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15 pages, 2091 KiB

Open AccessEditor’s ChoiceArticle

Application of Encapsulation Technology: In Vitro Screening of Two Ficus carica L. Genotypes under Different NaCl Concentrations

by Irene Granata, Luca Regni, Maurizio Micheli, Cristian Silvestri and Maria Antonietta Germanà

Horticulturae 2023, 9(12), 1344; https://doi.org/10.3390/horticulturae9121344 - 16 Dec 2023

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Abstract

Salinity stress represents an increasing issue for agriculture and has a great negative impact on plant growth and crop production. The selection of genotypes able to tolerate salt stress could be a suitable solution to overcome the problem. In this context, in vitro [...] Read more.

Salinity stress represents an increasing issue for agriculture and has a great negative impact on plant growth and crop production. The selection of genotypes able to tolerate salt stress could be a suitable solution to overcome the problem. In this context, in vitro cultures can represent a tool for identifying the NaCl tolerant genotypes and quickly producing large populations of them. The possibility of exerting selection for tolerance to NaCl by using encapsulation technology was investigated in two genotypes of fig: ‘Houmairi’ and ‘Palazzo’. The effects of five concentrations of NaCl (0, 50, 100, 150 and 200 mM) added to the artificial endosperm were tested on the conversion of synthetic seeds and on the growth of derived shoots/plantlets. Moreover, proline (Pro) and malondialdehyde (MDA), the enzymatic activities of catalase (CAT), guaiacol peroxidase (POD), and EL (Electrolytic Leakage), as well as the chlorophyll content, flavanols, anthocyanins, and Nitrogen Balance Index (NBI) were determined on shoots/plantlet. The obtained results clearly showed that ‘Houmairi’ and ‘Palazzo’ could tolerate salt stress, although a strong difference was found depending on each specific physiological pathway. Indeed, ‘Houmairi’ was revealed to be more tolerant than ‘Palazzo’, with different response mechanisms to salt stress. The use of encapsulated vitro-derived explants proved to be a useful method to validate the selection of genotypes tolerant to salinity stress. Further investigation in the field must validate and confirm the legitimacy of the approach. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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14 pages, 1944 KiB

Open AccessArticle

The Role of Silver Nanoparticles in Response of In Vitro Boysenberry Plants to Drought Stress

by Sevinç Şener and Hülya Sayğı

Horticulturae 2023, 9(11), 1177; https://doi.org/10.3390/horticulturae9111177 - 27 Oct 2023

Cited by 1 | Viewed by 1102

Abstract

Drought is one of the leading abiotic factors limiting morphological and physiological activities in blackberry cultivation. In this study, the effects of silver nanoparticles (AgNPs) on some morphological and biochemical processes in boysenberry (Rubus ursinus Chamisso and Schlenhtendal) under drought stress were [...] Read more.

Drought is one of the leading abiotic factors limiting morphological and physiological activities in blackberry cultivation. In this study, the effects of silver nanoparticles (AgNPs) on some morphological and biochemical processes in boysenberry (Rubus ursinus Chamisso and Schlenhtendal) under drought stress were investigated. The experiment was performed with three drought stress levels simulated with Polyethylene Glycol (PEG) (0, 4, 8%) and three AgNP treatments (0, 0.1, 0.2 mg L⁻¹) in vitro conditions. Drought stress reduced root and shoot development of boysenberry plants grown in vitro. The addition of AgNPs significantly alleviated the adverse effect of drought stress and increased the plant growth parameters. Antioxidant activity of superoxide dismutase (SOD) and catalase (CAT) enzymes increased in boysenberry leaves when treated with AgNPs under drought conditions, while the amount of malondialdehyde (MDA) decreased. As a result of the study, 0.1 mg L⁻¹ AgNPs is recommended as the most effective dose to promote the growth and development of in vitro boysenberry plants under drought stress. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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13 pages, 4935 KiB

Open AccessFeature PaperArticle

In Vitro Micropropagation, Rooting and Acclimatization of Two Agastache Species (A. aurantiaca and A. mexicana)

by Andrea Copetta, Paolo Mussano, Poonam Devi, Angelo Lanteri, Arianna Cassetti, Carlo Mascarello, Angela Bisio and Barbara Ruffoni

Horticulturae 2023, 9(10), 1065; https://doi.org/10.3390/horticulturae9101065 - 22 Sep 2023

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Abstract

Agastache aurantiaca ‘Sunset Yellow’ and A. mexicana ‘Sangria’ are aromatic plants with edible flowers characterized with good flavors, nutritional values and pleasant aromas. The aim of this study was to establish a successful protocol to ensure fast shoot propagation, rooting and in vivo [...] Read more.

