Thin Cell Layer Explants Research Articles

Changes in macromolecular movement accompany organogenesis in thin cell layers of Torenia fournieri

A range of fluorescently labelled probes of increasing molecular weight was used to monitor diffusion via the symplast in regenerating thin cell layer (TCL) explants of Torenia fournieri. An increase in intercellular movement of these molecules was associated with the earliest stages of vegetative shoot regeneration, with the movement of a 10 kDa dextran (FD 10000) observed between epidermal cells prior to the appearance of the first cell divisions. A low frequency of dextran movement in thin cell layers maintained under non-regenerating conditions was also observed, indicating a possible wound induced increase in intercellular movement. Dextran movement between epidermal cells reached a peak by day 4 of culture and then declined as cell division centres (CDCs) formed, became meristematic regions and finally emerged as adventitious shoots. Within CDCs, testing with small fluorescent probes (CF: carboxyfluorescein, mw 376 Da and F(Glu)3: fluorescein-triglutamic acid, mw 799 Da) revealed a mosaic of cell isolation and regions of maintained symplastic linkage. Within shoots, surface cells of the presumptive apical meristem permitted the intercellular movement of 10 kDa dextrans but epidermal cells of the surrounding leaf primordia did not permit dextran movement. In some cases, intercellular movement of CF was maintained within leaf primordia. Symplastic movement of labelled dextrans during regeneration in Torenia thin cell layers represents a significant increase in the basal size exclusion limit (SEL) of this tissue and reveals the potential for intercellular trafficking of developmentally related endogenous macromolecules.

Effect of 2,4-D and 4-CPPU on somatic embryogenesis from stigma and style transverse thin cell layers of Citrus

Callus induction, somatic embryogenesis and plant regeneration were obtained in lemon [Citrus limon (L.) Burm. cv `Femminello'] and sweet orange [C. sinensis (L.) Osb. cv `Washington Navel GS'] from cultures of stigma and style transverse thin cell layer explants [(t)TCLs]. Explants were cultured on 16 different media, based on the nutrients and vitamins of Murashige and Tucker medium (MT) supplemented with different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N′-phenylurea (4-CPPU). Sucrose (146 mM) was used as the sole carbon source. Somatic embryos arose from callus at the surface of stigma and style (t)TCLs 3–5 months after culture initiation of both sweet orange and lemon. The percentages of embryo formation from style (t)TCLs ranged from 0% (the media containing 2,4-D) to 16.0% (the medium supplemented with 4 μM 4-CPPU) for C. limon. Better results were obtained when stigma (t)TCLs from C. limon were used; in fact, percentages ranged from 0% on the media containing 2,4-D, with the only exception for the medium supplemented with 0.4 μM 2,4-D, to 24.8% on medium with 4 μM 4-CPPU. The embryogenic response of lemon (t)TCLs was usually higher than for sweet orange (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies ranging from 53% to 75% for sweet orange and lemon style derived embryos, respectively.

Zearalenone and flower bud formation in thin-cell layers of Nicotiana tabacum L.

Three lines of evidence indicated a connectionbetween zearalenone (ZEN) and flower bud formationin thin cell layer (TCL) explants of Nicotianatabacum L. cv. Samsun. (1) There were two peaks inthe endogenous ZEN level during the formation offlower buds. (2) The specific inhibitor of ZENbiosynthesis, malathion (MAL), inhibited thebiosynthesis of endogenous ZEN and at the same timeflower bud neoformation. (3) Exogenous ZEN inducedflower bud neoformation.

