Mitochondrial DNA polymorphism and phylogenetic relationships inHevea brasiliensis

Using fourteen random mitochondrial DNA probes, we have examined restriction fragment length polymorphism (RFLP) in wild and cultivatedHevea brasiliensis. A total of 395 accessions, including 345 from various prospectings collected in Brazil, Colombia and Peru and 50 cultivated clones, were analyzed. Two other species (H. benthamiana andH. pauciflora) were also included in the study for comparison. The high level of mitochondrial polymorphism allowed us to divide all the accessions analyzed into 212 distinct genotypes. The genetic variability of cultivated clones was limited to four genotypes forming two clusters. In contrast, considerable genetic variation was found in the wild collections. In almost all cases, accessions displaying the same RFLP profile were restricted to the same geographical area (same or neighbor administrative districts). In addition, accessions whose genetic closeness was predicted by RFLP profiles were also clustered according to geographical origin. In a few cases, however, similar RFLP profiles were found for accessions originating from geographically distant districts. This discrepancy can be explained either by seed dispersion (by river) or possibly by similar genetic events occurring independently in different geographical locations. Chloroplast DNA RFLP was also analyzed in 217 accessions, representative of 126 distinct mitochondrial genotypes. Very few differences were found, indicating that the chloroplast genome is more highly conserved than the mitochondrial genome.     

Effects of carbohydrate addition on the induction of somatic embryogenesis in Hevea brasiliensis

Plant Cell, Tissue and Organ Culture (PCTOC) - The effect of different carbohydrates was tested on early somatic embryogenesis of Hevea brasiliensis. Sucrose was replaced with maltose, fructose or...     

Ultrastructure of P-protein inHevea brasiliensis during sieve-tube development and after wounding

Summary P-protein and the changes it undergoes after wounding of sieve tubes of secondary phloem in one- to two-year old shoots ofHevea brasiliensis has been studied using electron microscopy. The P-protein in the form of tubules with a diameter of 8–9 nm and a lumen of 2–2.5 nm occurred in differentiating sieve elements and appeared as compact bodies which consisted of small aggregates of the tubules. As the sieve elements matured, these P-protein bodies dispersed with a disaggregation of the tubules before they turned into striated fibrils, 10–11 nm in diameter. In wounding experiments, as the mature sieve elements collapsed after cutting, their striated P-protein converted into tubules. These tubules were the same in ultrastructure as the tubules in differentiating sieve elements and they often were arranged in crystalline aggregates.     

Histology of somatic embryogenesis from floral tissues cocoa

Somatic embryogenesis fromTheobroma cacao L. flower buds, as previously reported on five Forastero hybrid genotypes, was tested on several other genotypes, belonging to the three cocoa-tree groups: Forastero, Trinitario and Criollo. The results gave evidence of genotypic efficiencies. Explants were cultivated under two successive conditions: callogenesis and expression media. The morphological and histological responses were different for embryogenic or non-embryogenic genotypes. For embryogenic genotypes, only staminodes and stamen filaments were able to produce somatic embryos: after a few days on the expression medium, groups of callus cells went through the meristematic and then embryonic stages, and finally formed somatic embryos. Many of them showed abnormalities. Simultaneously, some embryogenic cells were visible. These started to divide to form pro-embryos which however were unable to evolve into proper somatic embryos.     

Water status of callus from Hevea brasiliensis during induction of somatic embryogenesis

To improve somatic embryogenesis in Hevea brasiliensis , the water and plant growth regulator status of the culture medium was studied. Induction of embryogenic tissue from the internal integument of immature seeds was clearly favored by stabilizing the water potential of the culture medium at –0.7 MPa, by using low and decreasing concentrations of 3,4-dichlorophenoxyacetic acid and benzyladenine or by incorporating 10^-7 M abscisic acid in the medium. Each of these changes in the medium favored a specific water status in the callus, namely a high relative water content (93 to 95%) and an elevated water potential (–0.9 MPa). These characteristics were apparently important for initiating somatic embryogenesis, and their decrease corresponded to the loss of embryogenic potential in the callus. Thus, the relative water content and water potential of callus appear to be good markers of its embryogenic state.     

