John F. Seelye

Sugar Regulation of Harvest-Related Genes in Asparagus

The signals controlling the abundance of transcripts up-regulated (pTIP27, pTIP31, and pTIP32) or down-regulated (pTIP20 and pTIP21) after harvest in asparagus (Asparagus officinalis L.) spears were examined. pTIP27 and pTIP31 are known to encode asparagine synthetase (AS) and a [beta]-galactosidase ([beta]-gal) homolog, respectively. The nucleotide sequences of pTIP20, pTIP21, and pTIP32 were determined, and they encode histone 3, histone 2B, and an unknown product, respectively. Changes in respiration, soluble sugars, and abundance of the five mRNAs were similar in the tips stored as 30-mm lengths or as part of 180-mm spears. We previously hypothesized that sugars may regulate the level of AS transcripts in asparagus tissue. Asparagus cell cultures were used to test the role of sugar status in regulating gene expression. Transcript abundance for AS, [beta]-gal, and pTIP32 was low in cells in sugar-containing medium but increased within 12 h after transferring cells to a sugar-free medium. Histone 3 and histone 2B transcripts were, in general, abundant in cells on sugar-containing medium but declined in abundance when transferred to sugar-free medium. When cells were returned to sugar-containing medium the abundance of transcripts for histone 3 and histone 2B increased, whereas that for AS, [beta]-gal, and pTIP32 decreased. Soluble sugar levels are known to decline rapidly in the tips of harvested spears. Metabolic regulation by sugar status may have a major influence on gene expression in asparagus spears and other tissues after harvest.

Opportunities for synthetic plant chimeral breeding: Past and future

Many plant periclinal chimeras are selected by horticulturalists due to their distinctive, valuable phenotypes, and because they are relatively stable. Most of these have arisen by induced or spontaneous mutation. Interspecific chimeras have been accidentally produced from graft unions of plants from a wide range of families. Early last century Winkler developed a technique to produce interspecific chimeras from graft unions (graft chimeras). More recently in vitro techniques have been developed to synthesize interspecific and intervarietal chimeras. However, these techniques have only been successful for species in the families Solanaceae and Cruciferae, and rarely assessed on plants in other families. Research is required to improve these techniques or develop new approaches so that the efficiency of chimeral shoot production is improved and the techniques are applicable to plants in a wide range of families. The unique characteristics of interspecific or intervarietal chimeras show the potential of chimeral breeding to produce new cultivars. If chimeral breeding techniques were improved, they could become a standard breeding approach for some horticultural crops.

Interspecific hybridisation betweenLimonium perigrinum Bergius andLimonium purpuratum L.

SummaryInterspecific hybrids betweenLimonium perigrinum andL. purpuratum were obtained usingL. perigrinum as the female parent. No hybrids were produced by the reciprocal cross. Twelve- to 15-day-old embryos were rescued and cultured within their embryo sacs on modified B5 or KM medium. After two to three days the embryos were excised from their embryo sacs and re-plated on to fresh medium. When the embryo-derived plantlets had attained a length of 1 cm they were transferred to a modified MS medium containing BA and NAA for shoot proliferation. Plantlets were transferred to modified MS medium supplemented with IBA for 24 hours for root initiation then to a modified growth-regulator-free MS medium for root growth. After a further 28 days the plantlets were transferred to soil-less medium for acclimatisation. The hybrid characteristics of one of the 15 embryo-derived plants were determined by flow cytometry and by examination of morphological features. The mean DNA contents of 2C nuclei fromL. perigrinum, the hybrid andL. purpuratum were 13.98 pg, 16.81 pg and 19.37 pg, respectively. Mitotic and meiotic chromosome counts fromL. perigrinum andL. purpuratum showed that both parents and their hybrids had identical chromosome numbers (2n=24), and that the species were closely related. Morphological analyses of leaves and flowers showed that the hybrids displayed a number of features intermediate between both parents.

