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In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants

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A special issue of Horticulturae (ISSN 2311-7524). This special issue belongs to the section "Propagation and Seeds".

Deadline for manuscript submissions: closed (10 December 2023) | Viewed by 35536

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Special Issue Editors

Dr. Jean Carlos Bettoni

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Independent Researcher, 35 Brasil Correia Street, Videira 89560510, Santa Catarina, Brazil
Interests: germplasm conservation; cryopreservation procedures; pathogen eradication; horticultural species; plant tissue culture; micropropagation
Special Issues, Collections and Topics in MDPI journals

Dr. Min-Rui Wang

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College of Horticulture, Northwest A&F University, Yangling 712100, China
Interests: plant tissue culture; cryopreservation; abiotic stress; pathogen eradication

Prof. Dr. Qiao-Chun Wang

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Guest Editor

College of Horticulture, Northwest A&F University, Yangling 712100, China
Interests: cryopreservation; plant regeneration; virus eradication

Special Issue Information

Dear Colleagues,

In vitro tissue culture technologies provide novel tools for improving plant production. Organogenesis and somatic embryogenesis are the two pathways for plant regeneration, and have been widely used for in vitro micropropagation and germplasm conservation of horticultural species.

Horticultural plants are usually vegetatively propagated by grafting, rooting of cuttings and layering. The disadvantages of the traditional propagation methods are that they are season-dependent and labor-consuming. Propagated materials are also exposed to attacks of pathogens and pests, and to severe environmental conditions such as drought and extreme temperatures. In vitro micropropagation can avoid these adverse factors and has been widely used for the production of propagating materials of horticultural crops (production of disease-free propagules), particularly perennial woody species and ornamental plants.

The availability of and easy access to genetic resources of diverse plant species is a prerequisite in the breeding of elite cultivars by both classical and novel biotechnological programs. In vitro culture technologies have been widely used to establish medium-term (in vitro conservation) and long-term (cryopreservation) preservation methods for the germplasms of horticultural plants. In vitro conservation and cryopreservation strategies avoid the exposure of stored materials to both adverse abiotic and biotic conditions, and are also cost-effective, which are otherwise involved in field gene collections. Cryopreservation is currently considered an ideal means for long-term storage of plant genetic resources.

This Special Issue publishes research articles and reviews addressing in vitro plant regeneration, micropropagation and germplasm conservation of horticultural plants.

Dr. Jean Carlos Bettoni
Dr. Min-Rui Wang
Prof. Dr. Qiao-Chun Wang
Guest Editors

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Keywords

cryopreservation
in vitro culture
in vitro conservation
micropropgation
plant regeneration

Published Papers (12 papers)

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Editorial

Jump to: Research, Review, Other

5 pages, 197 KiB

Open AccessEditorial

In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants

by Jean Carlos Bettoni, Min-Rui Wang and Qiao-Chun Wang

Horticulturae 2024, 10(1), 45; https://doi.org/10.3390/horticulturae10010045 - 02 Jan 2024

Viewed by 1474

Abstract

In vitro tissue culture technologies provide novel tools for improving plant production [...] Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

Research

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14 pages, 914 KiB

Open AccessArticle

Morpho-Anatomical and Physiological Assessments of Cryo-Derived Pineapple Plants (Ananas comosus var. comosus) after Acclimatization

by Ariel Villalobos-Olivera, José Carlos Lorenzo-Feijoo, Nicolás Quintana-Bernabé, Michel Leiva-Mora, Jean Carlos Bettoni and Marcos Edel Martínez-Montero

Horticulturae 2023, 9(7), 841; https://doi.org/10.3390/horticulturae9070841 - 24 Jul 2023

