J Plant Biotechnol (2025) 52:001-008
Published online January 22, 2025
https://doi.org/10.5010/JPB.2025.52.001.001
© The Korean Society of Plant Biotechnology
Correspondence to : N. C. N. Alam (✉)
e-mail: camellia@mardi.gov.my
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
There has been a recent increase in the need for planting materials for Coffea liberica. In this study, we induced somatic embryogenesis of C. liberica leaf explants on modified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ), and 6-benzylaminopurine (BAP). The explants developed embryogenic calluses after 6 weeks of culture in MS medium supplemented with 0 mg/L BAP, 1 mg/L BAP, and 1 mg/L BAP + 1 mg/L TDZ. However, the explants cultured on MS medium supplemented with 1 mg/L BAP + 0.5 mg/L 2,4-D and 1 mg/L TDZ + 0.5 mg/L 2,4-D demonstrated no callus formation and browning of the leaf disc. Moreover, after 26 weeks of culturing, most cultures across all treatments were compromised due to browning except those cultured in MS medium supplemented with 1 mg/L BAP. Of these, 25% of MKL 10 (previously known as 224) clones survived, while only 10% of the clones differentiated into somatic embryos after 40 weeks of culture. Cotyledonary embryos were transformed into fully developed plants after 60 weeks of culturing. These results indicate that somatic embryogenesis of C. liberica leaf explants occur exclusively on MS medium supplemented with 1 mg/L BAP. Additionally, our results suggest that MS media supplemented with 1 mg/L BAP and 1 mg/L BAP + 1 mg/L TDZ were optimal for the induction of MKL 8 and 10 somatic embryogenesis, respectively. Both MKL 8 and 10 clones produce somatic embryos through the indirect embryogenesis pathway, except for MKL 10, which generates somatic embryos through both direct and indirect paths.
Keywords Thidiazuron, Leaf disc, Auxin, Callogenesis, Indirect embryogenesis, Cytokinin
Coffee is a globally esteemed beverage, making it a significant commodity in the international market and contributing substantially to the economies of several countries. Coffee cultivation occurs in tropical and subtropical regions, with the main producers being Brazil, Colombia, the Ivory Coast, Ethiopia, Mexico, Guatemala, Costa Rica, India, El Salvador, Uganda, Ecuador, Honduras, Vietnam, Indonesia and the Philippines (Menéndez-Yuffa and Garcia 1996). The primary species involved in the production of commercial coffee are Coffea arabica L. (arabica coffee) and Coffea canephora Pierre cv. robusta (robusta coffee) (Menéndez-Yuffa and Garcia 1996; Manila-Fajardo and Cervancia 2020). Other minor commercial coffee species include Coffea liberica Bull ex Hiern. var. liberica (liberica coffee) and Coffea excelsa A. Chev. [syn. Coffea liberica var. dewevrei: (De Wild. & T. Durand) Lebrun] (excelsa coffee) (Manila-Fajardo and Cervancia 2020; Menéndez-Yuffa and Garcia 1996). All the species belonged to the family Rubiaceae. Among the four, Liberica coffee possesses the largest fruit and has the highest caffeine content (Manila-Fajardo and Cervancia 2020).
Coffea liberica is cultivated commercially in Southeast Asian countries, including Malaysia, Indonesia, and the Philippines (Lee 2023). In Malaysia, mainly two kinds of coffee are commercially cultivated: Liberica (90%) and Robusta (10%) (Nor Amna and Mohd Amirul 2016). Liberica coffee species is a medium-sized tree that reaches approximately 9 meters in height. It possesses robust leaves, yields sizable berries, exhibits tolerance to arid environments and elevated salinity, and may flourish in peat soil (Hulupi 2014; N’Diaye et al. 2005). It is a diploid (2n = 22) and self-incompatible (PCARR 1976).
In Malaysia, Liberica coffee has been planted mostly in Johor, Selangor and Sabah (Mohd Zaffrie et al. 2016). The Liberica beans is relatively expensive in the Malaysian market. As a result, demand for planting materials is rising. Due to the challenges associated with producing Liberica coffee using vegetative or clonal methods, it is commonly propagated by seeds. Seed propagation induces variability, resulting in progenies that do not possess identical traits to their parent. Clonal propagation is achieved through cutting, grafting, and tissue culture. In vitro culture has recently gained attention as a method of large-scale propagation.
Somatic embryogenesis is one of the tissue culture techniques that are employed in plant propagation (Aguilar et al. 2022). This method involves a somatic cell, cultured under controlled conditions, transforming into an embryogenic cell, which then generates a somatic (clonal) embryo genetically identical to the parent cell by a series of morphological and biochemical changes (Toonen and De Vries 1996). The first report on coffee somatic embryo (SE) was published by Staritsky (1970). Somatic embryogenesis can be either direct or indirect, with embryogenic cells arising from non-embryogenic, disordered mitotic cycles or directly from explant cells (Acuna 1993). Therefore, this study aimed to evaluate the effects of plant growth regulators and media modification on development of somatic embryos of Coffea liberica.
Healthy new scion of Coffea liberica new clones were acquired from the Malaysian Agricultural Research and Development Institute (MARDI) in Kluang, Johor. (Fig. 1). The experiments were then carried out in the MyGeneBank Complex in the cryopreservation lab at MARDI, Serdang (2°58'42.3"N 101°41' 17.4"E). Surface sterilization of the explant was conducted promptly.