Agastache aurantiaca ‘Sunset Yellow’ and A. mexicana ‘Sangria’ are aromatic plants with edible flowers characterized with good flavors, nutritional values and pleasant aromas. The aim of this study was to establish a successful protocol to ensure fast shoot propagation, rooting and in vivo acclimatization to obtain many vigorous plants for edible flower production. Micropropagation was tested with an agarized Murashige and Skoog (MS) medium enriched with different 6-benzylamino purine (BAP) levels. In vitro rooting trials were carried out using a half-strength MS medium comparing the traditional jar method with an agarized medium and the temporary immersion system (TIS) bioreactor with a liquid medium. After acclimatization in a greenhouse, the survival of plants and their development were recorded. Microscopical analyses were performed on both in-jar and in-TIS leaves at rooting stages and compared with in vivo leaves. In both species, the greatest number of new shoots was noted at 2.22 µM and 3.33 µM of BAP. In TIS, rooting started earlier, resulting in larger plants with more roots and longer roots than in the jar substrate. Any anatomical difference was observed in leaves collected from the jar and TIS during the rooting stage. The best acclimatization performances were recorded in plants coming from the TIS bioreactor. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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16 pages, 214584 KiB

Open AccessArticle

“In Vitro” Ovule Culture to Improve Genetic Variability in Hydrangea macrophylla

by Beatrice Nesi, Lisetta Ghiselli, Massimo Gori, Roberto Natale, Regina Tomiozzo, Andrea Mansuino and Stefano Biricolti

Horticulturae 2023, 9(9), 1028; https://doi.org/10.3390/horticulturae9091028 - 12 Sep 2023

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Abstract

In flowering plants, such as Hydrangea macrophylla, the main breeding objective is to increase genetic variability in ornamental traits. This study investigates in vitro techniques, through ovule culture, to overcome the hybridization barriers and increase the efficiency of crossing in Hydrangea macrophylla in [...] Read more.

In flowering plants, such as Hydrangea macrophylla, the main breeding objective is to increase genetic variability in ornamental traits. This study investigates in vitro techniques, through ovule culture, to overcome the hybridization barriers and increase the efficiency of crossing in Hydrangea macrophylla in which breeding has been hampered by a fairly long breeding cycle and lack of information about its genetic resources. Two different types of media were compared, Gamborg B5 and Murashige and Skoog basal salts, to verify the germination rate of immature ovules in different intraspecific crosses. The germination rate and viability of the seedlings were influenced by the parental genotypes in the different combinations of crossing, highlighting, in some cases, the poor compatibility between some of them. The crossing combination “Parental A × Parental B”, showed the highest germinated ovules percentage (78.3%). The media used seem to less affect the ovule germination while mainly influencing the development and growth of the young seedlings and in particular the number of leaves, the branching attitude, and root length, with the Gamborg medium determining up to a 30% increase, compared to MS medium. In addition, we tested the effectiveness of using SSR markers to assess the parentage of the putative hybrids even though only three out of twelve SSR markers showed allelism. Although the number of SSR markers was low, they were allowed to profile the parentage according to Mendelian laws. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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14 pages, 2113 KiB

Open AccessArticle

Establishment of an Efficient Somatic Embryogenesis Protocol for Giant Reed (Arundo donax L.) and Multiplication of Obtained Shoots via Semi-Solid or Liquid Culture

by Elif Aylin Ozudogru, Elif Karlik, Doaa Elazab and Maurizio Lambardi

Horticulturae 2023, 9(7), 735; https://doi.org/10.3390/horticulturae9070735 - 23 Jun 2023

Cited by 1 | Viewed by 884

Abstract

This study developed an efficient protocol for the in vitro propagation of giant reed (Arundo donax L.) biomass, defining a complete cycle of the induction of somatic embryogenesis from immature inflorescences, followed by the maturation of somatic embryos and the subsequent multiplication [...] Read more.

