A cellulose binding domain protein restores female fertility when expressed in transgenic Bintje potato

Abstract

Background: Expression of a gene encoding the family 1 cellulose binding domain protein CBD1, identified in the cellulosic cell wall of the potato late blight pathogen Phytophthora infestans, was tested in transgenic potato to determine if it had an influence on plant cell walls and resistance to late blight.

Results: Multiple regenerants of potato (cv. Bintje) were developed and selected for high expression of CBD 1 transcripts. Tests with detached leaflets showed no evidence of increased or decreased resistance to P. infestans, in comparison with the blight susceptible Bintje controls, however, changes in plant morphology were evident in CBD 1 transgenics. Plant height increases were evident, and most importantly, the ability to produce seed berries from a previously sterile cultivar. Immunolocalization of CBD 1 in seed berries revealed the presence throughout the tissue. While Bintje control plants are male and female sterile, CBD 1 transgenics were female fertile. Crosses made using pollen from the late blight resistant Sarpo Mira and transgenic CBD1 Bintje as the female parent demonstrated the ability to introgress P. infestans targeted resistance genes, as well as genes responsible for color and tuber shape, into Bintje germplasm.

Conclusions: A family 1 cellulose-binding domain (CBD 1) encoding gene from the potato late blight pathogen P. infestans was used to develop transgenic Bintje potato plants. Transgenic plants became female fertile, allowing for a previously sterile cultivar to be used in breeding improvement. Selection for the absence of the CBD transgene in progeny should allow for immediate use of a genetically enhanced material. Potential for use in other Solanaceous crops is proposed.

Keywords: Bintje; Cellulose binding domain; Potato fertility.

Fig. 1

Height of CBD1 transgenic and control plant beginning 5 weeks after transfer to soil in the greenhouse. Only certain lines had height increases noticeably exceeding the control plant

Fig. 2

Floral and seed berry formation on Bintje CBD 1 transgenic. a Earlier flowering seen by the presence of seed berries in transformed plant B7 that is the same age as control plants where they have just initiated flowering b abundant seed berries c largest seed berries were produced from B-48, producing as many as 200 seeds/berry after outcrossing

Fig. 3

Immunolocalization of CBD 1. Purple regions represent precipitation of AP substrate. a, b CBD 1 protein can be seen accumulating throughout the fleshy region of the ovary and locule. c, d No purple precipitant is observed with transgenic tissues when using rabbit pre-immune serum followed by goat-anti rabbit AP conjugated secondary antibody, then AP substrate exposure

Fig. 4

Genome walking identification of integration sites using CBD 1 gene and vector specific forward primers and four sets of ApaGene reverse primers labeled A, B, C and D. After three rounds of PCR a single band was evident for B7, while multiple bands were present in the B48 samples. Band sizes greater than 500 bp are needed to identify integration sequences. Large bold sequence represents the pBI121 T-DNA border region, underlined sequences represent unique potato sequence, remaining sequence is common to APAgene kit

Fig. 5

Detached leaflets were inoculated with Phytophthora infestans sporangia and zoospores, then incubated for 6 days. a Bintje controls are fully susceptible to P. infestans US 11. b Sarpo Mira is fully resistant. A subset of progeny from the B7x Sarpo Mira cross is fully resistant to late blight as shown in Table 1

Fig. 6

Cross between CBD 1 transgenic Bintje B7 and cultivar Sarpo Mira resulted in progeny with a wide range of tuber colors, suggesting complementation of an anthocyanin pathway. Cross between CBD 1 transgenic Bintje B7 and cultivar Peter Wilcox resulted in a range of tuber shapes and colors, representing potential for development of Bintje