Sex and ploidy of anther culture derived papaya (Carica papaya L.) plants

Summary To improve the efficiency of papaya anther culture, we investigated (1) hormonal medium conditions for inducing haploids or dihaploids; (2) identified the sex of established plantlets using a sex-specific DNA molecular marker and (3) estimated their ploidy by flow cytometry analysis of DNA content. Anthers with a mixture of uninucleate, mitotic, and binucleate microspores were collected from a male plant, and cultured on MS agar medium with different concentrations of CPPU and NAA. An embryo induction rate of 13.8% was attained on MS agar medium with 0.01 mg l⁻¹ CPPU and 0.1 mg l⁻¹ NAA. The induced embryos were subcultured on medium with 0.0025 mg l⁻¹ CPPU. Rooting of the developed shoots was promoted by treating their basal parts with 1500 mg l⁻¹ IBA in a 50% ethanol solution for about 10 seconds. All the embryo-derived plantlets (27 plants) were identified as female, implying that they were derived from microspores. In addition, 26 plants were determined to be triploids and one to be tetraploids. We also observed a wide range of morphological variation (e.g., in tree height and fruit size) among the established plants. Based on the results, we discussed a potential value of anther culture techniques for the breeding of papaya.     

Relatedness does not affect the mating effort of Eisenia fetida Sav. (Oligochaeta) despite evidence for outbreeding depression

Animals that can distinguish kin from non-kin may enhance their breeding success through "optimal outbreeding". Although relatedness can influence the breeding success of earthworms, no studies of kin discrimination have been conducted in any Oligochaeta species. We determined whether Eisenia fetida Sav. would discriminate kin and change breeding effort with them. The number of cocoons produced (fecundity), viable cocoons, and hatchlings produced by full sibling (inbreeding) and non-sibling (outbreeding) pairs, from different populations, were compared; in this species, fecundity is a reliable indicator of the occurrence and frequency of mating. There were no significant differences in fecundity between inbreeding and outbreeding pairs, so we obtained no evidence for differential investment based on kinship. However, sibling pairs hatched a significantly greater proportion of their cocoons than non-sibling pairs, suggesting that outbreeding depression may reduce reproductive success. We also provide the first evidence for the possibility of parthenogenesis in this species.     

Structural characterization and biological activities of an exopolysaccharide kefiran produced by Lactobacillus kefiranofaciens WT-2B(T)

Lactobacillus kefiranofaciens, isolated from kefir grains, produces an extracellular polysaccharide when cultured, not only in PYG10 medium but also in a liquid medium containing a rice hydrolysate that had been previously degraded by treatment with a glucoamylase. The maximum yield of the polysaccharide, using the rice hydrolysate as the medium, was 2.5 g/L after a 7-day culture period at pH 5.0 and 33 degrees C. Compositional analysis, methylation analysis, specific rotation, and (1)H and (13)C NMR spectroscopy revealed that the structures of polysaccharides obtained from these two different culture media are essentially identical. The polysaccharide is composed of a hexasaccharide repeating unit and, thus, is known as kefiran. The weight-average molecular weight and the z-average radius of gyration of a sample, purified from the rice hydrolysate medium, were determined to be 7.6 x 10(5) g/mol and 39.9 nm, respectively, by gel permeation chromatography equipped with a multiangle laser-light-scattering photometer. Changes in blood pressure and serum components were examined in SHRSP/Hos rats, using doses of 100 and 300 mg of kefiran/kg of rat. A suppression in the increase in blood pressure was observed in these rats after 30 days. This activity is discussed in terms of the concentration of serum components of the rat, with emphasis on lipid components such as cholesterols, triglycerides, and free fatty acids.     

Accumulation of Zinc and Copper in an Arable Field after Animal Manure Application

An experiment was conducted to examine the accumulation and mobility of heavy metals (Zn and Cu) at different depths in three types of arable soils (Brown Lowland soil, Andosol, and Brown Forest soil) amended with cattle and pig farmyard manures for 5 years. Nitric-perchloric acid digestion was performed for the determination of the total amounts of heavy metals, and 0.1 M hydrochloric acid extraction was performed for the determination of the amounts of soluble heavy metals. Results of the soil analysis indicated that pig farmyard manure application resulted in serious contamination of arable soils with Zn and potentially Cu. Especially, the Brown Forest soil displayed a high ability to accumulate heavy metals on the soil surface. Total-Zn concentration in surface soils was considerably affected by the holding capacity of soluble-Zn traction. Although the Andosol amended with pig farmyard manure showed higher concentrations of heavy metals related to the higher ability of retention on a weight basis, the soil did not contribute to high heavy metal accumulation because of its low bulk density. Heavy metals were easily leached in sandy soils such as Brown Lowland soil, and Cu was potentially stable compared with Zn. We suggest that long-term pig farmyard manure application to the Brown Lowland soil and Andosol with a light soil texture is associated with a higher risk of groundwater pollution than the application to the Brown Forest soil.     

