Hairy root transformation in alfalfa (Medicago sativa L.)

Summary The widely cultivated forage legume alfalfa (Medicago sativa L.) was transformed with the agropine type Agrobacterium rhizogenes NCPPB 1855. Sterile root and callus cultures were derived from tumorous hairy roots which were easily obtained independent of the plant variety or genotype. Plant regeneration, via somatic embryogenesis, was achieved only when a selected alfalfa line, characterized by high regenerative capability, was utilized. Genetic transformation was confirmed by the presence of agropine and T-DNA. Phenotypic alterations, mainly affecting the root system, were observed in transformed plants. The possibility that T-DNA-induced variations could be useful in the improvement of M. sativa is discussed.Research work was partially supported by Progetto Strategico Agrobiotecnologia C.N.R., Italy     

Effect of plant genotype on the transformation of cultivated alfalfa (Medicago sativa) by Agrobacterium tumefaciens

The trait for somatic embryogenesis is being introduced sexually into alfalfa (Medicago sativa) breeding populations to facilitate genetic transformation of this crop. Cocultivation experiments were conducted with an agronomically-improved embryogenic clone from one such population as well as with two other embryogenic clones, one of which was the source of the embryogenic trait in the breeding populations. Transgenic plants were produced from the agronomically-improved clone whereas none were produced from the other two clones. Among the 16 transgenic plants analyzed there was a range in both copy number and number of integration sites for the NPT-II gene; those plants regenerated after a prolonged selection phase in vitro generally had the highest numbers in both respects. There was no evidence of sectoral chimerism of the transgene in a subsample of transgenic plants analyzed by PCR.     

Microgametophytic selection in two alfalfa (Medicago sativa L.) clones

Summary Microgametophytic selection was investigated using two ecologically diverse autotetraploid clones of alfalfa. Several selection pressures (drying, aging, freezing, and high and low temperatures) were applied to microgametophytes at three stages of the life cycle, 1) during microsporogenesis, 2) post-anthesis, and 3) pollen tube growth. Pollen aging produced a progeny population with a greater mean plant size and a lower coefficient of variation than the control progeny. High temperature (29.5 °C) applied both during microsporogenesis and pollen tube growth resulted in progeny populations which were significantly taller and, in one case, had a larger leaf number than the control populations. In contrast, air dried pollen resulted in a progeny population which had significantly smaller character means and larger coefficients of variation than the control population. Also, low temperature (15 °C) during pollen tube growth yielded progeny with reduced branch number and a larger coefficient of variation than the control progeny. In cases where progeny derived from selected microgametophytes were found to differ from the control offspring, corresponding shifts in the reciprocal cross were not observed. For the temperature stress treatments, the lack of reciprocal differences may be related to the different temperature adaptations of the two ecotypes. These results suggest that microgametophytic selection can be effective in shifting the mean of the progeny generation; however, the results obtained will vary depending upon the selection pressure, stage of selection, and the parents used.     

Interactions of highly regenerative genotypes of alfalfa (Medicago sativa) and tissue culture protocols

Summary Highly regenerative alfalfa genotypes selected in three different laboratories from ‘Regen-S’/ ‘Saranac’ (3 genotypes) and ‘Rangelander’ (1 genotype) were culture using tissue culture and regeneration protocols from two of the laboratories. Regeneration of progerny from crosses among the genotypes indicated that genotypes from Regen-S/Saranac have the same genetic control of regeneration, but the Regen-S/Saranac system may be at least partially different from the one in Rangelander. Significant interactions occurred between genotypes and culture protocols for both embryoid formation and conversion into plantelts. Moreover, interactions involving genotypes from Regen-S/Saranac were as different from each other as they were from the Rangelander genotype. The hypothesis that genotypes from the Saranac genetic background would regenerates equally well on both protocols was rejected. Factors in addition to genes controlling regeneration clearly influence genotype X culture-protocol interactions. The importance of evaluating final plantlet formation along with embryoid formation is discussed. A hybrid of two Rege-S derivatives in which 90% of the F₁ progeny regenerate was identified and the hybrid seed is available to interested scientists.     

Fine root demography in alfalfa (Medicago sativa L.)

