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Plant Tissue Culture and Plant Regeneration
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Plant Tissue Culture and Plant Regeneration

A special issue of Plants (ISSN 2223-7747). This special issue belongs to the section "Plant Development and Morphogenesis".

Deadline for manuscript submissions: 30 June 2025 | Viewed by 25081

Special Issue Editors

Dr. Douglas A. Steinmacher

E-Mail Website
Guest Editor
Vivetech Agrociências, Marechal Cândido Rondon 85960-000, Brazil
Interests: plant tissue culture
Dr. Taras Pasternak

E-Mail Website
Guest Editor
Instituto de Bioingeniería, Universidad Miguel Hernández, 03202 Elche, Spain
Interests: physiology and cell biology; plant tissue culture; molecular biology; cytochemistry; microscopy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Plant tissue culture and plant regeneration constitute crucial facets of plant biotechnology, spanning across several scientific and industrial domains. In recent years, we have witnessed significant advancements in the comprehension of the underlying mechanisms controlling in vitro plant regeneration, accompanied by the rapid evolution of specialized equipment and strategies for enhancing this process. This development has garnered the attention of both plant scientists and industries. Nonetheless, there exists an ongoing demand to further develop our strategies in order to select and regenerate superior genotypes, thereby refining protocols for enhanced reproducibility. In this context, a meticulous fine-tuning process is imperative. In light of these developments, we are excited to announce a Special Issue welcoming contributions covering all aspects of plant tissue culture and biotechnological processes, including the use of algorithms for improving protocols, aspects of somatic embryogenesis and organogenesis, cryopreservation, the use of temporary immersion bioreactors and new gene-editing technology enhancing shoot proliferation.

This Special Issue will accept original research articles, short-communications as well as comprehensive reviews.

We look forward to receiving your contributions.

Dr. Douglas A. Steinmacher
Dr. Taras Pasternak
Guest Editors

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Keywords

  • plant biotechnology
  • micropropagation
  • organogenesis
  • somatic embryogenesis
  • temporary immersion system
  • data-driven model
  • shoot proliferation
  • gene edition

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Published Papers (6 papers)

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22 pages, 6251 KiB  
Article
Importance of Media Composition and Explant Type in Cannabis sativa Tissue Culture
by Rekhamani Das, Tobias Kretzschmar and Jos C. Mieog
Plants 2024, 13(18), 2544; https://doi.org/10.3390/plants13182544 - 10 Sep 2024
Cited by 1 | Viewed by 1938
Abstract
Producing uniform Cannabis sativa (Cannabis) for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from “mother plants” in the cannabinoid industry, which also faces significant limitations. Cannabis tissue culture (TC) methods have been developed to overcome [...] Read more.
Producing uniform Cannabis sativa (Cannabis) for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from “mother plants” in the cannabinoid industry, which also faces significant limitations. Cannabis tissue culture (TC) methods have been developed to overcome these challenges, but the long-term health and maintenance of Cannabis explants in TC have been largely overlooked in previous studies. The current study focused on the development of an efficient and optimized micropropagation protocol covering the entire process, with a specific focus on the health and performance in the multiplication stage. Multiplication media were formulated hormone-free to avoid longer-term vitrification issues, resulting in single-main-shoot cultures rather than multiple-shoot cultures. This instigated the use of stage II explant types different from the standard shoot tips previously used for multiple shoot cultures. Multiplication media were further improved from the basal salt composition via nitrogen and calcium additives. The optimized protocol was used on eight diverse Cannabis cultivars to test its applicability across various genetic backgrounds. Results indicated that the protocol was effective for conservation purposes across all cultivars and achieved good long-term multiplication rates for some but not all. The outcomes of this study mark a significant stride towards an efficient Cannabis TC methodology ready for more comprehensive industrial applications. Full article
(This article belongs to the Special Issue Plant Tissue Culture and Plant Regeneration)
►▼ Show Figures