Histology of somatic embryogenesis in cultured leaf segments of sugarcane plantlets

Calli have been initiated from young leaves of in vitro grown sugarcane shoots. Histological examination has shown that the two types of calli induced (nodular and friable) originated from different regions of the explants and were cytologically different.This study has shown an obvious relation between the developmental stage of the excised tissue and the potential of plant regeneration of the in vitro initiated callus culture. Nodular calli were obtained from bases of the fast-growing young leaves while their more mature parts of the older leaves only produced friable calli. High-frequency plant regeneration via somatic embryogenesis was obtained from nodular calli while friable calli rarely produced plantlets.     

Callus culture of Coronilla varia L. (crownvetch): plant regeneration through somatic embryogenesis

Callus culture and plant regeneration through somatic embryogenesis have been obtained in Coronilla varia. Media used were UM (25) supplemented with 2 mg/l 2,4-D followed by subculture on MS (18) containing 1 mg/l 2-iP and 0.1 mg/l IAA. Embryoids developed into complete plantlets on filter paper saturated with hormone-free MS medium.     

Somatic embryogenesis of safflower: influence of auxin and ontogeny of somatic embryos

Influence of auxin type and concentration on somatic embryogenesis from cotyledonary explants of safflower was investigated. Embryogenic frequency as well as number of somatic embryos was dependent on auxin type and concentration. NAA at 10.74 M (2 mg l^–1) was optimum for high frequency of somatic embryos, whereas IAA provided the maximum number of somatic embryos per responding culture. Somatic embryo development was asynchronous and strongly affected by auxin type and concentration. Maximum numbers of well developed somatic embryos at the cotyledonary stage were obtained with 5.37 M (1 mg l^–1 ) NAA + 2.22 M (0.5 mgl ^–1) BA. Histological studies confirmed the unicellular origin of somatic embryos from the adaxial epidermis of the cotyledon. Broad base attachment of somatic embryos to the epidermis indicated the absence of a suspensor.     

Evidence of somatic embryogenesis from root tip explants of the rattan Calamus manan

Summary Somatic embryogenesis of Calamus manan, a single-stemmed rattan species, in tissue culture was scientifically demonstrated for the first time. Root tips of in vitro plantlets produced friable callus when the explants were cultivated for several mo. on a Murashige and Skoog induction medium containing 7.5 mg Picloram per l (31.1 μM). Histological analyses established the presence of proembryos within the callus which differentiated subsequently into somatic embryos using the same culture medium. Histological examination revealed that these somatic embryos completely lacked starch and protein reserves, which did not prevent them, however, from germinating, and showing bipolar development. These somatic embryos further developed into young plants, similarly to zygotic embryos.     

Effect of elevated CO2 concentration on seedling growth rate and photosynthesis inHevea brasiliensis

To study the effect of elevated CO₂ concentration on plant growth and photosynthesis, two clones ofHevea brasiliensis were grown in polybags and exposed to elevated concentration (700±25ppm) for 60 days. There was higher biomass accumulation, leaf area and better growth when compared to ambient air grown plantso From A/Ci curves it is clear that photosynthetic rates increases with increase in CO₂ concentrations. After 60 days of exposure to higher CO₂ concentration, a decrease in the carbon assimilation rate was noticed.     

Initiation of gametophytic callus and somatic embryogenesis in Laminaria japonica (Phaeophyta) exposed to mutagenic agents

Zoospores of Laminaria at the stage of zoospore germination fixed to glass slides were irradiated by γ-rays in doses of 50, 100, or 250 Gy; or treated with colchicine at a concentration of 4 × 10^?5% for 5 days. The cultivation was conducted in vessels with seawater at a temperature of 12°N and illumination of 4000 lux for one month. Once a day, from day 22 to day 30, the temperature was reduced to 0°N for 12 h. As a result, in experimental samples gametophytes appeared that did not form gametangia; these appeared by the third day of cultivation, as plaques up to 2 cm in diameter (1–2 plaques per slide). In the same culture we found structures (1–2 per slide) consisting of strictly radially arranged rows of somatic cells attached to the slides. Later, these disks transformed into cones up to 0.5 cm in diameter. We recorded the development of a single-layered sporophyte of Laminaria arising from the center of such a cone.     