Fully opened young leaves, from the scion emerging from orthotropic branches, were collected and washed with Decon 90 and subsequently rinsed with tap water. The explants were subsequently cleaned under running tap water for 30 minutes, immersed in 20 ml of Dettol antiseptic liquid diluted in 500 ml of distilled water for 30 seconds to 1 minute, and then rinsed with distilled water. Explants were afterwards immersed in a 0.5 g/l solution of Kenlate fungicide (50% benomyl) for 30 minutes and then rinsed with distilled water. After that, under the horizontal laminar airflow cabinet (ESCO, USA), explants were transferred and immersed in 30% commercial bleach Clorox® (5.65% sodium hypochlorite) with three drops of Tween 20 for 25 minutes, followed by rinsing with double-distilled water. The explants were subsequently disinfected with 70% ethanol for 30 seconds and washed with double-distilled water. The explants were then sectioned into smaller fragments with a punch with a diameter of 0.6 cm. The leaf discs were subsequently grown in modified Murashige and Skoog (1962) (MS) medium, as per Yasuda et al. (1985). At the beginning of the investigation, clones 211 and 224 (MKL 10) were assessed with five treatments of plant growth regulator (PGR) supplementation, either individually or in combination, including 6-Benzylaminopurine (BAP), Thidiazuron (TDZ), and 2,4-Dichlorophenoxyacetic acid (2,4-D). Treatments were as follows: (T1) 0 mg/L BAP, (T2) 1 mg/L BAP, (T3) 1 mg/L BAP+ 1 mg/L TDZ, (T4) 1 mg/L BAP + 0.5 mg/L 2,4-D, (T5) 1 mg/L TDZ + 0.5 mg/L 2,4-D. All treatments were duplicated four times, and each replication consisted in five experimental units.
Following that, the study was expanded by assessing seven different media combinations across three clones: 213 (MKL8), 222 (MKL9), and 224 (MKL10). Treatments were detailed as follows: (T1) MS modification Yasuda et al. (1985) + 30 g/L sucrose +3 g/L gelrite + 1 mg/L BAP, (T2) MS modification Yasuda et al. (1985) + 30 g/L sucrose + 3 g/L gelrite + 1 mg/L BAP + 7% PEG 6000 (Almeida et al. 2008), (T3) MS modification Yasuda et al. (1985) + 30 g/L sucrose +3 g/L gelrite + 1mg/L BAP + 1 mg/L TDZ, (T4) MS modification Yasuda et al. (1985) + 30 g/L sucrose + 3 g/L gelrite + 1 mg/L BAP + 1 mg/L kinetin, (T5) MS modification Quiroz-Figueroa et al. (2006) + 30 g/L sucrose +2.5 g/L gelrite + 1.12 mg/L, (T6) MS modification Acuna (1993) + Vitamin B5 + 30 g/L sucrose + 3 g/L gelrite + 1 mg/L 2ip, and (T7) Half strength MS +20 g/L sucrose + 6.75 mg/L BAP + 2.5 g/L gelrite (Almeida and Silvarolla 2009). Each treatment was duplicated eight times with three experimental units.
These cultures were incubated in the dark for 2 weeks at room temperature of 27 ± 2°C. The sub-culture was done using the same media after every 4 to 6 weeks of incubation. Embryo quality was recorded qualitatively by determining the formation, colour and texture of callus and embryo after 6 weeks of culture. All data were examined using analysis of variance (ANOVA) with Statistical Analysis System Software (SAS) version 9.4. The experiment was conducted using a Completely Randomised Design (CRD). The significance of differences among means was assessed using Duncan’s Multiple Range Test (DMRT). Statistical significance was defined as P ≤ 0.05.
The clean explant achieved around 97% efficacy through the sterilisation procedure. This study indicates that Coffea liberica mostly engages in an indirect pathway comprising two phases: callogenesis and embryogenesis. A comparable result was documented by Ardiyani et al. (2020). The first study revealed that the explants developed embryogenic calluses after 12 weeks of culture only with the Yasuda et al. (1985) medium containing (T1) 0 mg/L BAP, (T2) 1 mg/L BAP, and (T3) a combination of 1 mg/L BAP and 1 mg/L TDZ, yielded both compact and friable calluses (Table 1, Fig. 1). Treatment T1 and T2 yielded green and white calluses, whereas T3 resulted in green, white, yellow, and brown calluses in clone 211 and green, white, transparent, and brown calluses in clone 224 (Table 1, Fig. 2). Non-embryogenic callus exhibited a more compact or spongy texture, characterised by a yellowish-brown hue and the absence of distinct cells, while embryogenic callus was more friable, displaying a yellowish-white colour and a glossy appearance (Ardiyani 2015). The breakdown of embryogenic callus cells by phenolic substances produced as a byproduct of the callus production process was one of the reasons why non-embryogenic callus was more common than embryogenic callus (Alemanno et al. 1996). Since non-embryogenic callus has less capacity for regeneration, it has a propensity to be brown in hue. The presence of phenol and the activity of polyphenoloxidase may also contribute to browning (Ardiyani 2015).