Figure 1

Figure 1
<p>(<b>a</b>) The media comparison without (MM8) and with (MM8 + H) 0.5 mg/L meta-topolin. (<b>b</b>) Effect of different media compositions on vitrification (N = 15 × 3). The means were compared using Dunn’s test with a significance level of adjusted <span class="html-italic">p</span>-value &lt; 0.05, significant differences are indicated by different letters for each treatment.</p> Full article ">Figure 2
<p>Effect of different media modifications and explant types on height (N = 15 × 3). (<b>a</b>) Height variation for predicted mean of all treatments without explant-type effect. (<b>b</b>) Effect of explant type (E1—primary shoot tips usually have many nodes with shorter internodal segments, E2—secondary shoot tips usually have fewer nodes with long internodal segments, E3—secondary shoot tip with primary node, E4—small secondary shoot tip with primary node, and E5—primary nodes with or without emerging bud) on height in MM8 and MM9 media in Media Trial 1. The means were compared using Tukey’s honest significant difference with <span class="html-italic">p</span> &lt; 0.05, significant differences are indicated by different letters for each treatment.</p> Full article ">Figure 3
<p>Different types of stage II explant types used in multiplication trials. E1—primary shoot tips usually have many nodes with shorter internodal segments, E2—secondary shoot tips usually have fewer nodes with long internodal segments, E3—secondary shoot tip with primary node, E4—small secondary shoot tip with primary node, and E5—primary nodes with or without emerging bud.</p> Full article ">Figure 4
<p>Effect of different media compositions on health, height, multiplication rate, rooting, and vitrification (N = 15 × 3). The means were compared using Dunn’s test with a significance level of adjusted <span class="html-italic">p</span>-value &lt; 0.05; significant differences are indicated by different letters for each treatment.</p> Full article ">Figure 5
<p>The traits measured in the multiplication trials (MT0-2). (<b>a</b>) plant height (cm), (<b>b</b>) multiplication rate (count), (<b>c</b>) plant rooting (score 5–1 based on the density of the roots from most densely rooted to no rooting), (<b>d</b>) plant health (score from 5 to 1, ranging from best to worst), and (<b>e</b>) vitrification (scored from 5 to 1, ranging from highly vitrified to no vitrification).</p> Full article ">Figure A1
<p>Tissue-culture images with different micro-propagules. Stage I explants: (<b>d</b>–<b>g</b>), Stage II explants: (<b>h</b>–<b>j</b>). (<b>a</b>) High-CBD cultivar mother plants in green house. (<b>b</b>) 6–8 cm shoot tip for tissue culture. (<b>c</b>) Surface sterilization treatment. (<b>d</b>) Shoot-tip (&gt;0.5 cm). (<b>e</b>) Nodal (1 cm–0.5 cm). (<b>f</b>) Microshoot tip (&lt;0.5 cm). (<b>g</b>) Meristem. (<b>h</b>) <span class="html-italic">Cannabis</span> culture. (<b>i</b>) <span class="html-italic">Cannabis</span> tissue culture plantlets. (<b>j</b>) Processed <span class="html-italic">Cannabis</span> plantlet.</p> Full article ">Figure A2
<p>Fungal and bacterial contamination in different stages of <span class="html-italic">Cannabis</span> tissue culture. (<b>a</b>,<b>b</b>)—Explants fungal contamination in stage I. (<b>c</b>,<b>d</b>)—Explants showing bacterial contamination stage I. (<b>e</b>,<b>f</b>)—Explants showing different types of fungal and bacterial contamination Stage II. (<b>g</b>,<b>h</b>)—Explants showing endophytic bacterial contamination during root development in containers with a 10 cm base diameter.</p> Full article ">Figure A3
<p>Dot-plot showing the average correlations of the MT2 measurement traits over all treatments and explant types. Negative correlations are shaded red, and positive correlations are shaded blue. The strength of the correlation is indicated by dot size and red or blue color saturation.</p> Full article ">Figure A4
<p>Deflasking and acclimatization of the <span class="html-italic">Cannabis</span> TC plantlets. (<b>a</b>)—<span class="html-italic">Cannabis</span> TC plantlet on media, (<b>b</b>)—TC plantlet, (<b>c</b>)—Trimming of the leaves and roots before placing in seed tray, (<b>d</b>)—planting in seed tray, (<b>e</b>)—TC generated plantlet ready for acclimatization, (<b>f</b>)—TC plantlets in mini greenhouse, (<b>g</b>)—rooted TC plantlet and cutting of Charlotte’s Angle, and (<b>h</b>)—rooted cuttings and acclimatized TC generated plants of high CBD cultivar potted in potting mix.</p> Full article ">
19 pages, 6228 KiB  
Article
Induction and Suspension Culture of Panax japonicus Callus Tissue for the Production of Secondary Metabolic Active Substances
by Siqin Lv, Fan Ding, Shaopeng Zhang, Alexander M. Nosov, Andery V. Kitashov and Ling Yang
Plants 2024, 13(17), 2480; https://doi.org/10.3390/plants13172480 - 4 Sep 2024
Viewed by 1209
Abstract
Using Panax japonicus as research material, callus induction and culture were carried out, and high-yielding cell lines were screened to establish a suspension culture system that promotes callus growth and the accumulation of the “total saponins” (total content of triterpenoid glycosides or ginsenosides). [...] Read more.
Using Panax japonicus as research material, callus induction and culture were carried out, and high-yielding cell lines were screened to establish a suspension culture system that promotes callus growth and the accumulation of the “total saponins” (total content of triterpenoid glycosides or ginsenosides). Using the root as an explant, the medium for callus induction and proliferation was optimized by adjusting culture conditions (initial inoculation amount, carbon source, shaking speed, hormone concentration, culture time) and a high-yielding cell line with efficient proliferation and high total saponins content was screened out. The conditions of suspension culture were refined to find out the most suitable conditions for the suspension culture of callus, and finally, the suspension culture system was established. We found that the lowest (5%) contamination rate was achieved by disinfecting the fresh roots with 75% alcohol for 60 s, followed by soaking in 10% NaClO for 15 min. The highest induction rate (88.17%) of callus was obtained using the medium MS + 16.11 μmol·L−1 NAA + 13.32 μmol·L−1 6-BA + 30.0 g·L−1 sucrose + 7.5 g·L−1 agar. The callus was loose when the callus subcultured on the proliferation medium (MS + 5.37 μmol·L−1 NAA + 13.32 μmol·L−1 6-BA + 30.0 g·L−1 sucrose + 3.8 g·L−1 gellan gum) for 21 days. The callus growth was cultured in a liquid growth medium (MS + 5.37 μmol·L−1 NAA + 13.32 μmol·L−1 6-BA + 30.0 g·L−1 sucrose) with an initial inoculation amount of 40 g·L−1, a shaking speed of 110 r/min and darkness. Cell growth was fastest with a culture period of 21 days. We replaced the growth medium with the production medium (MS + 5.37 μmol·L−1 NAA + 13.32 μmol·L−1 6-BA + 30.0 g·L−1 glucose) for maximum accumulation of total saponins. [Conclusion] A callus induction and suspension culture system for the root of P. japonicus was established. In this way, we can promote the accumulation of total saponins in callus cells and provide a basis for large-scale cell culture and industrial production of medicinal total saponins. Full article
(This article belongs to the Special Issue Plant Tissue Culture and Plant Regeneration)
►▼ Show Figures