High frequency shoot regeneration from trifoliate orange ( Poncirus trifoliata L. Raf.) using the thin cell layer method

A method for a high frequency and direct in vitro bud regeneration of a woody species, the trifoliate orange ( Poncirus trifoliata L. Raf), was designed. Transverse thin cell layer (tTCL) explants excised from the stem internodes of 1-year-old young plants of P. trifoliata regenerated bud in vitro on a medium containing 6-benzylaminopurine (BAP 1-50 μM) and N-phenyl-N'-1,2,3-thidiazol-5-ylurea (thidiazuron, TDZ) (0.1–10 μM). The optimal concentrations for bud induction were 25 μM BAP and 1 μM TDZ leading to 87 and 72 % of responsive tTCLs and 24 and 15 buds per tTCL, respectively. A higher percentage of responsive tTCLs and a higher frequency of bud regeneration were obtained with BAP and TDZ combined. With a combination of 10 μM BAP and 1 μM TDZ, 90 % of responsive tTCLs forming 37 buds per tTCL were obtained. Shoot elongation occurred after a transfer onto a medium containing 1 μM GA 3. Rooting of individual shoot was induced using 5 μM NAA. One hundred per cent of rooted shoots developed normally after transfer to the greenhouse; no phenotype variation was observed. High numbers of regenerated viable plants can be produced directly without callus formation from tTCL after 9 weeks of culture.

Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus

Callus induction, somatic embryogenesis and plant regeneration were obtained in six different citrus species [Citrus deliciosa Ten. (cv 'Avana'), C.limon (L.) Burm. (cv 'Berna'), C.madurensis Lour. (cv 'CNR P9'), C.medica L. (cv 'Cedro di Trabia'), C.tardiva Hort. ex Tan. (cv 'CNR P6'), C.sinensis (L.) Osb. (cv 'Ugdulena 7')] from cultures of pistil transverse thin cell layer explants [(t)TCL]. Explants were cultured on three different media: the nutrients and vitamins of Murashige and Skoog medium alone (MS) or MS supplemented with either 500 mg l–1 malt extract (MS I) or 500 mg l–1 malt extract and 13.3 μM 6-benzylaminopurine (MS II). Sucrose (146 mM) was used as the carbon source. Somatic embryos were visible 2–5 months after culture initiation. The different genotypes showed a different embryogenic frequency from stigma, style and ovary (t)TCL explants. All of the cultivars regenerated somatic embryos. Percentages of style (t)TCL explants producing somatic embryos ranged from 0% (C.deliciosa, C.madurensis, C.sinensis and C.tardiva on the three different media) to 5.2% (C.limon on MS II). Embryo formation in stigma (t)TCL explants ranged from 0% (C.madurensis on MS and MS I, C.sinensis on MS, C.deliciosa and C.tardiva on the three different media) to 42.4% (C.limon on MS II). Embryo formation in ovary (t)TCL explants ranged from 0% (C.deliciosa on MS, C.limon, C.medica, and C.sinensis on the three different media) to 9.3% (C.tardiva on MS I). After about 12 weeks somatic embryos developed into plantlets at a high frequency.

High frequency shoot regeneration from Rhynchostylis gigantea (orchidaceae) using thin cell layers

Often regeneration in orchids is only achieved through protocorms, i.e., the juvenile stage. In order to produce directly shoots via bud regeneration both rapidly and with a high frequency, a transverse thin cell layer (tTCL) method is extended to Rhynchostylis gigantea. Transverse thin cell layer (tTCL) explants (0.3--0.5 mm) excised along the stem from the basis to the shoot tip of one-year-old plantlets were cultured on Murashige and Skoog (1962) medium supplemented with different combinations of benzyladenine (BAP), 1-naphthaleneacetic acid (NAA), thidiazuron (TDZ) and 3% sucrose. The optimal combination for maximal bud regeneration was 3 µM BAP and 3 µM TDZ, giving rise to 11.7 buds per tTCL. Roots were obtained with 10 µM forchlofenuron (CPPU) and 1% sucrose. The in vitro plants (> 3 cm long) obtained 4 to 6 weeks after the tTCLs culture were transferred to the greenhouse; their morphology was normal. Efficient micropropagation of direct production of shoots without passing through protocorm stage of orchid species can be achieved using the thin cell layer (TCL) method.

Rhizogenesis in buckwheat thin-cell-layer explants: effect of plant oligosaccharides

Thin cell-layer explants from Fagopyrum esculentum Moench hypocotyls cultured in liquid medium without growth regulators have been shown to be able to form roots. The intensity of this process depended upon the size of the explant and the length of the hypocotyl. Six diverse oligosaccharide fractions separated from the pea shoot tissues differently effected the rhizogenesis in this system. One fraction from the cell wall pectin and two from the cell sap stimulated this process. Another fraction from the cell wall pectin and two other from the cell sap inhibited explant rooting. One fraction from the cell sap differently influenced two separate stages of root development. These results indicate that the oligosaccharide fragments can be regulators of rhizogenesis.