Hairy roots of Brassica napus: II. Glutamine synthetase overexpression alters ammonia assimilation and the response to phosphinothricin

SummaryHairy roots of Brassica napus (rape cv. Giant) have been produced that contain the cytosolic glutamine synthetase (GS) gene from Glycine max (soybean). Leaf explants were cocultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pLN16. This vector was constructed by inserting a soybean cytosolic GS cDNA into the multiple cloning site of pGA643, placing it under the control of the CaMV promoter. In addition, the T-DNA region of pLN16 contained a NPTII gene for selection of transformed cells. Transgenic hairy roots grew prolifically on hormone-free media containing a selective level of kanamycin. Southern and northern analyses confirmed the presence of soybean GS DNA and transcripts, respectively. These transformed hairy roots also have a greater abundance of the GS polypeptide, approximately 3–6 fold greater GS activity and lower levels of endogenous ammonia. Hairy roots provide a useful system for studying responses to phosphinothricin (PPT). Hairy roots grown in media containing PPT had lower GS activity, greater ammonia accumulation and slower growth than controls. The presence of the soybean GS gene in the hairy roots reduced these PPT-induced effects and resulted in higher GS activity, lower ammonia levels and faster growth than in PPT-treated controls. Greater tolerance of PPT was also seen in shoots regenerated from the hairy roots displaying elevated levels of GS activity.

Wide crosses in the Colchicaceae: Sandersonia aurantiaca (Hook.) × Littonia modesta (Hook.)

Intergeneric hybrids were obtained between Sandersonia aurantiaca and Littonia modesta using ovule culture. The embryos were rescued by culturing 14 to 30 day old ovules. The ovules were cultured on modified KM medium for varying lengths of time until they germinated. After germination the embryo-derived-plantlets were transferred to modified growth regulator-free MS medium on which they developed tubers and became quiescent. The quiescent tubers could be successfully transferred to soil. The hybrid nature of both the putative Sandersonia × Littonia and the Littonia × Sandersonia hybrids was indicated by flow cytometry that showed the hybrid plants had a DNA content midway between that of the two parents. Mitotic and meiotic chromosome counts from S. aurantiaca, L. modesta and the hybrids gave chromosome numbers of (2n=) 24, 22 and 23 respectively. Morphological analyses of the leaves and flowers showed that the hybrids displayed features that were intermediate between both parents. Hybrids were male and female sterile. No morphological differences were observed between the two hybrids.

Shoot regeneration from leaf discs of Limonium perigrinum using thidiazuron

Abstract Shoots were regenerated from 12‐mm diameter leaf discs derived from greenhouse plants of Limonium perigrinum (Bergius), with either zeatin or thidiazuron (TDZ), the latter producing five times the number of shoots. After 8 weeks exposure to a modified Murashige and Skoog basal medium (BM) supplemented with 3.0 mg/litre TDZ, an average of 13 shoots/disc formed, 90% of which were ≤ 1 mm long. There was no significant difference in the total number of shoots formed at 1.0–5.0 mg/litre TDZ, however at lower rates (1.0–2.0 mg/litre) a greater proportion of longer shoots (> 1 mm) developed. Shoots regenerated after 8 weeks from leaf discs exposed to BM with TDZ for from 2 h to 6 weeks before being transferred to growth‐regulator‐free medium. A mean of 4.8 shoots/disc formed from the shortest TDZ pulse period, with the majority being longer than 1 mm. Serial sections of discs pulsed with TDZ medium showed many suppressed shoot initials within the new tissue originating from the cut edge of the discs.

Bushiness and cytokinin sensitivity in micropropagated Zantedeschia

Bushiness, the development of short, multiple stems, has been reported in some commercial hybrids of Zantedeschia, particularly the yellow-flowered cultivar Florex Gold derived from tissue culture. This cultivar exhibited a greater sensitivity to 6-benzylaminopurine when compared to four other cultivars using an in vitro root length bioassay, suggesting it may be pre-disposed to bushiness. The carry-over effect of cytokinin from micropropagation on subsequent tuber formation and flower development, in three selections of the cultivar Florex Gold, was also investigated. Cytokinin levels used in commercial Zantedeschia micropropagation protocols did not alter tuber or flower development. However, excessively high levels did affect development, also indicating that cytokinin may influence bushiness. Multi-eyed tubers used to initiate in vitro cultures were shown to have a significant effect on first year tuber formation and subsequent flower weight, compared to few-eyed tubers, although these plants did not necessarily develop bushy symptoms. Plant location within the greenhouse also influenced flowering although these plants did not display multiple stemmed bushy symptoms.