Morphogenesis in callus tissue ofMedicago sativa: The role of ammonium ion in somatic embryogenesis

Exogenously supplied ammonium ion is critical to alfalfa morphogenesis in vitro. In alfalfa, the ability to induce the formation of either roots or somatic embryos provided an opportunity to examine the effects of ammonium ion on each pattern of morphogenesis. Somatic embryo formation required a minimum of 12.5 mM NH ₄ ⁺ in regeneration medium for optimal expression. Root formation was inhibited by NH ₄ ⁺ levels of 50 mM and above, and occurred in the absence of exogenous NH₄ ⁺. At high levels of NH ₄ ⁺ somatic embryos were formed from cells exposed to the root-inducing combination of hormones. This observation suggests that the growth regulators and exogenously supplied ammonium ion comprise an interactive system controlling the in vitro pattern of alfalfa morphogenesis.     

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.     

The analysis of differential gene expression in early somatic embryogenesis on Lycium barbarum

Direct exposure of calluses of Lycium barbarum L. to an auxin-free medium can induce somatic embryogenesis. Somatic embryogenesis of Lycium barbarum L. is controlled artificially by regulating 2,4-D concentration. The total RNA that was isolated from calluses, embryonic calluses and early somatic embryos was used for analyzing differential genes expression. We obtained three cDNAs from early somatic embryogenesis which were not found in calluses. The results indicate that these cDNAs were early embryogenesis-specific cDNAs and this gene expression was induced in cultured calluses after a transfer to an auxin- free medium. A cDNA library was constructed using poly(A)⁺-RNA derived from early somatic embryos of Lycium barbarism L. Two full-length cDNAs were isolated from the library by differential screening. Northern blot hybridization analysis indicated that the expression of the full-length cDNA only existed in embryogenic calluses and early somatic embryos of Lycium barbarum L. This revised version was published online in June 2006 with corrections to the Cover Date.     

In-vitro regeneration of olive tree by somatic embryogenesis

We induced somatic embryogenesis from the cotyledon segments ofOlea europaea (L) cvs. ‘Chetoui’, ‘Chemleli’, and ‘Arbequina’. Calli were established from all three cultvars on OMc media supplemented with IBA and 2i-R The greatest success was obtained with media that contained zero or low concentrations of growth regulators. High levels of hormones (i.e.,>0.5 mgL^-1 IBA and 2i-P) inhibited embryogenesis. Embryos at different maturation stages were observed with continuously proliferating secondary embryogenesis. Abnormally shaped embryos and teratoma were also noted. Four weeks was the optimal incubation period for inducing embryogenesis on the auxin-containing medium. In addition, 30 to 40 gL^-1 sucrose was more effective than glucose in stimulating the growth and maturation of somatic embryos. Embryogeic efficiency was also higher when multivariate combinations of nitrogen sources (inorganic and organic nitrogen forms) were used. The plantlets that were derived from our germinating somatic embryos were similar to those obtained from axillary buds.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Histology of embryogenic responses in soybean anther culture

In order to clarify the embryogenic responses in soybean anther culture, anthers of four cultivars were cultured under known conditions to trigger androgenic response. A histological study was performed with anthers in vivo and with approximately 100 explants sampled after 9, 12, 15, 18, 21, 30 and 45 days of culture. In vitro culture triggered the frequent accumulation of phenolic compounds on the locular and anther surfaces, and also caused the destruction of cells and tissues in complex structure such as the tapetum, microspores and pollen grains. Somatic embryogenesis of unicellular origin was observed from the epidermis and the middle layer, and of multicellular origin from connective calluses. No androgenic response could be observed in the anthers of these four soybean genotypes, in the medium and conditions indicated. We point out to the need of changing the approach to the study of androgenesis in soybean, either by using culture conditions unfavourable to the proliferation of diploid tissues, or by culturing isolated microspores.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of soil,plant and meteorological factors on water relations and yield inHevea brasiliensis

Influence of factors governing the soil-plantatmosphere system on components of water relations and yield was studied in two clones of rubber tree,Hevea brasiliensis, viz. RRII 105 and RRII 118. Clonal variations were evident in yield and yield components and associated physiological parameters in response to soil moisture status and meteorological factors. Observations made during different seasons indicatedvariations in yield are attributed to differences in plugging index and initial flow rates, to the major yield components and also variations in components of water relations as influenced by meteorological factors. Among the two clones, RRII 105 was found to be fairly drought tolerant compared to RRII 118. RRII 105 was found to respond well to dry weather through higher stomatal resistances, higher leaf water potentials, lowered transpirational water loss and lower relative transpiration ratios, while RRII 118 was susceptible to stress situations.