Next Article in Journal
Optimizing Green Globular Body Induction for Micropropagation of Microsorum pteropus ‘Windeløv’
Previous Article in Journal
The Influence of Different LED Light Treatments on the Growth and Salt Stress Tolerance of Cauliflower Microgreens In Vitro
Previous Article in Special Issue
Assessing Cadmium Stress Resilience in Myrtle Genotypes Using Machine Learning Predictive Models: A Comparative In Vitro Analysis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 10
Issue 7
10.3390/horticulturae10070674
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

In Vitro Micropropagation of the Vulnerable Chilean Endemic Alstroemeria pelegrina L.

by
Francesca Guerra
1,
Ricardo Cautín
2 and
Mónica Castro
1,*
1
Laboratorio de Propagación, Escuela de Agronomía, Facultad de Ciencias Agronómicas y de los Alimentos, Pontificia Universidad Católica de Valparaíso, La Palma S/N, Quillota 2260000, Chile
2
Laboratorio de Especies Leñosas, Escuela de Agronomía, Facultad de Ciencias Agronómicas y de los Alimentos, Pontificia Universidad Católica de Valparaíso, La Palma S/N, Quillota 2260000, Chile
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(7), 674; https://doi.org/10.3390/horticulturae10070674
Submission received: 3 June 2024 / Revised: 14 June 2024 / Accepted: 18 June 2024 / Published: 25 June 2024
(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)
Browse Figures
Versions Notes

Abstract

:
The Chilean rhizomatous geophyte Alstroemeria pelegrina (A. pelegrina), a species endemic to the Coquimbo (31°45′ S) and Valparaíso (33°12′ S) regions, is currently classified as vulnerable and experiencing population decline due to loss of habitat. This heightened threat underscores the need to develop effective mass propagation techniques to support its conservation efforts. Consequently, the present study aimed to establish an optimized in vitro propagation protocol tailored specifically for A. pelegrina to facilitate its large-scale propagation and promote the preservation of this vulnerable geophyte. In our experiment, explants obtained from in vitro germination were used. These explants were then cultured on a Murashige and Skoog (MS) medium solidified with agar (6 g L−1) and supplemented with 2.22 μM 6-Benzylaminopurine (BAP) and 30 g L−1 sucrose. The results indicated an average of 4.6 new shoots produced per initial explant. The implementation of light-emitting diode (LED) illumination with a red:blue ratio of 3:1 and 2.22 μM BAP resulted in an average shoot length of 6.2 cm. For rhizome induction, the addition of either 5.37 μM 1-naphthaleneacetic acid (NAA) or 5.37 μM indole-3-butyric acid (IBA) resulted in average rhizome lengths of 1.9 cm and 1.7 cm, respectively, with fresh weights of the vitroplants ranging from 2.9 g to 3.1 g, and rooting percentages between 100% and 96%, respectively. Notably, the survival rate of rooted plants obtained through in vitro propagation was 90% after a 4-week acclimatization period in a cold greenhouse, indicating the efficacy of the developed protocol for mass propagation and conservation efforts of this vulnerable geophyte species.

