeks) for decreasing hyperhydricity and increasing the multiplication coefficient (MC). For ‘Golden Delicious’, the highest MC were obtained either with explants placed directly on the bioreactor basket and immersed six times per day for 60 s in MS with 2.2 µM BA or explants placed between rockwool cubes cultivated with 4.4 µM BA (both yielding MC of 8.9 and 5–10% hyperhydricity). These results were superior to ‘Royal Gala’, which showed a MC of 7.3 and 23% of hyperhydricity when cultivated in MS with half nitrates, 1.55 µM BA and rockwool cubes. Both varieties rooted efficiently (96–100%), and resulting plantlets were successfully acclimated. This is the first report in the micropropagation of these two commercial fruiting cultivars in temporary immersion, demonstrating the potential of this technology to enhance large-scale plant production for the apple nursery industry. Full article

(This article belongs to the Special Issue Development and Application of In Vitro Culture Techniques in Plants)

► Show Figures

Figure 1

26 pages, 28140 KB

Open AccessArticle

Response to In Vitro Micropropagation of Plants with Different Degrees of Variegation of the Commercial Gymnocalycium cv. Fancy (Cactaceae)

by Carles Cortés-Olmos, Vladimir Marín Guerra-Sandoval, Carla Guijarro-Real, Benito Pineda, Ana Fita and Adrián Rodríguez-Burruezo

Plants 2025, 14(7), 1091; https://doi.org/10.3390/plants14071091 - 1 Apr 2025

Cited by 1 | Viewed by 2184

Abstract

This study aims to establish efficient in vitro propagation protocols for Gymnocalycium cv. Fancy, an ornamental cactus with variegated variants, by evaluating the effects of cytokinin type and explant source on the organogenic response. Plants with different degrees of variegation (0–100%) were classified [...] Read more.

This study aims to establish efficient in vitro propagation protocols for Gymnocalycium cv. Fancy, an ornamental cactus with variegated variants, by evaluating the effects of cytokinin type and explant source on the organogenic response. Plants with different degrees of variegation (0–100%) were classified by size to obtain different explant types (apices, central discs, epicotyls, and hypocotyls). The effects of 6-Benzylaminopurine (BAP, 8 µM), Kinetin (KIN, 4 µM), and Thidiazuron (TDZ, 1 µM) were assessed on shoot production, callus formation, and rhizogenesis. Additionally, we studied the relationship between initial plant variegation and the productivity of the variegated shoots. The best shoot production results were obtained for central discs treated with 1 µM TDZ. Furthermore, a correlation was observed between the activated areole type (green, mixed, or fully colored) and shoot color percentage, enabling precise explant selection. The appearance of differently colored shoots confirms the potential for selecting new lines from this cultivar too. These findings hold significant potential not only for the breeding and propagation of ornamental cacti but also for the cultivation of other edible cacti and their relatives. Full article

(This article belongs to the Special Issue Development and Application of In Vitro Culture Techniques in Plants)

► Show Figures

Figure 1

19 pages, 5097 KB

Open AccessArticle

Development of a Feasible and Efficient In Vitro Rescue Protocol for Immature Prunus spp. Embryos

by Maria Casanovas, Elisabet Claveria and Ramon Dolcet-Sanjuan

Plants 2024, 13(21), 2953; https://doi.org/10.3390/plants13212953 - 22 Oct 2024

Cited by 2 | Viewed by 1827

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Development and Application of In Vitro Culture Techniques in Plants)

► Show Figures

Figure 1

15 pages, 1906 KB

Open AccessEditor’s ChoiceArticle

Development, Chlorophyll Content, and Nutrient Accumulation in In Vitro Shoots of Melaleuca alternifolia under Light Wavelengths and 6-BAP

by Antony Cristhian Gonzales-Alvarado and Jean Carlos Cardoso

Plants 2024, 13(20), 2842; https://doi.org/10.3390/plants13202842 - 11 Oct 2024

Cited by 9 | Viewed by 3029

Abstract

In vitro cultivation of Melaleuca could contribute to the cloning of superior genotypes. Studies of factors affecting micropropagation are needed, such as the interaction with light-emitting diodes (LEDs) and plant growth regulators added to the culture media. This study aimed at better understanding [...] Read more.

In vitro cultivation of Melaleuca could contribute to the cloning of superior genotypes. Studies of factors affecting micropropagation are needed, such as the interaction with light-emitting diodes (LEDs) and plant growth regulators added to the culture media. This study aimed at better understanding the effects of spectra on the development and physiology of melaleuca cultivated in vitro, as well as the interaction of LEDs with the main cytokinin used in micropropagation, N6-Benzylaminopurine (6-BAP). 6-BAP, spectra, and their interaction had a significant effect on most of the variables analyzed, altering the in vitro development and chlorophyll concentrations in the plants, as well as changing different variables in the culture medium, such as pH, EC, and levels of Ca²⁺, Mg²⁺, and P, and nutrient accumulation in the shoots. The results demonstrate that the main effects of adding BAP to the in vitro cultivation of melaleuca are an increase in the number of shoots, which resulted in greater fresh and dry masses; a reduction in height and chlorophyll content; complete inhibition of adventitious rooting; higher consumption of Mg, and lower consumption of Ca and P from the culture medium; higher content of Fe, and lower content of P, S, Mn, Cu and B in the in vitro shoot tissues. Full article

(This article belongs to the Special Issue Development and Application of In Vitro Culture Techniques in Plants)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Development and Application of In Vitro Culture Techniques in Plants

Share This Special Issue

Special Issue Editors

Special Issue Information

Keywords

Benefits of Publishing in a Special Issue

Related Special Issue

Published Papers (8 papers)

Research

Further Information

Guidelines

MDPI Initiatives

Follow MDPI