Research Article

Split Viewer

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

Somatic Embryogenesis of Coffea liberica New Clone in Malaysia

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

Received: 7 October 2024; Revised: 12 December 2024; Accepted: 12 December 2024; Published: 22 January 2025.

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.

Plant Materials

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.

Fig. 1. Effect of plant growth regulators (PGRs) on the leaf disc of Coffea liberica at 12 weeks. (a) T1: 0 mg/L BAP, (b) T2: 1 mg/L BAP, (c) 1 mg/L BAP + 1 mg/L TDZ, (d) T4: 1 mg/L BAP + 0.5 mg/L 2,4-D, and (e) T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ

Somatic Embryogenesis Induction

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.

Experimental Design and Data Analysis

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.

Effect of PGR combinations on leaf disc explant of Coffea liberica

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).

Fig. 2. Morphologies of non-embryogenic and embryogenic calluses obtained from Coffea liberica leaf disc culture. (a) Compact green calluses, (b) compact green callus (black arrow) and friable yellowish green callus (white arrow), (c) callus with globular structures, (d) white callus with clusters of elongated crystalline structures; (e) watery transparent and white callus; and (f) spongy white callus and compact dry brown callus

Table 1 Quality of the embryogenic calluses of Coffea liberica across various combinations of plant growth regulators (PGRs)

ClonesTreatmentsExplants producing calluses (%)Callus formationCallus colorCallus texture at 12 weeks
6 weeks12 weeks6 weeks12 weeks
Clone 211T1: 0 mg/L BAP4489+++GG, WF, C
T2: 1 mg/L BAP75100++GG, WF, C
T3: 1 mg/L BAP + 1 mg/L TDZ100100++++G, WG, W, Y, BF, C
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D00NANANANA
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ00NANANANA
Clone 224T1: 0 mg/L BAP3789+++GG, WF, C
T2: 1 mg/L BAP4256++GG, WF, C
T3: 1 mg/L BAP + 1 mg/L TDZ4545+++W, GG, W, T, BF, C
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D55+NANAC
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ00NANANANA

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.

Effect of Murashige & Skoog modification medias and PGR combinations on Coffea liberica new clones explant

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).

Fig. 3. Developmental stages of Coffea liberica somatic embryos. (a) Globular stage; (b) torpedo stage; (c, d) cotyledon stage by indirect embryogenesis; (e) cotyledon stage with leaf primordia; (f) fully developed plant; (g) undeveloped plant without root formation; (h) cotyledon stage by direct embryogenesis; (i) simultaneous occurrence of globular, heart and torpedo phases; and (j) clusters of globular embryos (yellow) and embryogenic callus (white). Abbreviations: G, globular; H, heart; T, torpedo; B, browning; and ec, embryogenic callus

Fig. 4. Impact of plant growth on somatic embryo initiation and development of Coffea liberica MKL 8 clone at 12, 16, 20, 24, and 28 weeks after in-vitro culture

Fig. 5. Impact of plant growth on somatic embryos initiation and development of Coffea liberica MKL 10 clone at 12, 16, 20, 24, and 28 weeks after in-vitro culture

Table 2 MKL 8, 9, and 10 callus induction, callus formation, and potential somatic embryos on seven culture media

ClonesTreatmentsExplants producing callus (%) at 4 weeksCallus formationExplants producing SE (cotyledon) (%) at 28 weeks
(MKL 8) 213T196 ± 4.2 b++38 ± 18.3 b
T287 ± 6.2 b++0 ± 0 a
T396 ± 4.2 b+++0 ± 0 a
T496 ± 4.2 b++12.5 ± 12.5 a
T562 ± 11.7 a+0 ± 0 a
T676 ± 9.7 ab++0 ± 0 a
T783 ± 6.4 ab++12.5 ± 12.5 a
(MKL 9) 222T141 ± 10.4 ab+0 ± 0 a
T217 ± 8.8 a+0 ± 0 a
T362 ± 14.7 bcd++0 ± 0 a
T446 ± 8.8 abc+0 ± 0 a
T575 ± 5.6 cd++0 ± 0 a
T683 ± 9.0 d+++0 ± 0 a
T775 ± 8.4 cd++0 ± 0 a
(MKL 10) 224T162 ± 14.7 ab++0 ± 0 a
T246 ± 8.8 a+0 ±0 a
T371 ± 9.9 ab++63 ± 16.4 b
T454 ± 8.8 ab++0 ± 0 a
T570 ± 7.6 ab+38 ± 16.4 a
T683 ± 6.4 b+++12.5 ± 12.5 a
T783 ± 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.

