Structural conservation of WEE1 and its role in cell cycle regulation in plants.

The WEE1 kinase is ubiquitous in plant development and negatively regulates the cell cycle through phosphorylations. However, analogies with the control of the human cell cycle by tyrosine- (Tyr-) phosphorylation of cyclin-dependent kinases (CDKs) are sometimes questioned. In this in silico study, we assessed the structural conservation of the WEE1 protein in the plant kingdom with a particular focus on agronomically valuable plants, the legume crops. We analyzed the phylogenetic distribution of amino-acid sequences among a large number of plants by Bayesian analysis that highlighted the general conservation of WEE1 proteins. A detailed sequence analysis confirmed the catalytic potential of WEE1 proteins in plants. However, some substitutions of an arginine and a glutamate at the entrance of the catalytic pocket, illustrated by 3D structure predictions, challenged the specificity of this protein toward the substrate and Tyr-phosphorylation compared to the human WEE1. The structural differences, which could be responsible for the loss of specificity between human and plants, are highlighted and suggest the involvement of plant WEE1 in more cell regulation processes.

Flow cytometry enables identification of sporophytic eliciting stress treatments in gametic cells.

Flow cytometry was used to quantify the effect of individual and combined stress treatments on elicitation of androgenesis by analyzing the relative nuclear DNA content of in vitro cultured microspores of Pisum sativum L. Differences in relative nuclear DNA content of microspores within anthers after stress treatments were clearly evident from the flow cytometry profiles, and permitted us to predict whether a combination of stresses were elicitors or enhancers of androgenesis. This is the first report to assess the effect of various stress treatments in a plant species based on relative nuclear DNA content and to use this information to categorize them as 'elicitors' or 'enhancers'. Flow cytometry represents a simple, quick and reliable way to analyze and discriminate the effect of various stress treatments on elicitation of androgenesis. These results form a solid basis for further efforts designed to enhance responses and to extend double haploid technology to other legumes.

Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae).

To induce androgenesis in field pea, grass pea and the model legume species Medicago truncatula, isolated microspores of various genotypes of these three species were submitted to a range of abiotic stresses prior to and during their initial culture, in order to stimulate them to divide and form embryos. Some stress agents had a positive effect on androgenesis from the treated microspores. Submission of flower buds to a cold period prior to anther excision or microspore isolation, modifying the osmotic pressure of the medium during initial culture and electroporation of isolated microspores were the three major individual stress agents to have an impact on the efficiency of androgenetic proliferation and subsequent differentiation from the microspores of pea, grass pea and M. truncatula genotypes. A combination of osmotic and electric shocks significantly improved responses from isolated microspores and yielded microcalluses and then calluses, but only few underwent morphogenesis. Further work is under way to improve responses and extend them to other genotypes. The results reported here are, to the best of our knowledge, the first successful results from isolated microspores of these species.

Genetic analysis of agronomic and quality traits in mustard (Brassica juncea).

To develop an efficient mustard (Brassica juncea) breeding programme, a better knowledge of the genetic control and relationships of the main selected characters is needed. Thus, doubled haploid (DH) lines were evaluated over 2 years in the field. Days to flowering, plant height, thousand-seed weight, fatty acid composition, seed oil content, sinigrin, gluconapin and total glucosinolate contents were determined in the DH population. The influence of seed coat colour was estimated. Results showed significant differences between yellow and brown seeds only for oil and fatty acid contents. Molecular analysis revealed that seed coat colour is associated with two Mendelian trait loci: Bjc1 [on linkage group (LG) 3] and Bjc2 (on LG6). The quantitative trait loci associated with characters-detected by composite interval mapping-were not co-localised and revealed a genetic independence. The results obtained in this study show that the most important agronomic and quality traits of brown mustard could be bred independently. Correlation between the studied traits is also discussed.

Development of an AFLP-based linkage map and localization of QTLs for seed fatty acid content in condiment mustard (Brassica juncea).

