Generating maize × Tripsacum hybrids
Typically, the generation of a maize × Tripsacum hybrid is accomplished by covering the developing maize ear with a bag to prevent cross‐contamination with corn pollen. Within a few days, ample silks are available on the ears and the silks are cut back to approximate 10–15 cm in length. Tripsacum pollen is gathered and dusted onto the maize ear. Typically, pollen from tetraploid Tripsacum (2n = 4x = 72) provides superior seed set, but occasionally, a diploid Tripsacum genotype (2n = 2x = 36) can be identified which also conditions superior seed development. When an appropriate maize genotype is utilized, embryo rescue techniques are not necessary. Experience has shown that a “Supergold popcorn” accession (PI222648) available from the USDA‐ARS Plant Introduction Station, Ames, IA, gives abundant and viable F1 seeds (Kindiger and Beckett 1992) (Figure B6.2). Experimentation with other maize germplasm can provide similar if not superior results.
Figure B6.2 Hybrid seed set utilizing Ladyfinger popcorn as the maternal parent when pollinated by tetraploid T. dactyloides. Over 100 F1 seeds can be readily obtained when an appropriate maize parent is utilized in the cross.
The F1 hybrids are completely pollen sterile and microsporogenesis is associated with a varying array of meiotic anomalies (Kindiger 1993) and vary in seed fertility from completely sterile to highly seed fertile (Harlan and de Wet 1977). To date, all seed fertile hybrids generated from tetraploid Tripsacum dactyloides resources exhibit some level of apomictic expression, which following backcrossing with maize, is often lost. The most common or sexual pathway of genomic change in a series of maize‐Tripsacum backcross hybrids has been clearly described by Harlan and de Wet (1977). Comparative genetics and other approaches that may result in the transfer of Tripsacum traits to maize, including apomixis are described below.
Gene transfer from Tripsacum to maize
Recent and past research strongly suggests that there is little homeology between the genomes of Tripsacum and maize. Maguire (1962), utilizing a set of recessive phenotypic maize markers, suggested that only maize chromosomes 2, 5, 8, and 9 have a potential for pairing and recombination and for gene introgression with Tripsacum. Additional research has confirmed conservation of loci specific to pistil development between maize and Tripsacum genomes (Kindiger et al. 1995; Li et al. 1997). Maguire (1957, 1960) successfully generated and identified a naturally occurring recombination event between an unknown Tripsacum chromosome and the short arm of maize chromosome 2. Studies using B‐A translocation deletion lines suggested that the Mz9S region could pair and recombine with Tripsacum chromosome 5. Genomic in situ hybridization (GISH) studies have also strongly suggested that only three regions of maize chromosomes have homeology with the Tripsacum genome: the sub‐terminal regions of Mz2S, Mz6L, and Mz8L (Poggio et al. 1999). These regions correspond well with groups of conserved restriction fragment length polymorphism (RFLP) markers identified between maize and Tripsacum genomes (Blakey et al. 1994; Leblanc et al. 1995). As a consequence, few sites are available for Tripsacum introgression into the maize genome and, to date, only two instances are known where verifiable recombination/translocation events have occurred (Maguire 1962; Kindiger et al. 1996b).
Potential pathways for Tripsacum introgression
The 28→38→20 Non‐apomictic pathway
This pathway is the earliest known pathway of maize‐Tripsacum hybridization first reported by Mangelsdorf and Reeves (1939) and repeated by several others. When crossing a diploid maize (2n = 2x = 20Mz) by a diploid Tripsacum (2n = 2x = 36Tr), the F1 hybrid consists of 10Mz + 18Tr chromosomes. Backcrossing this hybrid by diploid maize typically results in the fertilization of an unreduced egg by the pollen source. This partially apomictic event (a 2n + n mating) results in what is called a BIII derived hybrid (Bashaw and Hignight 1990) and now possesses 20Mz + 18Tr chromosomes. This behavior is also commonly observed in apomictic tetraploid Tripsacum dactyloides (Kindiger and Dewald 1997) which raises an interesting question regarding the potential of diploid Tripsacum to possess, but not utilize the mechanisms of apomictic reproduction. In a subsequent backcross of this 38‐chromosome individual with diploid maize, individuals possessing 20Mz + 1 thru 17Tr. chromosomes are generated. In some instances, the maize constitution can also be slightly aneuploid, 20 + 1 or 2 maize chromosomes. At this point, the predisposition of these individuals is to rapidly lose most if not all of their Tripsacum chromosomes following additional backcrossing. The end result of continued backcrossing with maize is the recovery of maize, often completely lacking any level of Tripsacum genome introgression through homoeologous pairing and/or recombination. Though, in this pathway, Tripsacum introgression is rare, a method for enhancing the opportunity for introgression has been suggested but not pursued (Kindiger and Beckett 1989).
The 28→38 apomictic transfer pathway
This pathway has been described only once and has been little examined or discussed in the literature. Consequently, some detail regarding the results in this research will be presented. In 1958, Dr. M. Borovsky, Institute of Agriculture, Kishinev, Moldova, performed a series of hybridizations between a diploid popcorn line identified as Risovaia 645 and a sexual diploid (2n = 2x = 36) T. dactyloides clone with the first maize‐Tripsacum hybrids being generated in 1960 (Borovsky 1966; Borovsky and Kovarsky 1967). The F1 hybrids generated from the experiments possessed 28 chromosomes (10Mz + 18Tr). The F1 plants were completely male sterile and were highly seed sterile. Backcrossing with diploid maize identified that some of the F1 hybrids were approximately 1–1.5% seed fertile and resulted in the production of progeny possessing 28 chromosomes (10Mz + 18Tr) and 38 chromosomes (20Mz + 18Tr). When the F1 was backcrossed to the Tripsacum parent, the fertile F1's generated progeny with 28 chromosomes (10Mz + 18Tr) and 46 chromosomes (10Mz − 18Tr + 18Tr). The complete set of backcrosses with maize and Tripsacum resulted in a ratio of approximately 10 (28‐chromosome plants) to one (38‐ or 46‐chromosome plant). Phenotypic observations suggested that the 28‐chromosome progeny were not different from their 28‐chromosome parent while the 38‐ and 46‐chromosome progeny were clearly different. In addition, some seed generated by the 28‐chromosome F1's were polyembryonic. Additional evaluations on the 28‐chromosome F1 and its 28‐chromosome progeny suggested that these F1 plants and their progeny were apomictic. This early, non‐replicated experiment is to date the only report where a 28‐chromosome F1 hybrid was maintained by apomixis. Polyembryony was noted, and a diploid sexual Tripsacum was used to generate the interspecific hybrid.
The 46→56→38 non‐apomictic pathway
This sexual or non‐apomictic pathway, as discussed by Harlan and deWet, is believed to offer the greatest opportunity for Tripsacum introgression into maize and represents results of an early attempt to transfer apomixis to maize (Petrov et al. 1979, 1984). In this pathway, a diploid maize (2n = 2x = 20Mz) is crossed with a tetraploid Tripsacum resource (2n = 4x = 72Tr). The resultant F1 hybrid possesses 10Mz + 36Tr chromosomes. To date, published reports indicate all of these hybrids are pollen sterile and vary considerably in their levels of seed fertility. In this particular
