Horticulturae

2024

Jump to: 2023, 2022, 2021

15 pages, 4485 KiB

Open AccessArticle

Optimization of an Efficient Direct Shoot Induction System and Histological Evaluation of Shoot Formation in Cucurbita maxima Duch

by Huijuan Song, Zhongquan Chen, Jiali Yan, Hong Jiang, Sihui Dai, Hongbo Yang, Longjun Sun and Xiaowu Sun

Horticulturae 2024, 10(2), 165; https://doi.org/10.3390/horticulturae10020165 - 11 Feb 2024

Cited by 1 | Viewed by 1209

Abstract

Improving plant regeneration ability and shortening regeneration time can promote the development of genetic transformation breeding technology for horticultural crops. We optimized several culture conditions, including explant type, mother plant genotype, and medium, to improve shoot formation in winter squash (Cucurbita maxima [...] Read more.

Improving plant regeneration ability and shortening regeneration time can promote the development of genetic transformation breeding technology for horticultural crops. We optimized several culture conditions, including explant type, mother plant genotype, and medium, to improve shoot formation in winter squash (Cucurbita maxima Duch.). Histological analysis of the occurrence of shoots was also carried out. The results indicate that cotyledon was the most suitable explant for inducing the shoot regeneration of winter squash. We found that ‘Jin-li’ had a shorter shoot induction time and a higher average number of shoots. The highest induction rate of 95.23% among the five lines. The average shoot induction rate of five lines was the highest (84.85%) on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.2 mg/L indole-3-acetic acid (IAA). We also found that there was an interaction between genotypes and induction media, and their interaction had a greater impact on the shoot induction rate than individual effects. Histological observation revealed that the induced shoots of winter squash cotyledons originated from subepidermal cells. We also found that the optimal medium for de novo root regeneration was 1/2 MS. We acclimatized and cultivated regenerated plants and harvested their fruits, which maintained the characteristics of mother plants. These findings lay an important foundation for further research on direct shoot regeneration and accelerate its application in winter squash genetic transformation. Full article

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2023

Jump to: 2024, 2022, 2021

9 pages, 1015 KiB

Open AccessArticle

Optimal Growth and Biomass of Centella asiatica Using a Twin-Bottle Temporary Immersion Bioreactor

by Thanakorn Wongsa, Anupan Kongbangkerd and Boworn Kunakhonnuruk

Horticulturae 2023, 9(6), 638; https://doi.org/10.3390/horticulturae9060638 - 29 May 2023

Cited by 2 | Viewed by 2442

Abstract

Centella asiatica or gotu kola has long been used as a traditional medicinal plant. Here, immersion times and culture systems on growth and biomass production of C. asiatica were investigated using a twin-bottle, temporary immersion system. Results indicated that all immersion times gave [...] Read more.

Centella asiatica or gotu kola has long been used as a traditional medicinal plant. Here, immersion times and culture systems on growth and biomass production of C. asiatica were investigated using a twin-bottle, temporary immersion system. Results indicated that all immersion times gave 100% survival, with a 5 min immersion 12 times/day, providing the highest number of new shoots (3.6 shoots/explant), leaves (10.2 leaves/explant), roots (8.3 roots/explant), and fresh and dry weights of clumps (5.06 g fresh weight and 0.48 g dry weight/clump). The temporary immersion system resulted in more than a three-fold increase in biomass accumulation, with the highest average number of new shoots, leaves, and roots compared to a semi-solid system. Full article

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13 pages, 2322 KiB

Open AccessArticle

Optimization of Morphogenesis and In Vitro Production of Five Hyacinthus orientalis Cultivars

by Hany M. El-Naggar, Ashraf M. Shehata, Maneea Moubarak and Amira R. Osman

Horticulturae 2023, 9(2), 176; https://doi.org/10.3390/horticulturae9020176 - 30 Jan 2023

Cited by 1 | Viewed by 1763

Abstract

To maximize the economic benefits of Hyacinthus orientalis L., the micropropagation and morphogenesis induction of five Hyacinthus cultivars were investigated under four different concentrations of indole acetic acid (IAA) with two cytokinins, benzyl adenine (BA), or kinetin (Kin). Days for morphogenesis initiation and [...] Read more.

To maximize the economic benefits of Hyacinthus orientalis L., the micropropagation and morphogenesis induction of five Hyacinthus cultivars were investigated under four different concentrations of indole acetic acid (IAA) with two cytokinins, benzyl adenine (BA), or kinetin (Kin). Days for morphogenesis initiation and shoot formation in the red cultivars were fewer than in the blue and white cultivars. Blue cultivars showed an increase in fresh weight and chlorophyll content under either BA or Kin, but they showed an increase in shoot height in the BA treatments only. IAA at 1.5 mg/L caused a time reduction in days for morphogenesis induction and shoot formation and enhanced shoot height and fresh weight. Kin had a lesser impact than BA on all parameters at all concentrations. The number of shoots differed significantly among the cultivars. The Murashige and Skoog (MS) medium supplemented with 3.0 mg/L indole-3-butyric acid (IBA) produced the highest percentage of root formation (93.3%), number of roots/plantlet (5.26), and root length (1.10 cm). The Jan Bos cultivar at 3.0 mg/L IBA had the highest mean rooting percentage (100%) and number of roots per plantlet (6.66), while Pink Pearl had the highest root length (1.39 cm). Full article

