Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.     

Early growth responses of mangroves to different rates of nitrogen and phosphorus supply

Experiments were conducted in an outdoor facility to quantify growth responses of six mangrove species to rates of dissolved inorganic nitrogen and phosphorus supply mimicking the range of N and P mineralization rates in natural soils. Growth of all six species on nitrogen was nonlinear. Stem extension rates of Rhizophora apiculata and Xylocarpus granatum were enhanced to the highest rate of N supply (50 mmol m^− 2 d^− 1); Bruguiera gymnorrhiza, Avicennia marina, and Xylocarpus moluccensis stem growth leveled off by 10 mmol m^− 2 d^− 1. Stem growth of Ceriops tagal peaked at 24-26 mmol N m^− 2 d^− 1. Except for A. marina and C. tagal, rates of biomass increase declined at the highest supply rate, indicating NH₄⁺ toxicity. At different rates of P supply, stem extension rates and rates of biomass increase of R. apiculata and C. tagal best-fit Gaussian curves and B. gymnorrhiza stem growth and biomass increase best-fit sigmoidal and Gaussian curves, respectively; X. moluccensis stem and biomass growth increased linearly, but stem and biomass growth rates of A. marina did not vary in relation to P supply. Stem growth of X. granatum was Gaussian but rates of biomass increase best-fit a quadratic equation. Changes in leaf and root N and P content mirrored the growth responses. As rates of N and P mineralization in natural mangrove soils overlap with the lowest rates of N and P supplied in these experiments, the growth responses imply that mangroves are intrinsically nutrient-limited at mineralization rates often encountered in nature. Such species specificity may have significant implications for recruitment success and the establishment of species gradients within mangrove forests.     

磷、铜及其相互作用对三叶草种子萌发与幼苗生长的影响

通过水培实验研究了施加P对Cu2 胁迫下三叶草种子萌发活力、幼苗生物量、光合色素含量以及电导率的影响.重金属Cu2 (0～20mg·L-1)显著抑制了三叶草种子萌发和幼苗生长.但当施加0.06%的P后,明显提高了cu2 胁迫下的三叶草种子萌发率,促进了幼苗的生长,生物量、光合色素含量显著增加,同时幼苗叶片细胞膜透性极显著下降.可见,外施一定量P能缓解过量Cu2 对三叶草的毒害.     

The carbohydrate-binding sequences in lectins of the clovers Trifolium repens, T. pratense, and T. trichocephalum

The carbohydrate-binding sequences (CBS) in the lectin genes of Trifolium repens, T. pratense, and T. trichocephalum were sequenced. The gene regions encoding lectin CBS of T. pratense and T. repens displayed a considerable similarity; however, the CBS of these species differed essentially. Moreover, T. repens formed a compact cluster with Melilotus albus and M. officinalis in the phylogenetic trees constructed according to the nucleotide sequences and the corresponding CBS of legume lectins. T. trichocephalum does not fall into the group of the tribe Trifolieae members according to both the amino acid sequence of lectin carbohydrate-binding region and the nucleotide sequence of lectin gene.     

Growth and ion relations in response to combined salinity and waterlogging in the perennial forage legumes Lotus corniculatus and Lotus tenuis

Lotus tenuis (Wadst. & Kit.) is a perennial legume widely grown for pasture in the flood-prone and salt affected Pampa region of Argentina. The physiology of salt and waterlogging tolerance in L. tenuis (four cultivars) was evaluated, and compared with Lotus corniculatus (three cultivars); the most widely cultivated Lotus species. Overall, L. tenuis cultivars accumulated less Na⁺ and Cl⁻, and more K⁺ in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 days in aerated or in stagnant solutions. Root porosity was higher in L. tenuis cultivars due to greater aerenchyma formation. In a NaCl dose–response experiment (0–400 mM NaCl in aerated solution), L. tenuis (cv. Chaja) accumulated half as much Cl⁻ in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na⁺ in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl. L. tenuis (cv. Chaja) again accumulated about half as much Cl⁻ in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na⁺ concentrations in the various shoot tissues. The higher root porosity, and maintenance of lower shoot Cl⁻ and Na⁺ concentrations in L. tenuis, compared with L. corniculatus, contributes to the greater tolerance to combined salt and waterlogging stress in L. tenuis. Moreover, significant variation for tolerance to combined salinity and waterlogging stress was identified within both L. tenuis and L. corniculatus.     

