Assessing genetic identity of sporophytic offspring of the brown alga Undaria pinnatifida derived from mono-crossing of gametophyte clones by use of amplified fragment length polymorphism and microsatellite markers

The haploid stage of gametophytes of the subtidal brown alga Undaria pinnatifida can be vegetatively propagated under favorable conditions. This unique characteristic makes it possible to establish independent gametophyte cell lines that are zoospore-derived. Sporophytic offspring can be generated through hybridizing the male and female gametophytes, which are derived from different cell lines. Accumulated experiences in this and other species in Laminariales demonstrated the applicability of this novel way to breed desired strains for open-sea cultivation. Sporophytic offspring originated from mono-crossing of male and female gametophyte clones were shown to have similar morphological characteristics under identical ambient conditions. However, there has been no report to relate this similarity on molecular levels. In this report, amplified fragment length polymorphism (AFLP) and microsatellite markers were used to analyze the genetic identity of sporophytic offspring of U. pinnatifida originated from two mono-crossing lines (M1 and M2), two self-breeding lines (S1 and S2) and one wild population (W). Totally 318 AFLP loci were revealed by use of 11 primer sets, of which 4.7%, 0.3%, 17.9%, 16.4% and 36.5% were polymorphic in M1, M2, S1, S2 and W, respectively. The pairwise genetic identity among the individuals of the same line was assessed. It was shown that offspring from mono-crossing lines had a higher degree of identity (95.6-100%) than self-breeding lines (87.7-98.4%) and the wild population (81.5-92.1%). Analysis by use of six microsatellite loci also revealed a higher genetic identity among individuals of the mono-crossing line, further confirming the results of AFLP analysis. Results from this investigation support, on molecular levels, the novel way to produce and maintain strains in U. pinnatifida by use of different gametophyte cell lines.

Effect of light period on egg-discharge of gametophyte clones of Undaria pinnatifida (Phaeophyta)

A sporeling culture method using gametophyte clones developed in the early 1990s led to egg discharge occurring in the dark 5 min after the start of the dark period under growth under a 11:13 L-D photoperiod. The course of egg discharge could be disturbed by light, with irradiance as low as 5-6 mu mol photon m(-2) s(-1) causing 75-80% of the discharged eggs to detach from the oogonia and consequently to die within several hours. In order to enhance outgrowth rate of young sporophytes, a study was conducted to test the effect of controlling darkness in the period 2-3 h after dusk. When the slides were transferred from the standard 11:13 L-D regime to continuous light, eggs were discharged 5 min after the end of the light phase and peaked 5-l5 min later on first day after transfer, indicating that the female gametes "remember" the light-dark regime. This suggests the existence of an endogenous circadian rhythm. During the second and third days, very few eggs were discharged throughout the 11 h of the normal light phase of the L-D regime, indicating the inhibitory effect of continuous light and that the rhtyhm is easily damped by light.

Mass culture of Undaria gametophyte clones and their use in sporeling culture

Undaria pinnatifida (Harv.) Sur. is one of the three main seaweed species under commercial cultivation in China. In the mid-1990s the annual production was about 20000 tons dry. The supply of healthy sporelings is key to the success of commercial cultivation of Undaria. Previous studies demonstrated that instead of the zoospore collection method, sporelings can be cultured through the use of gametophyte clones. This paper reports the experimental results on mass culture of clones and sporeling raising in commercial scale. Light had an obvious effect on growth of gametophyte clones. Under an irradiance of 80 mumol m(-2) s(-1) and favorable temperature of 22-25degreesC, mean daily growth rate may reach as high as 37%. Several celled gametophyte fragments were sprayed onto the palm rope frame. Gametogenesis occurred after 4-6 days. Juvenile sporeling growth experiments showed that nitrate and phosphate concentrations of 2.9 10(-4) mol 1(-1) and 1.7 10(-5) mol 1(-1) were sufficient to enable the sporelings to maintain a high daily growth rate. Sporelings can reach a length of 1 cm in a month. Since 1997, extension of the clone technique has been carried out in Shandong Province. Large-scale production of sporelings for commercial cultivation of 14 and 31 hectares in 1997 and 1998 had been conducted successfully.

Expression of the lacZ reporter gene in sporophytes of the seaweed Laminaria japonica (Phaeophyceae) by gametophyte-targeted transformation

The seaweed Laminaria japonica (Phaeophyceae) has a two-generation life cycle consisting of haploid gametophytes and diploid sporophytes. Female and/or male gametophytes were transformed using particle bombardment and the histological LacZ assay was performed on sporophytes generated by either parthenogenesis or inbreeding. Female gametophyte-targeted transformation resulted in similar lower efficiencies in both parthenogenetic and zygotic sporophytes, and only a chimeric expression pattern was observed. Male gametophyte-targeted transformation led to a higher efficiency, with 3.5% of the zygotic sporophytes stained completely blue (all-blue), implying the integration of lacZ at the one-cell stage. Polymerase chain reaction analysis using primers specific for a lacZ-vector juncture fragment and subsequent blotting indicated the presence of the introduced gene in the sporophytes. The method reported here has a potential for seaweed transformation using spore-based bombardment followed by the developmental process.