Glutamine Synthetase Activity, Ammonium Accumulation and Growth of Callus Cultures of Asparagus officinalis L. Exposed to High Ammonium or Phosphinothricin

Summary Glutamine synthetase (GS) activity, ammonium accumulation and growth responses of callus cultures of Asparagus officinalis L. were investigated following 4 weeks exposure to media with added ammonium, and again after a further 4 weeks on a modified basal medium (MBM) containing no added ammonium. Calli grown on MBM supplemented with 40 or 160 mM ammonium for 4 weeks had reduced GS activity, greatly enhanced ammonium content and reduced growth compared with calli exposed to 10 mM added ammonium. When calli were transferred back to MBM, GS activity increased, ammonium content decreased, and growth was enhanced. Phosphinothricin (PPT) was used to endogenously alter the ammonium content of callus tissue. Exposing calli to 10 or 100 μM PPT for 4 weeks reduced GS activity, enhanced ammonium accumulation and reduced growth compared with calli not exposed to PPT, and markedly enhanced callus glutamine content. Calli exposed to 100 pM PPT did not regrow when transferred back to basal media (BM) without added PPT for an additional 4 weeks. We also separated the effect of PPT-induced ammonium accumulation from alterations in tissue amino acid concentrations by adding up to 25 mM glutamine in addition to PPT during the initial 4-week treatment period. Glutamine supplementation overcame the PPT-induced reduction in growth even though GS activity was severely reduced and ammonium accumulated to high concentrations in calli exposed to 100 μM PPT. All calli continued to grow vigorously when transferred back to BM for an additional 4 weeks. The results demonstrate that ammonium accumulation per se was not lethal to asparagus callus tissue, and suggest other effects of using PPT as a selective inhibitor of GS.

Bushiness and cytokinin profile in dormant and sprouting tubers of Zantedeschia

The commercial Zantedeschia industry faces a potentially serious problem with the cultivar Florex Gold periodically producing weak, poor performing “bushy” plants in their second growing season. As these plants are derived from tissue culture, it had been suggested that the use of cytokinin during the tissue culture process might be a causative factor. Endogenous cytokinin concentration and profile in dormant and sprouting Zantedeschia tuber eyes (buds) of the cultivars Florex Gold (prone to bushiness) and Best Gold (not prone to bushiness) were determined by high-performance liquid chromatography and radioimmunoassay. During sprouting the total cytokinin concentration in Florex Gold tuber eyes rose tenfold, with a 40-fold increase in the nucleotides. No role for endogenous cytokinin concentration or profile in the expression of the bushy syndrome was established, as there were no measurable differences between bushy and control Florex Gold samples, whether dormant or sprouting. However, sprouting tuber eyes from the cultivar Florex Gold had nearly twofold more cytokinin than sprouting eyes from the cultivar Best Gold, which may in some way predispose Florex Gold to bushiness.

Inhibition of hexokinase and expression of asparagine synthetase and p‐galactosidase genes during sugar feeding and starvation of asparagus (Asparagus officinalis) callus cultures

Abstract Hexokinase has an important role in hexose metabolism and in signalling sugar status in plants. The aim of this study was to inhibit hexose phosphorylation using a hexokinase inhibitor (glucosamine), and to determine the effects on expression of asparagine synthetase (AS) and (3‐galactosidase during glucose feeding and starvation of asparagus (Asparagus officinalis L.) callus cultures. After 48 h without glucose and a further 24‐h incubation with a range of hexoses and analogues, expression of both AS and β‐galactosidase was repressed by d‐glucose, d‐galactose, and 2‐deoxyglucose, but the genes were not repressed when glucose was absent, or when 3‐O‐methylglucose or L‐glucose were supplied. Glucose‐and fructose‐phosphorylating activity was determined in extracts from callus cultures which had been exposed to glucose, 2‐deoxyglucose, 2‐deoxyglucose with glucosamine or mannitol (as an osmotic control), or glucose‐free media. After 48 h on glucose‐free media and 48‐h incubation with 2‐deoxyglucose and glucosamine, glucose‐ and fructose‐phosphorylating activities were reduced by 68 and 83%, respectively. When glucose was present in the cultures, there was no expression of AS transcripts, but when glucose was absent, AS was highly expressed. AS expression was reduced when 2‐deoxyglucose was present for 48 h, even when glucosamine or mannitol was also present in the culture media. P‐galactosidase was highly expressed when glucose was absent, but expression was very low in all of the treatments which contained 2‐deoxyglucose (including the glucosamine and mannitol treatments). The results suggest AS and P‐galactosidase are sugar regulated, but inconsistencies, particularly reduced AS expression in the presence of glucosamine, are discussed in relation to the possibilities that multiple forms of hexokinases exist which might be differentially affected by glucosamine, and that uptake and distribution of 2‐deoxyglucose and glucosamine are limited.