Direct Somatic Embryoid Formation on Immature Embryos of Trifolium repens, T. pratense and Medicago sativa, and Rapid Clonal Propagation of T. repens

By direct somatic embryogenesis in vitro a clone of asepticplantlets can be raised from a single immature embryo of Trifoliumrepens (white clover) within about 6 weeks of pollination. Embryoidsare induced directly from intact zygotic embryonic tissue ona culture medium containing 0·025 or 0·05 mg 1^–1BAP and 1·0 g 1^–1 yeast extract. Similar directsomatic embryogenesis has also been achieved for Trifolium pratense(red clover) and Medicago sativa (lucerne). Applications ofembryo propagation by direct somatic embryogenesis are discussed,particularly in relation to multiple screening of host genotypesfor analysis of host/pathogen and legume/Rhizobium interactions. Trifolium repens L., Trifolium pratense L., Medicago sativa L., clover, lucerne, tissue culture, embryoid, somatic embryogenesis, legumes     

Within-population variation in localized and integrated responses of Trifolium repens to biotically patchy environments

Summary Genets of Trifolium repens (white clover) were collected from three patches of old permanent pasture dominated by Agrostis capillaris, Holcus lanatus or Lolium perenne. Plants derived from the genets were grown with plants of one grass species present on one side of each T. repens, and a different grass species on the other side, in all combinations of two of the three grasses. Different modules (a node with its associated internode, leaf, and axillary bud) on the same clover plant responded independently to the microenvironment provided by their own neighbouring grasses. In contrast, all apical meristems on the plant reacted similarly, showing a unified response and integrating the effects of the different microenvironments experienced by the whole clover plant. This is consistent with what is known both physiologically about the nutrition of meristems and modules, and ecologically about the exploratory growth habit of the species. Averaged over all associated grasses, there was no significant variation in the final dry weight of the different clover genets but these differed in their growth habit response to different grasses. In response to Agrostis as a neighbour, each meristem of T. repens rapidly produced many small modules. New modules were produced more slowly and were larger when Holcus or Lolium was the neighbour. The same pattern of differences occurred among clovers sampled from different backgrounds. Either genetic differences paralleled plastic responses, or plastic changes in phenotype that developed in response to different neighbours in the field persisted in the greenhouse. Plants taken from backgrounds of different grass species showed different responses to growing with those grass species. The differences were manifest primarily in a positive leading diagonal effect of Holcus or not-Holcus. They were the result primarily of differences in the dry weight per module and the probability of development of the axillary bud into a branch. This confirms earlier results, and implicates the central importance of branching as a means of local response to the microenvironment.     

Leaf nitrogen and phosphorus concentrations of woody plants differ in responses to climate,soil and plant growth form

Leaf chemistry is important in predicting the functioning and dynamics of ecosystems. As two key traits, leaf nitrogen (N) and phosphorus (P) concentrations set the limits for plant growth, and leaf N:P ratios indicate the shift between N‐ and P‐limitation. To understand the responses of leaf chemistry to their potential drivers, we measured leaf N and P concentrations of 386 woody species at 14 forest sites across eastern China, and explored the effects of climate, soil, and plant growth form on leaf N, P and N:P ratios. In general, leaf N and P were both negatively related to mean annual temperature and precipitation, and positively related to soil N and P concentrations. Leaf N:P ratios showed opposite trends. General linear models showed that variation in leaf N was mainly determined by a shift in plant growth form (from evergreen broadleaved to deciduous broadleaved to conifer species) along the latitudinal gradient, while variations in leaf P and N:P were driven by climate, plant growth form, and their interaction. These differences may reflect differences in nutrient cycling and physiological regulations of P and N. Our results should help understand the ecological patterns of leaf chemical traits and modeling ecosystem nutrient cycling.     

Pollination in vitro: effects on the growth of pollen tubes,seed set and gametophytic self-incompatibility in Trifolium pratense L. and T. repens L.

Summary Growth of pollen tubes and seed set were compared after hand pollination in situ and in vitro in two self-incompatible species, Trifolium pratense and Trifolium repens. Adhesion of pollen grains to the stigma was greater in vitro for both species. After cross-pollination, in vitro culture gave a significant increase in the cumulative growth of pollen tubes in pistils of T. pratense compared to in situ conditions. After selfing in T. repens, pollen tube growth was significantly increased by in vitro culture of florets. Seed set after crossing in situ and in vitro was similar for both species. Seed set after selfing in vitro was not increased in T. pratense. Several genotypes of T. repens were classified as very good, good and poor selfers based on their capacity for seed set following selfing in situ. In vitro pollination increased self seed formation by 1.7-, 18.0- and 31.0-fold for each class, respectively. Ovules located nearest to the style were fertilized more often after selfing than after crossing.     