Biological Activity of Reducing-End-Derivatized Oligogalacturonides in Tobacco Tissue Cultures1

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspension-cultured N. tabacum cv Xanthi cells, and (d) elicit H2O2 accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.

Rapid plant regeneration of a C4 dicot species: Amaranthus edulis

The effects of TDZ (N-phenyl-N′-1,2,3-thidiazol-5-ylurea) and CPPU (N-(2-chloro-4-pyridil)-N′-phenylurea) on bud initiation of Amaranthus edulis thin cell layers (tTCLs) explants were investigated. tTCLs (0.2–0.4 mm thick) excised from 1 week old seedlings were cultured on Murashige and Skoog (MS) basal salts medium supplemented with varying concentrations of TDZ or CPPU ranging from 1 to 20 μM and 1 week after culture, buds were initiated. The optimal concentrations were 2 μM for TDZ and 10 μM for CPPU. Buds developed into normal plants after transfer onto a MS basal salts medium containing BA to obtain normal internode elongation and onto a MS basal salts medium containing IBA or TDZ for rooting.

The Plant OncogenerolDStimulates Flowering in Transgenic Tobacco Plants

TheAgrobacterium rhizogenesT-DNA oncogenerolDunder the control of its own 5′ regulatory region was transferred to day-neutral tobacco plants. The main trait induced byrolDin transgenic plants is a striking precocity in flower setting and a strong enhancement of the flowering potential. InrolDplants, early flowering is followed by the very rapid growth of numerous lateral inflorescences. The analysis of several morphological and histological parameters suggests that some characteristic morphological abnormalities observed inrolDplants can be accounted for by their early reproductive phase transition and points to the involvement in the transition of a greater portion of the plant body than is the case for untransformed tobacco. Thein vitromorphogenic potential of tissues fromrolDplants was also tested. Superficial thin cell layer explants fromrolDplants show an earlier and much enhanced flower organogenesis, compared to controls, both on flowering and on hormone-free medium.

Biologically-active soluble oligosaccharides from pea stem tissues

Two active fractions of soluble oligosaccharides were isolated from pea (Pisum sativum L.) stem tissues. Both fractions are capable of affecting different phases of root development on buckwheat thin cell-layer explants (TCLs) and of inhibiting auxin-promoted growth of etiolated pea stem segments. The existence of non-wall bioactive oligosaccharides which may have a role in cell development in vivo is proposed.

Competence for Regeneration during Tobacco Internodal Development (Involvement of Plant Age, Cell Elongation Stage, and Degree of Polysomaty).

This study deals with internodal development in vegetative plants of Nicotiana tabacum cv Samsun NN and its reflection in changes of the cellular competence for regeneration. During elongation of the internodes, the cells of the epidermis, subepidermis, and cortex exclusively expanded and increased their DNA content cell type specifically, generally from 2C to 4C. Cells with the 8C DNA content were found mainly among the cortex cells of mature internodes. The frequency of shoot regeneration (directly from subepidermal and epidermal cells together) on thin cell layer explants increased to an optimum along with elongation of the internodes and decreased in mature internodes along with aging. The frequencies of diploid shoots among the regenerants from elongating and mature internodes were high (88 and 75% on the average, respectively), indicating that most cells that had achieved the 4C DNA content generally retained the G2 phase of the diploid cell cycle. Shoots regenerated from explants of young plant material mainly had a vitrified appearance. The occurrence of this type of malformed growth was already determined by the physiological state of the cells in the internode and did not interfere with their acquisition of competence. Vitrification was unrelated to the degree of polysomaty of the internodal tissue. Using the occurrence of tetraploid root regenerants (from intermediate cortex-derived callus), up to a frequency of 50%, we show that the position in the plant where a majority of the 4C cortex cells switched to the G1 phase of the tetraploid cell cycle was at the transition from the elongation phase to the mature phase.