Table 1 Quality of the embryogenic calluses of Coffea liberica across various combinations of plant growth regulators (PGRs)
Clones | Treatments | Explants producing calluses (%) | Callus formation | Callus color | Callus texture at 12 weeks | ||
---|---|---|---|---|---|---|---|
6 weeks | 12 weeks | 6 weeks | 12 weeks | ||||
Clone 211 | T1: 0 mg/L BAP | 44 | 89 | +++ | G | G, W | F, C |
T2: 1 mg/L BAP | 75 | 100 | ++ | G | G, W | F, C | |
T3: 1 mg/L BAP + 1 mg/L TDZ | 100 | 100 | ++++ | G, W | G, W, Y, B | F, C | |
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D | 0 | 0 | NA | NA | NA | NA | |
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ | 0 | 0 | NA | NA | NA | NA | |
Clone 224 | T1: 0 mg/L BAP | 37 | 89 | +++ | G | G, W | F, C |
T2: 1 mg/L BAP | 42 | 56 | ++ | G | G, W | F, C | |
T3: 1 mg/L BAP + 1 mg/L TDZ | 45 | 45 | +++ | W, G | G, W, T, B | F, C | |
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D | 5 | 5 | + | NA | NA | C | |
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ | 0 | 0 | NA | NA | NA | NA |
Note: NA = Not available; formation + = very few, ++ = few, +++ = moderate, and ++++ = Abundant; color G = green, W = white, Y = yellow, B = brown, T = transparent; and texture F = friable and C = compact
Embryogenic callus possesses the capability to develop into somatic embryos through an indirect pathway. C. liberica leaf explants grown on a medium including (T4) 1 mg/L BAP and 0.5 mg/L 2,4-D, as well as (T5) 1 mg/L TDZ and 0.5 mg/L 2,4-D, exhibited inhibition of callus formation and browning of the explants (Fig. 1). Only 5% of the explant exhibited callus formation in media enriched with 1 mg/L BAP and 0.5 mg/L 2,4-D when grown with clone 224 (MKL10) (Table 1). This may be attributable to the presence of 2-4 D. In contrast, Almeida (2020) stated that auxin 2,4-D is the most often utilized agent for inducing callus initiation in C. arabica explants. Ardiyani (2015) proved that the addition of single auxin 2,4-D enhanced explant development into embryogenic callus in C. liberica. Hatanaka et al. (1995) also discovered that auxins applied at varying dosages impeded direct somatic embryogenesis in C. canephora. The combination of cytokinins (BAP, TDZ) with auxin 2,4-D demonstrated suppression of callus formation and produced browning in the leaf explant. This specific combination of plant growth regulators is solely applicable for inducing somatic embryos from generated callus, as demonstrated in the research conducted by Ardiyani et al. (2020). After 26 weeks of incubation, most cultures across all treatments were compromised due to browning except for explants in a growth medium containing 1 mg/L BAP, of which 25% of clone 224 survived and only 10% differentiated into somatic embryos (SEs) after 40 weeks of culture. The transformation of the cotyledonary embryo into a fully developed plant occurred after 60 weeks of culture.
Based on the findings of the initial study, further research was undertaken to examine the response of explants to various combinations of plant growth regulators (PGR) and supplementary osmotic agents across several modifications of MS media in three novel clones (MKL 8, 9, and 10) released by the Malaysian Agricultural Research and Development Institute (MARDI) in the year 2023. All treatments applied to these three clones resulted in callus formation from the leaf explant. In MKL 8, only treatments 1, 4, and 7 yielded somatic embryos up to the cotyledon stage. In MKL 9, no treatments yielded somatic embryos, whereas in MKL 10, only treatments 3, 5, and 6 generated somatic embryos up to the cotyledon stage (Table 2). The optimal media for somatic embryo production of MKL 8 was the MS modification by Yasuda et al. (1985) with the inclusion of 1 mg/L BAP, yielding a 38% success rate, whereas for MKL 10, the MS modification by Yasuda et al. (1985) supplemented with 1 mg/L BAP and 1 mg/L TDZ achieved a 63% success rate (Table 2). Ardiyani et al. (2021) discovered that an additional of 1 mg/L BAP during the developing phase yielded the highest number and weight of somatic embryos in C. liberica. Direct somatic embryogenesis has also been observed during this treatment on MKL 10. However, only one somatic embryo was documented originating from the edge of the leaf disc (Fig. 3h). The somatic embryo development of C. liberica began with the production of globular, torpedo, and cotyledonary stages, culminating in the transformation of the cotyledonary embryo into a full plant (Fig. 3), similar to C. arabica and also C. canephora (Ibrahim et al. 2013; Santana-Buzzy et al. 2007). Globular stage somatic embryos were first observed at 12 weeks for both MKL 8 and MKL 10. The heart/torpedo stage was established earliest at 12 weeks for MKL 8 and 16 weeks for MKL 10, and transitioned to the cotyledon stage latest at 20 weeks for MKL 8 and 16 weeks for MKL 10 (Fig. 4 and 5).