Cited by 2 | Viewed by 1447

Abstract

Studies on the morpho-physiology of cryo-derived pineapple plants after acclimatization have been quite limited. Therefore, in the present study, the morpho-anatomical and physiological characteristics of cryo-derived Ananas comosus var. comosus ‘MD-2’ plants after acclimatization were investigated. Plants obtained from cryopreserved and non-cryopreserved shoot tips, as well as in vitro stock cultures (control), showed similar morphological development (viz. plant height, number of leaves, D leaf length, D leaf width, D leaf area, diameter of stem base, number of roots, plant fresh weight and plant dry weight) to conventionally micropropagated and non-cryopreserved plants. The pineapple plantlets developed efficient anatomical leaf structures that allowed them to adapt to the transition process from in vitro to ex vitro. In all groups of plants, the content of water and chlorophylls (a, a + b, a/b) decreased during the first 15 days of acclimatization and then remained constant until the end of the evaluation. The mesophilic succulence index increased to its maximum value after 15 days, then decreased and remained constant up to 45 days. Although physiological indicators fluctuated during the 45 days of acclimatization, no differences were observed in any of the indicators evaluated when plantlets obtained from cryopreserved shoot tips were compared with controls. The results of the plants from cryopreserved shoot tips show that they switched from C3 to Crassulacean acid metabolism, which denoted metabolic stability during acclimatization. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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15 pages, 35861 KiB

Open AccessArticle

Evaluation of Critical Points for Effective Cryopreservation of Four Different Citrus spp. Germplasm

by Damla Ekin Ozkaya, Fernanda Vidigal Duarte Souza and Ergun Kaya

Horticulturae 2022, 8(11), 995; https://doi.org/10.3390/horticulturae8110995 - 26 Oct 2022

Cited by 5 | Viewed by 1315

Abstract

The different pre- and post-treatments are critical in cryopreservation procedures and affect the shoot tip regrowth after freezing. In the present study, the long-term storage of four citrus cultivars [Bodrum Mandarin (Citrus deliciosa Ten.); Klin Mandarin (Citrus nobilis Lauriro); White grapefruit and Red grapefruit (Citrus paradisi L.)] were carried out by droplet vitrification methods, and the critical points for effective cryopreservation of these species were determined. In this study, we investigated the effect of explant size, cold hardening treatments, sucrose concentrations, and media combinations on shoot regrowth after cryopreservation. The highest shoot tip regrowth, ranging from 13.3 to 33.3%, was achieved when they were obtained from 0.3 to 0.7 mm explants excised from cold hardened seedlings at 4 °C for three days that were then precultured in a medium containing 0.25 M of sucrose and treated with PVS2 at 0 °C for 45 min. In addition, it has been determined that a regeneration medium containing boric acid (H₃BO₃) or ferric ethylenediaminetetraacetate (FeEDDHA) increased the regeneration up to 33.3% after cryopreservation. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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13 pages, 1439 KiB

Open AccessArticle

How to Get More Silver? Culture Media Adjustment Targeting Surge of Silver Nanoparticle Penetration in Apricot Tissue during in Vitro Micropropagation

by Cristian Pérez-Caselles, Nuria Alburquerque, Lydia Faize, Nina Bogdanchikova, Juan Carlos García-Ramos, Ana G. Rodríguez-Hernández, Alexey Pestryakov and Lorenzo Burgos

Horticulturae 2022, 8(10), 855; https://doi.org/10.3390/horticulturae8100855 - 20 Sep 2022

Cited by 4 | Viewed by 1540

Abstract

The use of silver nanoparticles (AgNPs) is increasing nowadays due to their applications against phytopathogens. Temporary Immersion Systems (TIS) allow the micropropagation of plants in liquid media. This work aims to develop an effective protocol for apricot micropropagation in TIS and to study the necessary conditions to introduce AgNPs in apricot plants, as well as the effect of its application on proliferation-related parameters. AgNPs were introduced in different media at a concentration of 100 mg L⁻¹ to test the incorporation of silver to plant tissues. Silver content analysis was made by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). The effect of initial shoot density and the addition of AgNPs on micropropagation were evaluated after four weeks in culture on TIS. Productivity, proliferation, shoot-length and leave surface were measured. The better micropropagation rate was obtained with 40 initial shoots, 2 min of immersion every 6 h and 3 min of aeration every 3 h. To introduce AgNPs in apricot plants it is necessary to culture them in liquid media without chloride in its composition. These results will contribute to the development of an in vitro protocol for virus inhibition by AgNPs application. This depends on the introduction of Ag nanoparticles within the plant tissues, and this is not possible if AgNPs after interaction with Cl^- ions precipitate as silver chloride salts. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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14 pages, 29763 KiB

