Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration

Our objective was to establish a cryopreservation protocol for alfalfa (Medicago sativa L.) cells and study the physiological changes occurring in cells during cryopreservation treatment. Cell cultures of Pioneer cvs. 5262 (fall-dormant, winter-hardy) and 5929 (non-dormant, non-hardy) plants initiated regrowth after cryopreservation by encapsulation-dehydration (ED). Pre-treatment of the encapsulated cells for 4 days in B5 medium containing 0.75 M sucrose and dehydration for 4 h in a laminar flow hood were necessary to achieve maximum cell viability after ED and cryopreservation in liquid N₂ (EDN). Viability (measured as triphenyl tetrazolium chloride reduction) of the cv. 5262 cells after cryopreservation was two- to three-fold greater than that of the cv. 5929 cells. Cold acclimation of the cells (10 days at 2°C) improved viability after cryopreservation. The addition of 7.6 µM ABA to the B5 medium enhanced viability in ED but did not improve cell cryopreservability. Cold-acclimated cells had higher protein concentrations, but neither ABA nor cold acclimation influenced protein composition of cold-acclimated cells determined using SDS-PAGE. Encapsulated cells pre-treated for 4 days in B5 medium containing 0.75 M sucrose showed an increased concentration of cell protein and an altered protein composition. Suspension cultures were re-initiated from both ED and EDN treatments by transferring beads sequentially to B5 media containing 0.75, 0.5, 0.25 M sucrose and then to fresh B5 medium. The ED cells resumed rapid growth after two subcultures, whereas EDN cells needed four or five subcultures to resume rapid growth.     

陇东野生紫花苜蓿的核型分析

采用0.15%秋水仙素和0.002mol/L 8-羟基喹啉的混合液对陇东野生紫花苜蓿萌发种子的根尖预处理3h,室温条件下用1%果胶酶和1%纤维素酶酶解1.5h,得到清晰染色体制片.核型分析结果显示,陇东野生紫花苜蓿染色体数目为32条,具有1对随体,为2B核型,染色体核型组成公式为2n=32=30m(2SAT)+2sm,染色体长度组成公式为2n=2L+18M2+8M1+4S.     

Premature cytokinesis in pollen mother cells of transgenic tobacco plants Nicotiana tabacum L

In majority species of dicotyledonous plants cytokinesis in PMCs occurs once after completion of two caryokinesis cycles, that is a simultaneous type. This paper represents cytological picture and frequency characteristics of abnormality which resulted in cytokinesis triggering after first meiotic division in a part of transgenic tobacco PMCs. It was shown that the main process of cytoskeleton reorganization typical for simultaneous cytokinesis remained without any alterations in such cells. However, in most cases premature cell division occurred with abnormalities such as membrane "tunnel" or "gash" formation. It was ascertained that initiation of additional round ofcytokinesis did not block nuclear cycle and cytokinesis after second meiotic division. Thus, transition of cell division from simultaneous type to successive one occurs under this abnormality.     

Liming of alfalfa (Medicago sativa L.)

Summary A growth-chamber experiment was conducted to study the effect of liming upon growth of alfalfa. The beneficial effects observed were related to changes in soil properties brought about by lime application. Reductions of aluminum and manganese toxicities were the major factors responsible for the increased yields and the decreased growth period required to reach harvest stage. Significant correlations between plant growth parameters and various measures of extractable aluminum were found.     

紫花苜蓿胚状体形成中的二次诱导

1 植物名称 紫花苜蓿（Medicago sativa L．）。     

Characteristics of the cytomictic channel formation in Nicotiana tabacum L. pollen mother cells

Electron-microscopic analysis of cytomictic channels formation in the pollen mother cells in tobacco at the stage of meiosis prophase I of anthers has been conducted. The cytomictic channels in the pollen mother cells in tobacco have been established to be formed under the basis of both single plasmodesmata and de novo with the involvement of specific electron-dense bodies. The role of cytomictic channels in microsporogenesis regulation is discussed.     

Induced androgenesis in alfalfa (Medicago sativa L.)

Summary Anthers of 10 alfalfa (Medicago sativa L.) lines were used as initial material for the production of androgenic haploids. More than 30 variants of nutrient media were tested. Twenty five different treatments with low temperatures and gamma rays were tried in order to find optimal conditions for callus induction and organogenesis.The genotype, stage of microspore development, phytohormonal composition of the nutrient media and pretreatment with physical agents, alone or in combination, affected the efficiency of organogenesis and regeneration in anther cultures of alfalfa.Plants exhibited a high degree of variability in their chromosome number. Haploids, dihaploids and mixoploids were obtained.Cytological studies of in vitro pollen development revealed the origin of the regenerants from microspores.Abbreviations BAP 6-Benzylaminopurine - 2-ip 6-(,-dimethylallylamino)Purine - IAA Indolylacetic Acid - NAA Naphthaleneacetic Acid - 2,4-D Dichlorophenoxyacetic Acid - CMS Cytoplasmic Male Sterility     

Microtubule reorganization accompanying preprophase band formation in guard mother cells ofAvena sativa L.