Table 2 MKL 8, 9, and 10 callus induction, callus formation, and potential somatic embryos on seven culture media
Clones | Treatments | Explants producing callus (%) at 4 weeks | Callus formation | Explants producing SE (cotyledon) (%) at 28 weeks |
---|---|---|---|---|
(MKL 8) 213 | T1 | 96 ± 4.2 b | ++ | 38 ± 18.3 b |
T2 | 87 ± 6.2 b | ++ | 0 ± 0 a | |
T3 | 96 ± 4.2 b | +++ | 0 ± 0 a | |
T4 | 96 ± 4.2 b | ++ | 12.5 ± 12.5 a | |
T5 | 62 ± 11.7 a | + | 0 ± 0 a | |
T6 | 76 ± 9.7 ab | ++ | 0 ± 0 a | |
T7 | 83 ± 6.4 ab | ++ | 12.5 ± 12.5 a | |
(MKL 9) 222 | T1 | 41 ± 10.4 ab | + | 0 ± 0 a |
T2 | 17 ± 8.8 a | + | 0 ± 0 a | |
T3 | 62 ± 14.7 bcd | ++ | 0 ± 0 a | |
T4 | 46 ± 8.8 abc | + | 0 ± 0 a | |
T5 | 75 ± 5.6 cd | ++ | 0 ± 0 a | |
T6 | 83 ± 9.0 d | +++ | 0 ± 0 a | |
T7 | 75 ± 8.4 cd | ++ | 0 ± 0 a | |
(MKL 10) 224 | T1 | 62 ± 14.7 ab | ++ | 0 ± 0 a |
T2 | 46 ± 8.8 a | + | 0 ±0 a | |
T3 | 71 ± 9.9 ab | ++ | 63 ± 16.4 b | |
T4 | 54 ± 8.8 ab | ++ | 0 ± 0 a | |
T5 | 70 ± 7.6 ab | + | 38 ± 16.4 a | |
T6 | 83 ± 6.4 b | +++ | 12.5 ± 12.5 a | |
T7 | 83 ± 9.0 b | ++ | 0 ± 0 a |
Note: Values are represented as means ± SE; values represented with the same letter in the same column were not significantly different (P > 0.05), as determined by Duncan’s multiple range test
The simultaneous occurrence of all developmental phases was seen in both MKL 8 and 10, indicating that the somatic embryo development process is asynchronous, as noted by Ardiyani et al. (2020) in C. liberica. The specific regulatory mechanisms controlling somatic embryogenesis in coffee plants remain unclear. Understanding the variables that affect somatic embryogenesis in C. liberica will help enhance its use, particularly the direct pathway. The presence or absence of competent cells in the explant, which are intrinsic to their totipotency, dictates the ability of the species for somatic embryogenesis (Almeida 2020). The transition from the cotyledon stage to a fully developed plant is very low and occurred over a long period (more than a year), utilising the same medium employed for somatic embryo induction. Additional research is required to enhance the conversion rate of cotyledons into fully developed plants for C. liberica.
An essential component affecting the induction of somatic embryogenesis in C. liberica is the application of cytokinins, specifically BAP and TDZ. Auxin 2,4-D shown ineffectiveness in inducing callus formation on explants when paired with BAP or TDZ. Somatic embryogenesis only occurs on MS media supplemented with 1 mg/L BAP. The best medium for somatic embryo formation was the MS modification by Yasuda et al. (1985) supplemented with 1 mg/L BAP for MKL 8, while the MS modification by Yasuda et al. (1985) enhanced with 1 mg/L BAP and 1 mg/L TDZ was used for MKL 10. Both clones generate somatic embryos by the indirect pathway of embryogenesis, except for MKL10, which produces somatic embryos through both direct and indirect pathways.
The authors express gratitude to the Malaysian Agricultural Research and Development Institute (MARDI) for the financial support provided for this project under the Twelfth Malaysia Plan (RMK 12-PIC 507). The authors would also like to thank Noor Syahira Nasarudin for providing the planting materials for this project.
J Plant Biotechnol 2025; 52(1): 1-8
Published online January 22, 2025 https://doi.org/10.5010/JPB.2025.52.001.001
Copyright © The Korean Society of Plant Biotechnology.
Noor Camellia Noor Alam · Nora’ini Abdullah · Mohd Rani Awang
Industrial Crop Research Center, Malaysian Agricultural Research and Development Institute, 43400 Serdang, Selangor, Malaysia
Correspondence to:N. C. N. Alam (✉)
e-mail: camellia@mardi.gov.my
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
There has been a recent increase in the need for planting materials for Coffea liberica. In this study, we induced somatic embryogenesis of C. liberica leaf explants on modified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ), and 6-benzylaminopurine (BAP). The explants developed embryogenic calluses after 6 weeks of culture in MS medium supplemented with 0 mg/L BAP, 1 mg/L BAP, and 1 mg/L BAP + 1 mg/L TDZ. However, the explants cultured on MS medium supplemented with 1 mg/L BAP + 0.5 mg/L 2,4-D and 1 mg/L TDZ + 0.5 mg/L 2,4-D demonstrated no callus formation and browning of the leaf disc. Moreover, after 26 weeks of culturing, most cultures across all treatments were compromised due to browning except those cultured in MS medium supplemented with 1 mg/L BAP. Of these, 25% of MKL 10 (previously known as 224) clones survived, while only 10% of the clones differentiated into somatic embryos after 40 weeks of culture. Cotyledonary embryos were transformed into fully developed plants after 60 weeks of culturing. These results indicate that somatic embryogenesis of C. liberica leaf explants occur exclusively on MS medium supplemented with 1 mg/L BAP. Additionally, our results suggest that MS media supplemented with 1 mg/L BAP and 1 mg/L BAP + 1 mg/L TDZ were optimal for the induction of MKL 8 and 10 somatic embryogenesis, respectively. Both MKL 8 and 10 clones produce somatic embryos through the indirect embryogenesis pathway, except for MKL 10, which generates somatic embryos through both direct and indirect paths.
