Comparative Water Relations of Phaseolus vulgaris L. and Phaseolus acutifolius Gray

Leaf area expansion, dry weight, and water relations of Phaseolus vulgaris L. and P. acutifolius Gray were compared during a drying cycle in the greenhouse to understand the characteristics which contribute to the superior drought tolerance of P. acutifolius. Stomates of P. acutifolius closed at a much higher water potential than those of P. vulgaris, delaying dehydration of leaf tissue. P. acutifolius had a more deeply penetrating root system, which also contributes to its drought tolerance. Root-shoot ratios did not differ between the two species either under well watered or water stressed conditions. Leaf osmotic potential was also similar in the two species, with no apparent osmotic adjustment during water stress. These results indicate that P. acutifolius postpones dehydration and suggest that sensitive stomates and a deeply penetrating root system are characteristics which, if incorporated into cultivated beans, might increase their drought tolerance.     

Temperature Effects on Mitochondrial Respiration in Phaseolus acutifolius A. Gray and Phaseolus vulgaris L

Electron transport, using succinate as a substrate, was measured polarographically in mitochondria isolated from Phaseolus vulgaris and P. acutifolius plants at 25°C and 32°C. Mitochondria isolated from P. vulgaris plants grown at 32°C had reduced electron transport and were substantially uncoupled. Growth at 32°C had no effect on electron transport or oxidative phosphorylation in P. acutifolius compared to 25°C grown plants. Mitochondria isolated from 25°C grown P. vulgaris plants measured at 42°C were completely uncoupled. Similarly treated P. acutifolius mitochondria remained coupled. The uncoupling of P. vulgaris was due to increased proton permeability of inner mitochondrial membrane. The alternative pathway was more sensitive to heat than the regular cytochrome pathway. At 42°C, no alternative pathway activity was detected. The substantially greater heat tolerance of P. acutifollus compared to P. vulgaris mitochondrial electron transport suggests that mitochondrial sensitivity to elevated temperatures is a major limitation to growth of P. vulgaris at high temperatures and is an important characteristic conveying tolerance in P. acutifolius.     

Development of an In vitro pod culture technique for young pods of Phaseolus vulgaris L.

Summary A research program to develop an in vitro culture technique adapted to very immature Phaseolus zygotic embryos indicated that osmolality within young pods in vivo ranged from 580 to 350 mosm during the first 10 d after pollination. Different culture techniques for 2-d-old Phaseolus pods are described using a modified Phillips medium for maturation. The application of high and variable osmolality conditions, similar to what is observed in vivo, during pod culture (1 wk) and before extracting the embryos, gave the best results in terms of ovule and embryo development. To solve contamination problems affecting pod development during these investigations, a sterilizing technique was tested combining various concentrations of polyoxyethylenesorbitan monolaurate (Tween 20) and a broad-spectrum industrial biocide, Plant Preservative Mixture (PPM). The PPM sterilizing method totally eliminated pod contamination and did not affect ovule growth, but significantly reduced the embryo germination rate.     

Interspecific hybridization of Trifolium alexandrinum with T. constantinopolitanum using embryo rescue

The embryo rescue technique was successfully used to raise hybrids between Trifolium alexandrinum and T. constantinopolitanum. As a result of its narrow genetic base, genetic improvement in Egyptian clover (syn. Berseem; T. alexandrinum), an important fodder crop in tropical and subtropical countries, is hampered, thereby making it imperative to introduce alien genes from related species. In a conventional interspecific hybridization program, hybrids could not be raised due to post-fertilization barriers. Of the several combinations tried, pollination 2 days after emasculation was found to be the best. Globular embryos were observed 5–7 days after pollination (DAP), followed by heart-shaped embryos 10–12 DAP. Embryos excised at the heart-shaped stage responded well to EC3 culture medium. Of 612 crosses, 33 healthy embryos could be excised and cultured on EC3 medium. The plumule emerged 8–12 days following inoculation. The embryo-rescued plants were hardened, inoculated with Rhizobium and transferred to the field. The hybrids showed intermediate morphological features with reduced pollen fertility (55–65%) and a chromosomal complement of 2n=16. Biochemical characterization using isozymes confirmed hybridity.Abbreviations ACP: Acid phosphatase - IAA: Indole-3-acetic acid - BA: 6-Benzyl adenine - DAP: Days after pollination - Est: Esterase - NAA: -Naphthaleneacetic acid - SOD: Superoxide dismutaseCommunicated by P.P. Kumar     

Apical Dominance in Phaseolus vulgaris L.