In perennial forages like alfalfa (Medicago sativa L.), repeated herbage removal may alter root production and mortality which, in turn, could affect deposition of fixed N in soil. Our objective was to determine the extent and patterns of fine-diameter root production and loss during the year of alfalfa stand establishment. The experiment was conducted on a loamy sand soil (Udorthentic Haploboroll) in Minnesota, USA, using horizontally installed minirhizotrons placed directly under the seeded rows at 10, 20, and 40 cm depths in four replicate blocks. We seeded four alfalfa germplasms that differed in N₂ fixation capacity and root system architecture: Agate alfalfa, a winter hardy commercially-available cultivar; Ineffective Agate, which is a non-N₂-fixing near isoline of Agate; a new germplasm that has few fibrous roots and strong tap-rooted traits; and a new germplasm that has many fibrous roots and a strongly branched root system architecture. Video images collected biweekly throughout the initial growing season were processed using C-MAP-ROOTS software.More than one-half of all fine roots in the upper 20 cm were produced during the first 7 weeks of growth. Root production was similar among germplasms, except that the highly fibrous, branch-rooted germplasm produced 29% more fine roots at 20 cm than other germplasms. In all germplasms, about 7% of the fine roots at each depth developed into secondarily thickened roots. By the end of the first growing season, greatest fine root mortality had occurred in the uppermost depth (48%), and least occurred at 40 cm (36%). Survival of contemporaneous root cohorts was not related to soil depth in a simple fashion, although all survivorship curves could be described using only five rates of exponential decline. There was a significant reduction in fine root mortality before the first herbage harvest, followed by a pronounced loss (average 22%) of fine roots at the 10- and 20-cm depths in the 2-week period following herbage removal. Median life spans of these early-season cohorts ranged from 58 to 131 days, based on fitted exponential equations. At all depths, fine roots produced in the 4 weeks before harvest (early- to mid-August) tended to have shorter median life spans than early-season cohorts. Similar patterns of fine root mortality did not occur at the second harvest. Germplasms differed in the pattern, but not the ultimate extent, of fine root mortality. Fine root turnover during the first year of alfalfa establishment in this experiment released an estimated 830 kg C ha^–1 and 60 kg N ha^–1, with no differences due to N₂ fixation capacity or root system architecture.     

Media and genotype effects on the development and conversion of somatic alfalfa (Medicago sativa L.) embryos

Summary Various preconditioning treatments of alfalfa (Medicago sativa L.) somatic embryos to improve embryo quality and conversion were studied. Four different regenerating genotypes were compared. Embryogenic cultures were established in liquid culture. Globular embryos were collected and plated on an embryo development medium until they reached cotyledonary stage. They were then exposed to three treatments: a standard embryo development medium (control), media supplementation with 1 μM abscisic acid (ABA), 50 mM glutamine and 5% sucrose (T), additional supplementation with 50 μM ABA (TT), and additional supplementation followed by desiccation (TTD). Treatments affected embryo conversion, but not uniformly for all genotypes. Embryo conversion was increased (P<0.05) by pretreatment (T), while only one exhibited any response to additional ABA (T vs. TT). Desiccation decreased (P<0.05) conversion of pretreated embryos (TT vs. TTD) of all genotypes. The effect of treatments on plantlet weight was less pronounced and inconsistent across genotypes.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

Dissolution of ferric phosphate by alfalfa (Medicago sativa L.) root exudates

Alfalfa (Medicago sativa L.) was grown in hydroponic culture to investigate adaptation to Fe-deficiency. Root exudates released into the nutrient solution from Fe-deficient plants were trapped and condensed on an amberlite XAD-4 resin column. The diethyl ether fraction of these exudates dissolved ferric phosphate remarkably. The dissolving capability was about 62 times higher than that of root exudates obtained from Fe-sufficient plants in complete nutrient solution. The Fe-dissolving compound was separated and identified. It was a new natural compound with molecular formula C₁₄H₁₀O₅ and was identified as 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran by means of mass spectrometry and ¹H-nuclear magnetic resonance. This new compound worked as a phytoalexin and inhibited completely the fungal growth of Fusarium oxysporum f. sp. phaseoli.     

Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.     

