Epigenetic programming of phenotypic variations in reproductive strategies in the rat through maternal care.

Studies across multiple organisms reveal considerable phenotypic variation in reproductive tactics. In some species, this variation is associated with maternal effects in which variation in maternal investment results in stable individual differences in reproductive function. Recent studies with the rat suggest that maternal effects can alter the function of neuroendocrine systems associated with female sexual behaviour as well as maternal behaviour. These maternal effects appear to be mediated by epigenetic modifications at the promoter for oestrogen receptor alpha (ERalpha) and subsequent effects on gene expression. The tissue-specific nature of such effects may underlie the co-ordinated variation in multiple forms of reproductive function, resulting in distinct reproductive strategies.

Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts.

A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0-5.0×106 were obtained following 4-6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 105 protoplasts/ml was optimal for subsequent division. An initial division frequency of 12-15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 µM), and zeatin (2.3 µM). Colonies consisting of 100-200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1-2 mm in diameter) were formed after an additional 4-6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 µM) and benzylaminopurine (2.2 µM). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 µM gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation.

Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts.

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 x 10(6) electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 x 10(-4). More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.

Regeneration of fertile plants from protoplasts of soybean (Glycine max L. Merr.): genotypic differences in culture response.

Fourteen soybean (Glycine max L.) genotypes were evaluated for their regenerability from protoplasts using a procedure previously descibed for the cultivar Clark 63. Protoplasts were isolated from immature cotyledon tissue and were cultured in liquid or agarose gelled KP8, MS or B5 medium with different sugars. Significant differences were observed in plating efficiency, which was as high as 63% in Jack and A-2396, and as low as 38% in X-3337. Upon regular dilution with K8 medium, 1-2 mm diameter colonies were formed in 5-6 weeks with all the genotypes tested. These colonies were then transferred onto MSB (MS salts; Murashige & Skoog, 1962 + B5 organics; Gamborg et al., 1968) medium with 0.5 mg l(-1) each of 2,4-D, BA and KN and 500 mg l(-1) CH for further growth. Once the colonies had become green, compact and nodular, and were 8-10 mm in size, they were transferred to regeneration medium. Upon regular subculturing, calli of six genotypes; A-2396, Chamberlain, Heilong-26, Jack, Resnick and XP-3015 developed shoots, with the regeneration frequency being highest 27% in Jack (52 calli out of 192 produced 8-12 shoots). The regenerated shoots from different genotypes were elongated and rooted. So far, sixty three complete plants have been obtained, including twelve A-2396, nineteen Chamberlain, fifteen Jack, nine Resnick and eight XP-3015. A total of thirty five plants have been transplanted into pots in the greenhouse. Sixteen have set seeds and others are producing flowers and pods.

Regeneration of transformed shoots from electroporated soybean (Glycine max (L.) Merr.) protoplasts.

Stable transformation of soybean (Glycine max (L.) Merr.) protoplasts isolated from immature cotyledons was achieved following electroporation with plasmid DNA carrying chimeric genes encoding ß-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transformed colonies were stringently selected by growing 15-day-old protoplast-derived cells in the presence of 40 µg/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid were co-introduced and co-expressed at a very high freguency. This transformation procedure reproducibly yields transformants at frequencies of 2.9-6.8 × 10(-4) (based on the number of protoplasts electroporated) or 23.0% (based on the number of control microcalli formed) counted after 6 weeks of selection. After repeated subculturing on regeneration medium, shoots were induced from 8.0% of the transformed calli. Southern hybridization confirmed the presence of both the GUS and hygromycin genes in the transformed calli and shoots.

Factors affecting transient gene expression in electroporated Glycine max protoplasts.

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 µF and a protoplast concentration of 1-3 × 10(6)/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.

Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.).

Protoplasts were isolated from immature cotyledons of Glycine max L. Merr. cv. Clark 63 and cultured in liquid or in agarose-gelled modified KP8 medium. Plating efficiencies of 45-50% were obtained in liquid medium and 55-60% in 1.2% (w/v) agarose beads. Upon regular dilution with K8 medium rapidly growing green microcalli (1-2 mm in size) were obtained in 5-6 weeks, which upon transfer to MSB medium with 0.5 mg 1(-1) each of 2,4-D, BA, Kn and 500 mg 1(-1) CH produced compact green calli in 4-6 weeks. After 3-4 regular subcultures of 14 days each on MSB medium containing 0.5 mg 1(-1) each of BA, Kn, ZT, 0.1 mg 1(-1) NAA and 500 mg 1(-1) CH, about 21% of the compact calli formed multiple shoots. Addition of glutamine, asparagine and GA3 enhanced shoot regeneration up to 30%. Shoots of 0.5-1.0 cm length were transferred to 1/2 MS medium with 0.01 mg 1(-1) TH and 0.5 mg 1(-1) GA3 for elongation. In 2 to 3 weeks, approximately 60% of the shoots were 2-3 cm in length. These shoots were rooted on 1/2 MS with 1% sucrose and 0.2 mg 1(-1) IBA or 0.5 mg 1(-1) NAA. So far, twenty six plants have been transferred to the greenhouse, where they all have set seed.

Development of laticifer cells in callus cultures of Calotropis procera (Ait.) R. Br.

Tissue cultures were established from stem explants of Calotropis procera, a hydrocarbon yielding desert shrub on Murashige and Skoog's medium supplemented with 1.5 mg. 1(-01) 2,4-D + 0.5 mg.1(-1) kinetin and polyvinylpyrrolidone. Laticifer cells were not present in young callus but were observed after 4 weeks of callus growth when examined histochemically. These young laticifers were detected in the 5th week of culture and were distinguished from surrounding cells by the presence of characteristic cytoplasm and thin walls. A group of cells with extensive branching was developed after 8 weeks of growth of the callus cultures. These cells were thick walled and contained latex particles in coagulated masses. Positive Liebermann-Burchard test proved the presence of terpenoids in these laticifers.