This study developed an efficient protocol for the in vitro propagation of giant reed (Arundo donax L.) biomass, defining a complete cycle of the induction of somatic embryogenesis from immature inflorescences, followed by the maturation of somatic embryos and the subsequent multiplication of the derived shoots in liquid culture in a temporary immersion system (TIS). The best explants were found to be 30 cm long immature inflorescences, preferably collected in spring. Such an explant type was easy to decontaminate, and the spikelets isolated from it provided over 100 embryogenic callus lines. Among the callus induction media tested, gelled MS medium supplemented with 1.1 mg/L 2,4-D provided the highest percentage of responsive spikelets and the highest density of embryogenic callus. Maturation of the embryogenic callus was easily triggered on gelled MS medium devoid of plant growth regulators. The obtained shoots could be further multiplied on previously optimized gelled DKW medium supplemented with 30 g/L sucrose, 5 mg/L BA, 0.1 mg/L IBA, and 6.8 g/L plant agar. Subsequent high multiplication of the developed shoots was achieved in liquid culture in TIS using a Plantform™ bioreactor, with an immersion cycle of 12 min every 8 h. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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14 pages, 2597 KiB

Open AccessArticle

Use of Liquid Culture with the ElecTIS Bioreactor for Faster Recovery of Blackberry (Rubus fruticosus L.) Shoots from Conservation at 4 °C

by Doaa Elazab, Maurizio Capuana, Elif Aylin Ozudogru, Monica Anichini and Maurizio Lambardi

Horticulturae 2023, 9(6), 680; https://doi.org/10.3390/horticulturae9060680 - 08 Jun 2023

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Abstract

ElecTIS is a new single container bioreactor which does not require forced air blowing, instead making the culture material mobile and the liquid medium stationary. The timed up-and-down movement of the basket containing the shoot culture ensures periodic contact with the liquid medium [...] Read more.

ElecTIS is a new single container bioreactor which does not require forced air blowing, instead making the culture material mobile and the liquid medium stationary. The timed up-and-down movement of the basket containing the shoot culture ensures periodic contact with the liquid medium positioned at the base of the container. In this study we tested for the first time its use in the recovery of blackberry shoot cultures (Rubus fruticosus L., cvs Thornfree and Chester), coming from 5 months of slow growth storage (SGS), at 4 °C and in the dark. The shoot recovery at standard culture conditions was performed on two different types of ElecTIS, i.e., one with a smaller basket (ElecTIS_S, 234 cm² of culture area), and one with a large basket (ElecTIS_L, 336 cm²), comparing the culture in TIS (cycle of 8 min every 6 h, equal to 32 min/day) with the traditional one in a gelled medium in glass jars (500 cc). After each one of the three 4-week subcultures, the shoot growth parameters and the relative growth rate highlighted a clear superiority of ElecTIS in promoting the recovery of shoot cultures coming from SGS. The analyses of chlorophyll content and stoma functionality confirmed the superior quality of shoots cultured in the ElecTIS bioreactor, and these shoots were afterwards easily rooted and acclimatized ex vitro. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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11 pages, 1774 KiB

Open AccessArticle

In Vitro Conservation and Regeneration of Potato (Solanum tuberosum L.): Role of Paclobutrazol and Silver Nanoparticles

by Obdulia Baltazar Bernal, José Luis Spinoso-Castillo, Eucario Mancilla-Álvarez, Rafael Arturo Muñoz-Márquez Trujillo and Jericó Jabín Bello-Bello

Horticulturae 2023, 9(6), 676; https://doi.org/10.3390/horticulturae9060676 - 07 Jun 2023

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Abstract

In vitro conservation and regeneration of potato germplasm is important in breeding programs. The objective of this study was to assess the combined effect of paclobutrazol (PAC) and silver nanoparticles (AgNPs) during reduced-growth conservation and their in vitro ability to regenerate S. tuberosum [...] Read more.