Effects of cell density, light intensity and mixing on Undaria pinnatifida gametophyte activity in a photobioreactor

An on-line controlled 7 1 sterilizable photobioreactor was used for the optimisation of a culture of gametophytes of Undaria pinnatifida. The gametophytes, which had been stored for three years in a culture cabinet at 16 degreesC, could rapidly grow in the photobioreactor under controlled conditions. The rate of increase of dissolved oxygen and pH were used to monitor the photosynthetic activity. Optimal gametophytes density changed varying the light intensity. The optimal cell densities were 3.24 and 3.45 g FW l(-1) when the cultures were exposed to 61.7 and 82.3 muE m(-2) s(-1), respectively. The optimal cell density was higher under a high photon flux density (PFD) than under low PFD. On the other hand, the optimal light intensities were different for different cell density cultures. The light saturation point was higher at high cell density cultures than at low cell density cultures. The optimal rotational speed was 150 rpm for high cell density culture in the photobioreactor. (C) 2003 Elsevier B.V. All rights reserved.

A new method of Laminaria japonica strain selection and sporeling raising by the use of gametophyte clones

One-celled female and male gametophytes of three Laminaria japonica strains were isolated, cultured and gametophyte clones were formed. A technique combining strain selection with sporeling raising by the use of these female and male gametophyte clones was studied. Experiments on 9 different crossing combinations was conducted in November of 1997 in Qingdao, P. R. China. The main economic characteristics, frond length and fresh weight, of sporophytes of different crossing combinations were measured. F-1 sporophytes of No. 2 showed a higher fresh weight and longer length, therefore, No. 2 (Wh860 + x Lid) was selected as a good combination. Its parental female and male gametophyte clones are being mass cultured for sporeling production. By this method, the time needed for strain selection was shortened from 5-6 to 2 years. As compared with the routine method of sporeling raising by the collection of zoospores, the time of sporeling raising of this method decrease by 50%, and the production cost is also reduced by 50%. It is believed that this method will be labour and time saving and a more economic way for strain selection and sporeling raising in L. japonica cultivation industry.

Rapid optimization of process conditions for cultivation of transgenic Laminaria japonica gametophyte cells in a stirred-tank bioreactor

Batch cultivation for transgenic kelp gametophyte cells was investigated in an online controlled 5 L stirred-tank photo-bioreactor to rapidly optimize the process conditions by monitoring the rate of increase of pH. The transgenic kelp gametophytes with heterologous gene encoding hepatitis B surface antigen (HBsAg) could rapidly grow in the bioreactor. Optimal temperature and agitation rate for bioreactor cultivation of gametophytes were 15 degrees C and 200 rpm. Optimal incident light intensities depended on the initial cell densities. (c) 2006 Elsevier B.V. All fights reserved.

Somatic meiosis and development of the juvenile gametophyte in members of the Batrachospermales sensu lato (Rhodophyta)

Seven populations (six in culture and one sampled directly from nature) of the freshwater red algal families Batrachospermaceae, Lemaneaceae and Thoreaceae were examined, involving three species of Batrachospermum, two of Paralemanea and one of Thorea. All 'Chantransia' stages ultimately produced juvenile gametophytes. The production of juvenile gametophytes in the three populations of Batrachospermum was generally most abundant at 15°C and low irradiances (47-68 μmol photons m-2 s-1). The most abundant gametophyte development in the Paralemanea species was observed at 10°C and low or high irradiances (47-142 μmol photons m-2 s-1). Gametophyte production in Thoreaceae occurred at higher temperatures (20°C) and also at low irradiances. In species of the Batrachospermaceae and Lemaneaceae, the 'elimination cells' can be situated on the basal or suprabasal cell of the juvenile gametophyte, but the position is usually fixed in individual species. The presence and position of the elimination cells remain to be established in Thoreaceae. Our results corroborate a previous study suggesting that the position of elimination cells is such a constant feature that it is of potential diagnostic value at the generic or infrageneric (sectional or specific) level. The characteristics observed in the development of the juvenile gametophytes in species of Batrachospermaceae and Lemaneaceae essentially agreed with general descriptions in the previous studies. The characteristics of the Thoreaceae, with a distinctive developmental pattern of the juvenile gametophyte and the occurrence of two morphological types in the 'Chantransia' stage, support the proposal to elevate it to the ordinal level. Two remarkable observations in Batrachospermum species were the production of numerous juvenile gametophytes from filaments of the same plant of the 'Chantransia' stage and the formation of a system of rhizoidal filaments or cell agglomeration of the juvenile gametophytes, which produced new gametophytes. These two characteristics potentially increase the formation of additional gametophytes under favourable conditions.