Figure 1

Figure 1
<p>Effects of NAA/6-BA on callus proliferation of different cell lines of <span class="html-italic">Panax japonicus.</span> Note: Different lowercase letters indicate that different ratios of NAA and 6-BA have significant effects on callus proliferation (<span class="html-italic">p</span> &lt; 0.05). Note: (<b>a</b>) is the effect of NAA/6-BA on the growth of L-1 cell line, (<b>b</b>) is the effect of NAA/6-BA on the growth of L-2 cell line, (<b>c</b>) is the effect of NAA/6-BA on the growth of L-3 cell line, (<b>d</b>) is shows the effect of NAA/6-BA on the growth of L-4 cell line, (<b>e</b>) is the effect of NAA/6-BA on the growth of L-5 cell line.</p> Full article ">Figure 2
<p>Effects of the concentration of NAA and 6-BA on <span class="html-italic">Panax japonicus</span> callus proliferation in different cells. Note: Different lowercase letters indicated that different concentrations of NAA and 6-BA had significant effects on callus proliferation (<span class="html-italic">p</span> &lt; 0.05). Note: (<b>a</b>) is the effects of the concentration of NAA and 6-BA on L-1, (<b>b</b>) is the effects of the concentration of NAA and 6-BA on L-2, (<b>c</b>) is the effects of the concentration of NAA and 6-BA on L-3, (<b>d</b>) is the effects of the concentration of NAA and 6-BA on L-4, (<b>e</b>) is the effects of the concentration of NAA and 6-BA on L-5.</p> Full article ">Figure 3
<p>Normal and semi-logarithmic growth curves of callus of different cell lines of <span class="html-italic">Panax japonicus</span>. Note: (<b>a</b>) Normal growth curve of callus of different cell lines, (<b>b</b>) semi-logarithmic growth curve of callus of different cell lines.</p> Full article ">Figure 4
<p>Comparison of proliferation coefficient and “total saponins” content of different cell lines of <span class="html-italic">Panax japonicus</span>. Note: different lowercase letters indicate significant differences in proliferation coefficients as well as saponin content between cell lines (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 5
<p>Effects of different curing agents on the growth of callus of <span class="html-italic">P. japonicus</span> L-1 cell line (cultured for 21 days). Note: (<b>a</b>) is the callus when the curing agent is agar, (<b>b</b>) is the callus when the curing agent is gellan gum.</p> Full article ">Figure 6
<p>Effect of carbon source on cell growth of <span class="html-italic">Panax japonicus</span> suspension culture. Note: (<b>a</b>) is the effect of carbon source on cell density; (<b>b</b>) is the effect of carbon source on cell viability; (<b>c</b>) is the effect of carbon source on suspension sedimentation volume; (<b>d</b>) is the effect of carbon source on fresh weight; (<b>e</b>) is the effect of carbon source on dry weight. Different lowercase letters indicate that different carbon sources had significant effects on the growth of <span class="html-italic">P. japonicus</span> cells. (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 7
<p>The effect of initial inoculation amount on cell growth in suspension culture of <span class="html-italic">Panax japonicus</span> Note: (<b>a</b>) is the effect of initial weight on cell density; (<b>b</b>) is the effect of initial weight on cell viability; (<b>c</b>) is the effect of initial contact weight on suspension colonization volume; (<b>d</b>) is the effect of initial weight on fresh weight; (<b>e</b>) is the effect of initial weight on dry weight. Different lowercase letters indicated that different amounts of initial inoculation had significant effects on the growth of <span class="html-italic">P. japonicus</span> cells (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 8
<p>Influence of shaking speed on cell growth in the suspension culture of <span class="html-italic">Panax japonicus</span>. Note: (<b>a</b>) is the effect of shaking speed on cell density; (<b>b</b>) is the effect of shaking speed on cell viability; (<b>c</b>) is the effect of shaker rotation speed on suspension colonization volume; (<b>d</b>) is the effect of shaking speed on fresh weight; (<b>e</b>) is the effect of shaking speed on dry weight. Different lowercase letters indicated that different shaking speeds had a significant effect on the cell growth of <span class="html-italic">P. japonicus</span> (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 9
<p>Effects of concentrations of NAA and 6-BA on cell growth in suspension culture of <span class="html-italic">Panax japonicus</span>. Note: (<b>a</b>) is the effect of NAA and 6-BA concentration on cell density; (<b>b</b>) is the effect of NAA and 6-BA concentration on cell viability; (<b>c</b>) is the effect of NAA and 6-BA concentration on suspension sedimentation volume; (<b>d</b>) is the effect of NAA and 6-BA concentration on fresh weight; (<b>e</b>) is the effect of NAA and 6-BA concentration on dry weight; different lowercase letters indicate that NAA and 6-BA concentration had a significant effect on cell growth of <span class="html-italic">P. japonicus</span> (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 10
<p>Growth curve of cell suspension culture of <span class="html-italic">Panax japonicus</span>. Note: (<b>a</b>) is the change curve of cell density; (<b>b</b>) is the cell density curve; (<b>c</b>) is the volume change curve of suspension colonization; (<b>d</b>) is the change curve of fresh weight; (<b>e</b>) is the change curve of dry weight. Different lowercase letters indicate that there is a significant difference in the growth of <span class="html-italic">Panax japonicus</span> cells on different culture days (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 11
<p>Semi-logarithmic growth curve of suspension-cultured cells of <span class="html-italic">Panax japonicus</span>.</p> Full article ">Figure 12
<p>Effects of carbon sources on the content of “total saponins” in suspension-cultured cells of <span class="html-italic">Panax japonicus</span>. Note: Different lowercase letters indicate that different carbon sources have significant effects on “total saponins” in <span class="html-italic">Panax japonicus</span> (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 13
<p>Effect of initial grafting weight on “total saponins” content in <span class="html-italic">Panax japonicus</span> cells cultured in suspension. Note: Different lowercase letters indicate that the different amount of initial inoculation has a significant effect on “total saponins” in <span class="html-italic">Panax japonicus</span> (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 14
<p>Effect of shaking speed on the content of “total saponins” in <span class="html-italic">Panax japonicus</span> cells cultivated in suspension. Note: Different lowercase letters indicate that different shaking speeds have a significant effect on “total saponins” in <span class="html-italic">Panax japonicus</span> (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 15
<p>Effects of NAA and 6-BA concentrations on “total saponins” content in suspended cells of <span class="html-italic">Panax japonicus</span>. Note: Different lowercase letters indicate significant effects of different concentrations of NAA and 6-BA on “total saponins” of suspended cells of <span class="html-italic">Panax japonicus</span> (<span class="html-italic">p</span> &lt; 0.05). Note: the lower concentration (2.32 μmol·L<sup>−1</sup> NAA + 6.66 μmol·L<sup>−1</sup> 6-BA), medium concentration (5.37 μM NAA + 13.32 μM 6-BA) and higher concentration (5.37 μM NAA + 19.98 μM 6-BA); see <a href="#sec4dot3dot5-plants-13-02480" class="html-sec">Section 4.3.5</a>.</p> Full article ">Figure 16
<p>Influence of cultivation days on the “total saponins” content in the callus tissue of <span class="html-italic">Panax japonicus</span> suspension cells. Note: Different lowercase letters indicate significant differences in the “total saponins” content of the suspended cells of <span class="html-italic">Panax japonicus</span> during the different cultivation days (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">
21 pages, 6232 KiB  
Article
Genome-Wide Association Analysis Identifies Candidate Loci for Callus Induction in Rice (Oryza sativa L.)
by Wintai Kamolsukyeunyong, Yeetoh Dabbhadatta, Aornpilin Jaiprasert, Burin Thunnom, Wasin Poncheewin, Samart Wanchana, Vinitchan Ruanjaichon, Theerayut Toojinda and Parichart Burns
Plants 2024, 13(15), 2112; https://doi.org/10.3390/plants13152112 - 30 Jul 2024
Viewed by 1516
Abstract
Callus induction (CI) is a critical trait for transforming desirable genes in plants. A genome-wide association study (GWAS) analysis was conducted on the rice germplasms of 110 Indica rice accessions, in which three tissue culture media, B5, MS, and N6, were used for [...] Read more.
Callus induction (CI) is a critical trait for transforming desirable genes in plants. A genome-wide association study (GWAS) analysis was conducted on the rice germplasms of 110 Indica rice accessions, in which three tissue culture media, B5, MS, and N6, were used for the CI of those rice panels’ mature seeds. Seven quantitative trait loci (QTLs) on rice chromosomes 2, 6, 7, and 11 affected the CI percentage in the three media. For the B5 medium, one QTL (qCI–B5–Chr6) was identified on rice chromosome 6; for the MS medium, two QTLs were identified on rice chromosomes 2 and 6 (qCI–MS–Chr2 and qCI–MS–Chr6, respectively); for the N6 medium, four QTLs were identified on rice chromosomes 6, 7, and 11 (qCI–N6–Chr6.1 and qCI–N6–Chr6.2, qCI–N6–Chr7, and qCI–N6–Chr11, respectively). Fifty-five genes were identified within the haplotype blocks corresponding to these QTLs, thirty-one of which showed haplotypes associated with different CI percentages in those media. qCI–B5–Chr6 was located in the same region as qCI–N6–Chr6.2, and the Caleosin-related family protein was also identified in this region. Analysis of the gene-based haplotype revealed the association of this gene with different CI percentages in both B5 and N6 media, suggesting that the gene may play a critical role in the CI mechanism. Moreover, several genes, including those that encode the beta-tubulin protein, zinc finger protein, RNP–1 domain-containing protein, and lysophosphatidic acid acyltransferase, were associated with different CI percentages in the N6 medium. The results of this study provide insights into the potential QTLs and candidate genes for callus induction in rice that contribute to our understanding of the physiological and biochemical processes involved in callus formation, which is an essential tool in the molecular breeding of rice. Full article
(This article belongs to the Special Issue Plant Tissue Culture and Plant Regeneration)
►▼ Show Figures

Figure 1

Figure 1
<p>The different types of scutellum-derived callus formation: no callus induction (<b>A</b>), partial callus induction with incomplete embryogenic callus (<b>B</b>), and complete embryogenic callus induction (<b>C</b>).</p> Full article ">Figure 2
<p>The callus induction (CI) percentages of the 110 rice accessions were evaluated across three tissue culture media: B5, MS, and N6. (<b>A</b>) The boxplot illustrates the percentage CI distribution analyzed using ANOVA and Tukey’s HSD test. (<b>B</b>) Correlation plots were generated to examine the relationships among the different media. (<b>C</b>) The histogram shows the distribution of the CI percentages in each medium.</p> Full article ">Figure 3
<p>Genetic structure of the panel of 110 rice accessions: (<b>A</b>) SNP density on rice chromosomes, (<b>B</b>) principal component analysis (PCA) and kinship relatedness analysis of the 110 genotypes, and (<b>C</b>) population structure of the 110 rice accessions.</p> Full article ">Figure 4
<p>Linkage disequilibrium decay across 12 chromosomes in 110 <span class="html-italic">O. sativa</span> accessions. Mean LD decay ranges between 70 and 259 kb.</p> Full article ">Figure 5
<p>Manhattan plots resulting from genome-wide association study (GWAS) results for CI in three tissue culture media: (<b>A</b>) B5, (<b>B</b>) MS, and (<b>C</b>) N6. Yellow lines indicate the cut-off threshold at −log<sub>10</sub> (p) of 5.0.</p> Full article ">Figure 6
<p>Analysis of associated regions and haplotype analysis within the qCI–B5–Chr6 region. (<b>A</b>) Manhattan plots and LD heatmap across the 475 kb region surrounding the significant SNP. The black triangle in the LD heatmap indicates the candidate haploblock. (<b>B</b>–<b>D</b>) Boxplots showing the distribution of %CI and haplotype analysis for the genes <span class="html-italic">Os06g0256600</span>, <span class="html-italic">Os06g0255200</span>, and <span class="html-italic">Os06g0254200</span>.</p> Full article ">Figure 7
<p>Analysis of associated regions and haplotypes within the qCI–MS–Chr2 (<b>A</b>) and qCI–MS–Chr6 (<b>B</b>) regions. The black triangles in the LD heatmaps indicate the candidate haploblock.</p> Full article ">Figure 8
<p>Analysis of associated regions and haplotypes within the qCI–N6–Chr6.1 region. (<b>A</b>) Manhattan plot and LD heatmap across the 475 kb region surrounding the significant SNP. The black triangle in the LD heatmap indicates the candidate haploblock. (<b>B</b>,<b>C</b>) Boxplots showing the distribution of %CI and haplotype analysis for the genes <span class="html-italic">Os06g0169600</span>, <span class="html-italic">Os06g0169800</span>, and <span class="html-italic">Os06g0170500</span>.</p> Full article ">Figure 9
<p>Analysis of associated regions and haplotype analysis within the qCI–N6–Chr6.2 region. (<b>A</b>) Manhattan plot and LD heatmap across the 476 kb region surrounding the significant SNP. The black triangle in the LD heatmap indicates the candidate haploblock. (<b>B</b>) Boxplot showing the distribution of %CI and haplotype analysis for the Os06g0254600 gene.</p> Full article ">Figure 10
<p>Analysis of associated regions and haplotype analysis within the qCI–N6–Chr7 region. (<b>A</b>) Manhattan plot and LD heatmap across the 248 kb region surrounding the significant SNP. The black triangle in the LD heatmap indicates the candidate haploblock. (<b>B</b>,<b>C</b>) Boxplots showing the distribution of %CI and haplotype analysis for the genes <span class="html-italic">Os07g0256200</span> and <span class="html-italic">Os07g0256866</span>.</p> Full article ">Figure 11
<p>Analysis of associated regions and haplotype analysis of the qCI–N6–Chr11 region. (<b>A</b>) Manhattan plot and LD heatmap across the 136 kb region surrounding the significant SNP. The black triangle in the LD heatmap indicates the candidate haploblock. (<b>B</b>,<b>C</b>) Boxplots showing the distribution of %CI and haplotype analysis for the genes <span class="html-italic">Os11g0637700</span> and <span class="html-italic">Os11g0637800</span>.</p> Full article ">
11 pages, 13009 KiB  
Article
An Improved and Simplified Agrobacterium-Mediated Genetic Transformation Protocol for Solanum nigrum with a Shorter Growth Time
by Qianqian Li, Xiuyuan Wang, Chong Teng, Xuxia He, Xinyue Fu, Wentao Peng, Yinglun Fan and Shanhua Lyu
Plants 2024, 13(15), 2015; https://doi.org/10.3390/plants13152015 - 23 Jul 2024
Viewed by 1038
Abstract
Solanum nigrum (Solanaceae family) is widely consumed as a fruit or local leafy vegetable after boiling; it also serves as a medicinal plant. Although Agrobacterium-mediated genetic transformation has been established in S. nigrum, the transformation period is long. Specifically, induction [...] Read more.
Solanum nigrum (Solanaceae family) is widely consumed as a fruit or local leafy vegetable after boiling; it also serves as a medicinal plant. Although Agrobacterium-mediated genetic transformation has been established in S. nigrum, the transformation period is long. Specifically, induction of roots takes approximately five weeks for tetraploid and hexaploid S. nigrum, and 7 weeks for diploid Solanum americanum. In this study, we developed an improved rooting-induced method that requires only about 1 week and avoids the use of tissue culture. After generating the transgenic shoots, they were directly transplanted into the soil to facilitate root formation. Remarkably, 100% of the transgenic shoots developed roots within 6 days. Our improved method is time-saving (saving more than 1 month) and simpler to operate. The improved rooting-induced step can be applied to induce roots in various plants using tissue culture, exemplified by the carnation (Dianthus caryophyllus L.). Furthermore, we applied the improved method to generate S. americanum plants expressing AcMYB110 from kiwifruit (Actinidia chinensis spp.). This method will contribute to speeding up gene functional analysis and trait improvement in S. nigrum and might have potential in fast plant molecular breeding processes in crops and rapid rooting induction in tissue culture. Full article
(This article belongs to the Special Issue Plant Tissue Culture and Plant Regeneration)
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<p>Diagram of the improved <span class="html-italic">Agrobacterium</span>-mediated genetic transformation protocol for <span class="html-italic">S. nigrum</span> compared with the traditional method.</p> Full article ">Figure 2
<p>The improved <span class="html-italic">Agrobacterium</span>-mediated genetic transformation protocol for <span class="html-italic">S</span>. <span class="html-italic">nigrum</span>. The improved genetic transformation protocol for diploid <span class="html-italic">S. americanum</span> (<b>A</b>–<b>E</b>,<b>H</b>), tetraploid <span class="html-italic">S. nigrum</span> (<b>F</b>,<b>I</b>), and hexaploid <span class="html-italic">S. nigrum</span> (<b>G</b>,<b>J</b>). Induction of transgenic shoots (<b>A</b>). Transgenic shoots were covered with a plastic bag for 4–5 d for acclimation to the environment after transplanting in the soil (<b>B</b>). As a control, transgenic shoots were transplanted in the rooting medium in the traditional transformation method at 6 dpt (<b>C</b>). The transgenic grown soil plantlets produced via the improved transformation method at 6 dpt (<b>D</b>). A comparison of the transgenic plantlets between our improved transformation method and the traditional method. The transgenic plantlets were grown at 6 dpt (<b>E</b>–<b>G</b>), 49 dpt (<b>H</b>), 38 dpt (<b>I</b>), and 38 dpt (<b>J</b>). Letters T and I indicate transgenic plant(s) produced via the traditional transformation method and improved transformation method, respectively. Bars = 1 cm.</p> Full article ">Figure 3
<p>A comparative analysis of the improved method and traditional transformation methods in three <span class="html-italic">S. nigrum</span> species. A comparative anslysis of total root lengths (<b>A</b>), dry root weights (<b>B</b>), and dry shoot weights (<b>C</b>). The data were measured at 49 dpt (diploid <span class="html-italic">S. americanum</span>), 38 dpt (tetraploid <span class="html-italic">S. nigrum</span>), and 38 dpt (tetraploid <span class="html-italic">S. nigrum</span>). ** <span class="html-italic">p</span> &lt; 0.01. Values are mean ± SD of three independent replicates. ITM: improved transformation method; TTM: traditional transformation method.</p> Full article ">Figure 4
<p>Observation of red fluorescence in <span class="html-italic">DsRed2</span>-transgenic <span class="html-italic">S. americanum</span> shoots. Shoots-induced were photographed under bright light (<b>A</b>) and excitation wavelength at 540 nm/emission wavelength at 600 nm, respectively (<b>B</b>). Strong fluorescence of DsRed2 was observed in the transgenic shoots-induced (shown in the rectangle box). Bars = 1 cm. PCR analysis of <span class="html-italic">DsRed2</span> gene (678 bp) in <span class="html-italic">S. americanum</span> (<b>C</b>). Lane 1, ddH<sub>2</sub>O used as a template (blank control); Lane 2, Wild-type plant used as a negative control; Lane 3, pR35BTR1 plasmid used as a template (positive control); Lane 4–18, <span class="html-italic">DsRed2</span>-positive independent transgenic plant transformed with pR35BTR1 plasmid; M, DL2000 bp DNA ladder (Sangon Biotech Co., Shanghai, China).</p> Full article ">Figure 5
<p>Rapid root generation in carnations using our improved method. Induction of shoots (<b>A</b>). Comparison of the transgenic plantlets generated using the improved method and the traditional method (<b>B</b>,<b>C</b>). The transgenic plantlets were grown at 10 dpt (<b>B</b>) and 75 dpt (<b>C</b>), respectively. Letters T and I indicate transgenic plant(s) produced via the traditional transformation method and the improved transformation method, respectively. Bars = 1 cm.</p> Full article ">Figure 6
<p>Ectopic expression of <span class="html-italic">AcMYB110</span> in <span class="html-italic">S. americanum</span> plants induced anthocyanin accumulation in the flowers and fruits. Wild-type flowers (<b>A</b>); flowers of <span class="html-italic">35S::AcMYB110</span> overexpression plant (<b>B</b>); fruits at different developmental stages; wild-type fruits (upper row) and <span class="html-italic">35S::AcMYB110</span> fruits (lower row) (<b>C</b>,<b>D</b>); section D shows cross-sections of the fruits. Bars = 0.5 cm.</p> Full article ">Figure 7
<p>RT-PCR analysis of transgenic <span class="html-italic">S. americanum</span> expressing <span class="html-italic">AcMYB110</span>. Lane 1, ddH<sub>2</sub>O used as a template (blank control); Lane 2, Wild-type plant used as a negative control; Lanes 3–7, <span class="html-italic">DsRed2</span>-positive independent transgenic plants transformed with <span class="html-italic">pBM110</span> plasmid; M, DL2000 bp DNA ladder (Sangon Biotech Co., Shanghai, China). SnEF-1α was used as the reference gene.</p> Full article ">
9 pages, 1268 KiB  
Article
High-Efficiency In Vitro Root Induction in Pear Microshoots (Pyrus spp.)
by Jae-Young Song, Jinjoo Bae, Young-Yi Lee, Ji-Won Han, Ye-ji Lee, Sung Hee Nam, Ho-sun Lee, Seok Cheol Kim, Se Hee Kim and Byeong Hyeon Yun
Plants 2024, 13(14), 1904; https://doi.org/10.3390/plants13141904 - 10 Jul 2024
Viewed by 920
Abstract
Extensive research has been conducted on the in vitro mass propagation of pear (Pyrus spp.) trees through vegetative propagation, demonstrating high efficiency in shoot multiplication across various pear species. However, the low in vitro rooting rates remain a significant barrier to the [...] Read more.
Extensive research has been conducted on the in vitro mass propagation of pear (Pyrus spp.) trees through vegetative propagation, demonstrating high efficiency in shoot multiplication across various pear species. However, the low in vitro rooting rates remain a significant barrier to the practical application and commercialization of mass propagation. This study aims to determine the favorable conditions for inducing root formation in the in vitro microshoots of Pyrus genotypes. The base of the microshoots was exposed to a high concentration (2 mg L−1) of auxins (a combination of IBA and NAA) for initial root induction at the moment when callus formation begins. The microshoots were then transferred to an R1 medium (1/2 MS with 30 g L−1 sucrose without PGRs) to promote root development. This method successfully induced rooting in three European pear varieties, one Asian pear variety, and a European–Asian hybrid, resulting in rooting rates of 66.7%, 87.2%, and 100% for the European pear (P. communis), 60% for the Asian pear (P. pyrifolia), and 83.3% for the hybrid pear (P. pyrifolia × P. communis) with an average of 25 days. In contrast, the control group (MS medium) exhibited rooting rates of 0–13.3% after 60 days of culture. These findings will enhance in vitro root induction for various pear varieties and support the mass propagation and acclimatization of pear. The in vitro root induction method developed in this study has the potential for global commercial application in pear cultivation. Full article
(This article belongs to the Special Issue Plant Tissue Culture and Plant Regeneration)
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<p>In vitro microshoots for initial root induction. (<b>A</b>) In vitro proliferation of microshoot cultures of pear grown on MS medium supplemented with 2 mg L<sup>−1</sup> BA and 0.2 mg L<sup>−1</sup> IBA. (<b>B</b>) Microshoots on MS medium before transfer to R0-IN medium with auxin. (<b>C</b>) Swollen base of microshoots on R0-IN medium. (<b>D</b>) Microshoot base before PGR treatment. (<b>E</b>) Swollen base of ‘Bartlett’ after 1–2 days on R0-IN medium. (<b>F</b>) Swollen base of ‘BaeYun No. 3’ after 10 days on R0-IN medium. (<b>G</b>) Swollen base of ‘Oharabeni’ after 3 days on R0-IN medium.</p> Full article ">Figure 2
<p>Effect of IBA, NAA, and their combination on the in vitro root formation of ‘Bartlett’ microshoots using a two-step treatment method. MS refers to the rooting rate of microshoots on the MS medium after 30 days of culture. R0-I, R0-N, and R0-IN indicate the rooting rates of microshoots after 1 day of exposure to R0-I (2 mg L<sup>−1</sup> IBA), R0-N (2 mg L<sup>−1</sup> NAA), and R0-IN (1 mg L<sup>−1</sup> each of IBA and NAA), respectively, followed by transfer to the R1 (PGR-free) medium for 14 days. Different letters on bars indicate significant differences at <span class="html-italic">p</span> &lt; 0.05 according to Duncan’s multiple range test. Twelve plants per treatment were used, with three replications.</p> Full article ">Figure 3
<p>In vitro rooting of five different pear genotypes. The microshoots cultured on the initial root induction medium (R0-IN) in the dark for specified days were moved to the medium (R1).</p> Full article ">

Review

Jump to: Research

24 pages, 1023 KiB  
Review
Plant Growth Regulation in Cell and Tissue Culture In Vitro
by Taras P. Pasternak and Douglas Steinmacher
Plants 2024, 13(2), 327; https://doi.org/10.3390/plants13020327 - 22 Jan 2024
Cited by 29 | Viewed by 16398
Abstract
Precise knowledge of all aspects controlling plant tissue culture and in vitro plant regeneration is crucial for plant biotechnologists and their correlated industry, as there is increasing demand for this scientific knowledge, resulting in more productive and resilient plants in the field. However, [...] Read more.
Precise knowledge of all aspects controlling plant tissue culture and in vitro plant regeneration is crucial for plant biotechnologists and their correlated industry, as there is increasing demand for this scientific knowledge, resulting in more productive and resilient plants in the field. However, the development and application of cell and tissue culture techniques are usually based on empirical studies, although some data-driven models are available. Overall, the success of plant tissue culture is dependent on several factors such as available nutrients, endogenous auxin synthesis, organic compounds, and environment conditions. In this review, the most important aspects are described one by one, with some practical recommendations based on basic research in plant physiology and sharing our practical experience from over 20 years of research in this field. The main aim is to help new plant biotechnologists and increase the impact of the plant tissue culture industry worldwide. Full article
(This article belongs to the Special Issue Plant Tissue Culture and Plant Regeneration)
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<p>Hypothetic mechanism of plant regeneration in tissue culture in vitro. In green are representative of organizer cell, controlling in vitro response through auxin biosynthesis.</p> Full article ">Figure 2
<p>Proposed flowchart for plant micropropagation based upon the characteristics and aspects presented.</p> Full article ">
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