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2022

Jump to: 2024, 2023, 2021

11 pages, 1419 KiB

Open AccessFeature PaperArticle

In Vitro Propagation by Axillary Shoot Culture and Somatic Embryogenesis of Daucus carota L. subsp. sativus, ‘Polignano’ Landrace, for Biodiversity Conservation Purposes

by Claudia Ruta, Angela Campanelli, Giuseppe De Mastro and Federica Blando

Horticulturae 2022, 8(12), 1150; https://doi.org/10.3390/horticulturae8121150 - 6 Dec 2022

Cited by 3 | Viewed by 2532

Abstract

Carrots are a multi-purpose vegetable; hence, they have become a very popular consumers’ choice, also thanks to their nutritional value with health benefits. Several varieties and landraces exist of carrot (Daucus carota L. ssp. sativus). In Apulia (Italy), for example, a [...] Read more.

Carrots are a multi-purpose vegetable; hence, they have become a very popular consumers’ choice, also thanks to their nutritional value with health benefits. Several varieties and landraces exist of carrot (Daucus carota L. ssp. sativus). In Apulia (Italy), for example, a multicolored carrot landrace is present, the Polignano carrot (PC), threatened by the risk of genetic erosion. With the purpose of verifying the possibility of using in vitro culture for ex situ conservation purposes of PC, a rapid and reliable protocol for micropropagation and a highly efficient somatic embryogenesis protocol have been set up. For micropropagation studies, axillary buds from taproot were used as initial explants. Supplementation on basal medium (BM, a Murashige–Skoog modified medium) with low cytokinine (6-benzyladenine, BA, 0.22 μM) dosage was enough to obtain a good multiplication rate, also depending by the PC color. Rooting of proliferated shoots occurred easily in hormone-free medium and acclimatization was successfully reached in 6 weeks. Somatic embryos were formed from embryogenic callus induced by taproot sections placed on 2,4-dichlorophenoxyacetic acid (2,4-D)-added medium. After in vitro conversion in hormone-free medium, somatic embryos were also successfully acclimatized. Both microplants and somatic embryos produced true-to-type taproots (multicolored carrots), demonstrating the suitability of in vitro techniques for biodiversity conservation purposes. Full article

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20 pages, 3146 KiB

Open AccessArticle

Morphological and Physiological Responses of In Vitro-Grown Cucurbita sp. Landraces Seedlings under Osmotic Stress by Mannitol and PEG

by Rana Panahi Tajaragh, Farzad Rasouli, Mousa Torabi Giglou, Seyed Morteza Zahedi, Mohammad Bagher Hassanpouraghdam, Mohammad Ali Aazami, Anna Adámková and Jiří Mlček

Horticulturae 2022, 8(12), 1117; https://doi.org/10.3390/horticulturae8121117 - 28 Nov 2022

Cited by 4 | Viewed by 1846

Abstract

Screening and identification of tolerant genotypes using osmotic materials under in vitro culture could be rapid, easy, and even accurate. In this research, three Iranian landraces of Cucurbita sp. included Tanbal Ajili (Cucurbita maxima Duch.), Ajili Razan (Cucurbita pepo L.), and [...] Read more.

Screening and identification of tolerant genotypes using osmotic materials under in vitro culture could be rapid, easy, and even accurate. In this research, three Iranian landraces of Cucurbita sp. included Tanbal Ajili (Cucurbita maxima Duch.), Ajili Razan (Cucurbita pepo L.), and Balghabakhi (Cucurbita moschata Duch.) seeds were cultured in ¼ MS medium. After germination, plantlets were transferred to MS media containing mannitol and PEG 6000. Mannitol and PEG at three concentrations of 0.1, 0.2, and 0.4 M and 0.009, 0.012, and 0.015 M, respectively, were added into the MS medium, while the MS medium without any adding was used as control. Our findings revealed that osmotic treatments significantly increased shoot and root dry weight (DW), malondialdehyde (MDA), and proline content, but significantly reduced coleoptile length, shoot and root fresh weight, and photosynthesis pigments content. Protein content, phenols, and flavonoids content, enzymatic and non-enzymatic antioxidant including ascorbate peroxidase (APX), guaiacol peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) activity, reduced ascorbate (AsA), reduced ascorbate/dehydroascorbic acid (AsA/DHA), reduced glutathione (GSH), dehydroascorbic acid (DHA) and oxidized glutathione (GSSG), and reduced glutathione/oxidized glutathione (GSH/GSSG) were significantly increased at moderate osmotic stress induced by mannitol and PEG. In contrast, the previous physiological parameters were significantly reduced at higher water deficit conditions. With respect to most attributes and concentrations, mannitol simulated osmotic stress better than PEG. Our results revealed that applying PEG and mannitol under in vitro conditions could be an efficient way to evaluate and screen cucurbit genotypes for future breeding programs. Full article

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10 pages, 278 KiB

Open AccessArticle

Eradication of PPV and PNRSV Viruses from Three Peach Cultivars Using Thermotherapy In Vitro, Including Optimization of Microshoots’ Multiplication and Rooting Medium

by Neda Hesari, Ali Haji Mohammadi, Reza Zarghami, Bratali Fakheri, Erzsébet Kiss-Bába, Anita Szegő, István Papp and Iman Mirmazloum

Horticulturae 2022, 8(10), 929; https://doi.org/10.3390/horticulturae8100929 - 10 Oct 2022

Cited by 2 | Viewed by 1413

Abstract

Peach cultivars (’Elberta’, ‘Red Top’, and ’Dixie Red’) were studied for their in vitro adoptability and performance in producing virus-free plantlets. The thermotherapy method with increasing temperatures (25 °C to 37 °C) was applied for the elimination of famous peach infecting plum pox [...] Read more.