Histology of the Morphogenic Response in Thin Cell Layer Explants from Vegetative Tobacco Plants

The morphogenic response of thin cell layers (TCLs) from vegetative tobacco (Nicotiana tabacum L.) plants can be directed very precisely by varying the concentrations of benzyladenine (BA) and α-naphthalene acetic acid (NAA) in the culture medium. Medium containing 1·6 μM BA and 0·5 μM NAA was optimal for shoot formation, concentrations of 0·5 μM BA and 1·6 μM NAA were optimal for the induction of shoots and roots on the same explant, whereas concentrations of NAA higher than 16 μM resulted in callus proliferation only. Polarity in the distribution of the shoot buds was observed, i.e. a switch from basal to apical shoot formation occurred with increasing NAA concentrations, suggesting basipetal transport of NAA. Histological examination of TCLs on shoot induction medium revealed that first cell divisions occurred within 2 d in cortical cells which were directly in contact with the medium along the longitudinal cut surface, and after 2 d in subepidermal cells along the lateral edges of the explants. Individual lateral buds originated from one subepidermal and one or more epidermal cells, while apical buds originated from single subepidermal or cortical cells located directly at the apical end of the explant. After culture of TCLs for 2-3 d on root/shoot induction medium cells in the regeneration-competent subepidermis elongated, while on callus induction medium subepidermal cells elongated and dedifferentiated. The regeneration system as described in this study will be used to identify cells competent for regeneration as well as for transformation.

The competence of cells for cell division and regeneration in tobacco explants depends on cellular location, cell cycle phase and ploidy level

This work concerns application of flow cytometry (FCM) and confocal laser scanning microscopy (CLSM) to investigate the competence of cells for cell division and regeneration. FCM analysis of freshly-cut thin cell layer (TCL) explants of Nicotiana tabacum, excised from upper internodes of vegetative plants, revealed that one-quarter of the cells had the 2C nuclear DNA content, whereas of the other cells most nuclei had the 4C and some had the 8C DNA content. Cytometric examination using CLSM showed that the 2C nuclei were mainly located in the epidermis and subepidermis, and the 4C nuclei predominantly in the cortical tissue. During culture of the explants, part of the cortical cells went through mitosis from the first day onwards, and formed callus from which predominantly diploid and some tetraploid roots regenerated at low frequency. Most cortical cells were thus in the G 2 phase of the diploid cell cycle. FCM analysis showed that another fraction of the 4C cortical cells was induced to endoreduplicate to 8C cells. These cells thus had previously switched to the G 1 phase of the tetraploid cell cycle. CLSM analysis revealed that subepidermal and epidermal cells, respectively, underwent cell division from the second and third day onwards. Shoot primordia developed from cells of both cell layers together. Most shoot regenerants were normal diploids, some were mixoploids or tetraploids. The combination of FCM and CLSM allowed identification of the cell cycle phase, the ploidy level, the position of the cell, and the cellular development. The results give insight into the involvement of these parameters in the competence for cell division and regeneration at the level of the individual explant cells, and are therefore relevant for cellular and molecular approaches to plant transformation.

Effects of inhibitors on phenocritical events of in vitro shoot organogenesis in tobacco thin cell layers

Phenocritical disruption of in vitro shoot regeneration in tobacco ( Nicotiana tabacum cv. Samsun) thin cell layers (TCL) was assessed using four exogenously supplied inhibitors. Specifically, the inhibitors were used to disrupt the events associated with the differentiation and determination of adventitious shoots in pre-induced tobacco TCL explants in a phenocritical manner. Shoot determination and development exhibited the greatest sensitivity to all four inhibitors (cycloheximide, ribose, thymidine and triiodobenzoic acid) between days 0 and 4 following induction. Therefore, protein synthesis, energy stores, DNA synthesis and auxin transport are all very important in the early stages of shoot meristem determination. Knowledge of phenocritical events in de novo shoot organogenesis in tobacco TCL explants will be helpful in isolating and characterizing genes involved in the determination and development of such meristems.

