Norman L. Taylor

Genetic variation in tissue cultures of red clover

SummaryDesign II matings were made among randomly selected clones of ‘Arlington’ red clover (Trifolium pratense L.). Progeny were evaluated in vitro on two regeneration media for callus growth and differentiation. Additive genetic variance was a significant source of variability for nearly all traits evaluated, including somatic embryogenesis. In vitro traits, such as rapid callus growth, colony vascularization, root initiation, chlorophyll production and embryogenesis were highly heritable and should respond to breeding and selection. Dominance genetic variance was significant for only a few in vitro characters. Maternal and cytoplasmic factors were significant primarily in the early subcultures. Highly significant additive genetic correlation of performance on two regeneration media was found. A population selected on one of the regeneration media for such characteristics as improved plantlet regeneration, rapid callus growth, long term colony viability or the frequency of root initiation should show correlated improvement on the other medium. No significant differences for embryogenesis were attributable to differences in the regeneration media used. Furthermore, no interaction of additive genetic effects with regeneration media were observed. These data indicate that improvement in the frequency of plantlet regeneration from callus of red clover could effectively be achieved by breeding and selection for embryogenic types.

Red Clover Breeding and Genetics

Publisher Summary Red clover, Trifolium pratense L., has been a significant forage legume among agricultural crops. Red clover is adapted to a wide range of soil types, pH levels, and environmental conditions. This plasticity has enabled red clover to retain its usefulness for hay, silage, pasture, and soil improvement in much of the temperate region of the world. Red clover is a nitrogen-fixing legume, thus contributing to the supply of nitrogen available in the soil for subsequent crops. Red clover is a self-incompatible cross-pollinated species that, under natural conditions, produces very few self-seed. The mechanism of self-incompatibility is the one-locus, gametophytic S-allele system in which plants with the same S-alleles are prevented from selfing by slow growth of pollen tubes through styles. The system also prevents cross-fertilization of plants that have the same S-allele genotype. Details of the S-allele systems in red clover have been investigated by inbreeding via pseudo-self-compatibility (PSC).

Kura Clover (Trifolium ambiguum M.B.) Breeding, Culture, and Utilization

Publisher Summary This chapter discusses the kura clover (Trifolium ambiguum M.B.) breeding culture and utilization. The chapter shows that T. ambiguum has the potential to become major forage crop in the temperate forage-producing areas of the world. It produces well, even under intensive utilization, and the forage is of excellent quality. It has the ability to respond to fertilizer applications after periods of disuse or misuse. The origin and distribution of T. ambiguum is described. The strongest attribute of T. ambiguum is its longevity. Although T. ambiguum has many desirable attributes, it has some serious shortcomings, and most breeding efforts have been designed to take these into consideration. These include seed and forage yield, drought resistance, seedling vigor, early flowering, second crop flowering, and symbiotic performance in association with Rhizobium strains.

Interspecific hybridization of red clover (Trifolium pratense L.) with T. sarosiense Hazsl. Using in vitro embryo rescue

SummaryInterspecific hybrid clover plants from the cross Trifolium sarosiense Hazsl. X T. pratense L. were obtained in the present investigation. Immature hybrid embryos were excised aseptically from the pistillate parent, T. sarosiense (2 n = 48), and cultured in vitro prior to in situ abortion. Agar-solidified nutrient media modified from that developed previously for tissue and cell cultures of red clover (2 n = 14) were used for embryo rescue.The heart shaped embryos obtained were cultured for 8 to 14 days on a medium containing a high level of sucrose, a moderate level of auxin, and low cytokinin activity. Viable embryos were then transferred to a standard medium with low auxin and moderate cytokinin levels for the direct germination of shoots. Some embryos produced only callus. Plants were regenerated from callus using an alternate culture scheme. Hybrid shoot numbers were increased on a low auxin, high cytokinin medium and subsequently rooted before transfer to soil in the greenhouse.About 10% of the hybrid embryos were rescued using the optimal culture sequence. Five full-sib families of the F1 hybrid were successfully grown to maturity. Root-tip cells of hybrid plants possessed the expected somatic chromosome number of 31. The genetically determined leaf-mark trait carried by the staminate parent and the rhizomatous root habit of the pistillate parent were expressed in hybrid plants.

Physiology and Morphology of Red Clover

Publisher Summary This chapter focuses on physiology and morphology of red clover. Red clover has long been an important forage legume in world agriculture. Through a symbiotic association with Rhizobium, red clover fixes nitrogen and contributes to the supply of N for companion grasses and subsequent crops. These characteristics have enabled red clover to be used for hay, silage, pasture, and soil improvement in many regions of the world. Stems originate from the crown and are hollow when fully developed. They have a trilacunar, three-trace nodal anatomy, and have about 10 vascular bundles in cross–section. Red clover has epigeal emergence. Germination begins about three days after imbibition of water by the seed. Imbibition rates are higher at 25 than at 6.7°C. The radical appears first and develops into a slender taproot. It grows in the range of temperature between 7 and 35–38°C, but the optimum temperature for growth is between 20 and 25°C.

Effect of temperature on pseudo-self-compatibility in Trifolium pratense L.