Ecology and morphological characterization of gametophyte and 'Chantransia' stages of Sirodotia huillensis (Batrachospermales, Rhodophyta) from a stream in central Mexico

The morphology and phenology of Sirodotia huillensis was evaluated seasonally in a central Mexican first-order calcareous stream. Water temperature was constant (24-25°C) and pH circumneutral to alkaline (6.7-7.9), and calcium and sulfates were the dominant ions. The gametophyte stages were characterized by the presence of a distinctive mucilaginous layer, a marked difference in phycocyanin to phycoerythrin ratio between female and male plants, and the presence of a carpogonia with a large trichogyne (>60 μm). Occasionally three capogonia were observed on a single basal cell. The 'Chantransia' stages were morphologically similar to those described for the other members of Batrachospermales. A remarkable observation was the formation of dome-shaped structures, consisting of prostrate filaments that are related with the development of new gametophytes. Chromosome numbers were n = 4 for fascicle cells, cortical filament cells and dome-shaped cells, and 2n = 8 for gonimoblast filament cells and 'Chantransia' stage filaments. Gametophytes and 'Chantransia' stages occurred in fast current velocities (60-170 cm/s) and shaded (33.1-121 μmol photons/m2/s) stream segments. The population fluctuated throughout the study period in terms of percentage cover and frequency: the 'Chantransia' stages were most abundant in the rainy season, whereas gametophytic plants had the highest frequency values during the dry season. These results were most likely a result of fluctuations in rainfall and related changes in current velocity. Some characteristics of this population can be viewed as probable adaptations to high current velocities: the mucilaginous layer around plants that reduces drag; potential increase in fertilization by the elongate and plentiful trichogynes and abundant dome-shaped structures producing several gametophytes.

KINESIN MOTOR PROTEINS ARE ESSENTIAL FOR MALE GAMETOPHYTE DEVELOPMENT IN MARSILEA VESTITA

The male gametophyte of the semi-aquatic fern, Marsilea vestita, produces multiciliated spermatozoids in a rapid developmental sequence that is controlled post-transcriptionally when dry microspores are placed in water. Development can be divided into two phases, mitosis and differentiation. During the mitotic phase, a series of nine successive division cycles produce 7 sterile cells and 32 spermatids in 4.5-5 hours. During the next 5-6 hours, each spermatid differentiates into a corkscrew-shaped motile spermatozoid with ~140 cilia. This document focuses on the role of motor proteins in the regulation of male gametophyte development and during ciliogenesis. In order to study the mechanisms that regulate spermatogenesis, RNAseq was used to generate a reference transcriptome that allowed us to assess the abundance of transcripts at different stages of development. Over 120 kinesin-like sequences were identified in the transcriptome that represent 56 unique kinesin transcripts. Members of the kinesin-2, -4, -5, -7, -8, -9, -12, -13, and -14 families, in addition to several plant specific and ‘orphan’ kinesins are present. Most (91%) of these kinesin transcripts change in abundance throughout gametophyte development, with 52% of kinesin mRNAs enriched during the mitotic phase and 39% enriched during differentiation. Functional analyses show that the temporal regulation of kinesin transcripts during gametogenesis directly correlates with kinesin protein function. Specifically, Marsilea makes one kinesin-2 (MvKinesin-2) and two kinesin-9 (MvKinesin-9A and MvKinesin-9B) transcripts, which are present during spermatid differentiation and ciliogenesis. Silencing experiments showed that MvKinesin-2 and MvKinesin-9A are required for ciliogenesis and motility in the Marsilea male gametophyte; however, these kinesins display atypical roles during these processes. In contrast, spermatozoids produced after the silencing of MvKinesin-9B exhibit normal morphology. MvKinesin-2 is necessary for cytokinesis as well as for regulating ciliary length and MvKinesin-9A is needed for the correct orientation of basal bodies, events not typically associated with these proteins. In addition, Marsilea makes motile, ciliated gametophytes without the help of IFT dynein, outer arm dynein, or the BBsome. These results are the first to investigate the kinesin-linked mechanisms that regulate ciliogenesis in a land plant.

An egg apparatus-specific enhancer of Arabidopsis, identified by enhancer detection

Despite a central role in angiosperm reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer-detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis (Arabidopsis thaliana). The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5' and 3' deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp. Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This EASE works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE. The potential application of the EASE element and similar control elements as part of an open-source biotechnology toolkit for apomixis is discussed.