Summary In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell formation by mesh-like to radial microtubules that extend throughout the cytoplasm. Many of the Mts are also grouped in bundles. Gradually, this intermediate array is succeeded by longitudinal elements of the PPB. Thus, preprophase band formation is accompanied by a 90° shift in Mt orientation, with a radial arrangement serving as an intermediate stage. The micrographs are most consistent with the rearrangement of intact Mts, although changes in Mt assembly are possible as well. The role of the IMB in guard mother cells is also discussed.Abbreviations GMC guard mother cell - IMB interphase microtubule band - Mt microtubule - PPB preprophase band     

Intranuclear fibrillar material in cereal pollen mother cells

Ultrastructural studies of cereal anthers found intranuclear bundles of microfilaments in pollen mother cells (PMCs) but not elsewhere. The ultrastructure, distribution, and behaviour of this fibrillar material (FM) are described. FM was seen in all 19 genotypes studied comprising Aegilops, Triticum, Secale, Hordeum and Avena species, which together included haploid, diploid and allo-and autopolyploid, and natural and synthetic polyploid examples. Detailed studies in diploid S. cereale, and hexaploid T. aestivum and Triticale showed that FM was present in PMC nuclei during premeiotic interphase, leptotene and zygotene but not at pachytene and later meiotic stages. Moreover, it was most abundant at late premeiotic interphase in T. aestivum, and at leptotene in S. cereale and Triticale, when it occurred in up to 100% of sampled PMC nuclei in an anther. Although FM and synaptonemal complex (SC) occurred together in some PMC nuclei at later stages, FM was present long before SC, and reached its peak of abundance before SC did. Bundles of FM often formed links at their ends between either two masses of chromatin, or more rarely, between chromatin and the nuclear membrane. Individual bundles of FM varied in length but showed roughly similar ranges of lengths and widths in these three species. They were up to about 0.2 m in diameter and about 3 m in length, equivalent to about 20% of the maximum diameter of the nuclei containing them. Reconstructions of PMC nuclei indicated that FM was never associated with centromeres but was sometimes, and perhaps usually, associated with telomeric or sub-telomeric chromosome segments.The function of FM is unknown but its possible role is discussed in relation to (1) previously described intranuclear inclusions in meiocytes and (2) the cytogenetics and developmental behaviour of meiotic nuclei in the wheat comparium. As FM was a constant and characteristic structural component of PMC nuclei, its presence is probably of functional significance to the meiotic process. If so, it may function before, and over greater distances, than SC in establishing or maintaining the coorientation of chromosomes prerequisite for normal chromosome pairing. As FM was most abundant at stages when major chromosome movements occur, yet its distribution was non-centromeric, it is suggested that it may function in the attachment and movement of telomeres at the nuclear membrane formed after premeiotic mitosis. The possibility that a bundle of FM normally links corresponding sites on two homologues is considered.     

Starch Grain Distribution in Taproots of Defoliated Medicago sativa L

Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore, during foliar regrowth there is a spatial separation in starch degradation and synthesis in alfalfa taproots. Amyloplasts from alfalfa taproots contained numerous starch grains, prolamellar-, and electron-dense bodies. The high starch line had 23% more cross-sectional area as ray cells in wood tissues when compared to the low starch line, which may explain part of the difference in starch accumulation between these alfalfa lines.     

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.     

Role of Potassium in Carbon Dioxide Assimilation in Medicago sativa L

Alfalfa was grown hydroponically in 0, 0.6, and 4.8 millimolar K in order to determine the influence of tissue level of K on photosynthesis, dark respiration, photorespiration, stomatal and mesophyll resistance to CO₂, photosystem I and II activity, and synthesis and activity of ribulose 1,5-bisphosphate carboxylase (RuBPc).     

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     

Synaptonemal complex formation in pollen mother cells of Tradescantia

Seven nuclei of Tradescantia sp. (2n=12) were prepared using Gillies' modification of the Counce-Meyer whole-mount spreading technique. These nuclei were examined for the type (homologous and nonhomologous), amount, and location of synaptonemal complex (SC) formation. Estimates of the total potential number of discrete initiations of SC formation ranged from 251–299 per nucleus. The average distance between sites of initiation ranged from 7.3 to 11.2 m in the different nuclei. Two groups of Tradescantia were used. Of the T. ohiensis var. paludosa plants, the one instance of irregularity in synapsis was associated with a pairing partner switchpoint. In the other plants derived from Mericle's Clone 02, all nuclei, even those in early zygotene, had several examples of foldback synapsis. The significance of the location of SC formation, and the occurrence of foldback synapsis is considered and a model of SC formation is proposed. In this model, a recognition of homology step is not considered necessary for SC formation; homologous synapsis is usually accomplished by spatial and temporal coordination of zygotene DNA synthesis.     

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.     

Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Amylase was found in high activity (193 international units per milligram protein) in the tap root of alfalfa (Medicago sativa L. cv. Sonora). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (heavy amylase) and 41,700 (light amylase). Activity staining of electrophoretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the light amylase fraction. Three amylase isozymes with electrophoretic mobilities identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and light amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze pullulan or β-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato β-amylase, while that of bacterial α-amylase was considerably higher. The identification of maltose and β-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all β-amylases.     

Abnormal macrosporogenesis in five alfalfa (Medicago sativa) mutants producing 4n pollen

Summary An analysis of micro- and macrosporogenesis in five diploid alfalfa mutants was carried out using a stain-clearing technique. All plants produced tetranucleated microspores and jumbo pollen due to the complete failure of the postmeiotic cytokinesis as well as bi- and trinucleated macrospores. The latter was due to the absence of cytokinesis after the first and second meiotic division of macrosporogenesis. Only one out of the five clones analyzed formed tetranucleated macrospores as a consequence of the total lack of cytokinesis after both meiotic divisions. The fusion of nuclei within binucleated macrospores resulted in 2n macrospores of the SDR type, recognizable on the basis of nucleolus dimension, confirming the ability of jumbo pollen (jp) mutants to produce 2n eggs at a high frequency. Nuclear fusion was also observed within tri- and tetranucleated macrospores. Although having the same genetic background, the five clones showed significant variability in the expression of abnormal cytokinesis during macrosporogenesis.Research supported by National Research Council of Italy, special project RAISA, sub-project no. 2, paper no. 527