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14 pages, 1600 KiB

Open AccessArticle

Conservation of Green and White Ash Germplasm Using the Cryopreservation of Embryogenic Cultures

by Mason Richins, Cristian Montes and Scott Merkle

Plants 2024, 13(3), 352; https://doi.org/10.3390/plants13030352 - 24 Jan 2024

Viewed by 673

Abstract

Green ash (Fraxinus pennsylvanica) and white ash (F. americana) populations are currently experiencing major declines across their native ranges in North America due to infestation by the exotic insect pest emerald ash borer (Agrilus planipennis). The development [...] Read more.

Green ash (Fraxinus pennsylvanica) and white ash (F. americana) populations are currently experiencing major declines across their native ranges in North America due to infestation by the exotic insect pest emerald ash borer (Agrilus planipennis). The development of a reliable method for the long-term storage of green and white ash germplasm in the form of embryogenic cultures using cryopreservation would be a considerable aid to ash conservation efforts. We compared recovery percentages of cryopreserved green and white ash embryogenic cultures using vitrification versus slow cooling methods. Three Plant Vitrification Solution 2 (PVS2) exposure durations (40, 60, and 80 min) for vitrification and three DMSO concentrations (5%, 10%, and 15%) for slow cooling were tested for their effects on the percentage of cultures that regrew following cryostorage. Vitrification resulted in a higher overall culture recovery percentage (91%) compared to cultures that were cryostored using the slow cooling approach (39%), and a more rapid initiation of regrowth (5 days versus 2–3 weeks) resulted. Recovery from cryostorage by cultures using the slow cooling approach varied significantly (p < 0.05) between experiments and with genotype (p < 0.05). The recovery of vitrified tissue from cryostorage did not vary with genotype, species, or PVS2 exposure duration (p > 0.05). The vitrification cryopreservation protocol provides a reliable and versatile alternative to the traditional slow cooling method, strengthening our ability to preserve valuable ash germplasm for conservation and restoration. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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23 pages, 9785 KiB

Open AccessArticle

First Report on Mesophyll Protoplast Isolation and Regeneration System for the Duboisia Species

by Yuxin Xue, Jayeni Chathurika Amarathunga Hiti-Bandaralage, Zhangpan Hu, Zizhu Zhao and Neena Mitter

Plants 2024, 13(1), 40; https://doi.org/10.3390/plants13010040 - 21 Dec 2023

Viewed by 1327

Abstract

The Duboisia species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, [...] Read more.

The Duboisia species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, employing both conventional breeding methods and advanced biotechnology tools. The primary objective of this research was to establish a highly efficient method for isolating mesophyll protoplasts and facilitating their regeneration, thereby laying a robust foundation for the application of various advanced plant biotechnology tools in the pursuit of genetic enhancement. The mesophyll protoplast isolation process was developed for hybrid D. myoporoides × D. hopwoodii with careful optimisation of the following parameters: leaf strip size; incubation conditions; physical treatment; and enzyme concentration. The optimal parameters were combined in each individual step; the best enzyme concentration was determined to be 2% (w/v) cellulysin and 0.5% (w/v) macerase. Protoplast yield was found to be greatly affected by the enzyme concentrations. The isolated protoplasts were cultured at a density of 0.5 × 10⁵ to best sustain the highest cell division (33.2%) and a microcalli induction frequency of 17.9%. After 40 days of culture in a modified KM8P medium at 25 °C in darkness, visible microcalli were transferred to a solidified Murashige and Skoog (MS) medium with 1 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction under a 16 h photoperiod. After 30 days of culture, compact organogenic calli were transferred into a solid MS medium with 6-benzylaminopurine (BA) alone or thidiazuron (TDZ) alone or in combination with BA or naphthalene acetic acid (NAA) for shoot regeneration. The maximum shoot regeneration frequency (63.3%) was observed in the medium with 1.5 mg L⁻¹ TDZ alone. For the first time, a reliable protoplast isolation and regeneration system from mesophyll cells was established for Duboisia with high protoplast viability, successful microcalli formation, and intact plant regeneration. This innovation will significantly contribute towards the genetic enhancement of the Duboisia species. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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20 pages, 5156 KiB

