Following pollination of AZ 564-2 with several diploid T. occidentale plants, the frequency of pod development was high (approx. 80–90 %) and considerable numbers of small green heart-stage embryos were formed. Although large numbers of these were cultured, only six produced explants that were transplanted into potting mix in pots. Two of these explants died in a few weeks, but four grew strongly and produced robust mature flowering plants. Three (Table ) were confirmed as hybrids between 2x T. ambiguum and 2x T. occidentale but the fourth, which had apparently arisen from a self-pollination of T. ambiguum, was discarded.
General description of the plants
The general characteristics of the plants are summarized in Table which shows that traits were inherited from both parent species. All three plants were similar in morphology and intermediate between the parent species. The hybrids had inherited stolons from T. occidentale and rhizomes from T. ambiguum. However, close scrutiny revealed that young petioles were hairy (a T. occidentale trait), but leaves were dull on the lower surface (a T. ambiguum trait). Stems were abbreviated and densely branched (rather than elongated like T. occidentale). Inflorescences were axillary, as in T. occidentale, but, like T. ambiguum, the florets were white, turning to pink after anthesis.
Spring measurements (non-destructive)
Detailed morphological comparisons of the plants during spring are given in Table . The T. occidentale plant was more highly branched (vegetative growing points) and more leafy (leaves per plant) than the T. ambiguum parent. The hybrid plants were intermediate in branching and leafiness between the parents. The hybrids were also intermediate for petiole length where the T. ambiguum parent was considerably larger than the T. occidentale plant. Trifolium occidentale had markedly thicker leaves than T. ambiguum and the hybrids were similar to or thinner than the T. ambiguum parent. Trifolium occidentale had thinner stems than T. ambiguum and the hybrids were generally as thick as T. ambiguum. However, for plant spread, the hybrids were smaller than the poorer parent, T. ambiguum. Trifolium occidentale and the hybrids were earlier flowering and produced more flowers than T. ambiguum (Table ). One hybrid (65) flowered more profusely than both parents.
Below ground, the hybrids showed positive transgressive expression for numbers of roots per plant, the hybrids having more roots than either parent. Trifolium ambiguum roots were considerably thicker than those of T. occidentale, and the hybrids were intermediate in thickness, for both the original root and the thickest new roots. (The three thickest new roots were measured and all gave similar results. Data were presented only for the thickest.) All of the hybrids inherited the ability to form rhizomes from the T. ambiguum parent. However, the numbers of rhizomes were significantly lower than this parent. Rhizomes of hybrid 65 were thinner than those of the other hybrids.
Summer measurements (destructive harvest)
Results of this harvest are given in Table . As before, the T. occidentale plant was markedly more leafy and highly branched than the T. ambiguum parent, with the hybrids in between, but tending to be closer to T. ambiguum. By this time, flowering of T. occidentale was nearly finished, and flower numbers were markedly higher for the hybrids than for either parent. Shoot dry weights, both total and above cutting height, were higher for the T. occidentale plant than for the hybrids and T. ambiguum, which, within the group, did not significantly differ. By contrast, the T. ambiguum plant root system was approximately five times larger than that of the T. occidentale plant and more than twice hybrid 65. All hybrids produced rhizomes, but these contributed only 2–3 % to the dry weight of the total root-rhizome system, as opposed to 16 % in T. ambiguum. The total below-ground dry weight of the T. ambiguum plant was approx. 2·5 times that of the best hybrid. Total plant dry weight (stem + root + rhizomes) was highest for T. ambiguum, while T. occidentale and the hybrids were markedly smaller.
Somatic chromosome numbers of the three hybrids were 2n
= 16 (Figs A, A and B). In hybrid 65, two NORs were present but, in many cells, one NOR tended to be condensed and difficult to identify. FISH analysis using 18S and 5S rDNA probes (Fig. ) was used to distinguish the NOR chromosomes, where the T. occidentale
-derived chromosome carried 5S rDNA and the T. ambiguum
-derived chromosome lacked 5S rDNA (Ansari et al., 1999
). A 5S rDNA-carrying chromosome from T. ambiguum
was identified by a large signal, and a second 5S chromosome from T. occidentale
carried a very small signal (Ansari et al., 1999
Fig. 1. Somatic and meiotic chromosome preparations: (A) T. ambiguum × T. occidentale hybrid 65 root tip cell (2n = 2x = 16); (B) T. ambiguum × T. occidentale hybrid 65 metaphase I (eight bivalents); (C) T. ambiguum × T. occidentale hybrid (more ...)
Fig. 2. Somatic chromosome preparations of T. ambiguum × T. occidentale hybrids. (A) DAPI-stained highly condensed late metaphase cell of hybrid 65 in grey scale, (B) the same cell counterstained with DAPI and FISH with 5S rDNA (red) and 18S rDNA (green). (more ...)
Meiotic configurations (Table and Fig. ) in pollen mother cells of hybrid 65 were predominantly bivalent, with a low frequency of univalents and no multivalents. Disjunction at anaphase I was consistently 8–8.