Keywords: Thidiazuron, Leaf disc, Auxin, Callogenesis, Indirect embryogenesis, Cytokinin
Coffee is a globally esteemed beverage, making it a significant commodity in the international market and contributing substantially to the economies of several countries. Coffee cultivation occurs in tropical and subtropical regions, with the main producers being Brazil, Colombia, the Ivory Coast, Ethiopia, Mexico, Guatemala, Costa Rica, India, El Salvador, Uganda, Ecuador, Honduras, Vietnam, Indonesia and the Philippines (Menéndez-Yuffa and Garcia 1996). The primary species involved in the production of commercial coffee are Coffea arabica L. (arabica coffee) and Coffea canephora Pierre cv. robusta (robusta coffee) (Menéndez-Yuffa and Garcia 1996; Manila-Fajardo and Cervancia 2020). Other minor commercial coffee species include Coffea liberica Bull ex Hiern. var. liberica (liberica coffee) and Coffea excelsa A. Chev. [syn. Coffea liberica var. dewevrei: (De Wild. & T. Durand) Lebrun] (excelsa coffee) (Manila-Fajardo and Cervancia 2020; Menéndez-Yuffa and Garcia 1996). All the species belonged to the family Rubiaceae. Among the four, Liberica coffee possesses the largest fruit and has the highest caffeine content (Manila-Fajardo and Cervancia 2020).
Coffea liberica is cultivated commercially in Southeast Asian countries, including Malaysia, Indonesia, and the Philippines (Lee 2023). In Malaysia, mainly two kinds of coffee are commercially cultivated: Liberica (90%) and Robusta (10%) (Nor Amna and Mohd Amirul 2016). Liberica coffee species is a medium-sized tree that reaches approximately 9 meters in height. It possesses robust leaves, yields sizable berries, exhibits tolerance to arid environments and elevated salinity, and may flourish in peat soil (Hulupi 2014; N’Diaye et al. 2005). It is a diploid (2n = 22) and self-incompatible (PCARR 1976).
In Malaysia, Liberica coffee has been planted mostly in Johor, Selangor and Sabah (Mohd Zaffrie et al. 2016). The Liberica beans is relatively expensive in the Malaysian market. As a result, demand for planting materials is rising. Due to the challenges associated with producing Liberica coffee using vegetative or clonal methods, it is commonly propagated by seeds. Seed propagation induces variability, resulting in progenies that do not possess identical traits to their parent. Clonal propagation is achieved through cutting, grafting, and tissue culture. In vitro culture has recently gained attention as a method of large-scale propagation.
Somatic embryogenesis is one of the tissue culture techniques that are employed in plant propagation (Aguilar et al. 2022). This method involves a somatic cell, cultured under controlled conditions, transforming into an embryogenic cell, which then generates a somatic (clonal) embryo genetically identical to the parent cell by a series of morphological and biochemical changes (Toonen and De Vries 1996). The first report on coffee somatic embryo (SE) was published by Staritsky (1970). Somatic embryogenesis can be either direct or indirect, with embryogenic cells arising from non-embryogenic, disordered mitotic cycles or directly from explant cells (Acuna 1993). Therefore, this study aimed to evaluate the effects of plant growth regulators and media modification on development of somatic embryos of Coffea liberica.
Healthy new scion of Coffea liberica new clones were acquired from the Malaysian Agricultural Research and Development Institute (MARDI) in Kluang, Johor. (Fig. 1). The experiments were then carried out in the MyGeneBank Complex in the cryopreservation lab at MARDI, Serdang (2°58'42.3"N 101°41' 17.4"E). Surface sterilization of the explant was conducted promptly.
Fully opened young leaves, from the scion emerging from orthotropic branches, were collected and washed with Decon 90 and subsequently rinsed with tap water. The explants were subsequently cleaned under running tap water for 30 minutes, immersed in 20 ml of Dettol antiseptic liquid diluted in 500 ml of distilled water for 30 seconds to 1 minute, and then rinsed with distilled water. Explants were afterwards immersed in a 0.5 g/l solution of Kenlate fungicide (50% benomyl) for 30 minutes and then rinsed with distilled water. After that, under the horizontal laminar airflow cabinet (ESCO, USA), explants were transferred and immersed in 30% commercial bleach Clorox® (5.65% sodium hypochlorite) with three drops of Tween 20 for 25 minutes, followed by rinsing with double-distilled water. The explants were subsequently disinfected with 70% ethanol for 30 seconds and washed with double-distilled water. The explants were then sectioned into smaller fragments with a punch with a diameter of 0.6 cm. The leaf discs were subsequently grown in modified Murashige and Skoog (1962) (MS) medium, as per Yasuda et al. (1985). At the beginning of the investigation, clones 211 and 224 (MKL 10) were assessed with five treatments of plant growth regulator (PGR) supplementation, either individually or in combination, including 6-Benzylaminopurine (BAP), Thidiazuron (TDZ), and 2,4-Dichlorophenoxyacetic acid (2,4-D). Treatments were as follows: (T1) 0 mg/L BAP, (T2) 1 mg/L BAP, (T3) 1 mg/L BAP+ 1 mg/L TDZ, (T4) 1 mg/L BAP + 0.5 mg/L 2,4-D, (T5) 1 mg/L TDZ + 0.5 mg/L 2,4-D. All treatments were duplicated four times, and each replication consisted in five experimental units.
Following that, the study was expanded by assessing seven different media combinations across three clones: 213 (MKL8), 222 (MKL9), and 224 (MKL10). Treatments were detailed as follows: (T1) MS modification Yasuda et al. (1985) + 30 g/L sucrose +3 g/L gelrite + 1 mg/L BAP, (T2) MS modification Yasuda et al. (1985) + 30 g/L sucrose + 3 g/L gelrite + 1 mg/L BAP + 7% PEG 6000 (Almeida et al. 2008), (T3) MS modification Yasuda et al. (1985) + 30 g/L sucrose +3 g/L gelrite + 1mg/L BAP + 1 mg/L TDZ, (T4) MS modification Yasuda et al. (1985) + 30 g/L sucrose + 3 g/L gelrite + 1 mg/L BAP + 1 mg/L kinetin, (T5) MS modification Quiroz-Figueroa et al. (2006) + 30 g/L sucrose +2.5 g/L gelrite + 1.12 mg/L, (T6) MS modification Acuna (1993) + Vitamin B5 + 30 g/L sucrose + 3 g/L gelrite + 1 mg/L 2ip, and (T7) Half strength MS +20 g/L sucrose + 6.75 mg/L BAP + 2.5 g/L gelrite (Almeida and Silvarolla 2009). Each treatment was duplicated eight times with three experimental units.
These cultures were incubated in the dark for 2 weeks at room temperature of 27 ± 2°C. The sub-culture was done using the same media after every 4 to 6 weeks of incubation. Embryo quality was recorded qualitatively by determining the formation, colour and texture of callus and embryo after 6 weeks of culture. All data were examined using analysis of variance (ANOVA) with Statistical Analysis System Software (SAS) version 9.4. The experiment was conducted using a Completely Randomised Design (CRD). The significance of differences among means was assessed using Duncan’s Multiple Range Test (DMRT). Statistical significance was defined as P ≤ 0.05.
The clean explant achieved around 97% efficacy through the sterilisation procedure. This study indicates that Coffea liberica mostly engages in an indirect pathway comprising two phases: callogenesis and embryogenesis. A comparable result was documented by Ardiyani et al. (2020). The first study revealed that the explants developed embryogenic calluses after 12 weeks of culture only with the Yasuda et al. (1985) medium containing (T1) 0 mg/L BAP, (T2) 1 mg/L BAP, and (T3) a combination of 1 mg/L BAP and 1 mg/L TDZ, yielded both compact and friable calluses (Table 1, Fig. 1). Treatment T1 and T2 yielded green and white calluses, whereas T3 resulted in green, white, yellow, and brown calluses in clone 211 and green, white, transparent, and brown calluses in clone 224 (Table 1, Fig. 2). Non-embryogenic callus exhibited a more compact or spongy texture, characterised by a yellowish-brown hue and the absence of distinct cells, while embryogenic callus was more friable, displaying a yellowish-white colour and a glossy appearance (Ardiyani 2015). The breakdown of embryogenic callus cells by phenolic substances produced as a byproduct of the callus production process was one of the reasons why non-embryogenic callus was more common than embryogenic callus (Alemanno et al. 1996). Since non-embryogenic callus has less capacity for regeneration, it has a propensity to be brown in hue. The presence of phenol and the activity of polyphenoloxidase may also contribute to browning (Ardiyani 2015).
Table 1 . Quality of the embryogenic calluses of Coffea liberica across various combinations of plant growth regulators (PGRs).
Clones | Treatments | Explants producing calluses (%) | Callus formation | Callus color | Callus texture at 12 weeks | ||
---|---|---|---|---|---|---|---|
6 weeks | 12 weeks | 6 weeks | 12 weeks | ||||
Clone 211 | T1: 0 mg/L BAP | 44 | 89 | +++ | G | G, W | F, C |
T2: 1 mg/L BAP | 75 | 100 | ++ | G | G, W | F, C | |
T3: 1 mg/L BAP + 1 mg/L TDZ | 100 | 100 | ++++ | G, W | G, W, Y, B | F, C | |
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D | 0 | 0 | NA | NA | NA | NA | |
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ | 0 | 0 | NA | NA | NA | NA | |
Clone 224 | T1: 0 mg/L BAP | 37 | 89 | +++ | G | G, W | F, C |
T2: 1 mg/L BAP | 42 | 56 | ++ | G | G, W | F, C | |
T3: 1 mg/L BAP + 1 mg/L TDZ | 45 | 45 | +++ | W, G | G, W, T, B | F, C | |
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D | 5 | 5 | + | NA | NA | C | |
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ | 0 | 0 | NA | NA | NA | NA |
Note: NA = Not available; formation + = very few, ++ = few, +++ = moderate, and ++++ = Abundant; color G = green, W = white, Y = yellow, B = brown, T = transparent; and texture F = friable and C = compact.