1. Introduction

The genus Alstroemeria, belonging to the Alstroemeriaceae family, is native to South America, comprising approximately 90 species distributed across Chile, Brazil, Argentina, Peru, Bolivia, Paraguay, and Venezuela [1,2,3]. This genus has gained substantial commercial importance as an ornamental crop, extensively cultivated for its captivating floral forms and diverse color palette, which has driven its global production and trade both as cut flowers and potted plants [4,5,6,7].
Propagation of this genus primarily relies on vegetative techniques such as rhizome division. However, these methods are associated with several drawbacks, including low efficiency, time-consuming procedures, risks of disease dissemination (e.g., fungal and viral infections), reduced productivity, and seasonal limitations [8,9]. In contrast, seed-based propagation is not widely adopted due to challenges related to embryonic and combined dormancy, genotypic and physical constraints, and consequently diminished germination capacity in most Alstroemeria species [10,11].
However, the wild and endemic Chilean species A. pelegrina seeds undergo physical dormancy. Scarification techniques were employed to enhance its germination capacity, leading to the development of a successful in vitro germination protocol [12]. Currently, in vitro propagation techniques, particularly those associated with interspecific hybridization, mutagenesis, polyploidization, and in vitro embryogenesis, are used in this genus to address the need for commercial variety development and the difficulties associated with seed germination [13,14,15,16,17,18].
Micropropagation of Alstroemeria can be achieved through embryo rescue. Following manual pollination, seeds are collected 10–23 days later. Excised ovules are then cultured on Murashige and Skoog (1962) (MS) medium in darkness to promote embryo development. The culture medium is optimized to enhance this process [13,14,15,16,17,18]. Regeneration rates reported in vitro vary between 2 and 30%, with a culture period of 6–11 months [19]. Various explants for in vitro multiplication, including stems, leaves, flower buds, and fragmented rhizomes/meristems, were tested, and meristematic explants were the most successful, achieving multiplication rates of 2–3 shoots [20,21,22,23,24].
Apical bud dominance was identified as a major challenge in micropropagation. The highest shoot induction rate was achieved by incubating of Alstroemeria sp. explants in darkness for 15 days at 8 °C, followed by 15 days at 25 °C, under a 16/8 h light/dark cycle [20,21,22,23,24]. In contrast, the use of greenhouse-grown rhizome-derived plants cultured in vivo on a substrate present significant contaminations that were difficult to overcome [25]. In addition, in vitro propagation studies in this genus focused on optimizing the effect of gelling agent concentration, using between 3 and 7 g L−1. Conversely, the use of liquid medium resulted in hyperhydricity or vitrification, reducing growth, causing explant translucency, and potentially leading to necrosis. Cytokinins are the most commonly used growth regulators, with 6-benzyladenine and 6-benzylaminopurine at concentrations of 2.22 to 8.88 μM being particularly effective in promoting shoot growth and bud multiplication [26,27,28,29,30].
Chile harbors a high diversity of Alstroemeria plant genus, with 58 taxa documented (including 37 species, 11 subspecies, and 10 varieties). Notably, 82% of these taxa are endemic to the Chilean Mediterranean climate zone [31]. However, these endemic wild plants face threats due to human activities (urbanization) and climate change, which lead to a decline in their reproductive capacity [32,33]. One such threatened species is Alstroemeria pelegrina. It is found along the Chilean coast, distributed from the Coquimbo Region (31°45′ S) south to Quintay in the Valparaíso Region (33°12′ S). It typically occurs at heights up to 120 m a.s.l. (above sea level), inhabiting coastal cliffs, rocky areas, and mountain slopes. A. pelegrina is currently listed as vulnerable by the Chilean Ministry of the Environment due to overcollection and desertification, factors that diminish its natural habitat [32,33,34].
In vitro seed germination offers significant advantages for the conservation of vulnerable plant species. Compared to traditional seed germination, in vitro seed germination reduces germination time and allows more uniform seedling emergence [35,36]. In addition, micropropagation increases overall germination percentages and provides a rapid obtaining of plantlets. This contributes to maintaining genetic diversity in ex situ conservation programs by incorporating a broader range of genotypes, ultimately enhancing the viability of existing vulnerable plant populations. Micropropagation of shoots obtained from seeds germinated in vitro ensures this by preserving genetic variability within the propagated material, effectively reducing the risk of genetic bottlenecks that can occur with traditional seed-based propagation [35,36]. This leads to populations with greater adaptability, which is crucial for their long-term survival in the face of environmental challenges, and reduces the risk of extinction.
Given the critical need to preserve Chile’s unique biological heritage, developing methods for large-scale propagation of this Alstroemeria genus is essential. In vitro culture provides a viable option for mass propagation. Therefore, this study aimed to develop a protocol for the in vitro propagation of the vulnerable species, A. pelegrina.

2. Materials and Methods

Plant material
Alstroemeria pelegrina fruits were collected in January and February 2021 in the city of Quillota (32°54′ S, 71°16′ W), Chile, from a collection of 15 plants maintained at the Propagation Laboratory of the Pontificia Universidad Católica de Valparaíso. Thirty fruits of A. pelegrina at physiological maturity were randomly collected [12]. The seeds were manually taken out for germination.
Explant acquisition
Explants for in vitro propagation were obtained from seedlings of A. pelegrina following an established protocol for seed germination and seedling establishment [12]. This protocol involved breaking the physical dormancy of the seeds by transversally cutting the apical zone to a depth of 0.1 to 0.5 mm and then establishing them on a medium composed of half-strength MS [37] mineral salts. The pH was adjusted to 5.7 ± 0.1 and the medium was solidified with 6 g L−1 agar (Algas Marinas S.A., Santiago, Chile).

2.1. In vitro Multiplication Stage

To investigate the influence of light and BAP concentration on shoot multiplication of A. pelegrina, shoots obtained from in vitro germination, characterized by the presence of their first true leaf were used (Figure 1). Three artificial light sources were evaluated: L1: cool white fluorescent lamps (Philips TL-D 36W/54) emitting light with a wavelength range of 400–700 nm and a photosynthetic photon flux density (PPFD) of 5.27 μmol m⁻2 s⁻1. L2: red and blue LED light in a 3:1 ratio (PARALED PARALED_FRRB 55W system, Ciencia Pura SpA. Santiago, Chile). L3: red and blue LED light in a 1:1 ratio (PARALED PARALED_FRRB 55W system, Ciencia Pura SpA. Santiago, Chile). To avoid light contamination, each treatment was separated by a white screen. The effects of these light sources were assessed in combination with three different concentrations of 6-Benzylaminopurine (BAP): BAP1 (0 μM), BAP2 (2.22 μM), and BAP3 (4.44 μM). The PPFD and red:blue ratios for each light source were measured using a spectrophotometer (Lighting Passport Standard Pro model, Allied Scientific ProTM, Gatineau, QC, Canada). In addition, the PPFD photon irradiation provided by each light source was measured in the blue (400–499 nm) and red (600–699 nm) spectral regions. Detailed information on the PPFD measurements is provided in Table 1.
The plant material was cultured on MS medium supplemented with vitamins and sucrose (30 g L−1) for 30 days. The pH of the medium was adjusted to 5.7 ± 0.1 and gelled with 6 g L⁻¹ agar. Four plantlets were cultured per 250 mL flask and maintained in a growth chamber under controlled conditions of 25 ± 1 °C temperature and a 16:8 h photoperiod (light/dark cycle).
A completely randomized factorial design with a 3 × 3 arrangement was employed. Each treatment consisted of 20 shoots with three replicates. Shoot length and number of shoots were evaluated. Data were analyzed using two-way analysis of variance (ANOVA) and variance component analysis. Differences between treatments were determined using Tukey’s test with a significance level of p ≤ 0.05. Minitab 19 statistical software (Minitab Inc., State College, PA, USA) was used for the analyses.

