Development of plants resistant to Papaya leaf distortion mosaic virus by intergeneric hybridization between Carica papaya and Vasconcellea cundinamarcensis

Abstract

In this study, we confirmed that Vasconcellea cundinamarcensis resists Papaya leaf distortion mosaic virus (PLDMV), and used it to produce intergeneric hybrids with Carica papaya. From the cross between C. papaya and V. cundinamarcensis, we obtained 147 seeds with embryos. Though C. papaya is a monoembryonic plant, multiple embryos were observed in all 147 seeds. We produced 218 plants from 28 seeds by means of embryo-rescue culture. All plants had pubescence on their petioles and stems characteristic of V. cundinamarcensis. Flow cytometry and PCR of 28 plants confirmed they were intergeneric hybrids. To evaluate virus resistance, mechanical inoculation of PLDMV was carried out. The test showed that 41 of 134 intergeneric hybrid plants showed no symptoms and were resistant. The remaining 93 hybrids showed necrotic lesions on the younger leaves than the inoculated leaves. In most of the 93 hybrids, the necrotic lesions enclosed the virus and prevented further spread. These results suggest that the intergeneric hybrids will be valuable material for PLDMV-resistant papaya breeding.

Keywords: embryo rescue; flow cytometric analysis; monodehydroascorbate reductase gene; trnD-trnY.

Fig. 1

Symptoms on the inoculated leaves. (A) C. papaya with severe mosaic symptoms. (B) V. cundinamarcensis with no symptoms. (C) Intergeneric hybrid with no symptoms. (D) Intergeneric hybrid with necrotic lesions. (A) to (D) are the younger leaves than the inoculated leaves 3 months after inoculation with PLDMV. Boxes (5 mm × 5 mm) indicate the locations used for detection of PLDMV by RT-PCR. In (A) to (C), positions 1 to 5 and positions 6 to 10 represent veins and random positions, respectively. In (D), positions 1 to 5 and positions 6 to 10 represent veins and necrotic lesions, respectively. (E) C. papaya with leaf distortion and mosaic symptoms 6 months after the inoculation. (F) Intergeneric hybrid with no symptoms 6 months after the inoculation.

Fig. 2

Detection of the partial coat protein gene of PLDMV (748-bp) by RT-PCR. Lanes 1 to 10 correspond to positions 1 to 10, respectively, in Fig. 1. In (D), positions 1 to 5 represent veins and positions 6 to 10 represent necrotic lesions. As a control, the partial cDNA fragment of the actin gene (632-bp) was amplified.

Fig. 3

Production of intergeneric hybrid plants of C. papaya × V. cundinamarcensis. (A) Multiple embryos from a seed 90 to 100 days after pollination. (B) Somatic embryogenesis from a zygotic embryo. (C) Shoot elongation. (D) Root development in vermiculite medium. (E) An intergeneric hybrid plant. (F) Pubescent petioles of an intergeneric hybrid plant.

Fig. 4

Confirmation of intergeneric hybridization by flow cytometry to determine the relative nuclear DNA content. Peaks for C. papaya were standardized to a value of 100. (A) C. papaya. (B) V. cundinamarcensis. (C) Intergeneric hybrid plant. (D) Mixture of C. papaya and V. cundinamarcensis. (E) Mixture of C. papaya, V. cundinamarcensis, and the intergeneric hybrid. Peaks I, II, and III originated from C. papaya, V. cundinamarcensis and the intergeneric hybrid, respectively.

Fig. 5

Confirmation of intergeneric hybridization by PCR. M and N are 100-bp DNA Ladder Marker (New England BioLabs, Ipswich, MA, USA) and a negative control, respectively. C represents the female parent (C. papaya, cultivar ‘Sunrise Solo’). V represents the male parent (V. cundinamarcensis). (A) The PCR results for the trnD-trnY intergenic spacer region (Supplemental Fig. 2) showed amplification of a 577-bp fragment from the female parent and the intergeneric hybrids, and a 533-bp fragment from the male parent. (B) The PCR results for MDAR gene (Supplemental Fig. 2) showed amplification of a 480-bp fragment from C. papaya, 529- and 531-bp fragments from V. cundinamarcensis. From the intergeneric hybrids, 480- and 529-bp fragments or, 480- and 531-bp fragments were amplified.