SummaryA relatively high temperature treatment, applied during anthesis, was shown to enhance self-seed production through pseudo-self-compatibility in normally self-incompatible red clover (Trifolium pratense L.). The self-seeds were produced in cultures of excised stems held in 2.5 percent sucrose. The stems were excised when petal color was beginning to appear in the buds. During anthesis the cultures were incubated with the flower heads at 40 ° and the stems at 25 °C. When most of the florets per head had opened the cultures were transferred to 20 °C and held at that temperature during the period of pollen growth through the styles and also during seed development. The addition of calcium nitrate and boric acid to the culture medium did not enhance anthesis, seed weight, or the number of seeds produced.Plant genotype and the environment provided before anthesis were the primary factors influencing the number of self-seed produced. Although not all attempts to produce self-seed have been successful, with repeated trials all clones we tested produced some seed.ZusammenfassungDurch eine Behandlung normalerweise selbstunverträglichen Rotklees (Trifolium pratense L.) mit verhältnismäßig hohen Temperaturen während der Anthesis ergab sich eine Erhöhung des Samenertrages durch Pseudo-Selbstverträglichkeit. Die Samen aus der Selbstbefruchtung wurden an Kulturen abgeschnittener Stengel erzielt, die in 2.5% Sucrose gehalten wurden. Die Stengel waren zu dem Zeitpunkt, an dem die Petalenfarbe in den Knospen sichtbar wurde, abgeschnitten worden. In einem Brutraum wurden die Blütenköpfe während der Anthesis bei 40 °C und die Stengel durch Eintauchen der Kulturgläser in ein Wasserbad bei 25 °C gehalten. Sobald sich die Mehrzahl der Blütchen geöffnet hatte, wurden die Kulturen in 20 °C übergeführt und in dieser Temperatur während des Pollenwachstums durch die Griffel und auch während der Samenentwicklung belassen. Eine Beigabe von Kalziumnitrat und Borsäure zum Kulturmedium steigerte weder die Anthesis noch das Samengewicht und die Anzahl der erzeugten Samen. Der Genotyp der Pflanze und die Umwelt vor der Anthesis waren die Primärfaktoren, die die Anzahl der Samen aus Selbstbefruchtung beeinflußten. Obwohl nicht alle Versuche, Samen aus Selbstbefruchtung zu erzielen, erfolgreich waren, erzeugten in wiederholten Versuchen doch alle untersuchten Klone etwas Samen.

Genotype-dependent whole plant regeneration from protoplasts of red clover (Trifolium pratense L.)

Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.

Methods of overcoming interspecific barriers in Trifolium.

SummaryBarriers to interspecific hybridization in Trifolium were investigated by manipulation of mentor pollen treatments, ploidy levels, and compatibility and male sterility systems. Crosses involving the addition of mentor pollen produced fewer seeds and hybrids than crosses involving normal pollination. Lower seed set with mentor pollen was deduced to result from the use of less viable pollen, approximately half the pollen having been killed by alcohol. Pollinations at the diploid level resulted in more hybrids than at the tetraploid level, perhaps because genes for male sterility produced higher female sterility in the tetraploids. The self-compatible stock produced more seeds, mostly selfs, than the self-incompatible stock, but produced more hybrids only in one cross, T. pratense L. × T. diffusumEhrh. The use of male-sterile female parents reduced selfing but produced fewer hybrids than male-fertile female parents. Techniques of this study were designed to affect prefertilization barriers, but the lack of effect may indicate that postfertilization barriers in Trifolium are of greater importance.

Genetic system relationships inTrifolium

Examination of species of Trifolium in the Kentucky collection revealed that annual species generally have simple tap roots, low chromosome numbers (2n = 10−32), are usually self-pollinating and many were introduced from a Mediterranean type climate. Perennial species generally are tap-rooted, stoloniferous, or rhizomatous, possess higher chromosome numbers than annuals (2n = 12− 130), are mostly cross-pollinating and do not have specific climate-habitat relationships. Species introduced from Eurasia are more numerous and more diverse in base chromosome number (n = 5−8) than from other origins. Only species with diploid chromosome numbers of 16 or higher are stoloniferous or rhizomatous. Rhizomatous species, mostly cross-pollinated, were introduced from Eurasia, North and South America, but not from Africa, and not often from Mediterranean climates. Self- and cross-pollinated species occur in all origins. Different flower colors and leafmarks are not associated with origins, climates, and other morphological and physiological characteristics.

Self-incompatibility genotype identification and stability as influenced by inbreeding in red clover (Trifolium pratense L.)

SummaryS allele genotypes of I1 progenies from eight I0 red clover (Trifolium pratense L.) clones were determined under isolated field conditions. Each I1 progeny was vegetatively increased and isolated under a cage for pollination by honey bees. Clones within each I1 progeny producing relatively large and small amounts of seed were classified homozygous and heterozygous, respectively, for S allele genotype. S allele genotypes were verified by extensive sib crosses in the greenhouse, and almost complete agreement was found with the field classification. I2 progenies were reciprocally test-crossed with their parental I0 clones to detect any changes in S specificity and also to confirm the previous S genotype classifications in the I1 generation. It was concluded that the reliability of field and greenhouse sib classification of S genotypes is based on the strength of the incompatibility reaction in each particular clone. Most I1s showed a strong incompatibility reaction as evidenced by low seed set for heterozygous S genotypes, but one progeny showed a weak incompatibility reaction which resulted in S genotype misclassifications. An S specificity was changed in one I2 progeny.