Somatic chromosome numbers and meiotic configurations in 2x hybrid 65 and colchicine-doubled 4x hybrid 65-20
Fertility and seed-set
Pollen stainability results for all three hybrids indicated low male fertility. Hybrid 65 showed poor pollen dehiscence and was assessed for pollen stainability on three occasions, giving 5/300, 3/400 and 25/400 for a mean of 3·0 %. Hybrids 118-1 and 118-2 were similar at 2·5 % and 2·0 %, respectively. Female fertilities were determined from seed-sets following open-pollination and hand crosses.
Seed-set following open pollination
Plants open pollinated in the presence of both parent species, along with T. repens and 4x and 6x T. ambiguum plants, were observed to develop pods. A few of these pods were harvested while green and the majority were left to develop normally. The green pods contained a few developing seeds from which the embryos were extracted and placed in tissue culture, as for the original crosses. These produced six mature progeny plants which flowered.
The pods that were left to develop normally on hybrid 65 yielded seeds at a frequency of 119/149 (0·8) seeds/head. To date, several hundred OP seeds have been harvested. Hybrids 118-1 and 118-2 were less studied and have each produced OP seeds.
Seed-set following hand crosses
None of the hybrids produced seeds when self pollinated or when inter-crossed among themselves. The only successful hand crosses were between the hybrids as female and diploid T. occidentale as male. Hybrid 118-2 gave no seeds when pollinated with T. occidentale (seven heads), 118-1 gave two seeds (from six heads) and hybrid 65 produced 30 seeds from 19 heads. A few backcrosses (eight heads) to diploid and tetraploid T. ambiguum were unsuccessful. Crosses between hybrid 65 and one white clover plant produced a few empty testas but no full seeds.
Plants derived from embryos rescued after open-pollination
Two of the six progeny from embryo rescue were relatively highly fertile, one having pollen stainability of 48 %, and both producing hundreds of seeds when open-pollinated. Three produced no seeds, and the last plant (with pollen stainablity of 28 %) gave a few seeds which produced very weak plants with variegated leaves (green/yellow or green/white).
Progeny from seeds set naturally after open-pollination
OP progeny of hybrid 65 were extremely variable in phenotype, ranging from small compact plants through slender stoloniferous plants to robust plants with very thick stolons and roots and some apparent rhizomes. One group of 12 OP progeny plants is summarized in Table . Based on flow cytometry analyses of leaf tissue, three of these were probable triploids (FC = 2·8–2·9), consistent with pollination by diploid T. occidentale (next section). The remainder had variable higher ploidal levels (FC = 4·9–6·6) and these have not been verified or further analysed. The putative triploids all had estimated pollen fertilities of 20–30 % and exhibited slender stoloniferous growth. Very few seeds (approx. 1·0 per 500 florets) were produced following pollination with white clover. Several of the plants with apparent higher ploidies were robust with thick stolons and roots and pollen fertilities of 18–72 % (median 37 %) among the seven that flowered. Only two of these plants (–2, –3) produced selfed seeds but six produced low numbers of seeds (1–9 seeds per 100 florets) when pollinated with white clover.
Characteristics of 12 random OP progeny plants from hybrid 65
Plants from crosses with diploid T. occidentale
The progeny plants tested so far from crosses between hybrids 65 and 118-1 with 2n T. occidentale (OCD) were triploid (2n = 24; Figs D, C and D). There were three satellites (Fig. D), two of which were distal to extended NOR regions and one to a condensed NOR. FISH (Fig. D) showed the genomic constitution of one plant, hybrid (65 × OCD)-9 to be AOO (1 ambiguum: 2 occidentale), indicating that an unreduced female gamete (AO) from hybrid 65 had fused with a haploid male gamete (O) from T. occidentale. FISH also showed (Fig. C and D) that the two NORs from T. occidentale were decondensed and the NOR from T. ambiguum was condensed. These plants morphologically resembled hybrid 65. However, the flower heads were creamy-pink rather than white and the standard petals tended to have frilled edges. Pollen stainability results indicated higher male fertilities than hybrid 65 (Table ). No seeds resulted following self- or cross-pollination among this triploid progeny group. However, seeds were sometimes obtained from hand-crosses as female with T. occidentale (0–3 seeds per head). A few reciprocal crosses, as male, produced some pod and seed development but these remain unverified. One seed was obtained from pollination with T. repens (Table ) and the resulting plant was verified using leaf marks.
Pollen fertility and seed-set of triploid first backcross (BC1) hybrids of hybrid 65 (T. ambiguum × T. occidentale) × OCD (T. occidentale)
Chromosome doubled hybrid 65-20
The somatic chromosome number of the colchicine-doubled hybrid 65-20 was confirmed as 2n = 4x = 32 (Fig. E). The plant was very similar in phenotype to the F1 hybrid 65 but with more spread and thicker stems (Table ). Pollen stainability increased markedly from <10 % in hybrid 65 to 50–60 % in 65-20. Meiotic chromosome pairing in 65-20 had predominantly bivalent pairing along with low but regular incidences of univalents and multivalents (Table and Fig. F). Chromosome disjunction at anaphase I was predominantly 16–16 but some irregular disjunction was observed (Table ). No seeds formed when 65-20 was self pollinated, but it was female fertile and set seeds with various pollen sources. Confirmed hybrids were obtained by embryo rescue following crosses of 65-20 as female and genetically marked white clover. Pollination of white clover with 65-20 has produced one confirmed hybrid without the need for embryo rescue.