Pollination of 6x T. ambiguum with 2x T.occidentale led to a change of the maternal standard petal colour from white to pink within 24-48 hours. This was interpreted as evidence that fertilization had occurred. The frequency of florets developing pods was low (10-20%) and fewer than 1% produced detectable embryos. Dissection of hundreds of pods produced useful numbers of embryos that were green and at the early-torpedo stage. Eighteen of these were grown on agar plates into plantlets that were transferred to potting mix in the greenhouse. (A poor batch of medium led to losses of an unknown number during culture). Three months after transfer, 16 putative hybrid plants were growing in the greenhouse. Ten of these were verified as hybrids and are listed in Table 1 with their parentage. Six of these grew to maturity, flowered and three were studied in more detail (Table 1).
Early observations were that the hybrids plants were large plants, vegetatively closer in size to T. ambiguum than to the diminutive T. occidentale. They were similar to T. ambiguum in leaf surface texture and flowering pattern. However, they had morphological characteristics from both the parent species (Table 2), being semi-prostrate or semi-erect, with robust horizontal stems with adventitious roots only at the basal nodes. Leaflets were large and pointed and flower heads resembled those of T. ambiguum. Detailed measurements on pot-grown mature plants of three hybrids confirmed that the morphology of the hybrids was closer in shape and size to T. ambiguum than to T. occidentale (Tables 3, 4). Petioles of the hybrids were approximately double the length of those of T. occidentale and the leaflets were elongated and similar in shape to the leaflets of T. ambiguum. Stem thickness, leaf thickness and numbers of leaves and growing points per plant were also closer to those of T. ambiguum, while plant spread was inferior to both parents. Dry weights of all plant parts, except inflorescences (heads), were inferior to both parent controls (Table 4). Inflorescence numbers and DWs were transgressively much higher than both parent species. Flowering was terminal, as in T. ambiguum, rather than axillary, as in T. occidentale, and so the flowering stems were determinate, dying back to the basal nodes after seed maturity.
Below ground measurements on mature plants grown in pots (Table 3) showed that the three hybrids studied had thick roots similar in diameter to T. ambiguum. Rhizomes were present in all three hybrids but were fewer in number and, in two cases, thinner than those of T. ambiguum. However, the dry weights of the roots and rhizomes were markedly lower than T. ambiguum (Table 4).
Cytogenetic analyses of the F1 hybrids
Somatic chromosome preparations of hybrids 5, 33, 34, 51 and 140 showed 2n=4x =32, each consistent with derivation from a n=3x=21 gamete from 6x T. ambiguum and a n=x=7 gamete from T. occidentale. A cell of hybrid 5 is shown in Figure 1a, revealing four satellited chromosomes and a vague indication of a 3:1 ratio in chromosome sizes.
FISH using 35S and 5S rDNA as probes showed that the chromosome complement of hybrids 33 and 140 were consistent with expectations  following fusion of normal gametes from both species. Figures 2a, b show a somatic cell of hybrid 33. One NOR-bearing chromosome with a 5S signal on the opposite arm was from T. occidentale and three NOR-chromosomes lacking 5S signals were from T. ambiguum. There were three chromosomes with large 5S signals from T. ambiguum and one with a very small 5S signal from T. occidentale. The T. occidentale-derived NOR was observed to be always decondensed while one or two of the T. ambiguum-derived NORs were often, but not always, condensed.
Meiotic configurations in F1 hybrids
Metaphase I meiotic configurations were analysed for hybrids 5 and 34 (Table 5). The results for both plants were similar, showing high numbers of univalents per cell, a few bivalents and trivalents and very few quadrivalents. Hybrid 5 was also observed at anaphase I where it showed frequent laggards and only infrequent 16-16 disjunction.
Fertility of the F1 hybrids
Pollen fertility of the F1 hybrids was extremely low, with pollen staining ranging from 3-13% (Table 6). Based on the very low pollen fertility and unsuccessful attempts to obtain seed by hand pollination, cloned copies of six hybrids were grown in pots outside and were open-pollinated by bees. Surrounding plants included diverse genotypes of both parent species along with diploid and tetraploid T. ambiguum and white clover. Five of the hybrids produced seed from open-pollination. Seed-set was determined on four hybrids and ranged from zero to 14 seeds per 100 heads (i.e. up to approximately 0.28 seeds per 100 florets, assuming an average of 50 florets per head) (Table 6). Seed-lots from hybrids 5, 33 and 229 were germinated and the progeny were investigated.
Descriptions of OP progeny plants from hybrid 5
Hybrid 5 produced only one small normal OP seed and five very small seeds. Only the normal seed developed into a mature plant, designated 5OP-1. The seedling of 5OP-1 was raised in a greenhouse in winter and was abnormal, with a pale green unifoliolate leaf and a yellow first trifoliolate leaf. Subsequent trifoliolate leaves were pale green with very narrow elongate leaflets. The mature plant had very narrow leaflets with double-V white markings. The plant produced multiple short stems that were rooted at the basal nodes. Flower heads were T. ambiguum-like but were white rather than pink at the base. Mature foliage was light green while leaves produced in some seasons in an unheated greenhouse were yellow-green, suggesting sensitivity to cool temperatures.
5OP-1 was moderately fertile with pollen stainability of 35% and a somatic chromosome complement of 2n=48 (Figure 1b). The number of satellite chromosomes was five (Figure 1b) as would be expected if white clover was the male parent.