Embryogenic callus possesses the capability to develop into somatic embryos through an indirect pathway. C. liberica leaf explants grown on a medium including (T4) 1 mg/L BAP and 0.5 mg/L 2,4-D, as well as (T5) 1 mg/L TDZ and 0.5 mg/L 2,4-D, exhibited inhibition of callus formation and browning of the explants (Fig. 1). Only 5% of the explant exhibited callus formation in media enriched with 1 mg/L BAP and 0.5 mg/L 2,4-D when grown with clone 224 (MKL10) (Table 1). This may be attributable to the presence of 2-4 D. In contrast, Almeida (2020) stated that auxin 2,4-D is the most often utilized agent for inducing callus initiation in C. arabica explants. Ardiyani (2015) proved that the addition of single auxin 2,4-D enhanced explant development into embryogenic callus in C. liberica. Hatanaka et al. (1995) also discovered that auxins applied at varying dosages impeded direct somatic embryogenesis in C. canephora. The combination of cytokinins (BAP, TDZ) with auxin 2,4-D demonstrated suppression of callus formation and produced browning in the leaf explant. This specific combination of plant growth regulators is solely applicable for inducing somatic embryos from generated callus, as demonstrated in the research conducted by Ardiyani et al. (2020). After 26 weeks of incubation, most cultures across all treatments were compromised due to browning except for explants in a growth medium containing 1 mg/L BAP, of which 25% of clone 224 survived and only 10% differentiated into somatic embryos (SEs) after 40 weeks of culture. The transformation of the cotyledonary embryo into a fully developed plant occurred after 60 weeks of culture.
Based on the findings of the initial study, further research was undertaken to examine the response of explants to various combinations of plant growth regulators (PGR) and supplementary osmotic agents across several modifications of MS media in three novel clones (MKL 8, 9, and 10) released by the Malaysian Agricultural Research and Development Institute (MARDI) in the year 2023. All treatments applied to these three clones resulted in callus formation from the leaf explant. In MKL 8, only treatments 1, 4, and 7 yielded somatic embryos up to the cotyledon stage. In MKL 9, no treatments yielded somatic embryos, whereas in MKL 10, only treatments 3, 5, and 6 generated somatic embryos up to the cotyledon stage (Table 2). The optimal media for somatic embryo production of MKL 8 was the MS modification by Yasuda et al. (1985) with the inclusion of 1 mg/L BAP, yielding a 38% success rate, whereas for MKL 10, the MS modification by Yasuda et al. (1985) supplemented with 1 mg/L BAP and 1 mg/L TDZ achieved a 63% success rate (Table 2). Ardiyani et al. (2021) discovered that an additional of 1 mg/L BAP during the developing phase yielded the highest number and weight of somatic embryos in C. liberica. Direct somatic embryogenesis has also been observed during this treatment on MKL 10. However, only one somatic embryo was documented originating from the edge of the leaf disc (Fig. 3h). The somatic embryo development of C. liberica began with the production of globular, torpedo, and cotyledonary stages, culminating in the transformation of the cotyledonary embryo into a full plant (Fig. 3), similar to C. arabica and also C. canephora (Ibrahim et al. 2013; Santana-Buzzy et al. 2007). Globular stage somatic embryos were first observed at 12 weeks for both MKL 8 and MKL 10. The heart/torpedo stage was established earliest at 12 weeks for MKL 8 and 16 weeks for MKL 10, and transitioned to the cotyledon stage latest at 20 weeks for MKL 8 and 16 weeks for MKL 10 (Fig. 4 and 5).
Table 2 . MKL 8, 9, and 10 callus induction, callus formation, and potential somatic embryos on seven culture media.
Clones | Treatments | Explants producing callus (%) at 4 weeks | Callus formation | Explants producing SE (cotyledon) (%) at 28 weeks |
---|---|---|---|---|
(MKL 8) 213 | T1 | 96 ± 4.2 b | ++ | 38 ± 18.3 b |
T2 | 87 ± 6.2 b | ++ | 0 ± 0 a | |
T3 | 96 ± 4.2 b | +++ | 0 ± 0 a | |
T4 | 96 ± 4.2 b | ++ | 12.5 ± 12.5 a | |
T5 | 62 ± 11.7 a | + | 0 ± 0 a | |
T6 | 76 ± 9.7 ab | ++ | 0 ± 0 a | |
T7 | 83 ± 6.4 ab | ++ | 12.5 ± 12.5 a | |
(MKL 9) 222 | T1 | 41 ± 10.4 ab | + | 0 ± 0 a |
T2 | 17 ± 8.8 a | + | 0 ± 0 a | |
T3 | 62 ± 14.7 bcd | ++ | 0 ± 0 a | |
T4 | 46 ± 8.8 abc | + | 0 ± 0 a | |
T5 | 75 ± 5.6 cd | ++ | 0 ± 0 a | |
T6 | 83 ± 9.0 d | +++ | 0 ± 0 a | |
T7 | 75 ± 8.4 cd | ++ | 0 ± 0 a | |
(MKL 10) 224 | T1 | 62 ± 14.7 ab | ++ | 0 ± 0 a |
T2 | 46 ± 8.8 a | + | 0 ±0 a | |
T3 | 71 ± 9.9 ab | ++ | 63 ± 16.4 b | |
T4 | 54 ± 8.8 ab | ++ | 0 ± 0 a | |
T5 | 70 ± 7.6 ab | + | 38 ± 16.4 a | |
T6 | 83 ± 6.4 b | +++ | 12.5 ± 12.5 a | |
T7 | 83 ± 9.0 b | ++ | 0 ± 0 a |
Note: Values are represented as means ± SE; values represented with the same letter in the same column were not significantly different (P > 0.05), as determined by Duncan’s multiple range test.
The simultaneous occurrence of all developmental phases was seen in both MKL 8 and 10, indicating that the somatic embryo development process is asynchronous, as noted by Ardiyani et al. (2020) in C. liberica. The specific regulatory mechanisms controlling somatic embryogenesis in coffee plants remain unclear. Understanding the variables that affect somatic embryogenesis in C. liberica will help enhance its use, particularly the direct pathway. The presence or absence of competent cells in the explant, which are intrinsic to their totipotency, dictates the ability of the species for somatic embryogenesis (Almeida 2020). The transition from the cotyledon stage to a fully developed plant is very low and occurred over a long period (more than a year), utilising the same medium employed for somatic embryo induction. Additional research is required to enhance the conversion rate of cotyledons into fully developed plants for C. liberica.
An essential component affecting the induction of somatic embryogenesis in C. liberica is the application of cytokinins, specifically BAP and TDZ. Auxin 2,4-D shown ineffectiveness in inducing callus formation on explants when paired with BAP or TDZ. Somatic embryogenesis only occurs on MS media supplemented with 1 mg/L BAP. The best medium for somatic embryo formation was the MS modification by Yasuda et al. (1985) supplemented with 1 mg/L BAP for MKL 8, while the MS modification by Yasuda et al. (1985) enhanced with 1 mg/L BAP and 1 mg/L TDZ was used for MKL 10. Both clones generate somatic embryos by the indirect pathway of embryogenesis, except for MKL10, which produces somatic embryos through both direct and indirect pathways.
The authors express gratitude to the Malaysian Agricultural Research and Development Institute (MARDI) for the financial support provided for this project under the Twelfth Malaysia Plan (RMK 12-PIC 507). The authors would also like to thank Noor Syahira Nasarudin for providing the planting materials for this project.
Table 1 . Quality of the embryogenic calluses of Coffea liberica across various combinations of plant growth regulators (PGRs).
Clones | Treatments | Explants producing calluses (%) | Callus formation | Callus color | Callus texture at 12 weeks | ||
---|---|---|---|---|---|---|---|
6 weeks | 12 weeks | 6 weeks | 12 weeks | ||||
Clone 211 | T1: 0 mg/L BAP | 44 | 89 | +++ | G | G, W | F, C |
T2: 1 mg/L BAP | 75 | 100 | ++ | G | G, W | F, C | |
T3: 1 mg/L BAP + 1 mg/L TDZ | 100 | 100 | ++++ | G, W | G, W, Y, B | F, C | |
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D | 0 | 0 | NA | NA | NA | NA | |
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ | 0 | 0 | NA | NA | NA | NA | |
Clone 224 | T1: 0 mg/L BAP | 37 | 89 | +++ | G | G, W | F, C |
T2: 1 mg/L BAP | 42 | 56 | ++ | G | G, W | F, C | |
T3: 1 mg/L BAP + 1 mg/L TDZ | 45 | 45 | +++ | W, G | G, W, T, B | F, C | |
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D | 5 | 5 | + | NA | NA | C | |
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ | 0 | 0 | NA | NA | NA | NA |
Note: NA = Not available; formation + = very few, ++ = few, +++ = moderate, and ++++ = Abundant; color G = green, W = white, Y = yellow, B = brown, T = transparent; and texture F = friable and C = compact.
Table 2 . MKL 8, 9, and 10 callus induction, callus formation, and potential somatic embryos on seven culture media.
Clones | Treatments | Explants producing callus (%) at 4 weeks | Callus formation | Explants producing SE (cotyledon) (%) at 28 weeks |
---|---|---|---|---|
(MKL 8) 213 | T1 | 96 ± 4.2 b | ++ | 38 ± 18.3 b |
T2 | 87 ± 6.2 b | ++ | 0 ± 0 a | |
T3 | 96 ± 4.2 b | +++ | 0 ± 0 a | |
T4 | 96 ± 4.2 b | ++ | 12.5 ± 12.5 a | |
T5 | 62 ± 11.7 a | + | 0 ± 0 a | |
T6 | 76 ± 9.7 ab | ++ | 0 ± 0 a | |
T7 | 83 ± 6.4 ab | ++ | 12.5 ± 12.5 a | |
(MKL 9) 222 | T1 | 41 ± 10.4 ab | + | 0 ± 0 a |
T2 | 17 ± 8.8 a | + | 0 ± 0 a | |
T3 | 62 ± 14.7 bcd | ++ | 0 ± 0 a | |
T4 | 46 ± 8.8 abc | + | 0 ± 0 a | |
T5 | 75 ± 5.6 cd | ++ | 0 ± 0 a | |
T6 | 83 ± 9.0 d | +++ | 0 ± 0 a | |
T7 | 75 ± 8.4 cd | ++ | 0 ± 0 a | |
(MKL 10) 224 | T1 | 62 ± 14.7 ab | ++ | 0 ± 0 a |
T2 | 46 ± 8.8 a | + | 0 ±0 a | |
T3 | 71 ± 9.9 ab | ++ | 63 ± 16.4 b | |
T4 | 54 ± 8.8 ab | ++ | 0 ± 0 a | |
T5 | 70 ± 7.6 ab | + | 38 ± 16.4 a | |
T6 | 83 ± 6.4 b | +++ | 12.5 ± 12.5 a | |
T7 | 83 ± 9.0 b | ++ | 0 ± 0 a |
Note: Values are represented as means ± SE; values represented with the same letter in the same column were not significantly different (P > 0.05), as determined by Duncan’s multiple range test.
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