Embryo inviability in crosses of tetraploid (2n=40) X diploid (2n=20) can be overcome by embryo rescue
--María Dina García and María del Carmen Molina

In many unsuccessful crosses, fertilization and early embryo development occurs but some irregular events subsequently take place, mainly the failure of the endosperm to develop properly, resulting in embryo abortion and seed collapse (Raghavan V., In "Plant Cell, Tissue and Organ Culture", J. Reinert and Y.P.S. Bajaj, eds., p. 375, Springer-Verlag, Berlin, 1977). Embryo culture can be used to overcome this problem.

The aim of this work was to obtain triploid plants (2n=30) from crosses between tetraploids (2n=40) and diploids (2n=20) by embryo rescue.

Immature embryos were obtained from two crosses: inbred line N103A (2n=40) x cv. Colorado Klein (2n=20), and inbred line N104A (2n=40) x cv. Ever Green (2n=20). N103A and N104A were kindly supplied by Dr. E. B. Patterson in 1992.

Plants were grown in the greenhouse during spring. Caryopses were harvested 12, 13, 17 and 22 days after pollination (DAP) and surface sterilized. Embryos were aseptically excised, transferred to 10 ml culture medium and incubated at 28-30?C in 16 h photoperiod.

Embryos were cultured on the basic medium (García et al., 1991 MNL 65:76-77, 1991) supplemented with different concentrations of plant growth regulators (Table 1).

Caryopses collected at 22 DAP from N103A x Colorado Klein crosses looked shrivelled because of defective endosperm development. The embryos were excised from these caryopses at 1.8 to 2.2 mm length and cultured on A, B, C and D media. Of the embryos 48% showed coleoptilar growth but only 12% exhibited radicle development. One month after culture initiation, plantlets looked very weak and a few of them gave rise to adventitious roots (36%); subsequently no roots developed on rooting medium (basic medium + 1 mg.L-1 alpha-naphtaleneacetic acid). After two months of culture, 64% of the plantlets had died in vitro. The remainder were potted but they didn't survive.

Caryopses from N103A x Colorado Klein and N104A x Ever Green crosses, harvested at 12 and 13 DAP respectively, showed normal appearance. Embryo length ranged from 0.6 to 1.5 mm for both crosses. Caryopses from N103A x Colorado Klein collected at 17 DAP showed defective endosperm development and looked shrivelled. The embryo lengths were from 1.5 to 2.3 mm. These embryos were excised and transferred on E, F, G and H media (Table 1). During the first week of culture 100% of the embryos germinated without deformities on each culture medium. Plants showed narrow and slighty curly leaves and a few adventitious roots after 20 days of culture. Plants were potted and transferred to the greenhouse about 30 or 40 days after culture initiation. Few flowering plants were recovered (8%). These plants flowered from 90 to 170 days after culture initiation and gave morphologically normal tassels and ears but no seeds were obtained from self pollinated plants. Mature plant height varied from 40 cm to 130 cm.

Table 1. Culture media used for plating immature triploid embryos.
Plant growth regulators (mg L-1)
Culture media Picloran Kinetin 2,4,-D IBA
A - - - -
B 0.05 - - -
C 0.05 0.05 - -
D - 0.05 - -
E 0.10 0.05 - -
F - 0.05 0.10 -
G - 0.05 - 0.10
H - - - 0.10
Abbreviations: 2,4-D = 2,4-Dichlorophenoxyacetic acid. IBA = Indole-3-butyric acid.

Pollen viability ranged from 11% to 55% and cytogenetic analysis revealed a chromosome number 2n=30. At diakinesis and metaphase I chromosomes formed 10iii in 10% of the cells. The rest of the cells had different numbers of i, ii and iii. Cells at pachynema showed two nucleolar organizers and one or more persistent nucleoli at metaphase and anaphase. In 40% of the cells analysed at anaphase 15 chromosomes migrated towards each pole and in the remaining 60% different numbers of chromosomes migrated towards each pole. Abnormalities like lagging chromosomes, chromatid bridges or micronuclei were not observed.

In conclusion, caryopses from tetraploid (2n=40) x diploid (2n=20) crosses showed apparently normal embryos but endosperm growth failures. These caryopses were inviable, but mature plants (2n=30) were obtained from the embryos excised and cultured on nutrient media, although in vitro development of triploid embryos showed some abnormalities compared with the diploid ones under the same conditions (García et al., MNL 65:76-77, 1991; Van Lammeren, Acta Bot. Neerl. 37(1):49-61 1988). 

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