Interspecific Hybridization of Trifolium repens with T. hybridum Using In Ovulo Embryo and Embryo Culture

In ovulo embryo culture followed by culture of excised immatureembryos produced interspecific hybrids between Trifolium repensL. (white clover) and autotetraploid T. hybridum L. (alsikeclover). Ovules containing hybrid embryos were excised 12–14 dafter pollination and cultured on Nitsch (1951) medium supplementedwith 15% young cucumber juice for 5–6 d. Embryos weresubsequently excised and transferred to hormone-free EG medium,a medium suitable for the culture of immature embryos. A total of 118 hybrid seedlings were obtained from 1978 reciprocalpollinations. All seedlings produced showed various chlorophylldeficiencies, either totally albino or albino with green sectors.Transmission electron microscope studies were carried out toinvestigate plastid development in embryos and seedlings. Someembryos produced only callus. Plants were regenerated from sevencalli. Two semi-albino plants survived transfer to soil, andone plant produced flowers. Backcrosses to T. repens producedone green plant. Hybridity is supported by analysis of morphological characters,karyotype and the gel electrophoretic separation of leaf isozymes. Pollen irradiated with 40 Gy of gamma rays was also used forpollinations. Results indicate that in certain cases ionizingradiation might be useful in overcoming hybrid inviability. Trifolium repens, Trifolium hybridum, clover, interspecific hybridization, in ovulo embryo culture, irradiation     

狗牙根、白三叶草对淡剑纹灰翅夜蛾生长发育和繁殖的影响

利用室内饲喂法,以高羊茅分别为对照,研究狗牙根(Cynodon dacyton)、白三叶草(Trifolium repens)对淡剑纹灰翅夜蛾Spodoptera depravata(Butler)存活、生长发育及繁殖的影响。结果表明,用狗牙根、白三叶草饲喂淡剑纹灰翅夜蛾的化蛹率、幼虫成蛾率均显著低于对照组。幼虫历期显著长于对照组。用狗牙根、白三叶草饲喂幼虫时,淡剑纹灰翅夜蛾的蛹期、产卵前期、产卵期、产卵量、♀成虫寿命与对照组均无显著差异,但幼虫龄期明显增加。狗牙根、白三叶草饲喂的淡剑纹灰翅夜蛾种群增长参数与对照组相比存在明显差异:(1)世代净生殖率(R0);(2)内禀增长率(rm);(3)周限增长率(λ);(4)种群数量趋势指数(I)明显降低;世代平均周期(T)和种群加倍时间(DT)明显延长。由此可见,狗牙根和白三叶草并不是淡剑纹灰翅夜蛾喜好的寄主植物。     

Transfer of clover cyst nematode resistance from Trifolium nigrescens Viv. to T. repens L. by interspecific hybridisation

 Resistance to clover cyst nematode (Heterodera trifolii) has been successfully transferred from Trifolium nigrescens to T. repens by interspecific hybridisation. A sterile triploid hybrid (H-6909-5, 2n=3x=24) was initially produced with the aid of embryo culture. The hybrid was chromosome-doubled from axillary meristems by an in vitro colchicine method. Three chromosome-doubled plants were obtained, and these showed a marked increase in pollen stainability from 10% in 3x H-6909-5 to an average of 89% (range 88–91%) in 6x H-6909-5. T. nigrescens was a source of clover cyst nematode resistance. A mean of 23 (range 0–150) cysts per plant was recorded for T. nigrescens in comparison to a mean of 150 (range 50–240) cysts per plant for T. repens. The 3x and 6x interspecific hybrids were shown to be as resistant as the most resistant. T. nigrescens genotype and were significantly lower in cyst number per gram of root dry weight than the susceptible T. nigrescens and T. repens genotypes. Received: 11 July 1997 / Accepted: 15 July 1997     

Development of a fertile genetic bridge between Trifolium ambiguum M. Bieb. and T. repens L.

  Trifolium ambiguum M. Bieb and T. repens L. are taxonomically related but very difficult to cross. The rare hybrids so far reported between these two species were obtained only by embryo culture. This difficulty has been overcome in the present research by the creation of a “fertile bridge” between T. ambiguum and T. repens. Characters of interest can now be transferred from T. ambiguum to T. repens by using this “fertile bridge” without the use of sophisticated techniques. An array of backcross progenies was generated from crosses between a T. ambiguum×T. repens F₁ hybrid (8x H-435) and its parental species. The 8x hybrid was cross-fertile only with T. repens and resulted in 145 seeds from 1578 reciprocal crosses. Eleven of nineteen initially grown BC₁F₁ plants were all hexaploid with an average pollen stainability of 41.6%. A high frequency of multivalents at metaphase-I indicated that both autosyndetic and allosyndetic pairing occurred. Backcrosses of 6x BC₁F₁ plants to T. repens resulted in 5x BC₂F₁ plants with an average pollen stainability of 59.3%. On the other hand, 6x BC₁F₁×6x T. ambiguum crosses did not produce any seed and only two pentaploid plants were obtained from 6x BC₁F₁×4x T. ambiguum crosses. The difficulty encountered in generating 6x backcross progeny with 6x T. ambiguum was overcome by intercrossing the 6x BC₁F₁ plants and producing 6x BC₁F₂ plants with an average pollen stainability of 65.8%. One of these 6x BC₁F₂ plants was cross-compatible as a female with 6x T. ambiguum and resulted in CBC₂ plants that were all cross-compatible with 6x T. ambiguum. The 6x BC₁F₂ plants are likely to be superior to 6x BC₁F₁ progeny, as they have exhibited better expression of the combined rhizomatous and stoloniferous growth habit, improved fertility, more frequent nodal rooting and heavier nodulation. Consequently, the 6x BC₁F₂ plants can either be used directly in the selection programme or as a “fertile bridge” between the two parental species. The present work has resulted in the development of a series of fertile hybrids by the manipulation of chromosome numbers, combining the agronomic characteristics of the parent species in varying genome balances and at a range of ploidy levels. It is concluded that the initial sterility of the primary interspecific hybrids need not be a barrier to successful inter-breeding. Received: 2 August 1996 / Accepted: 4 April 1997     

The effect of source,concentration and time of application of nitrogen on the growth,nodulation and nitrogen fixation ofLotus pedunculatus andTrifolium repens

Summary Studies under growth cabinet conditions investigated the effect of source and concentration of nitrogen and timing of nitrogen application on the growth and nitrogen fixation byLotus pedunculatus cv. Maku andTrifolium repens cv. S184. KNO₃, NaNO₃ and NH₄NO₃ were added at transplanting at the following rates: 3.33, 7.78 and 13.33 mg N/plant. KNO₃ was added at 3.33 and 7.78 mg N/plant at 0, 6, 12, 18, 24 or 30 days after transplanting.Lotus shoot weight increased with all increasing nitrogen sources but clover only responded to KNO₃ and NaNO₃. The root weight of both species increased with increasing KNO₃ and NH₄NO₃. The percentage increase in lotus and clover shoot growth was greater than that of root growth when KNO₃ was added within a week of transplanting. Increases in growth by both species resulted from added nitrogen except with lotus when NaNO₃ was applied where increased nitrogen fixation also contributed to increased growth.Weight and number of effective nodules on both species were increased with 3.33 mg N per plant as KNO₃ but nitrogen fixation was not affected. Addition of 13.33 mg N as NaNO₃ reduced weight and number of effective nodules in both species and also nitrogen fixation by lotus.KNO₃ increased growth and nodulation of both species when applied within one week after transplanting. Nodulated lotus plants responded to KNO₃ by increasing growth but not nodulation.KNO₃ appeared to affect infection and development of nodules on lotus and may affect the growth of existing nodules on clover.     

Effect of lime,phosphorus and mycorrhizal fungi on growth,nodulation and nitrogen fixation by white clover (Trifolium repens) grown in UK hill soils

Summary The responses of white clover (cv NZ Grasslands Huia grown in four UK hill soil types) to additions of lime and P, to inoculation with Rhizobium and mycorrhizal fungi, and to differences in soil water status were assessed in pot and field experiments. With a deep peat soil in pots, shoot production, nodulation and N fixation by clover were increased by 160, 130 and 85% respectively following inoculation with mycorrhiza, but in the field, despite a doubling of root infection, there was no response in growth. On a brown earth soil in the field inoculation with one endophyte (Glomus mosseae L1) out of four tested depressed production of white clover shoots by 42% but enhanced that of leeks (Allium porrum) by 50%; the others were without effect. With dry peaty podzol and brown earth soils in pots, clover shoot production was highest with added P when a water holding capacity of 80% was maintained, but roots from the latter had only 2.6 compared to 68 nodules per plant from the former. Further work is required to explain poor nodulation in the brown earth soils.     

Nitrogen fixation, stomatal response and transpiration in Medicago sativa, Trifolium repens and T. subterraneum under water stress and recovery

Stomatal behaviour, transpiration and nitrogen fixation were investigated in Medicago sativa L. (cvs. Tierra de Campos and Aragon, Hidalgo-Maynar 1966), Trifolium repens L. (cv. Aberystwyth S-184) and Trifolium subterraneum L. (cv. Clare) subjected to drought by withholding water and then to three days’ recovery after rewatering. Dawn leaf water potential was measured with pressure chamber, stomatal response with a diffusion porometer and nitrogen fixation by using acetylene reduction technique. At low water potentials, the leaf resistance was higher in Medicago than in Trifolium. As water stress developed all species decreased their transpiration, T. subterraneum being the one most affected by moderate deficits. During water stress ‘Tierra de Campos’ always maintained higher acetylene reduction levels than ‘Aragon’ and the Trifolium species, except for the lowest water potentials. During recovery from water stress only ‘Tierra de Campos’ reached predeficit transpiration rates. In ‘Tierra de Campos’ acetylene reduction recovery after rewatering was more rapid and intense than in ‘Aragon’. It is concluded that, of the plants investigated, ‘Tierra de Campos’ was best adapted to water deficits.