Descriptions of OP progeny plants from hybrid 33
Sixteen seeds harvested from hybrid 33 after OP were germinated and 12 mature plants were obtained. Several of these flowered and showed varying degrees of fertility as shown by pollen stainability and seed-set from controlled crosses (Table 7). There was strong phenotypic evidence that to form some of these progeny, hybrid 33 had been pollinated by white clover (Table 7). For example, progeny plants 33OP-1, 3, 13, 16, 18, 19, 20 all were stoloniferous and nodally rooted. Plants 33OP-13 and -16 also carried purple leaf mark alleles from white clover. Progeny plant 33OP-17 was apparently rhizomatous, while several plants with horizontal stems and nodal roots only at the base could not be described as stoloniferous and might not have had a white clover parent. Plant 33OP-1 and its descendants were subjected to further study.
Plant 33OP-1 was robust and stoloniferous with elongated leaves that were obtuse, rather than acute, at the tips. The leaves carried two white V markings and the texture was thinner and less stiff than that of 6x T. ambiguum and hybrid 33. Flowers produced in the first spring had a strong scent of coconut oil. The anthers were small, and possibly indehiscent and, while pollen was not abundant, it showed moderate stainability (Table 5). The plant was also partially female fertile, giving 1-4 seeds/inflorescence when both open-pollinated and self-pollinated.
Flow cytometry indicated that this plant was near-6x, suggestive of the union of a near-4x (i.e. unreduced) female gamete from hybrid 33 and a 2x=16 male gamete. Somatic preparations confirmed that 33OP-1 had a chromosome complement of 2n=51. FISH using 35S and 5S rDNA as probes (Figure 2c, d) showed that, in addition to the full complement of marker chromosomes described above for hybrid 33, this plant had one more NOR-bearing chromosome with a 5S signal on the opposite arm, and a chromosome with a medium-sized 5S signal, as expected if white clover was the provider of the 2x=16 male gamete . In addition, there was a fourth, T. ambiguum-derived, chromosome carrying a large 5S signal.
Seeds and progeny from hybrids 31, 34 and 229
OP seeds were obtained from all three of these hybrids and was placed in storage. Nineteen OP progeny plants from hybrid 229 were grown in pots in a greenhouse. Only a few plants thrived, generally showing low fertility. A few had nodally rooted stolons and rounded leaves consistent with having had white clover as the male parent.
Progeny of 5OP-1 from controlled crosses
5OP-1 produced 3-4 seeds/head on self-pollination and 2-3 seeds/head when pollinated with two different white clover plants. One white clover head was pollinated with 5OP-1 and this resulted in 14 seeds.
Three seeds were germinated from the pollination of 5OP-1 with a white clover plant (BRI x WIL-1)-902, carrying the dominant Rl allele for purple leaf colour in heterozygous form. All three of the progeny showed white clover traits, being stoloniferous with nodal roots and axillary inflorescences and one had purple leaves, confirming the white clover paternity. A further five progeny plants were obtained after pollination of 5OP-1 with a second white clover plant and four of these were stoloniferous with nodal roots, again indicating successful fertilization by white clover. All eight plants obtained from crosses by white clover were partially fertile when open-pollinated among themselves, but seed-sets were low, the best two plants averaging approximately 10 and 14 seeds/head. Most of this seed was stored but a few (BC1F2) plants were grown and further backcrossed to white clover to produce putative BC2 seed that was also placed in storage.
Progeny from controlled crosses of OP plants from hybrid 33
Plants 33OP-1, 11, 14, 15, 16 and 20 produced small numbers of seeds following self-pollination (Table 7). Plants 33OP-1, 14 and 16 were inter-fertile with white clover, and 33OP-14 also set seed following pollination by 6x T. ambiguum. The progeny of 33OP-1 were investigated further.
Self-pollination of 33OP-1 gave about 2 seeds/head (Table 7). Twenty self seeds were germinated and these gave nine mature plants, with the remainder having major abnormalities and not thriving. All of the survivors expressed predominantly T. ambiguum traits (elongated leaflets, semi-erect stems) but also had basal nodal roots, a T. repens/T. occidentale trait. Six were robust plants, with terminal inflorescences and low pollen staining (13-36%).
Hand-pollination of 33OP-1 by a white clover plant heterozygous for red and diffuse red leaf markings (Rl/Rld) produced seeds that gave rise to verified progeny carrying one or the other leaf marking. Most of the progeny had pale green or variegated yellow/green leaflets, especially in the early seedling stages, and only about one third survived to maturity (25 seeds gave 22 seedlings of which eight grew to maturity and flowered). The mature plants were robust with thick, densely branched horizontal stems that lacked or had only basal nodal roots. Flow cytometry showed that they were near-5x. Chromosome counts on two of them confirmed numbers of 2n=42 and 41-42. The latter of these plants, carrying a white V mark and the diffuse red leaf allele was used as the pollen parent on an unmarked white clover plant. Nine verified progeny were obtained from 10 seeds sown (one plant was a self on the white clover mother plant). All nine progeny were strong, stoloniferous, plants resembling white clover. Apart from one that failed to flower, they were moderately fertile with a mean pollen staining of 43% (range 29-69%) and none showed the pale green foliage sectors that had been characteristic of the previous generation. Flow cytometry estimates averaged 2n=4.7 and ranged from 4.5-5.0. These plants were the products of at least two (probably three) crosses of hybrid 33 (AAAO) with white clover. Advanced generation progenies were produced for further investigation.
Progeny from controlled crosses of OP plants from hybrid 229
One OP progeny plant from hybrid 229 (229 OP-2) was crossed as male with white clover and produced nine seeds from two inflorescences. Nine progeny plants were grown and eight produced some seeds after open pollination. These plants showed variable expression of nodal rooting of the horizontal stems, ranging from no roots to strong rooting at all nodes.