The Effect of Environmental Conditions on the Expression of Disease in Cultured Tissues of Brassica napus ssp. oldfera Infected with Leptosphaeria maculans

AbstractAs a basis for devising an in vitro screening programme, culture conditions were optimized so that tissue cultures from two resistant cultivars of Brassica napus ssp. oleifera (Mikado, Bienvenu) and two susceptible cultivars (Lesira, Ceres) could be differentiated using a disease scoring scheme, when inoculated with Leptosphaeria maculans. Tissues inoculated included thin cell layer explants from soil‐grown plants and in vitro‐grown shoot cultures and callus tissue formed on such explants. The period of incubation and the incubation temperature were of importance in the development of differential disease reactions. Increasing temperature generally resulted in an increase in infection and too great an incubation period resulted in total overgrowth of the tissue. Increasing concentrations (1 × 10−6 M‐1 ×10−4 M) of the auxins 1‐naphthylacetic acid (NAA), 2,4‐dichlorophenoxyacetic acid (2,4‐D) and mdole‐3‐acetic acid (IAA) in the culture medium, resulted in a decrease in disease score of the thin cell layer (TCL) explants from soil‐grown plants. The cytokinins examined 6‐benzyl‐aminopurine (BAP) and 6‐4‐hydroxy‐3‐methyl‐2‐enylaminopurine (zeatin), reduced the extent of infection of the TCL explants when used in combination with the auxin NAA. Medium containing NAA at a concentration of 1 × 10−6 M in combination with BAP at a concentration of 1× 10−6 or 1 × 10−4 M allowed differentiation of the disease reactions of the resistant and susceptible cultivars, when the explants were incubated for 10 days at 20 °C after inoculation. Similar conditions of incubation and the addition of NAA (1 × 10−6 M) combined with BAP (1 × 10−6 M) to the medium also allowed the differentiation of the disease reactions on TCL explants from stems of in vitro shoot cultures of the cultivars Mikado and Lesira. Increasing concentrations of the auxin NAA and the cytokinin BAP resulted in a reduction in the mean disease score of the callus tissue produced on TCL explants from soil‐grown plants, and NAA (1 × 10−5 M) combined with BAP (1 × 10−6 or 1 × 10−5 M) allowed differentiation of resistance and susceptibility in callus tissues when incubated for 5 days at 20 °C. 2,4‐D did not allow differentiation of the cultivars. This was in contrast to the inoculation of callus tissue attached to TCL explants of in vitro shoot cultures, where combinations of 2,4‐D and BAP at concentrations of 1 × 10−6 M allowed differentiation of the resistant and susceptible cultivars.These findings provide a basis for designing selection protocols of value in both traditional as well as in vitro breeding programmes to select lines of oilseed rape with resistance/novel resistance to L. maculans.

The Use of Ergosterol as a Quantitative Measure of the Resistance of Cultured Tissues of Brassica napus ssp. oleifera to Leptosphaeria maculans

AbstractA simplified method for the quantitative assessment of the fungal lipid ergosterol was used to assess the levels of infection in tissue cultures of oilseed rape (Brassica napus ssp. oleifera) inoculated with Leptosphaeria maculans. The growth of L. maculans in liquid culture throughout a 36‐day period correlated well (r = 0·92) with the amount of ergosterol extracted from the mycelium. There were significant differences (P < 0·05) in the amount of ergosterol extracted from infected thin cell layer (TCL) explants and callus tissue of two resistant and three susceptible cultivars of oilseed rape. Amounts of ergosterol extracted from resistant cultivars were < 100 (g and from susceptible > 100 (g. The mean amounts of ergosterol extracted from shoot cultures of two resistant and four susceptible cultivars were similar to those for TCL explants and callus tissue, although the values obtained were variable. This technique can be used in in vitro breeding programmes to accurately assess the resistance of tissue cultures of B. napus to L. maculans and could also have value in conventional breeding programmes.