根癌农杆菌介导真菌遗传转化的研究及应用

根癌农杆菌介导转化法（Agrobacterium tumefaciens-mediated transformation,ATMT）具有转化效率高、遗传稳定、适用范围广等诸多优点,已成为真菌遗传转化研究中的强有力手段,在真菌基因资源开发、真菌性疾病研究和外源蛋白表达研究中发挥巨大作用。本文概述了根癌农杆菌转化法在真菌转化中的研究进展、技术优缺点、转化机制、实验方法和应用现状,着重介绍影响其转化效率的因素并对优化方法进行探讨,展望了该技术在真菌基因资源发掘、基因编辑等方面的应用前景,为今后真菌的遗传转化研究提供参考。     

Restriction endonuclease studies on the chloroplast and mitochondrial DNAs of alfalfa (Medicago sativa L.) protoclones

Summary Alfalfa protoclones were regenerated from the mesophyll protoplasts of two cloned source plants (parents), RS-K1 and RS-K2, initiated from Regen S seed. Because of the high frequency of karyotypic upset previously observed in these plants, chloroplast DNAs (cpDNA) from 23 protoclones and mitochondrial DNAs (mtDNA) from 20 protoclones were examined by restriction endonuclease analysis in order to assess recombination in their cytoplasmic genomes. Seven and four endonucleases were separately used for cpDNA and mtDNA analysis, respectively. Data were consistent with no, or a low frequency of, major sequence rearrangements in either the chloroplast or the mitochondrial genomes as a result of protocloning. However, two types of cpDNA were detected in the 23 protoclones, with only one protoclone possessing the cpDNA type of the cloned parental populations sampled. Possible explanations include a preferential selection during protocloning for one of two parental cpDNA types, an in planta sorting out of cpDNA types in the parental material or both.     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

Expression patterns of three genes in the stem of lucerne (Medicago sativa)

We have identified three stem abundantly expressed genes in lucerne (alfalfa, Medicago sativa). A cDNA library, constructed from lucerne stem polyadenylated RNA, was screened by differential hybridization. From this screening, cDNA clones that correspond to genes which are preferentially, or specifically, expressed in the stem were isolated. MsaS1 encodes an unidentified protein, MsaS2 encodes an S-adenosyl-homocysteine hydrolase and MsaS3 encodes an extensin-like protein. Northern blot analysis of RNA isolated from individual stem internodes indicated that the three corresponding genes show differing developmental patterns of expression. The expression of MsaS1 was confined to the youngest stem tissue and may be regulated by sucrose. In stem tissue the level of RNA for the three genes decreased in response to wounding. Tissue print hybridization analysis was used to localize the expression of the genes to the xylem side of vascular bundles in lucerne stems.     

Efficient transformation of Korean rice cultivars (Oryza sativa L) mediated byAgrobacterium tumefaciens

The purpose of this study was to improve transformation efficiency for three Korean rice cultivars, Ilpum, Dasan, and Namyang. Using two different media with or without light, efficiencies of callus induction, regeneration, and transformation of the Korean cultivars were compared to Japanese cultivar, Nipponbare, as a control. Immature cv. Nipponbare seeds produced 35.5% and 16.1% regeneration efficiency on CIM and N6D media, respectively. Among the Korean cultivars, only cv. Ilpum induced on CIM in the dark was actively regenerated with efficiency of 8.2%. With LBA4404 (pTOK233), no difference for the efficiency of transformation was found between mature and immature seeds of cv. Ilpum. This result reveals that mature seeds can be substituted for this study with no difference. The anther-derived calli of cv. Namyang inoculated with either LBA4404 (pTOK233) or EHA101 (pSMABuba) showed regeneration efficiencies of 14.5% and 20.9%, respectively, even though efficiency of transformation did not differ with these two vectors. We suggest that the anther-derived calli are better-materials for transformation experiment due to their genotype-independent regeneration. In the assay of GUS, all of the calli that survived on the second selection medium were strongly stained. PCR-Southern blot analyses confirmed that T-DNA was stably transformed into all tissues selected. Cvs. Nipponbare and Namyang transformed by LBA4404 (pTOK233) showed positive color in the NPTII ELISA.     

Effect of age and type of tissue on genetic transformation of Lotus corniculatus by Agrobacterium tumefaciens

Various experiments of Lotus corniculatus cv. Leo were infected with Agrobacterium tumefaciens strains C58 (wild-type) and GV3101 (control). A maximum of 83 per cent of cotyledons excised from 7 day old seedlings and 63 per cent of leaves excised from seedlings grown in vitro formed galls in culture. The genotype of the seedling had an effect on the response.