In vitro conservation and regeneration of potato germplasm is important in breeding programs. The objective of this study was to assess the combined effect of paclobutrazol (PAC) and silver nanoparticles (AgNPs) during reduced-growth conservation and their in vitro ability to regenerate S. tuberosum after conservation treatments. For the conservation system, apices were used as explants in Murashige and Skoog culture media with different combinations of PAC (0, 0.5, 1 and 2 mg L⁻¹) and AgNPs (0, 50, 100 and 200 mg L⁻¹). At six months of culture, plant length, number of lateral branches, number of leaves and roots, root length, stomatal density and total chlorophyll content were assessed. For regeneration, explants were internodes cultured in medium with only 2 mg L⁻¹ benzyladenine (BA). At 60 d of culture, the response percentage, number of shoots per explant and shoot length were assessed. For in vitro conservation, the combination of 2 mg L⁻¹ PAC and 50 mg L⁻¹ AgNPs was the best treatment, whereas for in vitro regeneration, the highest number of shoots was in explants that were treated with 1 or 2 mg L⁻¹ PAC with 50 mg L⁻¹ AgNPs. In conclusion, PAC and AgNPs are alternatives for in vitro S. tuberosum conservation, and their evaluation in other species recalcitrant to the effect of ethylene is recommended. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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17 pages, 3690 KiB

Open AccessArticle

In Vitro Long-Term Cultures of Papaya (Carica papaya L. cv. Solo)

by Carlos López Encina, Manuel Lopez Granero and José Javier Regalado

Horticulturae 2023, 9(6), 671; https://doi.org/10.3390/horticulturae9060671 - 06 Jun 2023

Cited by 1 | Viewed by 2607

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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13 pages, 2247 KiB

Open AccessArticle

Short-Term Conservation of Juglans regia L. via Synthetic Seed Technology

by Valbona Sota, Carla Benelli, Matilda Myrselaj, Efigjeni Kongjika and Nazim S. Gruda

Horticulturae 2023, 9(5), 559; https://doi.org/10.3390/horticulturae9050559 - 08 May 2023

Cited by 2 | Viewed by 1297

Abstract

Juglans regia L. is a crucial species as a forest tree and for its nutritional and medicinal values. It is also included in the list of endangered species in Albania and thus, there is a need to find methodologies to ensure its rapid [...] Read more.

Juglans regia L. is a crucial species as a forest tree and for its nutritional and medicinal values. It is also included in the list of endangered species in Albania and thus, there is a need to find methodologies to ensure its rapid regeneration and ex situ conservation. This research, investigated the regeneration of plantlets from synthetic seeds containing shoot tips of four native walnut varieties: ‘Përmet’, ‘Korçë’, ‘Peshkopi’, and ‘Tropojë’. First, in vitro-derived shoot tips from walnut seedlings are encapsulated using sodium alginate. After that, the regeneration potential of the encapsulated shoot tips and the influence of incubation conditions are evaluated. The synthetic seeds were incubated at either 25 °C or 8 °C, with and without dehydration treatment, in 0.5 M sucrose solution for 3 h. The synthetic seeds in both temperature regimes (25 °C and 8 °C) develop plantlets and provid conservation potential without the need for subcultures for 4 and 3.5 months, respectively. Furthermore, all walnut varieties incubated in these conditions achiev a high regeneration rates. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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7 pages, 907 KiB

Open AccessCommunication

An Optimized Protocol for In Vitro Regeneration of Ocimum basilicum cv. FT Italiko

by Sara Barberini, Chiara Forti, Marina Laura, Roberto Ciorba, Carlo Mascarello, Annalisa Giovannini, Barbara Ruffoni and Marco Savona

Horticulturae 2023, 9(3), 407; https://doi.org/10.3390/horticulturae9030407 - 22 Mar 2023

Cited by 2 | Viewed by 1639

Abstract

Sweet basil (Ocimum basilicum L.; Fam. Lamiaceae) is an annual herbaceous plant with a high economic value used in folk medicine, pharmacology, and food production. In Italy, most of the varieties are used to produce the famous “pesto” sauce; however, almost all [...] Read more.

Sweet basil (Ocimum basilicum L.; Fam. Lamiaceae) is an annual herbaceous plant with a high economic value used in folk medicine, pharmacology, and food production. In Italy, most of the varieties are used to produce the famous “pesto” sauce; however, almost all of them are susceptible to basil downy mildew (BDM) disease, strongly decreasing the growth of the fresh leaves and the survival of the whole plant. Nowadays, CRISPR/Cas9 technology is recognized to be a prominent way to enhance basil genetic breeding. In this work, we present an optimized protocol for in vitro direct regeneration of an elite cultivar, which is the major limiting factor for the transformation of O. basilicum. Regeneration has been obtained from different explants (leaves, cotyledons, cotyledonary nodes); the highest frequency has been obtained from cotyledonary nodes of seedlings germinated on MS medium containing TDZ. This protocol may be used for biotechnological applications as genome editing techniques to obtain basil-downy-mildew-disease-resistant clones. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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18 pages, 3864 KiB