  1. Acuna MEA (1993) Somatic embryogenesis induced by culture on single media in coffee plants from crosses of Coffea arabica by Timor hybrid. In: Proceedings of the 15th Colloquium of International Coffee Science Association, ASIC, 24-30 July; Montpellier. 1993. pp. 790-889
  2. Aguilar ME, Wang XY, Escalona M, Yan L, Huang LF (2022) Somatic embryogenesis of Arabica coffee in temporary immersion culture: Advances, limitations, and perspectives for mass propagation of selected genotypes. Front Plant Sci 13:994578
    Pubmed KoreaMed CrossRef DOAJ
  3. Alemanno L, Berthouly M, Michaux-Ferriere N (1996) Histology of somatic embryo-genesis from floral tissues cocoa. Plant Cell, Tissue and Organ 46:187-194
    CrossRef
  4. Almeida JAS (2020) Observations on somatic embryogenesis in Coffea arabica L. DT Castanheira, (Ed.). Coffee-production and research. IntechOpen. London, United Kingdom pp. 1-20
  5. Almeida JAS, Machado DFSP, Silvarolla MB, Machado EC (2008) Effect of PEG on the somatic embryogenesis of Coffea arabica genotypes. In: Proceedings of the 22nd Colloquium of International Coffee Science Association, ASIC, 14-19 September. Brazil: Campinas: pp. 1020-1023
  6. Almeida JAS, Silvarolla MB (2009) Induction of somatic embryos of Coffea arabica genotypes by 6-benzyladenine. International J Plant Dev Biol 53:5-8
  7. Ardiyani F, Utami ESW, Purnobasuki H (2021) Optimation of Auxin and Cytokinin on Enhanced Quality and Weight of Coffea liberica Somatic Embryos. Pelita Perkebunan 37(1):1-12
    CrossRef
  8. Ardiyani F, Utami ESW, Purnobasuki H, Paramita SA (2020) Development and regeneration of somatic embryos from leaves-derived calli of Coffea liberica. Biodiversitas 21(12):5829-5834
    CrossRef
  9. Ardiyani F (2015) Morphological characterization and identification of Coffea Liberica callus of somatic embryogenesis propagation. Pelita Perkebunan 31(2):81-89
    CrossRef DOAJ
  10. Hatanaka T, Sawabe E, Azuma T, Uchida N, Yasuda T (1995) The role of ethylene in somatic embryogenesis from leaf discs of Coffea canephora. Plant Sci 107(02):199-204
    CrossRef
  11. Hulupi R (2014) Varietas kopi Liberika anjuran untuk lahan gambut. Warta Pusat Penelitian Kopi dan Kakao Indonesia 26(1):1-6
  12. Ibrahim MSD, Rubiyoa RSH, Purwitoc A; Sudarsono (2013) Direct and indirect somatic embryogenesis on arabica coffee (Coffea arabica). Indones J Agric Sci 14(2):79-86
    CrossRef
  13. Lee KWT (2023) Liberica Coffee Development and Refinement Project in Sarawak Malaysia. Proceedings 89(1):15
    CrossRef DOAJ
  14. Manila-Fajardo A, Cervancia C (2020) Nectar Biology and its Influence on the Pollination of Coffea liberica W. Bull ex Hiern var liberica 1:14-22
  15. Menéndez-Yuffa A, Garcia E (1996) Coffea species (Coffee). Biotechnology in agriculture and forestry. YPS Bajaj, (Ed.). Trees IV. Springer. Berlin Heidelberg New York pp. 95-119
    CrossRef
  16. Mohd Zaffrie MA, Hairazi R, Nor Amna AMN, Mohd Amirul MAW, Azahar H (2016) Consumers perception and behaviour towards coffee in Malaysia. Economic and Technology Management Review 11a:37-51
  17. Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Plant Physiol 15:473-497
    CrossRef
  18. N'Diaye A, Poncet V, Louarn J, Hamon S, Noirot M (2005) Genetic differentiation between Coffea liberica var. liberica and C. liberica var. Dewevrei and comparison with C. canephora. Plant Syst Evol 253:95-104
    CrossRef
  19. Nor Amna AMN, Mohd Amirul MAW (2016) Exploring the Potentials of Coffee Industry in Malaysia. FTTC Agricultural Policy Platform. Food and Fertilizer Center for The Asian and Pacific Region
  20. Philippine Council for Agriculture and Resources Research (PCARR) (1976) The Philippines Recommends for Coffee. Los Baños, Laguna, Philippines. p. 62
  21. Quiroz-Figueroa FR, Rojas-Herrera R, Galaz-Avalos RM, Loyola-Vargas VM (2006) Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell Tissue Organ Cult 86:285-301
    CrossRef
  22. Santana-Buzzy N, Herrera RR, Ávalos RMG, Ku-Cauich JR, Mijangos-Cortés J, Gutiérrez-Pacheco LC (2007) Advances in coffee tissue culture and its practical applications. In Vitro Cell Dev Biol Plant 3:507-520
    CrossRef
  23. Staritsky G (1970) Embryoid formation in callus cultures of coffee. Acta Bot Neerl 19:509-514
    CrossRef
  24. Toonen MAJ, De Vries SC (1996) Initiation of somatic embryos from single cell. T.L. Wang, A. Cuming, (Eds.). Embryogenesis: the generation of a plant. BIOS Scientific Publishers limited. Oxford pp. 173-177
  25. Yasuda T, Fujii Y, Yamaguchi T (1985) Embryogenic callus induction from Coffea arabica leaf explants by benzyladenine. Plant Cell Physiol 26(3):595-597
    CrossRef

