Coffea arabica L. Resistant to Coffee Berry Borer (Hypothenemus hampei) Mediated by Expression of the Bacillus thuringiensis Cry10Aa Protein

Abstract

Coffea spp. are tropical plants used for brewing beverages from roasted and grounded seeds, the favorite drink in the world. It is the most important commercial crop plant and the second most valuable international commodity after oil. Global coffee trade relies on two Coffea species: C. arabica L. (arabica coffee) comprising 60% and C. canephora (robusta) comprising the remaining 40%. Arabica coffee has lower productivity and better market price than robusta. Arabica coffee is threatened by disease (i.e., coffee leaf rust), pests [i.e., Hypothenemus hampei or coffee berry borer (CBB) and nematodes], and susceptibility to climate change (i.e., drought and aluminum toxicity). Plant biotechnology by means of tissue culture inducing somatic embryogenesis (SE) process, genetic transformation, and genome editing are tools that can help to solve, at least partially, these problems. This work is the continuation of a protocol developed for stable genetic transformation and successful plant regeneration of arabica coffee trees expressing the Bacillus thuringiensis (Bt) toxin Cry10Aa to induce CBB resistance. A highly SE line with a high rate of cell division and conversion to plants with 8-month plant regeneration period was produced. To validate this capability, gene expression analysis of master regulators of SE, such as BABY BOOM (BBM), FUS3, and LEC1, embryo development, such as EMB2757, and cell cycle progression, such as ETG1 and MCM4, were analyzed during induction and propagation of non-competent and highly competent embryogenic lines. The particle bombardment technique was used to generate stable transgenic lines after 3 months under selection using hygromycin as selectable marker, and 1 month in plant regeneration. Transgenic trees developed fruits after 2 years and demonstrated expression of the Bt toxin ranging from 3.25 to 13.88 μg/g fresh tissue. Bioassays with transgenic fruits on CBB first instar larvae and adults induced mortalities between 85 and 100% after 10 days. In addition, transgenic fruits showed a seed damage lower than 9% compared to 100% of control fruits and adult mortality. This is the first report on stable transformation and expression of the Cry10Aa protein in coffee plants with the potential to control CBB.

Keywords: Bacillus thuringiensis; Cry10Aa; coffee berry borer; coffee transformation; somatic embryogenesis.

Figure 1

Bioassay using first instar larvae of Hypothenemus hampei. (A) Females (1.6–1.9 mm long) recovered from a laboratory colony. (B) Individualization of H. hampei females in 24-well titration dishes. (C) Close-up of coffee berry borer (CBB) drills in green coffee seed pieces (0.7 mm). (D) First instar larvae (0.019 mm) and egg (0.001 mm) after 20 days of infestation. (E) First instar larvae fed with 1 μl of transgenic plant extracts. (F) First instar larvae fed with 1 μl of control plant extract. Bars: 0.5 mm.

Figure 2

Gene expression analysis by qPCR during SE induction in highly competent embryogenic line (HC-SE) and non-competent line (NC-SE). Relative expression levels were plotted based on Log2 values, normalized with RPL39 (ribosomal protein L39), ACT (β-actin), and 24S (Ribosomal protein 24S).

Figure 3

(A) Transgenic Coffea arabica plants grown under greenhouse conditions after 24 months. (B) Flowering of transgenic coffee trees after 18 months under greenhouse conditions. (C,D) Fruit development from transgenic coffee plants after 22 months under greenhouse conditions.

Figure 4

Southern blot and hybridization analysis of transgenic coffee fruits. (A) Electrophoresis of DNA digest of transgenic fruits and SEM of Coffea arabica digested with EcoR1/HindIII. (B) Southern blot analysis hybridized with cry10Aa-biotin of 1,993 bp probes. Lane 1, DNA from wild-type fruits; lane 2, wild-type SEMs; lane 3, transgenic fruits, event E1; lane 4, transgenic SEM event E1; lane 5, transgenic fruits, event E2; lane 6, partially digested transgenic SEM, event E2; lane 7, transgenic fruits, event E3; lane 8, partially digested transgenic SEM, event E3.

Figure 5

Mortality of coffee berry borer (CBB) first instar larvae. (A) Average morphology of first instar larvae in 24-well titration plates at 10 days. R1, R2, R3, and R4: Replicates of bioassays with larvae fed with Cry10Aa extract. Vertical gray line, water-fed larvae. Bar: 1 mm. (B) Mortality curve for Cry 10Aa concentrations found in SEMs and leaves from three transformation events of coffee (μg/g fresh weight).

Figure 6

Fruits 125 days after anthesis from Cry10Aa transgenic plants infested with coffee berry borer (CBB) adults. Three transformation events. Events 1, 2, and 3 and a wild-type plant (A–D) were selected for the performance of bioassays. At the right-hand side, the evaluation of damage by cross section of fruits of F1 in the three events and the wild-type fruits after 30 days.