Citrus triploid recovery based on 2x × 4x crosses via an optimized embryo rescue approach

Seedlessness is a primary breeding objective for citrus fresh fruit market, and triploids have been proven to have great value to produce seedless fruits. In this study, aiming to produce triploid plants for developing some seedless cultivars, four 2x × 4x interploid crosses were conducted using three elite but seedy cultivars as seed parents and one newly flowered doubled diploid and two allotetraploid somatic hybrids as pollen parents. As a result, a total of 1454 developed and 3409 undeveloped seeds from 341 fruits were obtained. Using an optimized embryo rescue approach, 669 developed and 1301 undeveloped seeds germinated in vitro, with an average germination rate as 52.5% for the crosses using ‘Nadorcott’ tangor and ‘Bendizao’ tangerine (polyembryonic) as seed parents and 31.0% for the crosses using ‘Orah’ mandarin (monoembryonic) as seed parent. Then by shoot and root induction, totally 1354 plantlets were regenerated, among which 401 and 54 plants were proved to be triploids and tetraploids by flow cytometry (FCM) analysis and chromosome counting. Hybrid nature of the selected triploid progenies, as well as two different origins (doubled diploid and hybrid origins) for the selected tetraploid progenies was further confirmed by single nucleotide polymorphism (SNP) markers.