2.2. Rhizome and Root Induction Stage

Uniform 3 cm long shoots obtained from the multiplication stage were used. The effect of MS basal medium supplemented with different concentrations of 1-Naphthaleneacetic acid (NAA) and Indole-3-Butyric Acid (IBA) was evaluated. Five treatments were employed: T1: control (no growth regulator); T2: 2.69 μM NAA; T3: 5.37 μM NAA; T4: 2.69 μM IBA; and T5: 5.37 μM IBA. Ten shoots per treatment with three replicates were cultured in a growth chamber under controlled conditions: 25 ± 1 °C temperature, cool white fluorescent lamps (Philips TL-D 36W/54) providing light with a wavelength range of 400–700 nm and a PPFD of 5.27 μmol m−2 s−1, and a 16:8 h photoperiod (light/dark cycle).
Rhizome length, fresh weight (g) per plant, and rooting percentage were evaluated. Rooting percentage was calculated as the ratio of the number of rooted plants (with a root length of 0.5 cm) to the total number of shoots used for each treatment. Percentage values were transformed by natural logarithm for analysis.
The experiment was designed completely at random. One-way analysis of variance (ANOVA) and variance component analysis were performed. Differences between treatments were determined using Tukey’s test with a significance level of p ≤ 0.05. Minitab 19 statistical software (Minitab Inc., State College, PA, USA) was used for the analyses.

2.3. Ex Vitro Acclimatization

After rooting, all plants were transplanted into 200 cm³ containers filled with a 1:1 (v/v) mixture of peat moss and perlite. Each plant was covered with a transparent 200 cm³ container and transferred to a cold greenhouse for acclimatization. Temperatures inside the cold greenhouse fluctuated between 18 and 30 °C. The transparent containers were gradually removed in four discrete steps over a four-week period, allowing for a stepwise decrease in relative humidity. After four weeks, the containers were completely removed.
During this acclimatization stage, the survival rate (%) of A. pelegrina plants was evaluated. Survival rate was calculated as the percentage of living plants relative to the total number of acclimated plants.

3. Results

3.1. In Vitro Multiplication Stage

Analysis of variance (ANOVA) revealed a significant interaction (p ≤ 0.05) between light source and BAP application on shoot length (SL) (Table 2). The interaction of L2BAP2 (red:blue light 3:1 with 2.22 μM BAP) and L1BAP2 (white light with 2.22 μM BAP) resulted in the greatest shoot length, reaching 6.2 cm and 5.4 cm, respectively (p ≤ 0.05). Interactions L3BAP2 (red:blue light 1:1 with 2.22 μM BAP), L2BAP3 (red:blue light 3:1 with 4.44 μM BAP), L1BAP3 (white light with 4.44 μM BAP), L3BAP3 (red:blue light 1:1 with 4.44 μM BAP), L2BAP1 (red:blue light 3:1 with 0 μM BAP), and L3BAP1 (red:blue light 1:1 with 0 μM BAP) promoted an average shoot length of 4.5 cm, 4.1 cm, 3.7 cm, 3.5 cm, 3.3 cm, and 2.7 cm, respectively. Notably, L1BAP1 (white light with 0 μM BAP) combination produced the shortest shoots, averaging 2.4 cm. These findings indicate that shoot height in A. pelegrina is positively influenced by the synergistic effects of different light spectra and BAP concentrations.
In contrast to shoot length, shoot number (SN) was not significantly influenced by the different light spectra (p ≥ 0.05) (Table 2) and was solely determined by BAP concentration (p ≤ 0.05) (Table 3). The highest SN value (4.6 shoots; p ≤ 0.05) was achieved when 2.22 μM BAP was added to the culture medium, while BAP concentrations of 4.44 μM resulted in average SN values of 3.7. On the other hand, the lowest shoot number was obtained without BAP (Table 3 and Figure 2).

3.2. Rhizome and Root Formation Stage

After four weeks of culture on rhizome and root formation medium (Table 4), A. pelegrina seedlings significantly differed in rooting percentage, plant fresh weight, and rhizome length based on the auxin treatment applied. Treatments T3 (5.37 μM NAA) (Figure 3) and T5 (5.37 μM IBA) resulted in the highest rooting percentages, reaching 100% and 96%, respectively (p ≤ 0.05). Furthermore, these concentrations promoted significant increases in plant fresh weight (2.9 and 3.1 g) and average rhizome length (1.9 and 2.1 cm) compared to the control and other treatments (p ≤ 0.05). Treatments T2 (2.69 μM NAA) and T4 (2.69 μM IBA) allowed us to achieve moderate rooting percentages (53% and 51%), plant fresh weights (1.9 and 1.7 g), and rhizome lengths (1.4 and 1.6 cm), respectively (p ≤ 0.05). As expected, the control treatment (T1) lacking auxins caused the lowest rooting percentage (11%), with a significantly lower average fresh weight (1.3 g) and rhizome length (0.8 cm) compared to all auxin-supplemented treatments (p ≤ 0.05).