Peach cultivars (’Elberta’, ‘Red Top’, and ’Dixie Red’) were studied for their in vitro adoptability and performance in producing virus-free plantlets. The thermotherapy method with increasing temperatures (25 °C to 37 °C) was applied for the elimination of famous peach infecting plum pox viruses (PPVs) and prunus necrotic ringspot virus (PNRSV), and the DS-ELISA test and RT-PCR technique were performed to confirm the production of virus-free microshoots. The application of 30 mg L⁻¹ of Fe-EDDHA treatment resulted in the best performance for culture establishment of all cultivars, where the best subsequent morphological performance in terms of branch and leaf numbers was recorded for the ’Dixie Red’ cultivar in MS medium, supplemented with 0.5 mgL⁻¹ of gibberellic acid (GA₃) and 0.5 mg L⁻¹ of 6-Benzylaminopurine (BAP). At the regeneration stage, the highest (26.96 mm) and lowest (18.43 mm) shoot lengths were obtained from the ’Dixie Red’ cultivar treated with GA₃ (2 mg L⁻¹) + thidiazuron (TDZ) (2.5 mg L⁻¹) and the ’Red Top’ cultivar treated with GA₃ (1 mg L⁻¹) + TDZ (1 mg L⁻¹), respectively. The leaf numbers were affected by the application of growth regulators, where the ’Elberta’ cultivar under GA₃ (2 mg L⁻¹) + TDZ (2.5 mg L⁻¹) treatment showed the highest numbers and the ’Red Top’ cultivar under GA₃ (1 mg L⁻¹) + TDZ (1 mg L⁻¹) showed the lowest mean values. The thermotherapy treatment and micropropagation of shoot tips resulted in 100% virus-free plantlets, as confirmed by both applied diagnostic methods. The result of the application of the rooting stage with growth regulators on ’Elberta’ plantlets showed the best performance (90%) in ½ MS medium supplemented with 0.5–1 mg L⁻¹ of IBA, which was significantly higher than the same treatment in MS medium. The obtained results should constitute the basis for further optimization of the multiplication and rooting of virus-free peach plantlets to be served for nurseries and planation orchards. Full article

9 pages, 1608 KiB

Open AccessArticle

Encapsulation in Calcium Alginate of Nodes from Stolons of Mentha spicata L.

by Maurizio Micheli, Luca Regni and Daniel Fernandes da Silva

Horticulturae 2022, 8(5), 456; https://doi.org/10.3390/horticulturae8050456 - 19 May 2022

Cited by 2 | Viewed by 2224

Abstract

It is well known that the products of encapsulation (multifunctional beads and synthetic seeds) can be used as innovative technological tools to integrate micropropagation both for plant germplasm conservation and to simplify the management of propagation materials in nurseries. Nevertheless, the usual concept [...] Read more.

It is well known that the products of encapsulation (multifunctional beads and synthetic seeds) can be used as innovative technological tools to integrate micropropagation both for plant germplasm conservation and to simplify the management of propagation materials in nurseries. Nevertheless, the usual concept of encapsulation concerns the use of initial in vitro derived explants. In this study, for the first time, in vivo derived organs of Mentha spicata L., obtained through the excision of fragments (nodes) from stolons of cultivated mother plants, were employed. The artificial endosperm had a tenfold reduced concentration of Murashige and Skoog (MS) substrate, with the addition of sucrose (5 g L⁻¹), 6-benzyl-aminopurine (BAP) (0.1 mg L⁻¹) and 1-naphthalene acetic acid (NAA) (0.01 mg L⁻¹). Moreover, the calcium alginate matrix was enriched with different thiophanate-methyl (TM) concentrations (0, 10, 50, 100 and 200 mg L⁻¹) in order to prevent possible contamination during the conversion in nonsterile conditions. Interesting results were obtained encapsulating every single node of fresh stolon as a bipolar propagule able to develop a whole plantlet (conversion), as the coated seed in other species. The synthetic seeds of spearmint without TM in the artificial endosperm showed a satisfactory ability to convert (56.7%) into plantlets after sowing in soil under nonsterile conditions. TM at 100 and 200 mg L⁻¹ negatively affected the total emergence, which decreased to 30.0 and 33.3%, respectively. In general, in the artificial seeds without TM, higher values for most of the aboveground and belowground plants parameters were recorded compared to naked nodes. Full article

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18 pages, 8083 KiB

Open AccessArticle

Optimization of an In Vitro Embryo Rescue Protocol for Breeding Seedless Table Grapes (Vitis vinifera L.) in Italy

by Angelica Giancaspro, Andrea Mazzeo, Antonio Carlomagno, Agata Gadaleta, Stefano Somma and Giuseppe Ferrara

Horticulturae 2022, 8(2), 121; https://doi.org/10.3390/horticulturae8020121 - 29 Jan 2022

Cited by 16 | Viewed by 6304

Abstract

Conventional crossing of stenospermocarpic grapes for the obtainment of seedless cultivars presents some technical constraints causing embryo abortion in the early berry developmental stages. Embryo rescue technique partially overcomes these limitations, but the obtainment of viable plantlets relies on the optimization of several [...] Read more.