Article

Research Article

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.

Somatic Embryogenesis of Coffea liberica New Clone in Malaysia

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

Received: 7 October 2024; Revised: 12 December 2024; Accepted: 12 December 2024; Published: 22 January 2025.

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.

Abstract

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

Introduction

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.

Materials and Methods

Plant Materials

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.

Figure 1. Effect of plant growth regulators (PGRs) on the leaf disc of Coffea liberica at 12 weeks. (a) T1: 0 mg/L BAP, (b) T2: 1 mg/L BAP, (c) 1 mg/L BAP + 1 mg/L TDZ, (d) T4: 1 mg/L BAP + 0.5 mg/L 2,4-D, and (e) T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ

Somatic Embryogenesis Induction

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.

Experimental Design and Data Analysis

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.

Results and Discussion

Effect of PGR combinations on leaf disc explant of Coffea liberica

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).

Figure 2. Morphologies of non-embryogenic and embryogenic calluses obtained from Coffea liberica leaf disc culture. (a) Compact green calluses, (b) compact green callus (black arrow) and friable yellowish green callus (white arrow), (c) callus with globular structures, (d) white callus with clusters of elongated crystalline structures; (e) watery transparent and white callus; and (f) spongy white callus and compact dry brown callus

Table 1 . Quality of the embryogenic calluses of Coffea liberica across various combinations of plant growth regulators (PGRs).

ClonesTreatmentsExplants producing calluses (%)Callus formationCallus colorCallus texture at 12 weeks
6 weeks12 weeks6 weeks12 weeks
Clone 211T1: 0 mg/L BAP4489+++GG, WF, C
T2: 1 mg/L BAP75100++GG, WF, C
T3: 1 mg/L BAP + 1 mg/L TDZ100100++++G, WG, W, Y, BF, C
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D00NANANANA
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ00NANANANA
Clone 224T1: 0 mg/L BAP3789+++GG, WF, C
T2: 1 mg/L BAP4256++GG, WF, C
T3: 1 mg/L BAP + 1 mg/L TDZ4545+++W, GG, W, T, BF, C
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D55+NANAC
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ00NANANANA

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.

Effect of Murashige & Skoog modification medias and PGR combinations on Coffea liberica new clones explant

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).

Figure 3. Developmental stages of Coffea liberica somatic embryos. (a) Globular stage; (b) torpedo stage; (c, d) cotyledon stage by indirect embryogenesis; (e) cotyledon stage with leaf primordia; (f) fully developed plant; (g) undeveloped plant without root formation; (h) cotyledon stage by direct embryogenesis; (i) simultaneous occurrence of globular, heart and torpedo phases; and (j) clusters of globular embryos (yellow) and embryogenic callus (white). Abbreviations: G, globular; H, heart; T, torpedo; B, browning; and ec, embryogenic callus

Figure 4. Impact of plant growth on somatic embryo initiation and development of Coffea liberica MKL 8 clone at 12, 16, 20, 24, and 28 weeks after in-vitro culture

Figure 5. Impact of plant growth on somatic embryos initiation and development of Coffea liberica MKL 10 clone at 12, 16, 20, 24, and 28 weeks after in-vitro culture

Table 2 . MKL 8, 9, and 10 callus induction, callus formation, and potential somatic embryos on seven culture media.

ClonesTreatmentsExplants producing callus (%) at 4 weeksCallus formationExplants producing SE (cotyledon) (%) at 28 weeks
(MKL 8) 213T196 ± 4.2 b++38 ± 18.3 b
T287 ± 6.2 b++0 ± 0 a
T396 ± 4.2 b+++0 ± 0 a
T496 ± 4.2 b++12.5 ± 12.5 a
T562 ± 11.7 a+0 ± 0 a
T676 ± 9.7 ab++0 ± 0 a
T783 ± 6.4 ab++12.5 ± 12.5 a
(MKL 9) 222T141 ± 10.4 ab+0 ± 0 a
T217 ± 8.8 a+0 ± 0 a
T362 ± 14.7 bcd++0 ± 0 a
T446 ± 8.8 abc+0 ± 0 a
T575 ± 5.6 cd++0 ± 0 a
T683 ± 9.0 d+++0 ± 0 a
T775 ± 8.4 cd++0 ± 0 a
(MKL 10) 224T162 ± 14.7 ab++0 ± 0 a
T246 ± 8.8 a+0 ±0 a
T371 ± 9.9 ab++63 ± 16.4 b
T454 ± 8.8 ab++0 ± 0 a
T570 ± 7.6 ab+38 ± 16.4 a
T683 ± 6.4 b+++12.5 ± 12.5 a
T783 ± 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.

Conclusion

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.

Acknowledgement

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.

Fig 1.

Figure 1.Effect of plant growth regulators (PGRs) on the leaf disc of Coffea liberica at 12 weeks. (a) T1: 0 mg/L BAP, (b) T2: 1 mg/L BAP, (c) 1 mg/L BAP + 1 mg/L TDZ, (d) T4: 1 mg/L BAP + 0.5 mg/L 2,4-D, and (e) T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ
Journal of Plant Biotechnology 2025; 52: 1-8https://doi.org/10.5010/JPB.2025.52.001.001

Fig 2.

Figure 2.Morphologies of non-embryogenic and embryogenic calluses obtained from Coffea liberica leaf disc culture. (a) Compact green calluses, (b) compact green callus (black arrow) and friable yellowish green callus (white arrow), (c) callus with globular structures, (d) white callus with clusters of elongated crystalline structures; (e) watery transparent and white callus; and (f) spongy white callus and compact dry brown callus
Journal of Plant Biotechnology 2025; 52: 1-8https://doi.org/10.5010/JPB.2025.52.001.001

Fig 3.

Figure 3.Developmental stages of Coffea liberica somatic embryos. (a) Globular stage; (b) torpedo stage; (c, d) cotyledon stage by indirect embryogenesis; (e) cotyledon stage with leaf primordia; (f) fully developed plant; (g) undeveloped plant without root formation; (h) cotyledon stage by direct embryogenesis; (i) simultaneous occurrence of globular, heart and torpedo phases; and (j) clusters of globular embryos (yellow) and embryogenic callus (white). Abbreviations: G, globular; H, heart; T, torpedo; B, browning; and ec, embryogenic callus
Journal of Plant Biotechnology 2025; 52: 1-8https://doi.org/10.5010/JPB.2025.52.001.001

Fig 4.

Figure 4.Impact of plant growth on somatic embryo initiation and development of Coffea liberica MKL 8 clone at 12, 16, 20, 24, and 28 weeks after in-vitro culture
Journal of Plant Biotechnology 2025; 52: 1-8https://doi.org/10.5010/JPB.2025.52.001.001

Fig 5.

Figure 5.Impact of plant growth on somatic embryos initiation and development of Coffea liberica MKL 10 clone at 12, 16, 20, 24, and 28 weeks after in-vitro culture
Journal of Plant Biotechnology 2025; 52: 1-8https://doi.org/10.5010/JPB.2025.52.001.001

Table 1 . Quality of the embryogenic calluses of Coffea liberica across various combinations of plant growth regulators (PGRs).

ClonesTreatmentsExplants producing calluses (%)Callus formationCallus colorCallus texture at 12 weeks
6 weeks12 weeks6 weeks12 weeks
Clone 211T1: 0 mg/L BAP4489+++GG, WF, C
T2: 1 mg/L BAP75100++GG, WF, C
T3: 1 mg/L BAP + 1 mg/L TDZ100100++++G, WG, W, Y, BF, C
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D00NANANANA
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ00NANANANA
Clone 224T1: 0 mg/L BAP3789+++GG, WF, C
T2: 1 mg/L BAP4256++GG, WF, C
T3: 1 mg/L BAP + 1 mg/L TDZ4545+++W, GG, W, T, BF, C
T4: 1 mg/L BAP + 0.5 mg/L 2,4-D55+NANAC
T5: 0.5 mg/L 2,4-D + 1 mg/L TDZ00NANANANA

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.

ClonesTreatmentsExplants producing callus (%) at 4 weeksCallus formationExplants producing SE (cotyledon) (%) at 28 weeks
(MKL 8) 213T196 ± 4.2 b++38 ± 18.3 b
T287 ± 6.2 b++0 ± 0 a
T396 ± 4.2 b+++0 ± 0 a
T496 ± 4.2 b++12.5 ± 12.5 a
T562 ± 11.7 a+0 ± 0 a
T676 ± 9.7 ab++0 ± 0 a
T783 ± 6.4 ab++12.5 ± 12.5 a
(MKL 9) 222T141 ± 10.4 ab+0 ± 0 a
T217 ± 8.8 a+0 ± 0 a
T362 ± 14.7 bcd++0 ± 0 a
T446 ± 8.8 abc+0 ± 0 a
T575 ± 5.6 cd++0 ± 0 a
T683 ± 9.0 d+++0 ± 0 a
T775 ± 8.4 cd++0 ± 0 a
(MKL 10) 224T162 ± 14.7 ab++0 ± 0 a
T246 ± 8.8 a+0 ±0 a
T371 ± 9.9 ab++63 ± 16.4 b
T454 ± 8.8 ab++0 ± 0 a
T570 ± 7.6 ab+38 ± 16.4 a
T683 ± 6.4 b+++12.5 ± 12.5 a
T783 ± 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.


References

  1. Acuna MEA (1993) Somatic embryogenesis induced by culture on single media in coffee plants from crosses of Coffea arabica by Timor hybrid. In: Proceedings of the 15th Colloquium of International Coffee Science Association, ASIC, 24-30 July; Montpellier. 1993. pp. 790-889
  2. Aguilar ME, Wang XY, Escalona M, Yan L, Huang LF (2022) Somatic embryogenesis of Arabica coffee in temporary immersion culture: Advances, limitations, and perspectives for mass propagation of selected genotypes. Front Plant Sci 13:994578
    Pubmed KoreaMed CrossRef Etc
  3. Alemanno L, Berthouly M, Michaux-Ferriere N (1996) Histology of somatic embryo-genesis from floral tissues cocoa. Plant Cell, Tissue and Organ 46:187-194
    CrossRef
  4. Almeida JAS (2020) Observations on somatic embryogenesis in Coffea arabica L. DT Castanheira, (Ed.). Coffee-production and research. IntechOpen. London, United Kingdom pp. 1-20
  5. Almeida JAS, Machado DFSP, Silvarolla MB, Machado EC (2008) Effect of PEG on the somatic embryogenesis of Coffea arabica genotypes. In: Proceedings of the 22nd Colloquium of International Coffee Science Association, ASIC, 14-19 September. Brazil: Campinas: pp. 1020-1023
  6. Almeida JAS, Silvarolla MB (2009) Induction of somatic embryos of Coffea arabica genotypes by 6-benzyladenine. International J Plant Dev Biol 53:5-8
  7. Ardiyani F, Utami ESW, Purnobasuki H (2021) Optimation of Auxin and Cytokinin on Enhanced Quality and Weight of Coffea liberica Somatic Embryos. Pelita Perkebunan 37(1):1-12
    CrossRef
  8. Ardiyani F, Utami ESW, Purnobasuki H, Paramita SA (2020) Development and regeneration of somatic embryos from leaves-derived calli of Coffea liberica. Biodiversitas 21(12):5829-5834
    CrossRef
  9. Ardiyani F (2015) Morphological characterization and identification of Coffea Liberica callus of somatic embryogenesis propagation. Pelita Perkebunan 31(2):81-89
    CrossRef Etc
  10. Hatanaka T, Sawabe E, Azuma T, Uchida N, Yasuda T (1995) The role of ethylene in somatic embryogenesis from leaf discs of Coffea canephora. Plant Sci 107(02):199-204
    CrossRef
  11. Hulupi R (2014) Varietas kopi Liberika anjuran untuk lahan gambut. Warta Pusat Penelitian Kopi dan Kakao Indonesia 26(1):1-6
  12. Ibrahim MSD, Rubiyoa RSH, Purwitoc A; Sudarsono (2013) Direct and indirect somatic embryogenesis on arabica coffee (Coffea arabica). Indones J Agric Sci 14(2):79-86
    CrossRef
  13. Lee KWT (2023) Liberica Coffee Development and Refinement Project in Sarawak Malaysia. Proceedings 89(1):15
    CrossRef Etc
  14. Manila-Fajardo A, Cervancia C (2020) Nectar Biology and its Influence on the Pollination of Coffea liberica W. Bull ex Hiern var liberica 1:14-22
  15. Menéndez-Yuffa A, Garcia E (1996) Coffea species (Coffee). Biotechnology in agriculture and forestry. YPS Bajaj, (Ed.). Trees IV. Springer. Berlin Heidelberg New York pp. 95-119
    CrossRef
  16. Mohd Zaffrie MA, Hairazi R, Nor Amna AMN, Mohd Amirul MAW, Azahar H (2016) Consumers perception and behaviour towards coffee in Malaysia. Economic and Technology Management Review 11a:37-51
  17. Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Plant Physiol 15:473-497
    CrossRef
  18. N'Diaye A, Poncet V, Louarn J, Hamon S, Noirot M (2005) Genetic differentiation between Coffea liberica var. liberica and C. liberica var. Dewevrei and comparison with C. canephora. Plant Syst Evol 253:95-104
    CrossRef
  19. Nor Amna AMN, Mohd Amirul MAW (2016) Exploring the Potentials of Coffee Industry in Malaysia. FTTC Agricultural Policy Platform. Food and Fertilizer Center for The Asian and Pacific Region
  20. Philippine Council for Agriculture and Resources Research (PCARR) (1976) The Philippines Recommends for Coffee. Los Baños, Laguna, Philippines. p. 62
  21. Quiroz-Figueroa FR, Rojas-Herrera R, Galaz-Avalos RM, Loyola-Vargas VM (2006) Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell Tissue Organ Cult 86:285-301
    CrossRef
  22. Santana-Buzzy N, Herrera RR, Ávalos RMG, Ku-Cauich JR, Mijangos-Cortés J, Gutiérrez-Pacheco LC (2007) Advances in coffee tissue culture and its practical applications. In Vitro Cell Dev Biol Plant 3:507-520
    CrossRef
  23. Staritsky G (1970) Embryoid formation in callus cultures of coffee. Acta Bot Neerl 19:509-514
    CrossRef
  24. Toonen MAJ, De Vries SC (1996) Initiation of somatic embryos from single cell. T.L. Wang, A. Cuming, (Eds.). Embryogenesis: the generation of a plant. BIOS Scientific Publishers limited. Oxford pp. 173-177
  25. Yasuda T, Fujii Y, Yamaguchi T (1985) Embryogenic callus induction from Coffea arabica leaf explants by benzyladenine. Plant Cell Physiol 26(3):595-597
    CrossRef
JPB
Vol 52. 2025

Stats or Metrics

Share this article on

  • line

Related articles in JPB

Journal of

Plant Biotechnology

pISSN 1229-2818
eISSN 2384-1397
qr-code Download