Polyembryony in non-apomictic citrus genotypes

Abstract

Background and aims: Adventitious embryony from nucellar cells is the mechanism leading to apomixis in Citrus sp. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a consequence of 2x × 4x hybridizations and in vitro culture of isolated nucelli. The origin of the plants arising from the aforementioned processes remains unclear.

Methods: The genetic structure (ploidy and allelic constitution with microsatellite markers) of plants obtained from polyembryonic seeds arising from 2x × 4x sexual hybridizations and those regenerated from nucellus culture in vitro was systematically analysed in different non-apomictic citrus genotypes. Histological studies were also conducted to try to identify the initiation process underlying polyembryony.

Key results: All plants obtained from the same undeveloped seed in 2x × 4x hybridizations resulted from cleavage of the original zygotic embryo. Also, the plants obtained from in vitro nucellus culture were recovered by somatic embryogenesis from cells that shared the same genotype as the zygotic embryos of the same seed.

Conclusions: It appears that in non-apomictic citrus genotypes, proembryos or embryogenic cells are formed by cleavage of the zygotic embryos and that the development of these adventitious embryos, normally hampered, can take place in vivo or in vitro as a result of two different mechanisms that prevent the dominance of the initial zygotic embryo.

Fig. 1.

(A) Developmental stage of a seed used from nucellus culture in vitro extracted 13–15 weeks after pollination. (B) Seed with both integuments partially eliminated with the zygotic embryo inside. (C,D) Nucellus without integuments and zygotic embryo extracted at early cotyledonary stage.

Fig. 2.

(A) Developed seeds obtained in 2x × 4x hybridizations. (B) Undeveloped seeds obtained in 2x × 4x hybridizations. (C) Multiple embryos contained in undeveloped seeds. (D) Monoembryonic undeveloped seed. (E,F) Germination of multiple embryos from undeveloped seeds.

Fig. 3.

Types of seeds obtained in 2x × 2x hybridization. (A) Developed small seeds with triploid embryos. (B) Normal seeds.

Fig. 4.

Cluster analysis of SSR data from 2x × 4x progenies based on the neighbour-joining method and Dice dissimilarity. (A) Triploid plants obtained from undeveloped seeds originating from the pollinations between diploid clementines ‘Fina’ (Fin), ‘Hernandina’ (Her), ‘Clemenules’ (Nul) and tetraploid ‘Nova’ mandarin (Nov) male parent. (B) Triploid plants obtained from undeveloped seeds originating from pollinations between diploid clementines (Fin, Nul) and tetraploid ‘Orlando’ tangelo (Orl) male parent. (C) Triploid plants obtained from undeveloped seeds originating from the pollinations between diploid clementines ‘Bruno’ (Bru), ‘Tomatera’ (Tom) and tetraploid ‘Pineapple’ sweet orange (Pin) male parent.

Fig. 5.

Genetic analysis of clementine by tetraploid ‘Orlando’ tangelo with Ci02B07 SSR marker. Genotypes: 1, clementine; 2, tetraploid ‘Orlando’ tangelo; 3–5 and 6–9, triploid hybrids obtained from two different undeveloped seeds originating from the hybridization between ‘Fina’ clementine and tetraploid ‘Orlando’ tangelo; 10–13, triploid hybrids obtained from the same undeveloped seed originating from the hybridization between ‘Clemenules’ clementine and tetraploid ‘Orlando’ tangelo; 14 and 15, triploid hybrids obtained from the same undeveloped seed originating from the hybridization between ‘Fina’ clementine and tetraploid ‘Orlando’ tangelo; C, PCR negative control.

Fig. 6.

(A,B) Nucellus cultured in vitro with embryo development in the micropilar region. (C) Germination of embryos obtained from nucellus cultured in vitro. (D) Regenerated plants from embryos produced by nucellus cultured in vitro, showing trifoliate leaves.

Fig. 7.

Cluster analysis of SSR data from 2x × 2x progenies based on the neighbour-joining method and Dice dissimilarity. NulBec and ForBec correspond to plants recovered from nucellus culture in vitro obtained in the pollinations between ‘Clemenules’ clementine (Nul) and ‘Fortune’ mandarin (For) with P. trifoliata ‘Benecke’ (Bec). The first number indicates different seeds and in parentheses are the ploidy level of plants obtained from zygotic embryo and number and ploidy level of plants recovered from nucellus culture in vitro.

Fig. 8.

Genetic analysis with Mest 419 SSR marker of diploid plants obtained from nucellus culture in vitro and germination of zygotic embryo produced in ‘Clemenules’ clementine and P. trifoliata ‘Benecke’ hybridization. Z, plant obtained from germination of zygotic embryo; the other profiles of each group correspond to plants obtained from nucellus culture in vitro. Cl, ‘Clemenules’ clementine; P, P. trifoliata ‘Benecke’.

Fig. 9.

Histological section of seeds obtained in ‘Clemenules’ clementine by P. trifoliata ‘Benecke’ hybridization and sections stained by PAS. (A) Seed fixed 100 d after pollination. (B) Seed fixed 80 d after pollination. E, zygotic embryo; Ed, dominant embryo; Es, secondary embryo; En, endosperm; N, nucellus; Te, outer integument; Ti, inner integument.