Procedure for bovine ICSI, not sperm freeze-drying, impairs the function of the microtubule-organizing center

This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa.     

Challenging endeavour for preservation of freeze-dried mammalian spermatozoa

Freeze-drying (lyophilization) has been proposed as an alternative method for sperm preservation to overcome the disadvantages of the current cryopreservation method such as the high maintenance cost of frozen stocks, the problems associated with transportation of frozen materials and the potential risk of total loss of the frozen stock. Since freeze-dried spermatozoa after rehydration lose their motility, which is an essential requirement to complete physiological fertilization, a relatively difficult microinsemination technique must be applied to rehydrated spermatozoa. Theoretically, it has been supposed that freeze-dried spermatozoa could maintain their functions and abilities to interact with the oocyte cytoplasm after prolonged storage at refrigerator temperature. However, sufficient yield of transferable blastocysts and production of live offspring derived from freeze-dried sperm samples are still subjects to be challenged and overcome in large domestic species.     

Distribution of the Water-Soluble Astaxanthin Binding Carotenoprotein (AstaP) in Scenedesmaceae

Photooxidative stress-inducible water-soluble astaxanthin-binding proteins, designated as AstaP, were identified in two Scenedesmaceae strains, Coelastrella astaxanthina Ki-4 and Scenedesmus obtusus Oki-4N; both strains were isolated under high light conditions. These AstaPs are classified as a novel family of carotenoprotein and are useful for providing valuable astaxanthin in water-soluble form; however, the distribution of AstaP orthologs in other microalgae remains unknown. Here, we examined the distribution of AstaP orthologs in the family Scenedesmaceae with two model microalgae, Chlamydomonas reinhardtii and Chlorella variabilis. The expression of AstaP orthologs under photooxidative stress conditions was detected in cell extracts of Scenedesmaceae strains, but not in model algal strains. Aqueous orange proteins produced by Scenedesmaceae strains were shown to bind astaxanthin. The protein from Scenedesmus costatus SAG 46.88 was purified. It was named ScosAstaP and found to bind astaxanthin. The deduced amino acid sequence from a gene encoding ScosAstaP showed 62% identity to Ki-4 AstaP. The expression of the genes encoding AstaP orthologs was shown to be inducible under photooxidative stress conditions; however, the production amounts of AstaP orthologs were estimated to be approximately 5 to 10 times lower than that of Ki-4 and Oki-4N.     

Carotenoids in algae: distributions, biosyntheses and functions

For photosynthesis, phototrophic organisms necessarily synthesize not only chlorophylls but also carotenoids. Many kinds of carotenoids are found in algae and, recently, taxonomic studies of algae have been developed. In this review, the relationship between the distribution of carotenoids and the phylogeny of oxygenic phototrophs in sea and fresh water, including cyanobacteria, red algae, brown algae and green algae, is summarized. These phototrophs contain division- or class-specific carotenoids, such as fucoxanthin, peridinin and siphonaxanthin. The distribution of α-carotene and its derivatives, such as lutein, loroxanthin and siphonaxanthin, are limited to divisions of Rhodophyta (macrophytic type), Cryptophyta, Euglenophyta, Chlorarachniophyta and Chlorophyta. In addition, carotenogenesis pathways are discussed based on the chemical structures of carotenoids and known characteristics of carotenogenesis enzymes in other organisms; genes and enzymes for carotenogenesis in algae are not yet known. Most carotenoids bind to membrane-bound pigment-protein complexes, such as reaction center, light-harvesting and cytochrome b(6)f complexes. Water-soluble peridinin-chlorophyll a-protein (PCP) and orange carotenoid protein (OCP) are also established. Some functions of carotenoids in photosynthesis are also briefly summarized.