Stimulation of root development on buckwheat thin cell-layer explants by pectic fragments from pea stem cell walls

Buckwheat (Fagopyrum esculentum Moench.) thin cell-layers (TCLs) cultured individually in a liquid medium were used to test the root-inducing activity of pectic polysaccharides with a degree of polymerization (DP) of 20-25, isolated from pea (Pisum sativum L.) stem cell walls. These pectic fragments induced more rapid root formation on the explants in comparison with untreated controls. This pectic fragment treatment also promoted root growth as measured by both fresh and dry weights and about doubled the number of roots formed. This buckwheat TCL system is proposed as a new bioassay for oligosaccharins due to its sensitivity, reproducibility and ease of preparation.

Cellular changes in early development of regenerating thin cell layer‐explants of rapeseed analyzed by light and electron microscopy

Cellular changes in thin cell layer (TCL) explants of stem origin of Brassica napus L. cv. Vega were studied from 0 to 15 day by light and transmission electron microscopy. Apical and basal ends of the old explants were analysed separately. Quantitative and qualitative analyses showed that during the first culture day the parenchyma cells enlarged significantly as did the cytoplasm/vacuole ratio. The cytoplasm contained increased rough endoplasmic reticulum (RER), polysomes and dictyosomes associated with both coated and uncoated vesicles. The cell enlargement continued during the first 5 days of culture. The structural organization of the cell wall became somewhat loose and inhomogeneous. Parenchyma in the basal end divided frequently, resulting in several centres of division, while cell division in apical cells was less frequent and cells there remained enlarged. Starch accumulation started on the first day and increased until the third day. i. e. until cell divisions became more frequent. The starch content of dividing cells gradually decreased and starch was almost totally lacking in 15‐day‐old explants. Starch grains remained numerous, however, in the large non‐dividing apical cells, except in those cells adjacent to the medium. Cell divisions started close to medium in explants containing vascular tissue, but closer to the epidermis in the explants without vascular tissue.The results show how rapid (one day) striking changes in the cells take place and suggest that optimal hormone concentration and intertissue relations between epidermis and parenchyma and between parenchyma and vascular tissues as well as intercellular relations among parenchyma cells determine the first cell division sites and planes in the explants. Although the cells change from elongated to spheroid, their original polarity remains as evidenced by the formation of more numerous basal shoot primordia than in apical shoot primordia.

A Comparison of the Disease Reactions of Stems and Detached Leaves of Soil and in vitro Grown Plants and Regenerants of Oilseed Rape to Leptosphaeria maculans and Protocols for Selection for Novel Disease Resistance

AbstractProtocols for selecting plant tissues of winter oilseed rape (Brassica napus subsp. oleifera) with resistance to Leptosphaeria maculans by either stem or leaf inoculation of both soil and in vitro grown plant material are described. The stem inoculation procedure gave good correlation (r = 0. 92) between the 50 day stem disease scores of eight out of nine cultivars of soil grown winter oilseed rape inoculated with isolate 41A4 of L. maculans and the N. A. B. esistance ratings or resistance data from field trials. The exception was the cultivar Liradonna. Inoculation of stems of five cultivars with isolates 41A4, 433 and 478 indicated a range of isolate virulence 478 > 41A4 > 433. This was the inverse of that observed in leaf inoculations. Application of the stem inoculation procedure to in vitro shoot cultures allowed differentiation of resistant and susceptible cultivars, including the cultivar Liradonna, after 20 days incubation at 20°C. The protocol was also applicable to plantlets regenerated from thin cell layer explants grown in vitro. Inoculations with isolate 433 allowed the differentiation of resistant, intermediately resistant and susceptible leaf material of soil grown plants, when leaf discs from young leaves were incubated on water agar supplemented with BAP (1 × 10−5 M) at 25°C for 10 days. Intermediately resistant leaves were resistant after 10 days and susceptible after 15 days of incubation. Leaves of shoot cultures grown in vitro were more susceptible than the corresponding soil grown material. However, inoculation of old leaves with isolate 41A4 (an isolate of less virulence on leaves than 433) distinguished the cultivars after 15 days of incubation. These protocols allow the accurate assessment of resistance to L. maculans at the stem or leaf level and are of use in traditional as well as in vitro selection programmes.