Open AccessArticle

In Vitro Propagation of Three Populations of the Endangered, Greek Endemic Cerastium candidissimum and Short-Term Storability of Alginate-Encapsulated Shoot Explants for Exploitation and Conservation

by Konstantinos Bertsouklis and Stella Tsopela

Horticulturae 2023, 9(2), 273; https://doi.org/10.3390/horticulturae9020273 - 17 Feb 2023

Cited by 2 | Viewed by 1649

Abstract

Cerastium candidissimum Correns is an endangered species. Nevertheless, its ornamental characteristics make it an ideal candidate for commercial use as a new ornamental and landscape plant. As there are no references to the in vitro propagation of this species, the present research aims [...] Read more.

Cerastium candidissimum Correns is an endangered species. Nevertheless, its ornamental characteristics make it an ideal candidate for commercial use as a new ornamental and landscape plant. As there are no references to the in vitro propagation of this species, the present research aims to comparatively investigate the potential that in vitro propagation may have of three different populations of the plant, from Mounts Parnassos (PS), Parnitha (PR), and Hymettus (HM), respectively. The establishment of the initial cultures took place by cotyledonary- and stem-node explants, excised from in vitro grown seedlings, on Murashige and Skoog (MS) media, without hormone (Hf), or with 0.5 mg L⁻¹ 6-benzyladenine (BA). The shooting percentage was higher in the case of the PS population (97.6%). It was also higher for stem-node explants (93.9%). The multiplication phase took place on Hf MS, or on MS containing BA (0.5 and 1.0 mg L⁻¹), with or without 0.1 mg L⁻¹ 1-Naphthaleneacetic acid. The higher shoot production was observed in the PS and PR populations (88.0% and 76.9% shooting, with 3.1 and 2.7 shoots/explant, respectively). Micro-shoots rooted at high percentages on half-strength MS (80.0–100.0%). Successful acclimatisation (95.0%) was established on peat: perlite (1:1 v/v). Moreover, an efficient alginate-encapsulation procedure was developed from PS stem-node explants, using a 3.0% w/v sodium alginate and 14.7 g L⁻¹ calcium chloride solution. Alginate beads exhibited a higher shoot proliferation (3.8 shoots) after 60 days of storage at 4.0 °C and a 50-day growth on Hf MS than non-encapsulated in vitro node-explants did. The presented protocol of in vitro propagation of C. candidissimum could facilitate the exploitation of the species by the floricultural industry and contribute to conservation strategies. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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11 pages, 1591 KiB

Open AccessBrief Report

In Vitro Hypoxic Environment Enhances Volatile Compound Production in Persian Violet Flowers

by Sompoch Noichinda and Kitti Bodhipadma

Horticulturae 2023, 9(9), 981; https://doi.org/10.3390/horticulturae9090981 - 31 Aug 2023

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Abstract

Flowers of Persian violet (Exacum affine Balf. f. ex Regel) that are grown in nature typically produce a scent. However, whether Persian violet flowers developed inside sterile containers produce odors has yet to be studied. Therefore, this research aimed to study and [...] Read more.

Flowers of Persian violet (Exacum affine Balf. f. ex Regel) that are grown in nature typically produce a scent. However, whether Persian violet flowers developed inside sterile containers produce odors has yet to be studied. Therefore, this research aimed to study and compare the effects of ex vitro and in vitro environments on the volatile composition of Persian violet flowers. Persian violet flowers obtained from an in vitro culture and potted plants were analyzed for volatile constituents using gas chromatography–mass spectrometry (GC-MS). The main constituent of the volatile compounds in the Persian violet flowers grown in both conditions was alcohol, with 3-hexen-1-ol, which produces a grassy-green odor, being the dominant substance. In addition, the in vitro Persian violet flowers contained the highest amount of ethanol, which produces a wine aroma—followed by the terpene alcohol β-citronellol, which produces a rose scent. However, 3-carene (citrus odor), caryophyllene (floral odor), humulene (woody odor), and β-ionone (floral odor) were detected only in Persian violet flowers grown in natural conditions. Therefore, these results indicate that hypoxia possibly occurred during plantlet growth in the in vitro environment and caused some different volatile compound production from that in natural conditions. Full article

(This article belongs to the Special Issue In Vitro Propagation and Biotechnology of Horticultural Plants)

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