Conventional crossing of stenospermocarpic grapes for the obtainment of seedless cultivars presents some technical constraints causing embryo abortion in the early berry developmental stages. Embryo rescue technique partially overcomes these limitations, but the obtainment of viable plantlets relies on the optimization of several genetic and methodological issues. This work aimed to regenerate viable plants from immature ovules of stenospermocarpic table grape hybrids by applying a three-step in vitro culture protocol consisting of embryo development, embryo germination-rooting, and plantlet formation. The influence of parental genotypes (six “seedless × seedless” crosses), ovule sampling time (30, 40, 50 days after pollination (DAP)), and extent of embryo germination induction (4, 6, 8 weeks) was assessed on ovule fertilization, embryo development and germination, rooting, and plantlet formation to establish the best rescue time for each combination hybrid. Our optimized protocol included immature ovule isolation for 40 DAP and embryo germination induction for 8 weeks. As for genotypes, the most efficient embryo germination was recovered from hybrids of Thompson, Superior, and Regal cultivars, whereas the highest percentage of viable plants was derived from 50-DAP ovules of Luisa × Thompson progeny. Such an optimized protocol could be useful to maximize the efficiency of future breeding programs for grape seedlessness. Full article

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11 pages, 729 KiB

Open AccessArticle

Micropropagation of Feverfew (Tanacetum parthenium) and Quantification of Parthenolide Content in Its Micropropagated and Conventionally Grown Plants

by Huda E. Mahood, Majeed Kadhem Abbas and Nisar Ahmad Zahid

Horticulturae 2022, 8(1), 50; https://doi.org/10.3390/horticulturae8010050 - 5 Jan 2022

Cited by 4 | Viewed by 3138

Abstract

Feverfew (Tanacetum parthenium) is a well-known multi-functional plant with anti-inflammatory, cardiotonic, antiangiogenic, and anticancer effects. The therapeutic value of this plant is due to its phytochemical constitutes, especially parthenolide. Tissue culture techniques have been applied to improve the bioactive components of [...] Read more.

Feverfew (Tanacetum parthenium) is a well-known multi-functional plant with anti-inflammatory, cardiotonic, antiangiogenic, and anticancer effects. The therapeutic value of this plant is due to its phytochemical constitutes, especially parthenolide. Tissue culture techniques have been applied to improve the bioactive components of many herbal plants. Hence, this study, was carried out to establish a protocol for micropropagation of the feverfew plant and to quantify parthenolide content in its micropropagated and conventionally grown plants. To establish an aseptic culture, different concentrations of sodium hypochlorite (NaOCl) were investigated for seed surface sterilization. Besides, the effects of plant growth regulators (PGRs) on the callus induction, shoot organogenesis from callus and in vitro rooting were evaluated. Additionally, the parthenolide yield of the micropropagated and conventionally grown plants was determined by using high-performance liquid chromatography (HPLC). The results showed that surface sterilization of feverfew seeds with 6% NaOCl for 15 min obtained 65.00 ± 2.69% aseptic seeds. Murashige and Skoog (MS) medium supplemented with 0.4 mg/L thidiazuron (TDZ) and 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) resulted in 86.00 ± 1.72% callus induction. The highest number of shoots (5.00 ± 0.15) per explant was obtained in the treatment of MS medium supplemented with 5 mg/L zeatin. MS medium fortified with 3 mg/L indole-3-butyric acid (IBA) produced the maximum number of roots per plantlet (8.90 ± 0.35). A total of 90% of the micropropagated plantlets survived when planted in perlite + peat moss (1:1 v/v); the micropropagated plantlets were successfully established in the ex vitro conditions. According to parthenolide analysis, its level was significantly higher in the micropropagated plants than conventionally grown plants. Among different solvents, ethanolic extraction obtained the highest parthenolide content of the feverfew plant. Hence, it can be concluded that micropropagation of feverfew could be applied to produce disease-free planting materials and to improve the parthenolide content of the feverfew plant. Full article

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2021

Jump to: 2024, 2023, 2022

11 pages, 1396 KiB

Open AccessFeature PaperArticle

Embryo Culture, In Vitro Propagation, and Molecular Identification for Advanced Olive Breeding Programs

by Vito Montilon, Leonardo Susca, Oriana Potere, Vincenzo Roseti, Antonia Campanale, Antonia Saponari, Cinzia Montemurro, Valentina Fanelli, Pasquale Venerito and Giovanna Bottalico

Horticulturae 2022, 8(1), 36; https://doi.org/10.3390/horticulturae8010036 - 30 Dec 2021

Cited by 1 | Viewed by 4485

Abstract

The high biodiversity of the olive tree is an important opportunity to develop sustainable plans to control Xylella fastidiosa (Xf) through breeding programs. Olive tree breeding activities have been limited due to various features of this species including the long [...] Read more.

