Coffee Somatic Embryogenesis: How Did Research, Experience Gained and Innovations Promote the Commercial Propagation of Elite Clones From the Two Cultivated Species?

Abstract

Since the 1990s, somatic embryogenesis (SE) has enabled the propagation of selected varieties, Arabica F1 hybrid and Robusta clones, originating from the two cultivated coffee species, Coffea arabica and Coffea canephora, respectively. This paper shows how mostly empirical research has led to successful industrial transfers launched in the 2000s in Latin America, Africa, and Asia. Coffee SE can be considered as a model for other woody perennial crops for the following reasons: (i) a high biological efficiency has been demonstrated for propagated varieties at all developmental stages, and (ii) somaclonal variation is understood and mastered thanks to intensive research combining molecular markers and field observations. Coffee SE is also a useful model given the strong economic constraints that are specific to this species. In brief, SE faced four difficulties: (i) the high cost of SE derived plants compared to the cost of seedlings of conventional varieties, (ii) the logistic problems involved in reaching small-scale coffee growers, (iii) the need for certification, and (iv) the lack of solvency among small-scale producers. Nursery activities were professionalized by introducing varietal certification, quality control with regard to horticultural problems and somaclonal variation, and sanitary control for Xylella fastidiosa. In addition, different technology transfers were made to ensure worldwide dissemination of improved F1 Arabica hybrids and Robusta clones. Innovations have been decisive for successful scaling-up and reduction of production costs, such as the development of temporary immersion bioreactors for the mass production of pre-germinated embryos, their direct sowing on horticultural soil, and the propagation of rejuvenated SE plants by rooted mini-cuttings. Today, SE is a powerful tool that is widely used in coffee for biotechnological applications including propagation and genetic transformation. Basic research has recently started taking advantage of optimized SE protocols. Based on -omics methodologies, research aims to decipher the molecular events involved in the key developmental switches of coffee SE. In parallel, a high-throughput screening of active molecules on SE appears to be a promising tool to speed-up the optimization of SE protocols.

Keywords: bioreactors; cuttings; innovation; somaclonal variation; technology transfer.

FIGURE 1

Schematic representation of the Coffea arabica mass propagation process (24 months) through somatic embryogenesis (SE). The process is based on the use of liquid nutritive media for the embryogenic tissue multiplication and embryo regeneration phase and for the pre-germinated embryo and plantlet bulk production in temporary bioreactors (1L-RITA, 5L-MATIS and 3L-Twin-flasks). Direct sowing of germinated embryos and plantlets onto horticultural substrate avoids the use of gel media and requires less manpower in in vitro conditions.

FIGURE 2

Somatic embryogenesis of C. arabica: model of industrialization and professionalization. (A) Embryogenic cell suspension in liquid medium; (B) embryo differentiation in liquid medium (torpedo stage); (C) embryo development phase in TIS (temporary immersion system); (D) germinated embryos in Twin-Flask Bioreactor (above: cotyledonary embryos obtained in 1L-RITA); (E) Conversion into plantlets of pregerminated embryos onto horticultural substrate; (F) rooted cuttings in plug trays; (G) industrial hybrid nurseries with plastic pots and peat moss substrate. (H) Rooted cutting ready for field planting.

FIGURE 3

Illustration of the production, multiplication, and plant regeneration from embryogenic cells in coffee (C. arabica). (A) Formation of a primary callus made of meristematic cells at the edge of leaf explants 3 weeks following in vitro introduction; (B) embryogenic callus obtained 6 months after in vitro introduction; (C) regeneration of somatic embryos from the embryogenic callus; (D) proliferation of embryogenic cell suspensions and subsequent regeneration of somatic embryos in Erlenmeyer flasks agitated on orbital shakers in an industrial coffee micropropagation laboratory (CIRAD/ECOM unit, Sebaco, Nicaragua); (E) histological appearance of embryogenic cell clusters in suspension cultures; embryogenic cells have a high nucleus cytoplasm ratio and dense cytoplasm rich in soluble and reserve proteins; (F) mass regeneration of torpedo-stage somatic embryos from embryogenic cell suspensions; (G) coffee SE-derived plants in the nursery (Cartago, Costa Rica); (H) “Dwarf” phenotypic variant (on the right) compared to a normal plant on the left; (I) “Variegata” phenotypic variant (from Etienne et al., 2016).

FIGURE 4

Promoting participatory research and assistance to farmers with new hybrid plant material. (A) Farmer showing “his” Arabica hybrid plants in a farm located in Naranjo at 1300 m a.s.l., Alajuela province, Costa Rica; (B,C) visit of a farmer in San Ramon (1050 m a.s.l.), Alajuela province, Costa Rica; (D) planting of Arabica hybrids in “on farm” evaluation trials in the Sõn La province, Vietnam.

FIGURE 5

Technology transfer of C. arabica SE and democratization of the rooted mini-cuttings companion method. (A,B) Training in SE at CIRAD (France) for researchers from Cameroon and Vietnam. (C,D) Women’s mini-cuttings cooperative in Nicaragua at the “La Cumplida” farm, Matagalpa district.

FIGURE 6

Robusta somatic embryogenic tissues at different stages. (A) Primary embryogenic callus on a leaf explant, (B) embryogenic calli, (C) histological view of an embryogenic clump, (D) expression: torpedo-stage embryos at the end of the phase, (E) pre-germination: 10 L glass temporary immersion bioreactor (upper recipient), (F) Pre-germination: 10 L Box-in-Bag disposable bioreactor (upper recipient), (G) pre-germination: aspect of the cotyledonary-stage embryos, (H) selected cotyledonary embryos (pre-germinated embryos), (I) ex vitro germination test in the Nestlé R&D Tours greenhouse.

FIGURE 7

Notion of the “balance” and the effects of some factors on reaching the required equilibrium for the formation of embryogenic callus.

FIGURE 8

Mass propagation of selected Robusta clones at Nestlé R&D Center Tours.

FIGURE 9

Robusta SE plants produced in Nestlé’s French facility and distributed to farmers in the Philippines (Top); young trees in the field (Mindanao Island, Bottom Left); locally produced SE tree bearing fruits in a Mexican plantation (Chiapas, Bottom Right).

FIGURE 10

Coffee genetic transformation. Morphological (A1,B1,C1) and histological (A2,B2,C2) aspect of the different callus phenotypes observed in maintained embryogenic cultures assayed for coffee genetic transformation: (A1,A2) whitish type; (B1,B2) yellow type competent for genetic transformation comprising proembryogenic masses (PEMs); (C1,C2) gray type. (D) Transformation efficiency depending on the phenotype of maintained embryogenic callus cultures. (E) Effect of the age of the embryogenic callus culture on A. tumefaciens-mediated transformation efficiency (from Ribas et al., 2011).

FIGURE 11

The keys to successful high-throughput screening (HTS). Optimization of screening systems is evaluated according to 3 criteria: time, cost, and quality. S/N, signal-to-noise ratio; H/L, High-to-low ratio (sensitivity of the screening; adapted from Mayr and Fuerst, 2008).