3.3. Ex Vitro Acclimatization

Ex vitro acclimatization of rooted A. pelegrina shoots (Figure 4a) resulted in a high survival rate of 90% after four weeks of transfer to a 1:1 (v/v) peat moss/perlite substrate. Following six weeks of acclimatization (Figure 4b), continued growth and development of A. pelegrina plants were observed, including the emergence of new shoots and the expansion of the root system, with the formation of storage roots (Figure 4c).

4. Discussion

This study presents an efficient micropropagation protocol for A. pelegrina regeneration using MS medium with its corresponding vitamins, supplemented with 30 g L−1 sucrose, 6 g L−1 agar, and 2.22 μM BAP for shootmultiplication. Subsequently, rooting was achieved with the application of 5.37 μM NAA or 5.37 μM IBA during the rooting stage.
Previous studies reported micropropagation outcomes in various Alstroemeria species. In Alstroemeria var. Fiesta de 15 INTA, a regeneration rate of 76%, shoot length of 2.4 cm, and 3.4 leaves per explant were achieved using MS medium supplemented with 1.07 μM NAA [38]. In addition, the combination of 1.07 μM NAA and 4.44 μM BA resulted in 5.2 shoots [27]. For Alstroemeria pallida, an endemic Chilean species, the highest proliferation rate was observed in MS medium supplemented with 8.88 μM BAP and solidified with 3.5 g L⁻¹ agar, producing 3.5 shoots [19]. These findings are comparable to the results of this study, where an average of 4.6 shoots was obtained using 2.22 μM BAP. However, in other Alstroemeria genotypes, the application of 2.22 μM BAP for 3 weeks allowed us to obtain a 3.0-fold multiplication of shoots and a 2.3-fold increase in rhizomes [19].
In the Alstroemeria genus, the application of the cytokinin BAP was reported to promote shoot and rhizome multiplication [4]. In the case of the hybrid Alstroemeria ligtu, the application of 2.22 μM BAP and 2.69 μM NAA resulted in the development of 2.25 rhizomes and 2.75 roots per shoot [24]. In addition, BAP was demonstrated to reduce apical dominance in shoots [39], which could explain the observed increase in shoot number in A. pelegrina.
Light intensity and quality are crucial factors in micropropagation, influencing plant growth and development. In this study, the spectral energy distribution of blue light corresponded to an emission peak of 420–440 nm, while red light was 660 nm. Notably, these wavelengths coincide with the absorption spectra of chlorophyll A (430 and 663 nm) and chlorophyll B (453 and 642 nm), optimizing photosynthetic efficiency [40]. The combination of 3:1 red:blue LED lights and 2.22 μM BAP resulted in the highest shoot height (6.2 cm) among the tested treatments.
In contrast, studies in Cymbidium revealed that red light promoted leaf growth but decreased chlorophyll content [41]. However, Curcuma longa micropropagated under 2:1 red:blue LED lights (25 μmol m−2 s−1 red and 11 μmol m−2 s−1 blue) characterized with a proliferation rate of 5.12 plants per explant with an increased number of shoots [42]. Conversely, in Vaccinium corymbosum, blue LED lights promoted chlorophyll accumulation, while red LED lights reversed this effect [43].
The combination of blue and red light is essential for chlorophyll synthesis, enhancing plant growth and development in vitro [44]. Lycium barbarum micropropagated under RB LED lights exhibited a 68% increase in shoot number, a 41% increase in leaf number, and a 64% increase in shoot length compared to white fluorescent lights [45]. Similar findings were observed in Carica papaya and Bacopa monnieri, where the red:blue ratio strongly stimulated shoot number [46,47].
Red and blue light spectra serve as the primary energy sources for plant photosynthesis, enabling the assimilation of CO2. LEDs offer precise control over wavelengths, allowing them to match the absorption spectra of plant photoreceptors. Consequently, LEDs can influence plant morphology and metabolism, making them a valuable tool in in vitro culture technology [48,49,50]. By manipulating light spectra, precise control over plant growth, development, and nutritional quality can be achieved. The introduction of LEDs into in vitro culture represents a significant innovation in the field of plant propagation.
This study also evaluated the average fresh weight of micropropagated plants. After four weeks of culture on rooting medium supplemented with 5.37 μM NAA and 5.37 μM IBA, fresh weights of 2.9 and 3.1 g and rhizome lengths of 1.9 and 1.7 cm were achieved, respectively. These results differ from those obtained in A. pallida, where BAP concentrations of 4.44 and 8.88 μM were used, resulting in a fresh weight of 4.6 g after eight weeks [30].
Furthermore, the use of MS medium supplemented with 5.37 μM NAA and 5.37 μM IBA promoted rooting in A. pelegrina, giving 100% and 96% of rooted plants, respectively.