A genetic linkage map of Brassica juncea based on AFLP and RAPD markers was constructed using 131 F1-derived doubled-haploid (DH) plants from a cross between two mustard lines. The map included 273 markers (264 AFLP, 9 RAPD) arranged on 18 linkage groups, and covered a total genetic distance of 1641 cM; 18.3% of the AFLP markers showed a segregation distortion (P < 0.01). The markers with biased segregation were clustered on seven linkage groups. QTLs for oil contents, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), eicosenoic acid (20:1), and erucic acid (22:1), were mapped on the AFLP linkage map. Correlation studies among fatty acids in the DH population and the localization of QTLs involved in their control indicated that a major gene located on linkage group (LG) 2 controlled the elongation step of erucic acid.

Electroporation modulates the embryogenic responses of asparagus (Asparagus officinalis L.) microspores.

Microspores of three genotypes of Asparagus officinalis L. were mechanically isolated without affecting their viability and were submitted to electric fields in order to modulate their competence for embryogenesis. When a constant pulse length and various field strengths (250, 500, 750, 1000, 1500, and 2000 V/cm) were tested, the viability of electro-treated microspores decreased as the field strength increased, for all genotypes. Conversely, the embryogenic competence was genotype dependent and was enhanced by low voltages for two clones when microspores were cultured in the presence of auxin. When the effect of pulse duration was studied, despite a strong genotype effect on responses, a short pulse coupled with a low voltage appeared to improve the competence for proembryo formation compared with nonelectroporated microspores, while longer pulses significantly improved microspore division.

Improved microspore culture and doubled-haploid plant regeneration in the brown condiment mustard (Brassica juncea).

The availability of doubled haploids could greatly contribute to improving seed quality in condiment mustard (Brassica juncea). We have developed an efficient and reliable protocol of microspore culture, modified from that of Baillie et al. (1992), based on a modification of the sucrose concentration of culture media. A comparison of microspore culture media differing in their sucrose content showed that a decrease from 17% (w/v) sucrose during the first 48 h to 10% (w/v) thereafter favoured an increase in the production of embryos whatever the responding genotype tested. Thus, out of the 13 B. juncea genotypes studied, 12 gave rise to embryos, and seven of these embryos could be converted into plants. Doubled-haploid plants were produced after treatment with colchicine.

Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts.

Large numbers of viable protoplasts of pea (Pisum sativum) and grass pea (Lathyrus sativus) were efficiently and reproducibly obtained and, for the first time, fused. Different procedures for fusion were compared, based either on electrofusion (750, 1000, 1250 or 1500 V cm(-1)), or on the use of macro or micromethods with a polyethylene glycol (PEG 6000 or PEG 1540), or a glycine/high pH solution. Over 10% of viable heterokaryons were obtained, with PEG as the most efficient and reproducible agent for protoplast fusion (>20% of viable heterokaryons). Both the division of heterokaryons and the formation of small calluses were observed.

Plant regeneration from aged-callus of the woody ornamental species Lonicera japonica cv. "Hall's Prolific".

Callus cultures were initiated from in vitro grown leaf, stem and root segments of Lonicera japonica "Hall's Prolific", on a medium containing 10.7 µM α-naphthtylacetic acid and 2.7 µM benzyladenine, while media with 2,4-dichlorophenoxyacetic acid led to a rapid necrosis of explants. Shoot regeneration from true-callus (i.e. without any part of the original explant) was achieved for the three different source tissues within 12 weeks. The highest rate of regeneration was obtained by using benzyladenine (4.4 to 44.4 µM) as the sole hormone in the medium. The regenerated shoots were readily elongated and rooted on the same medium as used for multiplication, and plantlets were subsequently transferred to greenhouse conditions, where nearly 100% of them were successfully acclimatized. This is the first example of plant regeneration from aged (≥6 month-old) true-callus of a woody ornamental species.

Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus Xdomestica Borkh.).

Highly viable protoplasts were isolated in large numbers from in vitro-grown leaf and stem tissues of a haploid clone of the apple scion cultivar Golden Delicious (Malus Xdomestica Borkh.). Protoplasts from both sources divided rapidly to give microcallus, when cultured in a modified Kao and Michayluk-based medium. Following two successive subcultures for callusing, shoot buds were regenerated from such calli, on half-strength Murashige and Skoog medium with an increased concentration of group B vitamins and containing 5.0 mg.l(-1) 6-benzyl-aminopurine and 0.1 mg.l(-1) l-naphthaleneacetic acid (for the leaf protoplast-derived calli) or 4-indole-3yl-butyric acid (for stem protoplast-derived calli). The mesophyll protoplast-derived shoots were enfeebled and vitrified, in time with their ultimate death. Conversely, for those shoots deriving from the stem protoplasts, in vitro propagation was successfully achieved. This is the first report on the successful isolation, culture and organogenesis from stem protoplasts of a woody plant genotype.