Open AccessArticle

Transcriptomics and Physiological Analyses Reveal Changes in Paclitaxel Production and Physiological Properties in Taxus cuspidata Suspension Cells in Response to Elicitors

by Zirui Zhao, Yajing Zhang, Wenlong Li, Yuanhu Tang and Shujie Wang

Plants 2023, 12(22), 3817; https://doi.org/10.3390/plants12223817 - 10 Nov 2023

Viewed by 893

Abstract

In this research, the cell growth, physiological, and biochemical reactions, as well as the paclitaxel production, of Taxus cuspidata suspension cells after treatment with polyethylene glycol (PEG), cyclodextrin (CD), or salicylic acid (SA) (alone or in combination) were investigated. To reveal the paclitaxel [...] Read more.

In this research, the cell growth, physiological, and biochemical reactions, as well as the paclitaxel production, of Taxus cuspidata suspension cells after treatment with polyethylene glycol (PEG), cyclodextrin (CD), or salicylic acid (SA) (alone or in combination) were investigated. To reveal the paclitaxel synthesis mechanism of T. cuspidata suspension cells under elicitor treatment, the transcriptomics of the Control group and P + C + S group (PEG + CD + SA) were compared. The results show that there were no significant differences in cell biomass after 5 days of elicitor treatments. However, the content of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), and the activities of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) after elicitor combination treatments were decreased compared with the single-elicitor treatment. Meanwhile, the antioxidant enzyme activity (superoxide dismutase (SOD), catalase (CAT), and peroxidase (PO)) and the contents of soluble sugar and soluble protein were increased after combination elicitor treatments. Additionally, the paclitaxel yield after treatment with the combination of all three elicitors (P + C + S) was 6.02 times higher than that of the Control group, thus indicating that the combination elicitor treatments had a significant effect on paclitaxel production in T. cuspidata cell suspension culture. Transcriptomics analysis revealed 13,623 differentially expressed genes (DEGs) between the Control and P + C + S treatment groups. Both GO and KEGG analyses showed that the DEGs mainly affected metabolic processes. DEGs associated with antioxidant enzymes, paclitaxel biosynthesis enzymes, and transcription factors were identified. It can be hypothesized that the oxidative stress of suspension cells occurred with elicitor stimulation, thereby leading to a defense response and an up-regulation of the gene expression associated with antioxidant enzymes, paclitaxel synthesis enzymes, and paclitaxel synthesis transcription factors; this ultimately increased the production of paclitaxel. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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20 pages, 1023 KiB

Open AccessArticle

Investigation of the Influence of Polyamines on Mature Embryo Culture and DNA Methylation of Wheat (Triticum aestivum L.) Using the Machine Learning Algorithm Method

by Barış Eren, Aras Türkoğlu, Kamil Haliloğlu, Fatih Demirel, Kamila Nowosad, Güller Özkan, Gniewko Niedbała, Alireza Pour-Aboughadareh, Henryk Bujak and Jan Bocianowski

Plants 2023, 12(18), 3261; https://doi.org/10.3390/plants12183261 - 13 Sep 2023

Cited by 8 | Viewed by 1325

Abstract

Numerous factors can impact the efficiency of callus formation and in vitro regeneration in wheat cultures through the introduction of exogenous polyamines (PAs). The present study aimed to investigate in vitro plant regeneration and DNA methylation patterns utilizing the inter-primer binding site (iPBS) [...] Read more.

Numerous factors can impact the efficiency of callus formation and in vitro regeneration in wheat cultures through the introduction of exogenous polyamines (PAs). The present study aimed to investigate in vitro plant regeneration and DNA methylation patterns utilizing the inter-primer binding site (iPBS) retrotransposon and coupled restriction enzyme digestion–iPBS (CRED–iPBS) methods in wheat. This investigation involved the application of distinct types of PAs (Put: putrescine, Spd: spermidine, and Spm: spermine) at varying concentrations (0, 0.5, 1, and 1.5 mM). The subsequent outcomes were subjected to predictive modeling using diverse machine learning (ML) algorithms. Based on the specific polyamine type and concentration utilized, the results indicated that 1 mM Put and Spd were the most favorable PAs for supporting endosperm-associated mature embryos. Employing an epigenetic approach, Put at concentrations of 0.5 and 1.5 mM exhibited the highest levels of genomic template stability (GTS) (73.9%). Elevated Spd levels correlated with DNA hypermethylation while reduced Spm levels were linked to DNA hypomethylation. The in vitro and epigenetic characteristics were predicted using ML techniques such as the support vector machine (SVM), extreme gradient boosting (XGBoost), and random forest (RF) models. These models were employed to establish relationships between input variables (PAs, concentration, GTS rates, Msp I polymorphism, and Hpa II polymorphism) and output parameters (in vitro measurements). This comparative analysis aimed to evaluate the performance of the models and interpret the generated data. The outcomes demonstrated that the XGBoost method exhibited the highest performance scores for callus induction (CI%), regeneration efficiency (RE), and the number of plantlets (NP), with R² scores explaining 38.3%, 73.8%, and 85.3% of the variances, respectively. Additionally, the RF algorithm explained 41.5% of the total variance and showcased superior efficacy in terms of embryogenic callus induction (ECI%). Furthermore, the SVM model, which provided the most robust statistics for responding embryogenic calluses (RECs%), yielded an R² value of 84.1%, signifying its ability to account for a substantial portion of the total variance present in the data. In summary, this study exemplifies the application of diverse ML models to the cultivation of mature wheat embryos in the presence of various exogenous PAs and concentrations. Additionally, it explores the impact of polymorphic variations in the CRED–iPBS profile and DNA methylation on epigenetic changes, thereby contributing to a comprehensive understanding of these regulatory mechanisms. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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12 pages, 1932 KiB

Open AccessArticle

Improvements in Protoplast Isolation Protocol and Regeneration of Different Cabbage (Brassica oleracea var. capitata L.) Cultivars

by Ester Stajič

Plants 2023, 12(17), 3074; https://doi.org/10.3390/plants12173074 - 27 Aug 2023

Cited by 1 | Viewed by 2594

Abstract

Protoplasts are a versatile tool in plant biotechnology since they can be used for basic biological studies as well as for breeding strategies based on genome editing. An efficient protoplast isolation protocol is essential for conducting protoplast-based studies. To optimize the protoplast isolation [...] Read more.

Protoplasts are a versatile tool in plant biotechnology since they can be used for basic biological studies as well as for breeding strategies based on genome editing. An efficient protoplast isolation protocol is essential for conducting protoplast-based studies. To optimize the protoplast isolation protocol in cabbage (Brassica oleracea var. capitata L.), different enzyme solutions were tested for the isolation of leaf mesophyll protoplasts. In our experiments, the combination of 0.5% Cellulase Onozuka RS and 0.1% Macerozyme R-10 showed the best result. The optimized protocol proved suitable for the isolation of protoplasts from five different cabbage cultivars with yields ranging from 2.38 to 4.63 × 10⁶ protoplasts/g fresh weight (fw) and a viability of 93% or more. After three weeks in culture, protoplasts from all of the tested cultivars formed micro-calli, but further callus growth and shoot regeneration depended strongly on the genotype and regeneration protocol used. For shoot formation, 1 mg/L BAP in combination with auxin 0.2 mg/L NAA showed the best results with a regeneration of 23.5%. The results obtained will contribute to the development of different applications of cabbage protoplasts and facilitate the breeding process of this important horticultural crop. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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12 pages, 1480 KiB

Open AccessArticle

Development of a New Micropropagation Protocol and Transfer of In Vitro Plants to In Vivo Conditions for Cascade Hop

by Nicolò Iacuzzi, Francesco Salamone, Davide Farruggia, Noemi Tortorici, Lorena Vultaggio and Teresa Tuttolomondo

Plants 2023, 12(15), 2877; https://doi.org/10.3390/plants12152877 - 06 Aug 2023

Cited by 1 | Viewed by 2207

Abstract

The vegetative propagation of hops, despite being a reliable method, is not very common due to the unavailability of the plant material. In this study, the technique of in vitro propagation was applied to the Cascade variety of Humulus lupulus L. The plant [...] Read more.

The vegetative propagation of hops, despite being a reliable method, is not very common due to the unavailability of the plant material. In this study, the technique of in vitro propagation was applied to the Cascade variety of Humulus lupulus L. The plant material was collected from a private field in Sicily; the explants were subjected to sterilization before in vitro culture. Single-node explants were placed in in vitro culture in nine different culture media for multiplication. Thidiazuron (TDZ), Benzyladenine (BAP) and meta-Topoline (mT) were tested for multiplication phase. For the rooting phase, five types of different culture media were evaluated. Binodal cuttings coming from the previous multiplication test were placed in the culture. The rooting media differ from each other in the concentration and ratio of two auxin hormones: Indolo-3-acetic acid (IAA) and Indole-3-butyric acid (IBA). In vitro rooted plants obtained from the rooting phase were transferred to ex vitro conditions in a microbox with agri-perlite and a solution containing Murashige and Skoog (MS) basal medium at half concentration. With a culture medium containing the highest TDZ doses (H6) and combination with cytokinin (H8 and H9), the highest shoot percentage was obtained. After 3 months of in vitro culture, the highest shoot percentage was observed in the culture medium with 2 mL L⁻¹ of BAP. The highest rooting percentage, roots numbers and root length were found when the culture medium was supplemented with 1 mL L⁻¹ of IAA. The usage of agri-perlite and MS at half concentration, without PGR, allowed us to obtain a 99.1% survival rate. This micropropagation protocol is useful for obtaining virus-free plants and for the development of the brewery industry. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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22 pages, 2630 KiB

Open AccessArticle

Primary Metabolite Screening Shows Significant Differences between Embryogenic and Non-Embryogenic Callus of Tamarillo (Solanum betaceum Cav.)

by André Caeiro, Ivana Jarak, Sandra Correia, Jorge Canhoto and Rui Carvalho

Plants 2023, 12(15), 2869; https://doi.org/10.3390/plants12152869 - 04 Aug 2023

Viewed by 1061

Abstract

Tamarillo is a solanaceous tree that has been extensively studied in terms of in vitro clonal propagation, namely somatic embryogenesis. In this work, a protocol of indirect somatic embryogenesis was applied to obtain embryogenic and non-embryogenic callus from leaf segments. Nuclear magnetic resonance [...] Read more.

Tamarillo is a solanaceous tree that has been extensively studied in terms of in vitro clonal propagation, namely somatic embryogenesis. In this work, a protocol of indirect somatic embryogenesis was applied to obtain embryogenic and non-embryogenic callus from leaf segments. Nuclear magnetic resonance spectroscopy was used to analyze the primary metabolome of these distinct calli to elucidate possible differentiation mechanisms from the common genetic background callus. Standard multivariate analysis methods were then applied, and were complemented by univariate statistical methods to identify differentially expressed primary metabolites and related metabolic pathways. The results showed carbohydrate and lipid metabolism to be the most relevant in all the calli assayed, with most discriminant metabolites being fructose, glucose and to a lesser extent choline. The glycolytic rate was higher in embryogenic calli, which shows, overall, a higher rate of sugar catabolism and a different profile of phospholipids with a choline/ethanolamine analysis. In general, our results show that a distinct primary metabolome between embryogenic and non-embryogenic calli occurs and that intracellular levels of fructose and sucrose and the glucose to sucrose ratio seem to be good candidates as biochemical biomarkers of embryogenic competence. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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13 pages, 2646 KiB

Open AccessArticle

In Vitro Propagation and Genetic Uniformity Assessment of Manglietiastrum sinicum: A Critically Endangered Magnoliaceae Species

by Yiyang Luo, Keyuan Zheng, Xiaodi Liu, Jialu Tao, Xugao Sun, Yanwen Deng and Xiaomei Deng

Plants 2023, 12(13), 2500; https://doi.org/10.3390/plants12132500 - 30 Jun 2023

Cited by 2 | Viewed by 1208

Abstract

Manglietiastrum sinicum Y.W. Law is a critically endangered species with great ornamental and commercial value, which urgently requires protection. We tested different combinations of basal media and plant growth regulators to determine (i) the optimal conditions for bud induction and proliferation of explants [...] Read more.

Manglietiastrum sinicum Y.W. Law is a critically endangered species with great ornamental and commercial value, which urgently requires protection. We tested different combinations of basal media and plant growth regulators to determine (i) the optimal conditions for bud induction and proliferation of explants and (ii) optimal rooting conditions. RAPD- and ISSR-PCR were used to assess the genetic fidelity of regenerated plantlets. Murashige and Skoog medium (MS) supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.05 mg/L indole-3-butyric acid (IBA) is the optimal medium for bud induction (100% induction). MSM medium (a special basal medium for M. sinicum) was more suitable for the efficient proliferation and rooting of M. sinicum. Maximum bud proliferation rate (446.20%) was obtained on MSM, with 0.4 mg/L BA, 0.5 mg/L kinetin, and 0.06 mg/L IBA, while maximum root induction rate (88.89%) was obtained on MSM supplemented with 0.4 mg/L 1-naphthylacetic acid and 1.0 mg/L IBA with a 7-day initial darkness treatment. The rooted plantlets were transferred to a substrate containing peat soil, perlite, coconut chaff, and bark (volume ratio 2:1:1:1), with a resulting survival rate of 92.2%. RAPD and ISSR markers confirmed the genetic uniformity and stability of regenerated plants. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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17 pages, 3632 KiB

Open AccessArticle

How Scarification, GA₃ and Graphene Oxide Influence the In Vitro Establishment and Development of Strelitzia

by Patrícia Duarte de Oliveira Paiva, Diogo Pedrosa Correa da Silva, Bruna Raphaella da Silva, Israela Pimenta de Sousa, Renato Paiva and Michele Valquíria dos Reis

Plants 2023, 12(11), 2142; https://doi.org/10.3390/plants12112142 - 29 May 2023

Viewed by 1185

Abstract

The propagation of strelitzia plants can be carried out in vitro as an alternative to combine the aseptic conditions of the culture medium with the use of strategies to promote germination and controlled abiotic conditions. However, this technique is still limited by the [...] Read more.

The propagation of strelitzia plants can be carried out in vitro as an alternative to combine the aseptic conditions of the culture medium with the use of strategies to promote germination and controlled abiotic conditions. However, this technique is still limited by the prolonged time and low percentage of seed germination, which is the most viable explant source, due to dormancy. Thus, the objective of this study was to evaluate the influence of chemical and physical scarification processes of seeds combined with gibberellic acid (GA₃), as well as the effect of graphene oxide in the in vitro cultivation of strelitzia plants. Seeds were subjected to chemical scarification with sulfuric acid for different periods (10 to 60 min) and physical scarification (sandpaper), in addition to a control treatment without scarification. After disinfection, the seeds were inoculated in MS (Murashige and Skoog) medium with 30 g L⁻¹ sucrose, 0.4 g L⁻¹ PVPP (polyvinylpyrrolidone), 2.5 g L⁻¹ Phytagel^®, and GA₃ at different concentrations. Growth data and antioxidant system responses were measured from the formed seedlings. In another experiment, the seeds were cultivated in vitro in the presence of graphene oxide at different concentrations. The results showed that the highest germination was observed in seeds scarified with sulfuric acid for 30 and 40 min, regardless of the addition of GA₃. After 60 days of in vitro cultivation, physical scarification and scarification time with sulfuric acid promoted greater shoot and root length. The highest seedling survival was observed when the seeds were immersed for 30 min (86.66%) and 40 min (80%) in sulfuric acid without GA₃. The concentration of 50 mg L⁻¹ graphene oxide favored rhizome growth, while the concentration of 100 mg L⁻¹ favored shoot growth. Regarding the biochemical data, the different concentrations did not influence MDA (Malondialdehyde) levels, but caused fluctuations in antioxidant enzyme activities. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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22 pages, 4329 KiB

Open AccessArticle

Coconut Callus Initiation for Cell Suspension Culture

by Eveline Y. Y. Kong, Julianne Biddle, Sundaravelpandian Kalaipandian and Steve W. Adkins

Plants 2023, 12(4), 968; https://doi.org/10.3390/plants12040968 - 20 Feb 2023

Cited by 5 | Viewed by 2790

Abstract

The development of a cell suspension culture system for the scaling up of coconut embryogenic callus (EC) production would drastically improve efforts to achieve the large-scale production of high-quality clonal plantlets. To date, the hard nature of coconut EC appeared to be the [...] Read more.

The development of a cell suspension culture system for the scaling up of coconut embryogenic callus (EC) production would drastically improve efforts to achieve the large-scale production of high-quality clonal plantlets. To date, the hard nature of coconut EC appeared to be the main constraint for developing cell suspension cultures. Hence, this study attempted to acquire friable EC through the following approaches: The manipulation of (1) medium type and subculture frequency, (2) a reduced 2,4-dichlorophenoxy acetic acid concentration during subculture, (3) the nitrate level and the ammonium-to-nitrate ratio, and the addition of amino acid mixture, (4) the addition of L-proline, and (5) the reduction of medium nutrients. Unfortunately, none of these culture conditions produced friable coconut EC. Even though friable EC was not achieved via these approaches, some of the conditions were found to influence the formation of compact EC, therefore these results are important for further studies focused on somatic embryogenesis in coconut and other species. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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10 pages, 3908 KiB

Open AccessCommunication

A Simple but Effective Combination of pH Indicators for Plant Tissue Culture

by Bryn Funnekotter, Ricardo L. Mancera and Eric Bunn

Plants 2023, 12(4), 740; https://doi.org/10.3390/plants12040740 - 07 Feb 2023

Cited by 4 | Viewed by 3168

Abstract

The use of pH indicators provides a simple, semi-quantitative visual method for quickly assessing pH changes in tissue culture media; however, pH indicators are rarely used in routine plant tissue culture media. In this study, chlorophenol red, bromocresol purple, and bromocresol green were [...] Read more.

The use of pH indicators provides a simple, semi-quantitative visual method for quickly assessing pH changes in tissue culture media; however, pH indicators are rarely used in routine plant tissue culture media. In this study, chlorophenol red, bromocresol purple, and bromocresol green were tested to assess their functionality in the growth medium for plant shoot cultures. In addition, a combination of bromocresol green and bromocresol purple was tested to determine if they would widen the observable colour change to better assess pH changes in the medium. Varying the ratio of bromocresol green to bromocresol purple alters the pH at which the colour changes from blue to green to yellow, with a 1:3 ratio providing a useful pH range of 5–6.5, while a 1:1 ratio provides a useful pH range of 4.5–6. All the pH indicators showed no toxic side effects for the plant species tested in this study and were able to be autoclaved to ensure media sterility. The addition of these pH indicators to quickly assess media pH in large tissue culture collections can aid in routine maintenance. These pH indicators can be used as a ‘traffic light’ system, with blue indicating a high pH, green a normal pH, and yellow a low pH in the media. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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13 pages, 1137 KiB

Open AccessArticle

Optimized Recovery of Cryostored Dormant Buds of Mulberry Germplasm

by Ravish Choudhary, Surendra Kumar Malik, Rekha Chaudhury and Atmakuri Ananda Rao

Plants 2023, 12(2), 225; https://doi.org/10.3390/plants12020225 - 04 Jan 2023

Cited by 1 | Viewed by 1392

Abstract

A two-step freezing cryoprotocol preceded by desiccation to 15 to 25% moisture content was developed and successfully applied to winter dormant buds of mulberry (different Morus spp.) of a core set comprising 238 accessions studies in our laboratory. The survival and recovery percentage [...] Read more.

A two-step freezing cryoprotocol preceded by desiccation to 15 to 25% moisture content was developed and successfully applied to winter dormant buds of mulberry (different Morus spp.) of a core set comprising 238 accessions studies in our laboratory. The survival and recovery percentage of diverse accessions cryobanked for various periods were tested under in vitro conditions, and several factors were analyzed to determine their role in optimizing the recovery of low-viability accessions. The effect of rates of freezing and thawing (both fast and slow), were tested and recovery compared. Recovery conditions such as dark incubation and rehydration in sterile moist moss grass for different durations after cryopreservation led to a higher survival percentage compared to controls. Two different recovery culture media were compared for their efficiency in survival. On average, the survival under in vitro culture conditions using optimized conditions was high: above 60% in majority of the accessions. Dormant buds showed viability in the range of 25 to 100% with an average of 50.4%. The recovery percentage of winter dormant buds after cryopreservation via slow freezing and slow thawing with rehydration by moist moss grass for 2 h was recorded in the range from 63.3 to 90.9% with an average of 81.05%. Without rehydration, it ranged from 50 to 75% with an average of 60.4%. Regeneration of cryopreserved mulberry germplasm after 6 years of storage indicated no survival loss over different years of storage, and 33–40% of the accessions showed viability above 40%, up to a maximum of 100%. Maximum shoot formation (100%) was obtained from Morus alba. The majority of the accessions were rooted in vitro within 20–25 days of subculture in the auxin rich rooting media, except in wild species M. latifolia and M. laevigata, which took longer (45 to 60 days) for root development. All the rooted plantlets were then transferred to the field and successfully established in a glasshouse. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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Review

Jump to: Research

24 pages, 2207 KiB

Open AccessReview

Plant Heterotrophic Cultures: No Food, No Growth

by Roman K. Puzanskiy, Daria A. Romanyuk, Anastasia A. Kirpichnikova, Vladislav V. Yemelyanov and Maria F. Shishova

Plants 2024, 13(2), 277; https://doi.org/10.3390/plants13020277 - 17 Jan 2024

Viewed by 1208

Abstract

Plant cells are capable of uptaking exogenous organic substances. This inherited trait allows the development of heterotrophic cell cultures in various plants. The most common of them are Nicotiana tabacum and Arabidopsis thaliana. Plant cells are widely used in academic studies and [...] Read more.

Plant cells are capable of uptaking exogenous organic substances. This inherited trait allows the development of heterotrophic cell cultures in various plants. The most common of them are Nicotiana tabacum and Arabidopsis thaliana. Plant cells are widely used in academic studies and as factories for valuable substance production. The repertoire of compounds supporting the heterotrophic growth of plant cells is limited. The best growth of cultures is ensured by oligosaccharides and their cleavage products. Primarily, these are sucrose, raffinose, glucose and fructose. Other molecules such as glycerol, carbonic acids, starch, and mannitol have the ability to support growth occasionally, or in combination with another substrate. Culture growth is accompanied by processes of specialization, such as elongation growth. This determines the pattern of the carbon budget. Culture ageing is closely linked to substrate depletion, changes in medium composition, and cell physiological rearrangements. A lack of substrate leads to starvation, which results in a decrease in physiological activity and the mobilization of resources, and finally in the loss of viability. The cause of the instability of cultivated cells may be the non-optimal metabolism under cultural conditions or the insufficiency of internal regulation. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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20 pages, 363 KiB

Open AccessReview

From Petri Dish to Field: Plant Tissue Culture and Genetic Engineering of Oats for Improved Agricultural Outcomes

by Krishna Mohan Pathi and Thorben Sprink

Plants 2023, 12(21), 3782; https://doi.org/10.3390/plants12213782 - 06 Nov 2023

Cited by 2 | Viewed by 2506

Abstract

Oats (Avena sativa) hold immense economic and nutritional value as a versatile crop. They have long been recognized as an exceptional choice for human consumption and animal feed. Oats’ unique components, including proteins, starches, and β-glucans, have led to its widespread [...] Read more.

Oats (Avena sativa) hold immense economic and nutritional value as a versatile crop. They have long been recognized as an exceptional choice for human consumption and animal feed. Oats’ unique components, including proteins, starches, and β-glucans, have led to its widespread use in various food products such as bread, noodles, flakes, and milk. The popularity of oat milk as a vegan alternative to dairy milk has soared due to the increasing number of vegetarians/vegans and growing environmental awareness. Oat milk offers a sustainable option with reduced greenhouse gas emissions during its production, rendering it an appropriate choice for individuals who are lactose-intolerant or have dairy allergies. To ensure improved adaptability and enhanced nutrition, the development of new oat varieties is crucial, considering factors like cultivation, climate, and growing conditions. Plant cell culture plays a crucial role in both traditional and contemporary breeding methods. In classical breeding, plant cell culture facilitates the rapid production of double haploid plants, which can be employed to accelerate the breeding process. In modern breeding methods, it enables genetic manipulation and precise genome editing at the cellular level. This review delves into the importance of oats and their diverse applications, highlighting the advantages of plant cell culture in both classical and modern breeding methods. Specifically, it provides an overview of plant tissue culture, encompassing genetic transformation, haploid technology, protoplast technology, and genome editing. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

18 pages, 1319 KiB

Open AccessReview

Haploid Induction in Indica Rice: Exploring New Opportunities

by Ruwani Mayakaduwa and Tara Silva

Plants 2023, 12(17), 3118; https://doi.org/10.3390/plants12173118 - 30 Aug 2023

Cited by 2 | Viewed by 1716

Abstract

Haploid plants are of significant interest to crop breeders due to their ability to expedite the development of inbred lines. Chromosome-doubling of haploids, produced by either in vitro or in vivo methods, results in fully homozygous doubled haploids. For nearly five decades, in [...] Read more.

Haploid plants are of significant interest to crop breeders due to their ability to expedite the development of inbred lines. Chromosome-doubling of haploids, produced by either in vitro or in vivo methods, results in fully homozygous doubled haploids. For nearly five decades, in vitro methods of anther and microspore culture have been attempted in many crops. In rice, in vitro methods are used with some success in japonica cultivars, although indica types have remained recalcitrant to a large extent. This review aims to explore the reasons for the lack of success of in vitro methods in indica rice and discuss new advancements in in vivo haploid induction protocols in other cereals and their relevance to rice. In particular, the current level of understanding of in vivo haploid inducer systems that utilize MTL and CENH3 mutants is analyzed in detail. One notable advantage of in vivo haploid induction systems is that they do not require tissue culture competence. This makes these methods more accessible and potentially transformative for research, offering a pragmatic approach to improving indica rice cultivars. By embracing these in vivo methods and harnessing the power of gene editing technologies like CRISPR/Cas9 systems, breeders can reshape their approach to indica rice improvement. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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21 pages, 2042 KiB

Open AccessReview

Protoplast Technology and Somatic Hybridisation in the Family Apiaceae

by Ankush S. Ranaware, Nandkumar S. Kunchge, Smita S. Lele and Sergio J. Ochatt

Plants 2023, 12(5), 1060; https://doi.org/10.3390/plants12051060 - 27 Feb 2023

Cited by 5 | Viewed by 3804

Abstract

Species of the family Apiaceae occupy a major market share but are hitherto dependent on open pollinated cultivars. This results in a lack of production uniformity and reduced quality that has fostered hybrid seed production. The difficulty in flower emasculation led breeders to [...] Read more.

Species of the family Apiaceae occupy a major market share but are hitherto dependent on open pollinated cultivars. This results in a lack of production uniformity and reduced quality that has fostered hybrid seed production. The difficulty in flower emasculation led breeders to use biotechnology approaches including somatic hybridization. We discuss the use of protoplast technology for the development of somatic hybrids, cybrids and in-vitro breeding of commercial traits such as CMS (cytoplasmic male sterility), GMS (genetic male sterility) and EGMS (environment-sensitive genic male sterility). The molecular mechanism(s) underlying CMS and its candidate genes are also discussed. Cybridization strategies based on enucleation (Gamma rays, X-rays and UV rays) and metabolically arresting protoplasts with chemicals such as iodoacetamide or iodoacetate are reviewed. Differential fluorescence staining of fused protoplast as routinely used can be replaced by new tagging approaches using non-toxic proteins. Here, we focused on the initial plant materials and tissue sources for protoplast isolation, the various digestion enzyme mixtures tested, and on the understanding of cell wall re-generation, all of which intervene in somatic hybrids regeneration. Although there are no alternatives to somatic hybridization, various approaches also discussed are emerging, viz., robotic platforms, artificial intelligence, in recent breeding programs for trait identification and selection. Full article

(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)

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