5. Conclusions

This study reports the development of an efficient in vitro micropropagation protocol for the vulnerable species A. pelegrina. Shoots derived from in vitro seed germination were used as explants. MS medium supplemented with 2.22 μM BAP facilitated a multiplication rate of 4.6 plantlets per initial shoot. LED illumination further enhanced plant production potential. Following 4–5 months in culture, rooting success rates of 96–100% were achieved, accompanied by increased fresh weight and improved rhizogenic capacity. The protocol incorporates a critical acclimatization phase conducted in a cool greenhouse using a substrate. This final step resulted in a 90% survival rate after 6 weeks.
The successful establishment of this in vitro micropropagation protocol for A. pelegrina opens up exciting possibilities for the mass propagation of this vulnerable species. In vitro culture serves as a high-impact strategy for addressing conservation challenges associated with vulnerable or endangered plant species. The protocol’s efficiency, with a multiplication rate of 4.6 plantlets per initial shoot and high rooting success rates, makes it an attractive tool for large-scale production.

Author Contributions

Conceptualization, F.G. and M.C.; Methodology, F.G. and M.C.; Formal Analysis, F.G.; Research, F.G. and M.C.; Resources, M.C.; Writing—Original Draft Preparation, F.G.; M.C. and R.C.; Drafting: revising and editing, F.G., M.C. and R.C.; Acquisition of funds, M.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research has been funded by the Propagation Laboratory of the School of Agronomy, Pontificia Universidad Católica de Valparaíso, Chile.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors would like to thank the Propagation Laboratory the Pontificia Universidad Católica de Valparaíso, Chile, for the unconditional support given to this research.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

References

  1. Chiari, A.; Bridgen, M.P. Rhizome spiliting: A new micropropagation technique to increase in vitro propagula yield in Alstroemeria. Plant Cell Tissue Org. Cult. 2000, 62, 39–46. [Google Scholar] [CrossRef]
  2. Aagesen, L.; Sanso, M. The phylogeny of Alstroemeriaceae; based on Morphology; rps16 Intron; and rbcL sequence data. Syst. Bot. 2003, 28, 47–69. [Google Scholar]
  3. Chacón, J.; Sousa, A.; Baeza, C.M.; Renner, S.S. Ribosomal DNA distribution and a genus-wide phylogeny reveal patterns of chromosomal evolution in Alstroemeria (Alstroemeriaceae). Am.J. Bot. 2012, 99, 1501–1512. [Google Scholar] [CrossRef]
  4. Khaleghi, A.; Khalighi, A.; Sahraroo, M.; Karimi, A.; Rasoulnia, N.; Ghafoori, R. In vitro propagation of Alstroemeria cv. ‘Fuego’. Am.-Eurasian J. Agric. Environ. Sci. 2008, 3, 492–497. [Google Scholar]
  5. Hoshino, Y. Advances in alstroemeria biotechnology. In Floriculture, Ornamental and Plant Biotechnology; da Silva, J., Ed.; Advances and Topical Issues Vol. 5. Chapter 51; Global Science Books: Ikenobe, Japan, 2008; pp. 540–547. [Google Scholar]
  6. Cajas, D.; Baeza, C.; Ruíz, E.; Negritto, M. Análisis citogenético en poblaciones de Alstroemeria hookerilodd ssp. Hookeri (Alstroemeriaceae) en la región del Bio-Bio, Chile. Gayana Bot. 2009, 66, 117–126. [Google Scholar] [CrossRef]
  7. Hassani-Mehraban, A.; Dullemans, A.M.; Verhoeven, J. Alstroemeria yellow spot virus (AYSV): A new orthotospovirus species within a growing Eurasian clade. Arch. Virol. 2019, 164, 117. [Google Scholar] [CrossRef] [PubMed]
  8. Van Zaayen, A. Alstroemeria. In Virus and Virus-like Diseases of Bulb and Flower Crops; Loebenstein, G., Lawson, R.H., Brunt, A.A., Eds.; Wiley: West Sussex, UK, 1995; pp. 340–343. [Google Scholar]
  9. Lin, H.S.; De Jeu, M.J.; Jacobsen, E. Direct shoot regeneration from excised leaf explants of in vitro grown seedlings of Alstroemeria L. Plant Cell Rep. 1997, 16, 770–774. [Google Scholar] [CrossRef]
  10. King, J.; Bridgen, M. Environmental and Genotypic Regulation of Alstroemeria Seed Germination. HortScience 1990, 25, 1607–1609. [Google Scholar] [CrossRef]
  11. Hartmann, H.; Kester, D.; Davies, T.; Geneve, R. Principios y Prácticas de Propagación de Plantas, 7th ed.; Prentice Hall: Upper Saddle River, NJ, USA, 2002; pp. 367–374. [Google Scholar]
  12. Guerra, F.; Peñaloza, P.; Vidal, A.; Cautín, R.; Castro, M. Seed Maturity and Its In Vitro Initiation of Chilean Endemic Geophyte Alstroemeria pelegrina L. Horticulturae 2022, 8, 464. [Google Scholar] [CrossRef]
  13. Lu, C.; Bridgen, M.P. Effects of genotype; culture medium and embryo developmental stage on the in vitro responses from ovule cultures of interspecific hybrids of Alstroemeria. Plant Sci. 1996, 116, 205–212. [Google Scholar] [CrossRef]
  14. Kamstra, S.A.; Ramanna, M.S.; de Jeu, M.J.; Kuipers, A.G.J.; Jacobsen, E. Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea × A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes. Heredity 1999, 82, 69–78. [Google Scholar] [CrossRef]
  15. Burchi, G.; Mercuri, A.; Bianchini, C.; Bregliano, R.; Schiva, T. New interspecific hybrids of alstroemeria obtained through in vitro embryo-rescue. Acta Hortic. 2000, 508, 233–236. [Google Scholar] [CrossRef]
  16. De Benedetti, L.; Burchi, G.; Mercuri, A.; Pecchioni, N.; Faccioli, P.; Schiva, T. Random amplified polymorphic DNA (RAPD) analysis for the verification of hybridity in interspecific crosses of Alstroemeria. Plant Breed. 2000, 119, 443–445. [Google Scholar] [CrossRef]
  17. Bridgen, M.; Kollman, E.; Chunsheng, L. Interspecific hybridization of alstroemeria for the development of new; ornamental plants. Acta Hortic. 2009, 836, 73–78. [Google Scholar] [CrossRef]
  18. Aros, D.; Olate, E.; Valdés, S.; Infante, R. Gamma irradiation on Alstroemeria aurea G. in vitro rhizomes: An approach to the appropriate dosage for breeding purposes. Rev. Fac. Cienc. Agrarias. 2012, 44, 191–197. [Google Scholar]
  19. Lin, H.; Van Der Toorn, C.; Raemakers, K.J.J.M.; Visser, R.G.F.; De Jeu, M.; Jacobson, E. Genetic transformation of Alstroemeria using particle bombardment. Mol. Breed. 2000, 6, 369–377. [Google Scholar] [CrossRef]
  20. Kim, J.B.; Raemakers, C.J.; Jacobsen, E.; Visser, R.G.F. Efficient somatic embryogenesis in Alstroemeria. Plant Cell Tissue Organ Cult. 2006, 86, 233–238. [Google Scholar] [CrossRef]
  21. Pedraza-Santos, M.E.; López-Peralta, M.C.; González-Hernández, V.A.; Engleman-Clark, E.M.; Sánchez-García, P. In vitro regeneration of Alstroemeria cv. ‘Yellow king’ by direct organogenesis. Plant Cell Tissue Organ Cult. 2006, 84, 189–198. [Google Scholar] [CrossRef]
  22. Pumisutapon, P.; Visser, R.G.F.; De Klerk, G.J. Hormonal control of the outgrowth of axillary buds in Alstroemeria cultured in vitro. Biol. Plant. 2011, 55, 664–668. [Google Scholar] [CrossRef]
  23. Shahriari, A.G.; Bagheri, A.; Sharifi, A.; Moshtaghi, N. Efficient regeneration of ‘Caralis’ Alstroemeria cultivar from rhizome explants. Not. Sci. Biol. 2012, 4, 86–90. [Google Scholar] [CrossRef]
  24. Nasri, F.; Mortaza, S.N.; Ghaderi, N.; Javadi, T. Propagation in vitro of Alstroemeria ligtu hybrid through direct organogenesis from leaf base. J. Hortic. Res. 2013, 21, 23–30. [Google Scholar] [CrossRef]
  25. Pedersen, C.; Brandt, K. A method for disinfection of underground rhizome tips of Alstroemeria and Heliconia. Acta Hortic. 1992, 325, 499–504. [Google Scholar] [CrossRef]
  26. Ebrahim, M.; Ibrahim, I. Influence of medium solidification and pH value on in vitro propagation of Maranta leuconeura cv. Kerchoviana. Sci. Hortic. 2000, 86, 211–221. [Google Scholar] [CrossRef]
  27. Hamidoghli, Y.; Bohloli, S.; Abdollah, H. In vitro propagation of Alstroemeria using rhizome explants derived in vitro and in pot plants. Afr. J. Biotechnol. 2007, 6, 2147–2149. [Google Scholar] [CrossRef]
  28. De Klerk, G.J.; Ter Brugge, J. Micropropagation of Alstroemeria in liquid medium using slow release of medium components. Propag. Ornam. Plants 2010, 10, 246–252. [Google Scholar]
  29. Kyte, L.; Kleyn, J.; Scoggins, H.; Bridgen, M. Plants from Tubes: An Introduction to Micropropagation, 4th ed.; Timber Press: Portland, OR, USA, 2013; p. 269. [Google Scholar]
  30. Aros, D.; Vásquez, M.; Rivas, C.; Prat, M.L. An efficient method for in vitro propagation of Alstroemeria pallida Graham rhizomes. Chil. J. Agric. Res. 2017, 77, 95–99. [Google Scholar] [CrossRef]
  31. Muñoz, M.; Moreira, A. Alstroemerias de Chile: Diversidad; Distribución y Conservación; Taller La Era: Santiago, Chile, 2003; pp. 16–25. [Google Scholar]
  32. Ministerio del Medio Ambiente (MMA). Inventario Nacional de Especies de Chile. Available online: http://especies.mma.gob.cl/ (accessed on 15 March 2018).
  33. Ravenna, P.; Teillier, S.; Macaya, J.; Rodríguez, R.; Zöllner, O. Categorías de conservación de las plantas bulbosas nativas de Chile. Bol. Mus. Nac. Hist. Nat. 1998, 47, 47–68. [Google Scholar] [CrossRef]
  34. Finot, V.; Baeza, C.; Muñoz-Schick, M.; Ruiz, E.; Espejo, J.; Alarcón, D.; Carrasco, P.; Novoa, P.; Eyzaguirre, M. Guía de Campo Alstroemerias Chilenas, 1st ed.; Corporación Chilena de la Madera: Concepción, Chile, 2018; pp. 176–179. [Google Scholar]
  35. Pullman, G.S.; Bai, K.; Hane, M.; Ruland, D.; Cruse-Sanders, J.M.; Boyd, R.S.; Johnson, S. Seed cryopreservation and micropropagation of the federally threatened species; Price’s potato-bean (Apios priceana B.L. Robins.). In Vitro Cell. Dev. Biol. Plant 2019, 55, 558–568. [Google Scholar] [CrossRef]
  36. Issac, M.; Kuriakose, P.; Leung, S.; Costa, A.B.; Johnson, S.; Bucalo, K.; Stober, J.M.; Determann, R.O.; Rogers, W.L.; Cruse-Sanders, J.M.; et al. Seed Cryopreservation; Germination; and Micropropagation of Eastern Turkeybeard; Xerophyllum asphodeloides (L.) Nutt.: A Threatened Species from the Southeastern United States. Plants 2021, 10, 1462. [Google Scholar] [CrossRef]
  37. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962, 15, 473–497. [Google Scholar] [CrossRef]
  38. Tombion, L.; Soto, M.S.; Mori, M.; Mira, A.; Facciuto, G. PROPAGACIÓN IN VITRO DE Alstroemeria var. TIESTA DE 15 INTA’ A PARTIR DEL CULTIVO DE MERISTEMAS DE RIZOMA. Chil. J. Agric. Anim. Sci. 2020, 36, 110–116. [Google Scholar] [CrossRef]
  39. Ongaro, V.; Leyser, O. Hormonal control of shoot branching. J. Exp. Bot. 2008, 59, 67–74. [Google Scholar] [CrossRef] [PubMed]
  40. McCree, K. The action spectrum, absorptance and quantum yield of photosynthesis in crop plants. J. Agric. Meteorol. 1971, 9, 191–216. [Google Scholar] [CrossRef]
  41. Tanaka, M.; Takamura, T.; Watanabe, H.; Endo, M.; Yanagi, T.; Okamoto, K. In vitro growth of Cymbidium plantlets cultured under superbright red and blue light-emitting diodes (LEDs). J. Hortic. Sci. Biotechnol. 1998, 73, 39–44. [Google Scholar] [CrossRef]
  42. Marchant, M.J.; Molina, P.; Montecinos, M.; Guzmán, L.; Balada, C.; Fassio, C.; Castro, M. In Vitro Propagation of Easter Island Curcuma longa from Rhizome Explants Using Temporary Immersion System. Agronomy 2021, 11, 2121. [Google Scholar] [CrossRef]
  43. Hung, C.D.; Hong, C.H.; Kim, S.K.; Park, J.Y.; Nam, M.W.; Lee, H.I. LED light for in vitro and ex vitro efficient growth of economically important highbush blueberry (Vaccinium corymbosum L.). Acta Physiol. Plant. 2016, 38, 152. [Google Scholar] [CrossRef]
  44. Gupta, S.D.; Jatothu, B. Fundamentals and applications of light-emitting diodes (LEDs) in in vitro plant growth and morphogenesis. Plant Biotech. Rep. 2013, 7, 211–220. [Google Scholar] [CrossRef]
  45. De Oliveira Prudente, D.; De Souza, L.B.; Paiva, R. Goji berry (Lycium barbarum L.) in vitro multiplication improved by light-emitting diodes (LEDs) and 6-benzylaminopurine. In Vitro Cell. Dev. Biol.-Plant. 2019, 55, 258–264. [Google Scholar] [CrossRef]
  46. Da Silva, J.A.T. Photoauto-, Photohetero- and photomixotrophic in vitro propagation of papaya (Carica papaya L.) and response of seed and seedlings to light-emitting diodes. Tham Int. J. Sci. Technol. 2014, 19, 57–71. [Google Scholar]