Organogenesis from 'Passe Crassane' and 'Old Home' pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants.

Large numbers of highly viable mesophyll protoplasts were isolated from shoot cultures of the scion cv 'Passe Crassane' and the rootstock genotype 'Old Home' of common pear (Pyrus communis L.). Protoplasts were cultured for both genotypes either as liquid layers or as liquid-over-agar cultures, in ammonium-free MS medium with 0.5 M mannitol, 50 mg/l casein enzymatic hydrolysate (CEH), 2.0 mg/l NAA and 1.0 mg/l BAP, plus either 0.5 mg/l IAA (for 'Old Home') or 2.0 mg/l IAA (for 'Passe Crassane'). Protoplast microcalli, obtained by day 60 ('Passe Crassane') or day 80 ('Old Home'), were transferred for further growth to ammonium-free MS medium with 2.0 mg/l NAA and 1.0 mg/l BAP. Shoot bud regeneration from the protoplastderived callus was first attempted between 100 ('Passe Crassane') and 120 ('Old Home') days after protoplast isolation. For 'Passe Crassane', shoot buds were regenerated (day 130) on a half-strength MS medium with 0.1 mg/l IBA, 0.5 mg/l BAP, 50 mg/l CEH and 20 mg/l Ca-panthotenate. For 'Old Home', shoot but regeneration only occurred 30 days later and on the same medium as above, which was additionally supplemented with double the concentration of the group B vitamins found in the original MS formulation and 0.05 mg/l GA3. Following micropropagation and in vitro rooting of shoots, the plants were transferred to soil following standard procedures. Trueness-to-type of the regenerated plants was assessed by analysing their leaf isozyme banding profiles (for EST, AP, PRX, SOD, ENP, LAP, PGI, AAT, ADH, MDH and PGM) and comparing them to those corresponding to the original shoots that provided the protoplasts. No differences between the mother shoots and the protoclones were observed for any one of the 11 isozyme systems studied.

Tissue culture and top-fruit tree species.

The commercial cultivation of rosaceous fruit trees (e.g., pear, apple, cherry, peach, plum) relies heavily upon the quality and performance of the rootstocks. This is even more the case now that self-rooted scions produce larger trees with a longer juvenile phase (1). It would, therefore, be of special interest for the fruit breeder to have general purpose rootstocks with a wide ecophysiological adaptation and high compatibility coupled with early cropping. In addition, many of the older and highly adapted scion varieties of fruit trees could benefit greatly from the introduction of stable, yet minor changes in their genome. Fruit trees are generally highly heterozygous, outbreeding, and thus are asexually propagated (see Chapter 10 , this vol.). Consequently, genetic improvement is likely to be based on protoplast technology, and achieved mainly through somatic methods, such as somaclonal variation or somatic hybridization.

Plant regeneration from root callus protoplasts of sour cherry (Prunus cerasus L.).

Callus protoplasts of sour cherry clone CAB4D entered sustained division in Murashige and Skoog's (1962) medium with 1-naphthaleneacetic acid, 6-beneylaminopurine and zeatin. Further to callusing, organogenesis was induced from the protoplastderived callus, in a basal regeneration medium with these same growth regulators at 0.01 mg/l, 2,0 mg/l and 0.05 mg/l, respectively. The regeneration pathway, from such callus, could be altered by adding different organic compounds to this medium. Casein hydrolysate, added alone, promoted rhizogenesis, with shoot buds regenerated from the protoplast-derived roots, while in a basal regeneration medium with casein hydrolysate and a group B vitamin mixture direct caulogenesis occurred.

Evidence for quantitative responses during co-culture of Pyrus communis protoplasts and Erwinia amylovora.

Mesophyll protoplasts were isolated from axenic shoot cultures of pear cultivars, exhibiting different degrees of susceptibility to fire blight infection at the whole plant level and they were co-cultured with the wild-type strain CFBP 1430 of Erwinia amylovora, and with an avirulent transposon mutant of the former (PMV 6046). Results, as assessed in terms of the effects of bacteria on protoplast viability, the time to the onset of divisions, the percentage of the originally cultivated protoplasts that divided once and of those proliferating to give 10-cell colonies, correlated with field resistance to fire blight of the respective pear genotypes. These results might provide a model for a better understanding of the interaction between pear and E. amylovora.