The high biodiversity of the olive tree is an important opportunity to develop sustainable plans to control Xylella fastidiosa (Xf) through breeding programs. Olive tree breeding activities have been limited due to various features of this species including the long time required for seed germination caused by the inhibition effect of the woody endocarp, the seed integument, and the endosperm. Starting from F₁ seeds by cross-breeding, the embryo culture was compared with traditional seed germination, evaluating the effectiveness of in vitro multiplication of the plantlets for large-scale production. The isolated embryos were established on a new medium based on Rugini ‘84 macroelements, Murashige & Skoog ‘62 microelements, with Nitsch J. P. & Nitsch C. ‘69 vitamine and subcultured on Leva MSM modified. The results obtained confirmed that in vitro culture of olive embryos is a valid tool for increasing the percentage and speed of germination, helping to reduce the time of the olive breeding programs, offering the possibility to effectively propagate plantlets for further experiments. Full article

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18 pages, 3227 KiB

Open AccessArticle

Use of Biotechnological Methods to Support the Production of New Peach Hybrids

by Irina Mitrofanova, Nina Lesnikova-Sedoshenko, Valentina Tsiupka, Anatoliy Smykov and Olga Mitrofanova

Horticulturae 2021, 7(12), 533; https://doi.org/10.3390/horticulturae7120533 - 1 Dec 2021

Cited by 3 | Viewed by 2005

Abstract

Peach [Prunus persica (L.) Batsch] is among the most demanded fruit crops in the world. Biotechnological methods help to originate new hybrid forms in order to increase the cultivar diversity and create new valuable genotypes. Cross combinations between the cultivars Clyde Wilson, [...] Read more.

Peach [Prunus persica (L.) Batsch] is among the most demanded fruit crops in the world. Biotechnological methods help to originate new hybrid forms in order to increase the cultivar diversity and create new valuable genotypes. Cross combinations between the cultivars Clyde Wilson, Jerseyglo, Loadel, Summerglo and the promising cultivar ‘Nikitskiy Podarok’ have been done. The embryos of these hybrids germinated and formed plantlets after stratification at 4 °C for 45–60 days. The best regeneration rates in the hybrids ‘Loadel’ × ‘Nikitskiy Podarok’ and ‘Summerglo’ × ‘Nikitskiy Podarok’ (96.30% and 92.59%, respectively) were noted on hormone-free Monnier culture medium supplemented with 400.0 mg L⁻¹ casein hydrolyzate. When the newly formed plantlets had necrosis of the shoot apex or immature roots, nodal shoot segments were used. At the same time, a high regeneration capacity was noted in the hybrids ‘Summerglo’ × ‘Nikitskiy Podarok’ and ‘Loadel’ × ‘Nikitskiy Podarok’ on B5 culture medium with 0.75 mg L⁻¹ 6–benzyl–aminopurine (BAP) + 0.1 mg L⁻¹ indole–3–butyric acid (IBA). After the second subculture, the number of new adventitious shoots was 5.18 ± 0.18 and 4.95 ± 0.18 shoots per explant, respectively. The plants obtained from the hybrid embryos in a soil mixture soil: peat: sand (3:1:1) were adapted. The main morphological and anatomical features of the leaf blades in newly originated peach hybrids have been studied: the thickness of their tissues and the distribution of stomatal apparatus, as well as the physiological parameters of the photosystem II activity in regenerants cultured in vitro and during their in vivo acclimatization. The high capacity to post aseptic adaptation in the obtained hybrids has been shown. Full article

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13 pages, 1446 KiB

Open AccessArticle

Endophytic Bacteria in Banana In Vitro Cultures: Molecular Identification, Antibiotic Susceptibility, and Plant Survival

by Antar Nasr El-Banna, Mohammed Elsayed El-Mahrouk, Yaser Hassan Dewir, Mona Ali Farid, Doaa Mahmoud Abou Elyazid and Heinz Martin Schumacher

Horticulturae 2021, 7(12), 526; https://doi.org/10.3390/horticulturae7120526 - 28 Nov 2021

Cited by 11 | Viewed by 4398

Abstract

Microbial contamination is a common problem that causes significant losses in plant micropropagation systems. The present study reports on the identification and control of bacterial contaminants in banana in vitro cultures. Twelve isolates belonging to Bacillus pumilus (S2), Bacillus subtilis (R2 and M4), [...] Read more.

Microbial contamination is a common problem that causes significant losses in plant micropropagation systems. The present study reports on the identification and control of bacterial contaminants in banana in vitro cultures. Twelve isolates belonging to Bacillus pumilus (S2), Bacillus subtilis (R2 and M4), Geobacillus stearothermophilus (S1, S3, S4, P2, M3 and R3) and Paenibacillus spp. (P1, R1 and M2) were identified by sequencing of 16S rRNA, gyrA or gyrB genes. Antibiotic susceptibility testing was performed with the disk diffusion method on bacterial isolates using 36 antimicrobial agents. Some antibiotics, notably Ticarcillin, Penicillin, Ampicillin, Cefazolin and Imipenem, had a broader range of bactericidal activity than others did. When contaminated axillary shoot cultures of banana were treated with 100 or 200 mg·L⁻¹ of ticarcillin, ampicillin or penicillin the bacteria were eliminated, but a reduction in shoot multiplication and growth was observed. These findings contribute to minimizing the losses in the commercial micropropagation of banana. Full article

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11 pages, 6182 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Somatic Embryogenesis in Vitis for Genome Editing: Optimization of Protocols for Recalcitrant Genotypes

by Lucia Rosaria Forleo, Margherita D’Amico, Teodora Basile, Antonio Domenico Marsico, Maria Francesca Cardone, Flavia Angela Maria Maggiolini, Riccardo Velasco and Carlo Bergamini

Horticulturae 2021, 7(11), 511; https://doi.org/10.3390/horticulturae7110511 - 19 Nov 2021

Cited by 6 | Viewed by 2653

Abstract

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have [...] Read more.

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have proven to be very recalcitrant to regeneration via somatic embryogenesis. In this work, the development of a protocol for improved production of embryogenic calluses is described. Two sterilization protocols were tested: (a) a lower active chlorine concentration for a longer time (LS); and (b) a higher chlorine concentration for a shorter time (HS), in combination with the absence or presence of citric acid in the growing substrate in the first growth media. The embryogenic calluses formation in Chardonnay, a cv. with a high embryogenic response, was significantly higher in presence of citric acid in the initial growing substrate regardless of the sterilization protocol. In Aglianico, a cv. with a lower embryogenic response, no significant differences were observed. Instead, in a recalcitrant cv. as Italia, we obtained a 13-fold increase in embryogenic calluses formation performing sterilization of flowers with the HS protocol compared to LS. Full article

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10 pages, 2158 KiB

Open AccessFeature PaperArticle

Cryopreservation of Hazelnut (Corylus avellana L.) Axillary Buds from In Vitro Shoots Using the Droplet Vitrification Method

by Alessandra Sgueglia, Andrea Frattarelli, Adele Gentile, Gaia Urbinati, Simona Lucioli, Maria Antonietta Germanà and Emilia Caboni

Horticulturae 2021, 7(11), 494; https://doi.org/10.3390/horticulturae7110494 - 15 Nov 2021

Cited by 2 | Viewed by 2455

Abstract

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then [...] Read more.

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots. Full article

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9 pages, 1500 KiB

Open AccessFeature PaperArticle

In Vitro Adventitious Regeneration of Artemisia annua L. Influencing Artemisinin Metabolism

by Federica Blando, Francesca Rizzello, Miriana Durante, Angelo De Paolis, Sofia Caretto and Giovanni Mita

Horticulturae 2021, 7(11), 438; https://doi.org/10.3390/horticulturae7110438 - 28 Oct 2021

Cited by 5 | Viewed by 2489

Abstract

Artemisia annua L. is a herbaceous plant belonging to the Asteraceae family, known for producing, although at low levels, the sesquiterpene lactone artemisinin (AN), which is highly effective against malaria. In this study, an in vitro regeneration process of A. annua L. using [...] Read more.

Artemisia annua L. is a herbaceous plant belonging to the Asteraceae family, known for producing, although at low levels, the sesquiterpene lactone artemisinin (AN), which is highly effective against malaria. In this study, an in vitro regeneration process of A. annua L. using ‘Artemis’ progeny was established and the potential of tissue culture for inducing new variability in terms of AN metabolism of in vitro regenerated plants was investigated. Among the plant growth regulators tested, the cytokinin 6-benzyladenine (BA) at 4.4 μM in combination with the auxin indole-butyric acid (IBA) at 0.35 μM yielded the greatest frequency of shoot induction. The optimal multiplication medium contained BA at 0.9 μM and naphthaleneacetic acid (NAA) at 0.05 μM. Regenerated plants (RPs), after transferring to the greenhouse and subsequently to the field, were analyzed during the growth cycle at different sampling times, showing a peak of AN content 20 days before blossom. Variability among different RPs and sampling times, in terms of AN and its precursors dihydroartemisinic acid (DHAA) and artemisinic acid (AA) was observed. This suggests that adventitious shoot induction could provide a useful strategy to induce variability influencing artemisinin metabolism as a consequence of in vitro manipulation. Full article

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16 pages, 1889 KiB

Open AccessArticle

Morpho-Anatomical and Biochemical Characterization of Embryogenic and Degenerative Embryogenic Calli of Phoenix dactylifera L.

by Amal F. M. Zein El Din, Ola H. Abd Elbar, Saleh M. Al Turki, Khaled M. A. Ramadan, Hossam S. El-Beltagi, Hemmat A. Ibrahim, Ezzeldin G. Gadalla, Ibrahim M. Shams El-Din, Ibrahim S. Ibrahim, Reham Farag, Mohamed F. M. Ibrahim, Mona S. Abd El-Aal, Ahmed Abou El-Yazied, Mohamed M. El-Mogy, Mina S. F. Samaan and Yasmin M. R. Abdellatif

Horticulturae 2021, 7(10), 393; https://doi.org/10.3390/horticulturae7100393 - 12 Oct 2021

Cited by 6 | Viewed by 3050

Abstract

The study of morpho-anatomical aspects, metabolic changes of proteins, antioxidant substances, as well as phenolic compounds in embryogenic callus (EC) and degenerative embryogenic callus (DEC) was the aim of the present investigation. Ability to form somatic embryos (SEs) was associated with the softness [...] Read more.

The study of morpho-anatomical aspects, metabolic changes of proteins, antioxidant substances, as well as phenolic compounds in embryogenic callus (EC) and degenerative embryogenic callus (DEC) was the aim of the present investigation. Ability to form somatic embryos (SEs) was associated with the softness of the EC, which exhibited a white or creamy color and was composed of isodiametric cells containing dense cytoplasm, conspicuous nuclei and minimal vacuoles with observed mitotic activity. Furthermore, protein, reduced glutathione (GSH) and ascorbic acid (ASC) concentrations and the ratio between ASC and dehydroascorborbic acid (DHA) were increased significantly in the EC in comparison to the DEC. In addition, the phenolic extract of the EC was proved to have higher scavenging activity than the extract from the DEC. A loss of embryogenic competence in the DEC was correlated with the presence of more rigid clumps and such calli had a yellowish to brown color and no cell division could be observed in the cells of such aggregates as the cells had large vacuoles and they have very thick walls. Moreover, these morphological and anatomical observations of the DEC were accompanied by accumulations of the oxidized form of ascorbic acid (DHA), H₂O_2, total soluble phenolic compounds and overaccumulation of naringenin. Alternations in cellular metabolism can affect and regulate the morphogenesis of somatic embryos. Full article

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17 pages, 2225 KiB

Open AccessEditor’s ChoiceArticle

Screening of Suitable Plant Regeneration Protocols for Several Capsicum spp. through Direct Organogenesis

by Marina Martínez-López, Ana García-Pérez, Esther Gimeno-Páez, Jaime Prohens, Santiago Vilanova and Edgar García-Fortea

Horticulturae 2021, 7(9), 261; https://doi.org/10.3390/horticulturae7090261 - 25 Aug 2021

Cited by 7 | Viewed by 4008

Abstract

Peppers (Capsicum spp.) are recalcitrant to in vitro culture regeneration, making the application of in vitro-based breeding strategies difficult. We evaluated the impact of different combinations of auxins, cytokinins and micronutrients on the induction of direct organogenesis in cotyledon and hypocotyl explants [...] Read more.

Peppers (Capsicum spp.) are recalcitrant to in vitro culture regeneration, making the application of in vitro-based breeding strategies difficult. We evaluated the impact of different combinations of auxins, cytokinins and micronutrients on the induction of direct organogenesis in cotyledon and hypocotyl explants of C. annuum, C. baccatum and C. chinense. We found variation in the regeneration response among species and type of explant. In this way, the average numbers of shoots per cotyledon and hypocotyl explant were, respectively, 1.44 and 0.28 for C. annuum, 4.17 and 3.20 for C. baccatum and 0.08 and 0.00 for C. chinense. Out of the six media, the best overall results were obtained with the medium Pep1, which contained 5 mg/L BAP (6-benzylaminopurine), 0.5 mg/L IAA (indole-3-acetic acid) and 0.47 mg/L CuSO₄, followed by a subculture in the same medium supplemented with 10 mg/L AgNO₃ (medium Pep1.2). The best result for the Pep1 + Pep1.2 medium was obtained for C. baccatum using cotyledon explants, with 8.87 shoots per explant. The explants grown in medium Pep1 + Pep1.2 were the ones with greener tissue, while overall the hypocotyl explants were greener than the cotyledon explants. Our results indicate that there is wide variation among Capsicum species in terms of regeneration. Our results suggest that the synergistic effect of copper and silver resulted in a higher regeneration rate of Capsicum explants. Explants with shoots were transferred to different media for elongation, rooting and acclimatization. Although acclimatized plantlets were obtained for C. baccatum and C. chinense, an improvement in these latter stages would be desirable for a high throughput regeneration pipeline. This work contributes to the improvement of Capsicum regeneration protocols using specific combinations of medium, explant and genotype, reaching the levels of efficiency required for genetic transformation and of gene editing technologies for other crops. Full article

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14 pages, 5081 KiB

Open AccessEditor’s ChoiceArticle

Plant Production and Leaf Anatomy of Mertensia maritima (L.) Gray: Comparison of In Vitro Culture Methods to Improve Acclimatization

by Andrea Copetta, Miriam Bazzicalupo, Arianna Cassetti, Ilaria Marchioni, Carlo Mascarello, Laura Cornara, Laura Pistelli and Barbara Ruffoni

Horticulturae 2021, 7(5), 111; https://doi.org/10.3390/horticulturae7050111 - 13 May 2021

Cited by 9 | Viewed by 3798

Abstract

Mertensia maritima is a commercially interesting herb with edible leaves and flowers, characterized by oyster flavor and taste. Plant propagation and traditional cultivation are challenging for this species. Therefore, the main purpose of the present study was to establish successful protocols aimed at [...] Read more.

Mertensia maritima is a commercially interesting herb with edible leaves and flowers, characterized by oyster flavor and taste. Plant propagation and traditional cultivation are challenging for this species. Therefore, the main purpose of the present study was to establish successful protocols aimed at ensuring oyster plant shoot propagation, rooting and in vivo acclimatization. Both micropropagation and rooting were tested, comparing the traditional in vitro solid substrate in jar vs. the liquid culture in a temporary immersion system (TIS) bioreactor (Plantform™). A Murashige and Skoog (MS) medium added with 4-μM thidiazuron (TDZ) and 1-μM α-naphthaleneacetic acid (NAA) was employed for micropropagation, while a half-strength MS medium supplemented with 4-μM indole−3-butyric acid (IBA) was used for rooting. Different acclimatization conditions in the greenhouse or in growth chamber were tested. Morphometric and microscopical analyses were performed on the oyster plant leaves at the propagation, rooting and acclimatization stages both in a jar and in a TIS. Micropropagation in a TIS allowed to obtain large shoots, while a great number of shoots was observed in the jar. M. maritima shoots rooted in TIS produced more developed roots, leaves with more developed waxy glands and well-formed stomata; moreover, the plants coming from the TIS showed the best acclimatization performances. Full article

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13 pages, 3082 KiB

Open AccessArticle

In-Vivo In-Vitro Screening of Ocimum basilicum L. Ecotypes with Differential UV-B Radiation Sensitivity

by Haana Mosadegh, Alice Trivellini, Rita Maggini, Antonio Ferrante, Luca Incrocci and Anna Mensuali

Horticulturae 2021, 7(5), 101; https://doi.org/10.3390/horticulturae7050101 - 7 May 2021

Cited by 5 | Viewed by 2912

Abstract

Elevated UV-B radiation (UV-B) has been previously reported to affect plant development, physiology, and promote the biosynthesis of UV-absorbing compounds. Sweet basil (Ocimum basilicum L.) is an aromatic herb, widely cultivated worldwide for its use in the food, pharmaceuticals, and cosmetics industry. [...] Read more.

Elevated UV-B radiation (UV-B) has been previously reported to affect plant development, physiology, and promote the biosynthesis of UV-absorbing compounds. Sweet basil (Ocimum basilicum L.) is an aromatic herb, widely cultivated worldwide for its use in the food, pharmaceuticals, and cosmetics industry. This species exhibits high diversity among different ecotypes based on their geographical locations. There has been little research on intra-specific photosynthetic and metabolic differences in UV-B tolerance across ecotypes from different geographical areas. This study evaluated the protection responses to high UV-B radiation of nine O. basilicum accessions with different geographic origins. Specifically, the changes in chlorophyll a fluorescence parameters and the leaf rosmarinic acid (RA) compound were assessed using an “in vivo-vitro system” in a closed-type plant production system. Our results revealed a significant variation in UV-B protection mechanisms among accessions when plants were treated with high UV-B doses. The accumulation of RA increased significantly by UV-B light treatment in OCI142, OCI148, OCI30, OCI160, and OCI102, with the highest concentration measured in OCI160 plants. This ecotype showed the highest value of the Fv/Fm ratio, 0.70, after 48 h. Recovery of leaf functionality was more rapid in OCI160 than in other sweet basil accessions, which may indicate better photosynthetic capacity associated with enhanced biosynthesis of UV absorbing compounds. This study shows that the biosynthesis of the UV-absorbing compound (RA) represents an effective mechanism to reduce the photoinhibitory and photooxidative damage caused by high UV stress. Full article

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11 pages, 2157 KiB

Open AccessArticle

Superior Line from Anther Culture of Dendrocalamus latiflorus Selected after Field Trial

by Wei Zhang, Yujun Wang, Guirong Qiao, Huijin Fan, Kangming Jin, Biyun Huang, Wenmin Qiu, Yueguo Zou, Jinzhong Xie and Renying Zhuo

Horticulturae 2021, 7(5), 98; https://doi.org/10.3390/horticulturae7050098 - 3 May 2021

Cited by 1 | Viewed by 2792

Abstract

The selection of superior lines is extremely important to improve the utilization rate and economic value of bamboo. In this research, 120 anther-regenerated bamboo lines were planted in the field, and the survival rate reached 84.2% one year after planting. During five years [...] Read more.

The selection of superior lines is extremely important to improve the utilization rate and economic value of bamboo. In this research, 120 anther-regenerated bamboo lines were planted in the field, and the survival rate reached 84.2% one year after planting. During five years of observations, we continuously measured and recorded the number of shoots and the size of the new bamboo of these regenerated lines. The results showed that there were considerable differences in culm size and growth rate among the different lines. After comprehensive evaluation, we found that one of the lines (P82) had obvious advantages in culm size and growth rate compared with the others. The chromosome ploidy of line P82 and the other three lines (P38, P84, and P34) was detected. It was found that P82 was hexaploid, while the other three lines were dodecaploid. Nutritional components of the P82 shoots were further detected. The results showed that the content of soluble sugar was 1.4%, the content of free amino acid was 3.5 g·kg⁻¹ (FW, fresh weight), and the content of protein was 14.8 g·kg⁻¹ (FW), and there were no significant differences compared with the local wild mature bamboo. Anatomical analysis showed that the vascular bundle size of the line P82 (hexaploid) was significantly larger than that of line P38 (dodecaploid), and the length of parenchyma cells in the culm wall of line P82 was similar to that of line P38, however, the cell width of line P82 was significantly wider than that of line P38. In this study, the breeding of superior lines of regenerated bamboo plants from an anther culture was realized, which provided an example for a new method for selecting superior lines from an anther culture, and also enriched the resources of superior lines of D. latiflorus. Full article

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