Open AccessArticle

An Optimized Protocol for Indirect Organogenesis from Root Explants of Agapanthus praecox subsp. orientalis ‘Big Blue’

by Qianwen Tang, Xiangxin Guo, Yuanshan Zhang, Qingyun Li, Guanqun Chen, Huale Sun, Weiming Wang and Xiaohui Shen

Horticulturae 2022, 8(8), 715; https://doi.org/10.3390/horticulturae8080715 - 09 Aug 2022

Cited by 4 | Viewed by 1740

Abstract

Agapanthus praecox has become a burgeoning variety in the flower market due to its high ornamental value with unique large blue-purple inflorescence. For rapid entering into the market, tissue culture technology or organogenesis has an attractive application over the conventional reproduction approach. In this study, a highly efficient protocol based on indirect organogenesis has been successfully established for A. praecox subsp. orientalis ‘Big Blue’. Two types of explants, root tips versus root segments, were compared for callus induction frequency in response to the induction culture media. The induction media contain Murashige and Skoog’s (MS) Basal Salt supplemented with various concentrations of picloram (PIC), 2,4-Dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ), kinetin (KT) and naphthalene acetic acid (NAA). Of the two types of explants, root tips were found to be more effective for callus induction than root segments. Among the induction media tested, the highest callus induction rate (100.00%) was achieved when cultured on MS supplemented with 2.0 mg/L PIC, 1.5 mg/L KT and 0.1 mg/L NAA, which was probably accredited to higher endogenous phytohormone contents, especially of 3-indoleacetic (IAA). The optimal medium for callus proliferation was MS + 1.0 mg/L PIC + 1.0 mg/L 6-BA + 0.4 mg/L NAA, and the fresh weight increased by 72.74%. After being transferred onto the adventitious bud induction medium for 25 days, shoots were dedifferentiated from the surface of the flourishing callus, which then developed to the plantlet with roots in 90 days. The plantlets were transplanted in a greenhouse with a survival rate of 92.86%. This study innovatively established an indirect organogenesis tissue culture system of A. praecox with roots as explants, which provided a practical reference in its application. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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14 pages, 8843 KiB

Open AccessEditor’s ChoiceArticle

In Vitro and In Vivo Performance of Plum (Prunus domestica L.) Pollen from the Anthers Stored at Distinct Temperatures for Different Periods

by Milena Đorđević, Tatjana Vujović, Radosav Cerović, Ivana Glišić, Nebojša Milošević, Slađana Marić, Sanja Radičević, Milica Fotirić Akšić and Mekjell Meland

Horticulturae 2022, 8(7), 616; https://doi.org/10.3390/horticulturae8070616 - 07 Jul 2022

Cited by 5 | Viewed by 1846

Abstract

A study was conducted to investigate the effect of different storage periods and temperatures on pollen viability in vitro and in vivo in plum genotypes ‘Valerija’, ‘Čačanska Lepotica’ and ‘Valjevka’. In vitro pollen viability was tested at day 0 (fresh dry pollen) and after 3, 6, 9 and 12 months of storage at four different temperatures (4, −20, −80 and −196 °C), and in vivo after 12 months of storage at distinct temperatures. In vitro germination and fluorescein diacetate (FDA) staining methods were used to test pollen viability, while aniline blue staining was used for observing in vivo pollen tube growth. Fresh pollen germination and viability ranged from 42.35 to 63.79% (‘Valjevka’ and ‘Čačanska Lepotica’, respectively) and 54.58 to 62.15%, (‘Valjevka’ and ‘Valerija’, respectively). With storage at 4 °C, pollen viability and germination decreased over the period, with the lowest value after 12 months of storage. Pollen germination and viability for the other storage temperatures (−20, −80 and −196 °C) were higher than 30% by the end of the 12 months. Pollination using pollen stored at 4 °C showed that pollen tube growth mostly ended in the lower part of the style. With the other storage temperatures, pollen tube growth was similar, ranging between 50 and 100% of the pistils with pollen tubes penetrated into the nucellus of the ovule in the genotype ‘Čačanska Lepotica’. The results of these findings will have implications for plum pollen breeding and conservation. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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17 pages, 3442 KiB

Open AccessArticle

In Vitro Techniques for Shipping of Micropropagated Plant Materials

by Jingwei Li, Min He, Xiuhong Xu, Tingmin Huang, Huan Tian and Wanping Zhang

Horticulturae 2022, 8(7), 609; https://doi.org/10.3390/horticulturae8070609 - 06 Jul 2022

Cited by 2 | Viewed by 1780

Abstract

Shipping of in vitro micro-cuttings in tubes or jars is a frequently used method as the plants are more likely to quickly reproduce and comply with quarantine regulations in plant germplasm distribution. However, these containers are fragile during transportation. To diminish the risk associated with the long-distance shipping of in vitro plants, a safe and widely applicable packing and conservation technique based on microplate and slow growth was developed in this study. Potato cultivar ZHB and ginger cultivar G-2 were used to optimize the system with microplates (96 wells), vacuum-sealed packaging, and slow-growth techniques. Under regular culture conditions, packing in vacuum-sealed microplates reduced the survival of ZHB and G-2 micro-cuttings to 85.8% and 20.0%, respectively, and regeneration to 61.8% and 0%, respectively. Reducing the temperature to 10 °C maintained the survival of ZHB and G-2 micro-cuttings in the range of 83.3–100% after 60 days. Exposure to darkness decreased the survival of G-2 and inhibited regrowth. Thus, conservation in darkness at 10 °C is suggested. The effects of iron concentration and plant growth retardants were further assessed. The addition of 1/4 MS medium combined with 100 mg/L chlormequat chloride (CCC) resulted in full survival and growth inhibition of plantlets, without malformation identified. Finally, incubation with 1/4 MS medium supplemented with 100 mg/L CCC in vacuum-sealed microplates at 10 °C in the dark resulted in high survival and suppressed germination. Sweet potato HXS was incubated as well to test the broad-spectrum applications of the technique; 100% survival and 6.7% germination was gained. Morphological indices of released cuttings recovered to control levels after two cycles of subculture in MS medium. A 0.1–0.2% genetic variation was detected by SSR and ISSR, suggesting genetic stability of the conserved samples. Finally, micro-cuttings were safely transported to cities located thousands of kilometers away without package and sample damage. Our results enable easy distribution of in vitro plant germplasms. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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18 pages, 4104 KiB

Open AccessArticle

Micropropagation of Plum (Prunus domestica L.) in Bioreactors Using Photomixotrophic and Photoautotrophic Conditions

by Diego Gago, Conchi Sánchez, Anxela Aldrey, Colin Bruce Christie, María Ángeles Bernal and Nieves Vidal

Horticulturae 2022, 8(4), 286; https://doi.org/10.3390/horticulturae8040286 - 29 Mar 2022

Cited by 10 | Viewed by 3148

Abstract

In this study, we propagated two old Galician plum varieties in liquid medium using a temporary immersion system with RITA© bioreactors. Environmental variables including culture system, light intensity, CO₂ enrichment, immersion frequency and sucrose supplementation were evaluated in relation to in vitro proliferation, physiological status and ex vitro performance. Bioreactors were superior to jars for culturing shoots in photomixotrophic conditions, producing up to 2 times more shoot numbers and up to 1.7 times more shoot length (depending on the genotype) using shoot clusters. The number and quality of shoots were positively influenced by the sucrose concentration in the medium, plus by the light and gaseous environment. For individual apical sections the best response occurred with 3% sucrose, 150 µmol m⁻² s⁻¹ photosynthetic photon flux density and 2000 ppm CO₂, averaging 2.5 shoots per explant, 26 mm shoot length and 240 mm² leaf area, while with 50 µmol m⁻² s⁻¹ light and ambient CO₂ (400 ppm) values decreased to 1.2 shoots per explant, 14 mm of shoot length and 160 mm² of leaf area. Shoots cultured photoautotrophically (without sucrose) were successfully rooted and acclimated despite of showing limited growth, low photosynthetic pigments, carbohydrate, phenolic and antioxidant contents during the multiplication phase. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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13 pages, 1817 KiB

Open AccessArticle

In Vitro Culture of Eremurus spectabilis (Liliaceae), a Rare Ornamental and Medicinal Plant, through Root Explants

by Yeganeh Basiri, Nematollah Etemadi, Mahdi Alizadeh and Javad Alizargar

Horticulturae 2022, 8(3), 202; https://doi.org/10.3390/horticulturae8030202 - 25 Feb 2022

Cited by 4 | Viewed by 2659

Abstract

Eremurus spectabilis M. Bieb, a perennial herbaceous wild species, is commonly used in the horticultural, ornamental, and pharmaceutical markets. Studies on the tissue culture systems for this species would be beneficial for mass multiplication as well as for future breeding programs. An in vitro propagation technique was established here using tuberous root explants as unique and responsive starting materials for culture initiation. The proliferated calli were sub-cultured on shoot proliferation media and regenerated microshoots were assessed. The shoot proliferation rate, leaf number, leaf length, and chlorophyll and carotenoid contents were recorded. The highest callus induction per explant (76.67%), callus dry weight (10.25 mg), callus firmness ratio (3.97), and callus color intensity ratio (2.83) were observed in explants inoculated on Murashige and Skoog (MS) medium supplemented with 10.0 mgL⁻¹ 6-benzylaminopurine (BAP). The highest shoot proliferation rates were obtained when calli were sub-cultured on MS or Schenk and Hildebrandt (SH) basal media supplemented with 2.0 mgL⁻¹ BAP. The half-strength MS medium fortified with 4.0% sucrose + 2.0 mgL⁻¹ indole butyric acid (IBA) + 200 mgL⁻¹ activated charcoal was a superior combination for root emergence and rooting parameters. Regenerated plantlets were then successfully adapted to ex vitro conditions. The reported protocol can be exploited at a commercial scale following minor modification, or could be beneficial in the production of secondary metabolites in bioreactors where callus is required as fresh plant material. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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17 pages, 3271 KiB

Open AccessArticle

Development of an Improved Micropropagation Protocol for Red-Fleshed Pitaya ‘Da Hong’ with and without Activated Charcoal and Plant Growth Regulator Combinations

by Yu-Chi Lee and Jer-Chia Chang

Horticulturae 2022, 8(2), 104; https://doi.org/10.3390/horticulturae8020104 - 25 Jan 2022

Cited by 11 | Viewed by 3708

Abstract

Micropropagation protocols for red-fleshed Hylocereus species (Cactaceae) have been developed; however, these methods prolong the sprout duration from areoles and produce irregular micro-propagules in ‘Da Hong’ pitaya. Thus, the present study aimed to establish an improved micropropagation protocol for this cultivar. Shoot regeneration and root induction of self-pollinating seedling segments were evaluated in response to combinations of activated charcoal (AC; 200 mg/L), α-naphthaleneacetic acid (NAA; 0.05, 0.10, and 0.20 mg/L), and 6-benzylaminopurine (BAP; 1.00, 2.00, and 4.00 mg/L). The correlations among plantlet growth characteristics and plantlet survival rate after transplantation under field conditions were calculated. Increasing the NAA concentration increased the number of roots but reduced root length. The addition of AC enhanced shoot length and prevented the regeneration of dried-out, clustered, and abnormal shoots. Plantlets treated with 200 mg/L AC and 0.10 mg/L NAA produced the highest number of shoots, i.e., 4.1 shoots, which however, were shorter and lighter than those cultured with AC alone. Plantlets grown on medium supplemented with BAP showed no advantage in shoot number, shoot weight, plantlet surface area, or plantlet volume. The weight and shoot surface area of plantlets were strongly correlated. All plantlets grew well at 4 weeks post-transplantation. Overall, these results support this improved micropropagation method to regenerate robust ex vitro plantlets. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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Review

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20 pages, 1191 KiB

Open AccessReview

In Vitro Micrografting of Horticultural Plants: Method Development and the Use for Micropropagation

by Min-Rui Wang, Jean Carlos Bettoni, A-Ling Zhang, Xian Lu, Dong Zhang and Qiao-Chun Wang

Horticulturae 2022, 8(7), 576; https://doi.org/10.3390/horticulturae8070576 - 24 Jun 2022

Cited by 3 | Viewed by 10949

Abstract

In vitro micrografting is an important technique supporting the micropropagation of a range of plant species, particularly woody plant species. Over the past several decades, in vitro micrografting has become a strategy to facilitate shoot recovery and acclimatization of in vitro-grown horticultural species. This review focuses on studies on horticultural crops over the past two decades that cover the establishment of in vitro micrografting, discusses factors affecting the success of in vitro micrografting, and provides commentary on the contribution of micrografting applications to the field of micropropagation. Considering the important roles of micrografting in the restoration of vigor and rooting competence, in promotion of shoot recovery following somatic embryogenesis and organogenesis, and in facilitation of shoot regrowth after cryopreservation, the potential use of this technique in facilitation of genetic engineering and safe conservation of horticultural species are specially highlighted. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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Other

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11 pages, 1724 KiB

Open AccessEssay

Rapid and Efficient Regeneration of Rhododendron decorum from Flower Buds

by Hairong Wu, Qian Ao, Huie Li and Fenfang Long

Horticulturae 2023, 9(2), 264; https://doi.org/10.3390/horticulturae9020264 - 15 Feb 2023

Cited by 1 | Viewed by 1415

Abstract

Rhododendron decorum is a woody species with high ornamental and medical value. Herein, we introduce a novel in vitro regeneration method for R. decorum. We used flower buds to develop an efficient and rapid plant regeneration protocol. Sterile flower buds of R. decorum of a 2 cm size were used as explants to study the effects of the culture medium and plant growth regulators on the callus induction and adventitious shoot differentiation, proliferation, and rooting. According to the results, the optimal medium combination for callus induction was WPM + 1 mg/L TDZ + 0.2 mg/L NAA, and its induction rate reached 95.08%. The optimal medium combination for adventitious shoot differentiation from the callus was WPM + 0.5 mg/L TDZ + 0.1 mg/L NAA, and its differentiation rate reached 91.32%. The optimal medium combination for adventitious shoot proliferation was WPM + 2 mg/L ZT + 0.5 mg/L NAA, for which the proliferation rate reached 95.32% and the proliferation coefficient reached 9.45. The optimal medium combination for rooting from adventitious shoots was WPM + 0.1 mg/L NAA + 1 mg/L IBA, and its rooting rate reached 86.90%. The survival rates of the rooted regenerated plantlets exceeded 90% after acclimatization and transplantation. This regeneration system has the advantages of being simple and highly efficient, and it causes little damage to the shoots of the mother plants, laying a foundation for the plantlet propagation, genetic transformation, and new-variety breeding of R. decorum. Full article

(This article belongs to the Special Issue In Vitro Regeneration, Micropropagation and Germplasm Conservation of Horticultural Plants)

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