  47. Karataş, M.; Aasim, M. Efficient in vitro regeneration of medical aquatic plant water hyssop (Bacopa monnieri L. Pennell). Pak. J. Agric. Sci. 2014, 51, 667–672. [Google Scholar]
  48. Kasajima, S.-Y.; Inoue, N.; Mahmud, R.; Fujita, K.; Kato, M. Effect of light quality on developmental rate of wheat under continuous light at a constant temperature. Plant Prod. Sci. 2007, 10, 286–291. [Google Scholar] [CrossRef]
  49. Bourget, C.M. An Introduction to Light-emitting Diodes. HortScience 2008, 43, 1944–1946. [Google Scholar] [CrossRef]
  50. Massa, G.D.; Kim, H.-H.; Wheeler, R.M.; Mitchell, C.A. Plant Productivity in Response to LED Lighting. HortScience 2008, 43, 1951–1956. [Google Scholar] [CrossRef]
Horticulturae 10 00674 g001
Figure 1. A. pelegrina seedling with first true leaf obtained by in vitro germination. Scale bar 1 cm.
Figure 1. A. pelegrina seedling with first true leaf obtained by in vitro germination. Scale bar 1 cm.
Horticulturae 10 00674 g001
Horticulturae 10 00674 g002
Figure 2. Effect of 6-Benzylaminopurine (BAP) concentration on A. pelegrina. BAP1: 0 μM; BAP2: 2.22 μM; BAP3: 4.44 μM. Scale bar 1 cm.
Figure 2. Effect of 6-Benzylaminopurine (BAP) concentration on A. pelegrina. BAP1: 0 μM; BAP2: 2.22 μM; BAP3: 4.44 μM. Scale bar 1 cm.
Horticulturae 10 00674 g002
Horticulturae 10 00674 g003
Figure 3. Rooting of an A. pelegrina shoot. Treatment 3 (ANA 5.37 μM). Scale bar 1 cm.
Figure 3. Rooting of an A. pelegrina shoot. Treatment 3 (ANA 5.37 μM). Scale bar 1 cm.
Horticulturae 10 00674 g003
Horticulturae 10 00674 g004
Figure 4. A. pelegrina plant during ex vitro acclimatization. (a) Plant after 2 weeks of acclimatization. (b) Acclimatized plant after 6 weeks. (c) Acclimatized plant after 6 weeks with storage roots. Scale bar 1 cm.
Figure 4. A. pelegrina plant during ex vitro acclimatization. (a) Plant after 2 weeks of acclimatization. (b) Acclimatized plant after 6 weeks. (c) Acclimatized plant after 6 weeks with storage roots. Scale bar 1 cm.
Horticulturae 10 00674 g004
Table 1. Photosynthetic photon flux density (PPFD) and red:blue ratios of light sources used for the in vitro multiplication of A. pelegrina.
Table 1. Photosynthetic photon flux density (PPFD) and red:blue ratios of light sources used for the in vitro multiplication of A. pelegrina.
Light TypeRedBlueRed:Blue Ratio
PPFD, μmol m−2 s−1
LIGHT1111:1
LIGHT2114≈3:1
LIGHT3661:1
Table 2. Effect of light spectra and BAP concentration on A. pelegrina shoot length and shoot number.
Table 2. Effect of light spectra and BAP concentration on A. pelegrina shoot length and shoot number.
Led LightsBAP ConcentrationSL (cm)SN
L1 (white light)BAP1 (0 μM)2.4 e2.9 bc
L1 (white light)BAP2 (2.22 μM)5.4 a4.8 a
L1 (white light)BAP3 (4.44 μM)3.7 bc3.5 abc
L2 (3:1 red:blue)BAP1 (0 μM)3.3 cd3.1 abc
L2 (3:1 red:blue)BAP2 (2.22 μM)6.2 a *4.6 ab
L2 (3:1 red:blue)BAP3 (4.44 μM)4.1 bc4.1 abc
L3 (1:1 red:blue)BAP1 (0 μM)2.7 de2.6 c
L3 (1:1 red:blue)BAP2 (2.22 μM)4.5 b4.3 abc
L3 (1:1 red:blue)BAP3 (4.44 μM)3.5 cd3.6 abc
F lights × BAPp = 0.017p = 0.86
* Different letters indicate significant differences according to Tukey’s test (p ≤ 0.05). SL: shoot length. SN: Shoot number.
Table 3. Effect of 6-Benzylaminopurine (BAP) concentration on A. pelegrina shoot number.
Table 3. Effect of 6-Benzylaminopurine (BAP) concentration on A. pelegrina shoot number.
TreatmentBAP Concentration (μM)SN
BAP102.9 c
BAP22.224.6 a *
BAP34.443.7 b
FBAPp = 0.00
FLEDp = 0.32
FBAP × LEDp = 0.86
* Different letters indicate significant differences according to Tukey’s test (p ≤ 0.05). SN: Shoot number.
Table 4. Effect of different NAA and IBA concentrations on rooting, fresh weight, and rhizome length of A. pelegrina.
Table 4. Effect of different NAA and IBA concentrations on rooting, fresh weight, and rhizome length of A. pelegrina.
TreatmentsAuxin TypeAuxin Concentration (μM)Rooting (%)Plant Fresh Weight (g)Rhizome Length (cm)
T1Control011 ± 1.0 c1.3 ± 0.2 c0.8 ± 0.2 c
T2NAA2.6953 ± 1.5 b1.9 ± 0.1 b1.4 ± 0.1 b
T3NAA5.37100 ± 2.0 a *2.9 ± 0.1 a *1.9 ± 0.1 a *
T4IBA2.6951 ± 1.3 b1.7 ± 0.3 b1.6 ± 0.2 b
T5IBA5.3796 ± 1.7 a3.1 ± 0.2 a2.1 ± 0.3 a
* Letters indicate significant differences according to Tukey’s test (p ≤ 0.05). Mean ± standard deviation.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Guerra, F.; Cautín, R.; Castro, M. In Vitro Micropropagation of the Vulnerable Chilean Endemic Alstroemeria pelegrina L. Horticulturae 2024, 10, 674. https://doi.org/10.3390/horticulturae10070674

AMA Style

Guerra F, Cautín R, Castro M. In Vitro Micropropagation of the Vulnerable Chilean Endemic Alstroemeria pelegrina L. Horticulturae. 2024; 10(7):674. https://doi.org/10.3390/horticulturae10070674

Chicago/Turabian Style

Guerra, Francesca, Ricardo Cautín, and Mónica Castro. 2024. "In Vitro Micropropagation of the Vulnerable Chilean Endemic Alstroemeria pelegrina L." Horticulturae 10, no. 7: 674. https://doi.org/10.3390/horticulturae10070674

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop