High-frequency somatic embryogenesis in Coffea canephora Pierre ex A. Froehner: integrating in vitro propagation and genetic diversification for breeding advancement
摘要
Robusta coffee (Coffea canephora Pierre ex A. Froehner) is an economically important crop that provides livelihood to millions of farm households in the Global South. Genotype dependent responses in tissue culture remain a significant challenge for the in vitro propagation of elite cultivars and for the development of tissue culture induced variation (TCIV)-based novel germplasm. This work established a high-frequency somatic embryogenesis (HFSE) pipeline for C. canephora by evaluating different plant growth regulator (PGR) regimens and optimizing in vitro culture conditions. These comprised i) 2,4-dichlorophenoxyacetic acid (2,4-D; 1.0 to 3.0 mg L−1) with 6-benzylaminopurine (BAP; 1.0 mg L−1), ii) picloram (0.5 to 1.5 mg L−1) with BAP (2.0 mg L−1) and silver nitrate (AgNO₃; 6.8 mg L−1), and iii) 1-naphthaleneacetic acid (NAA; 0.05 mg L−1) with thidiazuron (TDZ; 2.0 to 5.0 mg L−1). The most effective regimens based on callus induction frequency (CIF) and friable embryogenic callus (FEC) yield were identified. These included i) 0.5 mg L−1 picloram with 2.0 mg L−1 BAP and 6.8 mg L−1 AgNO₃ (CIF: 85%, FEC: 57.2), ii) 2.0 mg L−1 2,4-D with 1.0 mg L−1 BAP (CIF: 84%, FEC: 56.4) and iii) 0.05 mg L−1 NAA with 2.0 mg L−1 TDZ with (CIF: 73%, FEC: 39.8). Picloram- and 2,4-D-based regimens induced higher callogenesis and yielded more FEC. In contrast, the NAA-TDZ regimen generated higher quality FEC and robust embryos. Moreover, somatic embryos obtained from the NAA-TDZ regimen achieved a 100% embling conversion rate, compared with 86.7% and 50.0% for the 2,4-D-BAP and picloram-BAP-AgNO₃ regimens, respectively. Genetic fidelity evaluation using ten microsatellite markers showed high similarity between in vitro derived plants and the mother genotype. However, sequence analysis of a mono-allelic locus revealed nucleotide level variation with an average sequence homology of 93.88%, suggesting the occurrence of TCIV. The established HFSE pipeline provided a dual platform for C. canephora propagation and novel germplasm generation, offering a scalable system to support genetic diversification and long-term production resilience.