Selection for salt and drought tolerance in protoplast- and explant-derived tissue cultures of Colt cherry (Prunus avium x pseudocerasus).

Colt cherry (Prunus avium x pseudocerasus) callus cultures were derived from leaf protoplasts, protoplasts of root cell suspension cultures, or by direct culture of leaf and root tissues. Survival of calli cultured on basal proliferation medium containing 25, 50, 100 or 200 mN (millinormal) NaCl, Na(2)SO(4) or KCl, or iso-osmotic (with NaCl) concentrations of mannitol ranged from 1 to 15%. After six transfers on the same medium, surviving cell lines were subjected to three cycles of direct recurrent selection; i.e., in each cycle, they were cultured alternately on basal proliferation medium, and on basal proliferation medium supplemented with NaCl, KCl, Na(2)SO(4) or mannitol. Salt- or mannitol-tolerant cell lines selected in this way had smaller cells than unselected cell lines, and they grew more rapidly and had higher callus and cell survival rates than unselected cell lines when cultured in the presence of salt or mannitol. Cells lines selected for tolerance to one agent (sodium salt, potassium salt or mannitol) showed minimal tolerance to another agent. However, when plants were regenerated from salt- or mannitol-tolerant callus and new cultures derived from them, the new cultures showed tolerance to all of the salts and mannitol. Plant regeneration from the new cultures was not achieved under the conditions that led to the regeneration of the parent plants from callus.

Somatic hybridization of sexually incompatible top-fruit tree rootstocks, wild pear (Pyrus communis var. pyraster L.) and Colt cherry (Prunus avium x pseudocerasus).

Mesophyll protoplasts of wild pear (Pyrus communis var. pyraster L., Pomoideae) were chemically fused with cell suspension protoplasts of cherry rootstock Colt (Prunus avium x pseudocerasus, Prunoideae), following an electroporation treatment of the separate parental protoplast systems. Fusion-treated protoplasts were cultured, on modified K8P medium, where it had been previously established that neither parental protoplasts were capable of division. Somatic hybrid calli were recovered and, following caulogenesis on MS medium with zeatin and after rooting of regenerated shoots, complete trees were obtained and grown in vivo. Hybridity of these trees was confirmed based on morphological characters, chromosome complement and isozyme analysis. Two separate cloned lines of this intersubfamilial rootstock somatic hybrid (wild pear (+) Colt cherry) were produced. This is the first report of the production of somatic hybrid plants of two woody species, of agronomic value, within the order Rosales.

Cell wall synthesis and salt (saline) sensitivity in cultured colt cherry (Prunus avium × pseudocerasus) protoplasts.

In order to investigate the cellular basis of salt tolerance, Colt cherry (Prunus avium ×pseudocerasus) protoplasts from mesophyll tissues and root cell suspension cultures were cultured in the presence of NaCl, KCl or Na2SO4, at normalities of 25, 50, 100 or 200 mN for each salt and with or without 2,6-dichlorobenzonitrile, an inhibitor of cell wall synthesis. Results showed that the acquisition of salt tolerance was concomitant with the onset of cell wall regeneration, with protoplasts exhibiting a greater salt tolerance than cells.

Long-term effect of electroporation on enhancement of growth and plant regeneration of colt cherry (Prunus avium × pseudocerasus) protoplasts.

Electric pulses applied to Colt cherry protoplasts enhanced the long-term growth and plant regeneration of protoplast-derived tissues. Protoplasts isolated from long-term cultured tissues derived from electroporated protoplasts retained the ability to enter division in culture earlier and with a higher frequency of plant regeneration than untreated cell suspension protoplasts.

Plant regeneration from mesophyll protoplasts of Williams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.).

Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Murashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAA and BAP and exhibited shoot regeneration on half-strength Murashige and Skoog medium supplemented with 0.2 mg 1(-1) 4-indole-3yl-butyric acid, 2.0 mg 1(-1) BAP, 0.2 mg 1(-1) gibberellic acid, 50 mg 1(-1) casein hydrolysate and 10 mg 1(-1) Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization.