EVOLUTION OF COTYLEDONARY AND NODAL VASCULATURE IN THE JUGLANDACEAE

Cotyledonary nodal patterns of the Juglandaceae range from 1-gap, 2-trace to multi-gap, multi-trace. The development of increased nodal complexity is associated with at least two independent evolutionary shifts from epigeal to hypogeal germination. The taxa with epigeal germination such as Engelhardia sect. Engelhardia, Engelhardia sect. Psilocarpeae, Platycarya, and Pterocarya all have 1-gap, 2-trace nodes. The change to hypogeal germination in Engelhardia sect. Oreomunnea and Alfaroa is correlated with the development of 1-gap, 3-trace cotyledonary nodes. The second line has led to large, heavy-fruited members with hypogeal germination and complex cotyledonary nodes ranging from 2–6 gaps. The diversity of nodal patterns is the result of variation on a common theme; five basic vascular strands in the cotyledon, undergoing variations in dichotomy, fusion, and separation, are associated with one to many gaps. Presumably the complex development of the cotyledonary node is a response to increased functional demands of hypogeous cotyledons.     

Studies on the leaf anatomy of Euphorbia III. The node

Anatomical studies of representatives of 11 groups of species of the genus Euphorbia , erected on the basis of leaf venation patterns, reveal the occurrence of 3 main types of nodal organization, viz. unilacunar 1-trace, bilacunar 2-trace and trilacunar 3-trace (in the majority of the species). In some instances, a deceptive multi-stranded condition is visualized if cleared leaves alone are studied. Euphorbia polygonifolia and E. peplis leaves have a variable vascular supply in the same plant, consisting of 2 or 3 strands in the former and 2 or 4 strands in the latter. No correlation was found between the type of node and either the habit and habitat of the plant or the leaf shape and size.     

Hydraulic architecture of sugarcane in relation to patterns of water use during plant development*

Hydraulic conductance was measured on leaf and stem segments excised from sugarcane plants at different stages of development. Maximum transpiration rates and leaf water potential (Ψ_L) associated with maximum transpiration were also measured in intact plants as a function of plant size. Leaf specific hydraulic conductivity (L_sc) and transpiration on a unit leaf area basis (E) were maximal in plants with approximately 0.2 m² leaf area and decreased with increasing plant size. These changes in Fand L_sc were nearly parallel, which prevented φ_L in larger plants from decreasing to levels associated with substantial loss in xylem conductivity caused by embolism formation. Coordination of changes in E and leaf hydraulic properties was not mediated by declining leaf water status, since φ_L increased with plant size. Hydraulic constrictions were present at nodes and in the node-leaf sheath-leaf blade pathway. This pattern of constrictions is in accord with the idea of plant segmentation into regions differing in water transport efficiency and would tend to confine embolisms to the relatively expendable leaves at terminal positions in the pathway, thereby preserving water transport through the stem.     

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differences in hormonal requirements, the various explants showed preferential growth in different basal media.     

Alteration of C-assimilate partitioning in leaves of soybeans having increased reproductive loads at one node

The objectives of this study were to determine if the partitioning of recently fixed carbon between starch and water-soluble compounds could be altered by increasing the pod load in the leaf axil, and if the presence of source leaves acropetal to such a node would influence the partitioning of carbon within the subtending leaf. Soybeans (Glycine max L. Merr. cv Hodgson 78) were grown to full-bloom in a controlled environment chamber, and then deflowered at all nodes except the eighth. This treatment resulted in an 83% increase in the number of pods at the eighth node. At 24 days after flowering, one-half of the treated plants were girdled above the untreated node. Forty-two hours later, the eighth trifoliolate was pulsed with ¹⁴CO₂ and sampled for radiolabeled starch and water-soluble compounds (WSC) at 0.5, 2, 4, 8, 12, and 24th after labeling.

When no girdling was applied above the increased pod load at the eighth node more label was accumulated by the pod walls (+6.9%) and seeds (+6.3%) when compared to the controls. Starch accumulation was not altered in the labeled leaf of the nongirdled plants. When the stem was girdled above the eighth node, significantly less starch was retained in the labeled leaf. Girdling also resulted in an increase in label accumulation by the pod walls (+5.4%) and seeds (+6.6%). These data suggest that the plant will change the distribution patterns of assimilate to supply added sink demand before altering the partitioning of recently fixed carbon in the subtending leaf.

     

COMPARATIVE MORPHOLOGY OF THE COCHLOSPERMACEAE. II. ANATOMY OF THE YOUNG VEGETATIVE SHOOT

The generic scope and systematic position of the Cochlospermaceae were evaluated using observations from the anatomy of the stem, node, and leaf. There are few basic differences in vegetative anatomy between Amoreuxia and Cochlospermum. Secretory cells and canals, dilated phloem rays, and banded phloem are unifying features. Mature nodal anatomy is 3-trace, trilacunar, and the leaves of both genera have elongate, unicellular, branched idioblasts in the spongy mesophyll. Bixa has some features in common with Amoreuxia and Cochlospermum but is distinctive in vascularization of the petiole, leaf anatomy, and vestiture. Rhopalocarpus is quite different from the above genera, and its placement in a separate family is justified on anatomical grounds. The Cochlospermaceae, consisting of Amoreuxia and Cochlospermum, seem more closely related to the Sterculiaceae and Tiliaceae than to the Flacourtiaceae, Cistaceae, or Violaceae.     

DEVELOPMENTAL STABILITY IN LEAVES OF CLARKIA TEMBLORIENSIS (ONAGRACEAE) AS RELATED TO POPULATION OUTCROSSING RATES AND HETEROZYGOSITY

Four natural populations of Clarkia tembloriensis, whose levels of heterozygosity and rates of outcrossing were previously found to be correlated, are examined for developmental instability in their leaves. From the northern end of the species range, we compare a predominantly selfing population (t? = 0.26) with a more outcrossed population (t? = 0.84), which is genetically similar. From the southern end of the range, we compare a highly selfing population (t? = 0.03) with a more outcrossed population (t? = 0.58). We measured developmental stability in the populations using two measures of within-plant variation in leaf length as well as calculations of fluctuating asymmetry (FA) for several leaf traits. Growth-chamber experiments show that selfing populations are significantly more variable in leaf length than more outcrossed populations. Developmental instability can contribute to this difference in population-level variance. Plants from more homozygous populations tend to have greater within-plant variance over developmentally comparable nodes than plants from more heterozygous populations, but the difference is not significant. At the upper nodes of the plant, mature leaf length declines steadily with plant age, allowing for a regression of leaf length on node. On average, the plants from more homozygous populations showed higher variance about the regression (MSE) and lower R² values, suggesting that the decline in leaf length with plant age is less stable in plants from selfing populations than in plants from outcrossing populations. Fluctuating asymmetry (FA) was calculated for four traits within single leaves at up to five nodes per plant. At the early nodes of the plant where leaf arrangement is opposite, FA was also calculated for the same traits between opposite leaves at a node. Fluctuating asymmetry is significantly greater in the southern selfing population than in the neighboring outcrossed population. Northern populations do not differ in FA. Fluctuating asymmetry can vary significantly between nodes. The FA values of different leaf traits were not correlated. We show that developmental stability can be measured in plants using FA and within-plant variance. Our data suggest that large differences in breeding system are associated with differences in stability, with more inbred populations being the least stable.     

星叶草属2隙节及2裂叶及其系统学意义

报道了存在于星叶草属植物中与单隙节共存的２隙节及与不分裂叶共存的２裂叶。通过分析后认为,这两个现象在该属植物中的出现并不是异常的。     

Vegetative micro-cloning to sustain biodiversity of threatened Moringa species

Efficient vegetative cloning in vitro requires definition of plant growth regulator regimes for each genotype, and therefore formulation of a uniform culture protocol for a genetically heterogeneous wild or uncultivated plant population is often impossible. The likelihood of cloning a wide array of plant genotypes by avoiding the use of plant growth regulator(s) was explored with Moringa oleifera Lamk., Moringa stenopetala (Baker f.) Cufod, and Moringa peregrina Forssk. ex Fiori tree seedlings. Propagation was achieved by multiple shoot regeneration from the cotyledonary node of decapitated seedlings, followed by axillary shoot growth from single node shoot segments and rooting of excised shoots. All steps were accomplished on basal Murashige and Skoog medium without plant growth regulator supplements. The results revealed competence for generation of multiple shoots from cotyledonary node tissue, stimulated by repeated shoot harvest, in seedlings of all three tree species. Tens of plants per seedling were regenerated in about 4 mo from culture initiation. In a given species clone size was seedling-dependent, which presumably stems from genotypic variability among seedlings in regeneration ability in vitro. By this means the laborious search for a plant growth regulator regime suitable for organogenesis induction and adapted per genotype became redundant, and biodiversity of the seed germplasm could be maintained. The approach ideally suits establishment of clones of wild plants of endangered species, like those of the Moringaceae, species with high ability for producing supplementary shoots, and without the need to add plant growth regulators, including the rooting stage.     

Use of in vitro cultures of Nicotiana benthamiana for the purification of grapevine virus A

Seedlings of Nicotiana benthamiana were inoculated with grapevine virus A (GVA). Three weeks later, upon systemic symptom expression, cultures were established in vitro using single nodes from these plants. GVA was purified from these cultures and from leaves of GVA-infected N. benthamiana plants maintained in the greenhouse. More virus was obtained from the proliferating in vitro node cultures than from the leaves. The use of in vitro cultures for virus purification represents a valuable practical application of plant tissue culture techniques.     

Differences in shoot regeneration response from cotyledonary node explants in Asiatic Vigna species support genomic grouping within subgenus Ceratotropis (Piper) Verdc.

The efficiency of any plant regeneration system lies in part in its wide applicability to diverse genotypes. In Asiatic Vigna, cotyledon and cotyledonary node explants from 4-day-old seedlings of 27 genotypes were cultured in a medium consisting of MS salts, B5 vitamins, 3.0% sucrose and 1.0 mg l^-1 BA. Direct and efficient multiple shoot regeneration (80–100%) from the cotyledonary nodes was obtained in all epigeal species namely radiata, mungo, aconitifolia, subspecies radiata var. sublobata, mungo var. silvestris and in the hypogeal but allotetraploid glabrescens. In contrast, two other hypogeal species V. angularis and V. umbellata failed to initiate shoots from the nodes. However, adventititious shoots developed at the basipetal cut (hypocotyl) in 35–67% of V. angularis explants. These results provide evidence in support of the existing genomic grouping within subgenus Ceratotropis, which designates AA, A₁A₁ and A₁A₁/- to epigeal, hypogeal and the allotetraploid species, respectively. Mean shoot production ranged from 3.3 to 10.4 shoots per explant during the first subculture and varied significantly among the responsive genotypes within 4 species. Additional shoots were obtained in all genotypes after subsequent subculture. However, cotyledons were not as regenerable as cotyledonary node explants. Although significant differences in rooting were observed among the shoots of the 15 genotypes, the response was generally higher in MS basal medium (MSO) than in MS with 1.0 mg l^-1 IAA. Regenerated plants were successfully transferred to soil (50–100% survival rate) and all surviving plants were reproductively fertile. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of Transgenic Soybean Plants with Two Anti-Fungal Protein Genes Via Agrobacterium and Particle Bombardment

Utilizing either Agrobacterium-mediated transformation or particle bombardment we obtained transgenic soybean [Glycine max (L.) Merr.] plants expressing the chitinase gene (chi) and the barley ribosome-inactivating protein gene (rip). Six regenerated plants were grown to maturity and set seed. The identification of transgenic soybean plants that co-integrated the two anti-fungal protein genes was determined by polymerase chain reaction (PCR) and Southern blot analysis. Protein detection from the soybean leaves demonstrated the expression of the chitinase (CHI) and the ribosome-inactivating protein (RIP) in the six R₀ transformants. Soybean cotyledonary nodes were transformed using the bivalent plant expression vector pBRC containing chi and rip both driven by the CaMV 35S double promoter. Following vacuum (0.06 MPa) infiltration treatment of the tissue for 5 min, Agrobacterium was co-cultivated with the cotyledonary nodes for 3 d on MSB medium (MS salts and B₅ vitamins) (pH 5.2), the transformation frequency reached a maximum of 1.33 %. The chi and rip genes were present in all the transgenic plants. Co-bombardment of immature cotyledons with plasmids pBchE (encoding chi) and pARIP (encoding rip) resulted in a maximum transformation frequency of 0.52 % with a 50 % co-integration rate. Our results demonstrate efficient co-transformation of multiple genes in soybean.     

Micropropagation of Crataeva nurvala

A simple protocol for mass multiplication of Crataeva nurvala, a medicinal tree, from seedling-derived explants is described. Six different types of explants (cotyledonary nodes, epicotyl nodes, hypocotyl segments, first pair of leaves, cotyledons, and root segments) developed shoots on Murashige and Skoog's (MS) basal medium or the same supplemented with different concentrations of 6-benzylaminopurine (BAP). Among the explants tested for caulogenic potential, only the epicotyl and cotyledonary nodal explants developed shoots on MS basal medium, while on BAP (0 – 2.0 mg dm⁻³) adjuvated media all the explants exhibited caulogenesis. The optimum concentration of BAP varied for these explants. The shoots could be rooted on half strength MS with 0.02 mg dm⁻³ α-naphthalene acetic acid to get plants, which have been transferred to soil. The explants from in vitro regenerated shoots also possessed a similar caulogenic potential. This revised version was published online in July 2006 with corrections to the Cover Date.     

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T₀ generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T₁ seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T₁ generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use.     

Competence for regeneration of cucumber cotyledons is restricted to specific developmental stages

The total duration of the plant regeneration process from cucumber (Cucumis sativus L.) cotyledonary explants was only six weeks, which included the induction of buds and their development into plants. Regeneration of shoots from cotyledons from three to five day-old seedlings ranged up to 100%. The regenerated plants were morphologically normal, flowered and set seed. The regeneration capacity of cotyledons from seven days-old and older seedlings was lowered dramatically. Most of those regenerated plants were polyploid/mixoploid and had an abnormal morphology. During seedling development (3 to 13 days), the DNA content of cotyledonary cells changed from 2C to 4C and more. The results show that the decrease in regeneration competence correspond with the change in DNA content of the cotyledonary cells.     

Crown Gall on Castor Bean Leaves: II. FORMATION OF SECONDARY TUMOURS

Secondary tumours were formed on the cotyledonary leaf petiole,the hypocotyl, and first true leaf of castor bean seedlingsafter inoculating the blades of the cotyledonary leaves withAgrobacterium tumefaciens. Depending on the strain of bacteriaemployed, 0 to 80 per cent of the plants developed secondarytumours. The ability of different strains to initiate secondarytumours was not obviously correlated with their relative effectivenessin initiating primary tumours. Though all produced primary tumours,five out of ten auxotrophic strains failed to yield secondarytumours, whereas only one out of 14 prototrophic strains failedto do so. Both the number of plants developing secondary tumoursand the frequency with which these tumours occurred on differentparts of the plant were positively correlated with the concentrationof the primary inoculum. Tumours also developed on the cotyledonaryleaf petiole and on the hypocotyl after vacuum infiltrationof A. tumefaciens into the blade of cotyledonary leaves. Inmost instances (9 out of 11 plants) no tumours were formed onthe blade of the infiltrated leaf. Thus, tumour formation equivalentto secondary tumours can occur in the absence of a primary tumouror an overt primary wound. Excision of inoculated leaves showedthat the stimulus for secondary tumour formation moves fromthe blade to the hypocotyl within 24 h. Attempts to demonstratethe presence of a sub-cellular tumour-initiating agent in homogenatesof inoculated leaves were unsuccessful. A. tumefaciens, however,was found in the petiole of the cotyledonary leaf and in thehypocotyl within 24 h of inoculation. The migrating agent responsiblefor secondary tumour formation in castor beans is concludedto be intact bacteria.     

A multi-needle-assisted transformation of soybean cotyledonary node cells

A new and simple method for wounding cotyledonary node cells of soybean [Glycine max (L) Merrill] was developed for obtaining a high frequency of transformants. Soybean seeds were germinated for 1 day, and the cotyledonary node cells of half-seeds were wounded mechanically by using a multi-needle consisting of thin 30 fibers. The wounded half-seeds were inoculated with Agrobacterium tumefaciens cells harboring a recombinant DNA that contained the bar and sgfp genes conferring phosphinothricin (PPT)-resistance and green fluorescent protein (GFP) activity, respectively. The inoculated explants were selected on medium containing 5 or 3 mg PPT/l. The transformation efficiency of soybean was up to 12%. Polymerase chain reaction and genomic Southern blot analysis confirmed stable integration of the transgenes in the genome of the PPT-resistant plants. GFP analysis revealed that the transgenes were highly expressed in the plantlets. Adult plants were resistant to 100 mg PPT/l applied on the leaves, demonstrating their herbicide-resistance.An erratum to this article can be found at     

In vitro propagation of Vicia faba L. by micro-cutting and multiple shoot induction

The influences of nitrogen sources, culture temperature and activated charcoal supplements were studied in relation to the rooting ability of V. faba cuttings. The interaction of these factors led to quantitative and qualitative modifications of the culture responses. Low temperatures (14–18°C) were suitable for in vitro culture, limiting the formation of phenolics in plant material and making activated charcoal supplement unnecessary. Nitrogen supplements contributed in modifying the different plant responses, in accordance with temperature. Multiple shoot formation was obtained from the cotyledonary node and from the stem nodes cultivated in the presence of 6-benzylaminopurine (BAP). BAP at 4 mg l^-1 was the most effective concentration in promoting high rates of shoot development. The original position of stem nodes was found to determine the explant response to plant growth regulator treatments, possibly due to the effect of residual apical dominance.     

Developing a rapid and highly efficient cowpea regeneration,transformation and genome editing system using embryonic axis explants

Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium-mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium-mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas-mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.