Although determinations of the ABA content of lateral buds ofPhaseolus vulgaris revealed no difference between decapitatedand intact control plants in the first 12 h following decapitation,a relative decrease in the ABA content of lateral buds of decapitatedplants was detectable 24 h following decapitation. Shoot decapitationwas also observed to result in a decrease in the ABA contentof stem tissue. The application of IAA to the stem of decapitatedplants prevented these changes and increased the ABA contentof stem tissue relative to that of intact plants. The levelsof IAA and ABA were also determined in the stem tissue fromthe nodes of intact bean plants. The possible interdependenceof these two plant hormones was further investigated by a studyof [2_–14ClABA metabolism. The results are discussed inrelation to the possible role of these hormones in apical dominance. Key words: Apical dominance, Abscisic acid, Indole-3-acetic acid     

Cytokinin metabolism in Phaseolus vulgaris L.

The major cytokinins in stems of decapitated, disbudded bean plants have been identified by enzymic degradation, Sephadex LH20 and reversed phase high performance liquid chromatography, and by combined gas chromatography-mass spectrometry as 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosylpurine (zeatin riboside), 6-(4-hydroxy-3-methylbutylamino)-9--D-ribofuranosylpurine (dihydrozeatin riboside), and the 5-phosphates of these compounds (zeatin ribotide and dihydrozeatin ribotide). Minor cytokinins in this tissue were tentatively identified as dihydrozeatin-O--D-glucoside and zeatin ribotide-O--D-glucoside. [8-¹⁴C-]Dihydrozeatin appeared to be rapidly metabolized to dihydrozeatin ribotide when supplied to segments of stems from decapitated plants. These results are discussed in relation to the metabolism and distribution of cytokinins in the whole plant.Abbreviations TEAB triethyl ammonium bicarbonate - UV ultra-violet - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - TMS trimethyl silyl     

Interspecific hybridization of perennial Medicago species using ovule-embryo culture

Summary New interspecific hybrids between alfalfa (Medicago sativa L.) and several perennial Medicago species were obtained by embryo rescue techniques. The methodology, designated ovule-embryo culture, involved preculturing the fertilized ovule (10 to 20 days post-pollination) for a period of six to 12 days followed by excision and direct culture of the embryo. Placement of the hybrid embryo directly onto culture medium without the interim ovule culture was unsuccessful. Ovule culture to germination without removing the embryo also was unsuccessful. Ovule-embryo culture was essential for recovering interspecific hybrids between diploid alfalfa (2n=2x=16) and the following diploid (2n=2x=16) species: M. hybrida Traut., M. marina L., M. papillosa Boiss., M. rhodopea Velen. and M. rupestris M.B. In addition, trispecies hybrids between M. sativa x M. dzhawakhetica Bordz. F₁ hybrids (2n=3x=24) and either M. cancellata M.B. (2n=6x=48) or M. saxatilis M.B. (2n=6x=48) were obtained from ovuleembryo culture. Media manipulations using M. sativa x M. rupestris F₁ and first backcross generation embryos demonstrated the optimum concentration of 12.5 mM NH₄ ⁺ for successful embryo rescue; ammonium salt formulation (whether chloride, nitrate or sulfate) was not critical. From a few thousand crosses, hybrids between M. sativa and either M. rhodopea or M. rupestris were recovered relatively efficiently with 157 and 66 hybrids, respectively. However, only 13 hybrids between M. sativa and M. papillosa were obtained from more than 2,000 crosses, and just two hybrids each have been recovered from the combinations M. sativa x M. hybrida and M. sativa x M. marina from 2,000 to 3,000 crosses. The predominant chromosome number between diploid alfalfa and the other diploid perennial species was 2n=2x=16. Morphology of the hybrids was generally intermediate. Electrophoretic analysis of the F₁ hybrids and parental clones on uniform or gradient polyacrylamide gels demonstrated that peroxidase phenotypes could be used to confirm hybridity. For all interspecific combinations there was at least one peroxidase isozyme unique to the wild species that was present in the F₁ interspecific hybrid.     

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

Summary Interspecific 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.The investigations reported in this paper (No. 81-3-151) were performed in connection with projects of the Kentucky Agricultural Experiment Station and the paper is published with the approval of the Director     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

Isozymes of Glutamine Synthetase in Phaseolus vulgaris L. and Phaseolus lunatus L. Root Nodules

The glutamine synthetase (GS) isozymes in the plant fraction of nodule extracts from 62 cultivars of Phaseolus vulgaris L. and one cultivar of Phaseolus lunatus L. were analyzed by polyacrylamide gel electrophoresis. All P. vulgaris nodule extracts displayed two GS activity bands: a nodule-specific band (GS_n1) and a band (GS_n2) similar to the single band (GS_r) present in root extracts. In nodule extracts of P. lunatus, the GS_n1 band was detected, but the GS_n2 band was barely detectable. In contrast to P. vulgaris, the GS_n2 band and the GS_r band of P. lunatus appeared to be different. The electrophoretic mobility of the GS_n1 band in P. vulgaris was governed by both the plant cultivar and the development stage of the nodule. In nodule extracts of P. vulgaris and P. lunatus, the zone of GS_n1 activity coincided with six to nine distinct protein bands as revealed after treatment of gels, which had previously been stained for GS activity, with Coomassie blue. All these protein bands were shown to consist of polypeptides of identical molecular weight (approximately 47,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that P. vulgaris continuously generates isozymes of GS_n1 of increasing electrophoretic mobility during the course of nodule development.     

Tissue Cultures of Phaseolus vulgaris L.

Abstract

Callus production and plant regeneration from different explants of Phaseolus vulgaris L. cv. Giza are reported. Calli cultures were induced from leaf, hypocotyl, embryo and root explants. Rapid growth of callus was achieved by leaf explants cultured on MS salts, B5 vitamins and supplemented with 2,4— dichlorophenoxyacetic acid (2, 4—D)+0.5 mg/l kinetin (kin). Addition of casein hydrolysate at 2 g/l to maintenance medium enhanced callus growth and hindered the early appearance of necrotic parts. This report also provides a detailed method for production of multiple shoots directly from the wounded edges of immature cotyledon explant via organogenesis on 1 mg/l benzyladenine (BA) or indirectly on 0.5 mg/l naphthaleneacetic acid (NAA)+2 mg/l BA. The regeneration of bean plants through the two ways described here (direct or indirect) may be of use in genetic improvement of bean.     

Ureide Metabolism in Non-nodulated Phaseolus vulgaris L.

The distribution of ureide-N was studied throughout vegetativeand reproductive growth of non-nodulated Phaseolus vulgarisL. (bushbean) grown in nitrate nutrient solution. Largest increasesin ureide-N per plant were correlated with flowering and earlypod formation and with seed filling. Highest amounts of ureidesper organ were measured in stems and axillary trifoliates. Highestconcentrations (µmol ureide-N g^–1 fr. wt.) weremeasured in young developing organs and stems. Seeds did notaccumulate ureides until the ureide content of pods had reacheda maximum. Results obtained using the inhibitor of xanthine oxidase, allopurinol,are consistent with the origin of ureides via purine degradationbut alternative pathways cannot be discounted. Leaves and stems were shown to have the ability to degrade allantoatevia an enzymic process.     

Interspecific hybridization between Vigna pubescens and V. unquiculata (L.) Walp through embryo rescue

Interspecific hybridization was achieved between cowpea (Vigna unquiculata [L.] Walp) and a hairy wild relative (V. pubescens). Hybrid embryos which otherwise would have shrivelled and failed to germinate were excised prematurely and cultured on a solidified MS medium. The F₁ plants were vigorous in growth, partially sterile, slightly hairy and showed some intermediate features between the two parental types. Cytological investigations on F₁ pollen mother cells showed average chromosome associations per cell of 0.66_I+10.00_II+0.34_IV.     

Ultrastructural Studies of Microsporogenesis in Phaseolus vulgaris L.

Microsporogenesis in dwarf Phaseolus vulgaris was studied under the electron microscope. Before meiosis the microspore mother cell had a lot of organelles especially plastids and ER in its cytoplasm. There were many osmiophilic granules adhering to the membranes of the plastids and vesicular ER until meiosis began. Some cytoplasmic channels were present between adjacent microsporocytes from pachytene to telophase Ⅱ. The organelles were at early stage in the early rnlcrospore, the plastids and mitochondria of which showed regional distribution. Original vacou[es were produced by smooth ER. The organelles in the tapetum cells were mainly mitochondria, plastids and ER. The ER was concentric circles in shape in transverse section.     

The influence of aluminium availability on phosphate uptake in Phaseolus vulgaris L. and Phaseolus lunatus L.

Aluminium toxicity is one of the major limiting factors of crop productivity on acid soils. High levels of available aluminium in soil may induce phosphorus deficiency in plants. This study investigates the influence of Aluminium (Al) on the phosphate (P_i) uptake of two Phaseolus species, Phaseolus vulgaris L. var. Red Kidney and Phaseolus lunatus L. The two bean species were treated first with solutions of Al at different concentrations (0, 25, 50 and 100 μM, pH 4.50) and second with solutions of P_i (150 μM) at pH 4.50. The higher the Al concentration the higher the Al concentration sorbed but P. vulgaris L var. Red Kidney adsorbed significantly more Al than P. lunatus L. Both species released organic acids: P. vulgaris L var. Red Kidney released fumaric acid and P. lunatus L. fumaric and oxalic acids which could have hindered further Al uptake.The two bean species showed a sigmoid P_i uptake trend but with two different mechanisms. P. vulgaris L var. Red Kidney showed a starting point of 3 h whereas P. lunatus L. adsorbed P_i immediately within the first minutes. In addition, P. vulgaris L var. Red Kidney presented significantly higher P_i uptake (higher uptake rate ‘k’ and higher maximum adsorption ‘a’ of the kinetic uptake model). The Al treatments did not significantly influence P_i uptake. Results suggest that P. lunatus L. might adopt an external Al detoxification mechanism by the release of oxalic acid. P. vulgaris L var. Red Kidney on the other hand seemed to adopt an internal detoxification mechanism even if the Al sorbed is poorly translocated into the shoots. More detailed studies will be necessary to better define Al tolerance and/or resistance of Phaseolus spp.     

Interspecific hybridization of phaseolus vulgaris with P. lunatus and P. acutifolius

Summary The influence of genotypic combinations on the growth of hybrid embryos between Phaseolus vulgaris and P. lunatus, and between P. vulgaris and P. acutifolius was examined. All embryos obtained from P. vulgaris × P. lunatus crosses developed only to a stage which appears to be comparable to the pre-heart-shape stage of selfed embryos. Reciprocal crosses were attempted, but pods abscised at a very early stage. Embryos derived from P. vulgaris × P. acutifolius and reciprocal crosses attained the cotyledon stage although no mature seeds were formed. A distinct characteristic of these embryos was the uneven development of the two cotyledons. The rate of growth and final size of these hybrid embryos seemed to be influenced by the genotypes of both parents.Immature embryos were cultured on defined medium and the effects of glutamine and gibberellin (GA₃) were examined. Glutamine was effective in increasing the survival rate; gibberellin had no apparent effect. Plants derived from cultured embryos of P. vulgaris × P. lunatus, P. vulgaris × P. acutifolius and P. acutifolius × P. vulgaris were obtained.Technical paper No. 00 of the Oregon Agricultural Experiment Station. Research was supported by the Oregon Agricultural Experiment Station, the Research Council of Oregon State University (National Institute of Health Biomedical Research Support Grant RR07079) and the Processor Research Council of Oregon. A.R. is supported by an African Graduate Fellowship from the African-American Institute     

A cytokinin glucoside from the leaves of Phaseolus vulgaris L.

Phaseolus vulgaris plants decapitated above the primary leaves accumulate high cytokinin activity. The major cytokinin in these leaves was identified by Sephadex LH 20 chromatography, sensitivity to -glucosidase and permanganate oxidation, and by combined gas chromatography-mass spectrometry as 6-(4-O--D-glucosyl-3-methylbutylamino) purine, (dihydrozeatin-O--D-glucoside). A possible reason for the persistance of this compound in the primary leaves is discussed.Abbreviations BSA Bis-(trimethylsilyl)-acetamide - DHZ dihydrozeatin - DHZOG dihydrozeatin-O--D-glucoside - TMS trimethylsilyl - Z zeatin