Fertility and growth in the field of Lolium perenne and Festuca rubra plants regenerated from suspension cultured cells and protoplasts

The effect of regeneration of Lolium perenne and Festuca rubra from embryogenic suspension cells and protoplasts on fertility and growth was evaluated. Embryogenic suspension cultures were either routinely subcultured or cryopreserved and re-established. Phenology, morphology and fertility of regenerated plants were studied for two growing seasons in a replicated field experiment. Most regenerated L. perenne and F. rubra plants showed a delay in inflorescence emergence, a reduced seed yield and differences in morphological traits when compared with seed-grown plants. For L. perenne, performance of plants regenerated from cryopreserved suspension cultures and protoplasts was similar to that of respective plants regenerated from routinely maintained suspension cultures. However, differences in performance were observed for respective regenerants in F. rubra. The phenotypic deviation observed was partly reflected in the randomly amplified polymorphic DNA (RAPD) analysis performed. However, regenerants of both species showing similar, or even superior performance to the seed-grown plants were also found. Embryogenic suspension cells and corresponding protoplasts of L. perenne and F. rubra have the potential for producing fertile, well-performing plants which can be integrated in breeding programs.     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

The inheritance of a basal branching type in guar

Summary Eight different genotypes of the F₁ hybrids between Oriental × Asiatic lily (Lilium) hybrids (2n = 2x = 24) were treated with nitrous oxide (N₂O) gas under pressure for 24 and 48 hours. At the time of treatment, all plants possessed early meiotic stages in the anthers of the oldest flower buds. The mature flowers from treated plants were monitored for fertility through pollen germination in vitro as well as by using them in crosses with diploid Asiatic hybrids (2n = 2x = 24) both as male and female parents. In five out of the eight genotypes of OA hybrids there was evidence for the production of 2n pollen which germinated in vitro from either one or both treatments. The 2n pollen from three genotypes was successfully used in crosses. In two cases, the treated plants were successfully used as female parents which indicated the formation of 2n (or 2x) egg cells. From an analysis of 41 sexual polyploid progenies obtained from N₂O treated plants it was shown that they were all euploids consisting of 34 triploids (2n = 3x = 36) and seven tetraploids (2n = 4x = 48). A detailed cytological analysis of 12 progeny plants through genomic in situ hybridization (GISH) proved that N₂O had induced first division restitution gametes in most cases and in two cases they produced gametes through indeterminate meiotic restitution. There was evidence for intergenomic recombination in three cases.     

Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.)

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.     

Triploid and Hexaploid Regenerants from Hexaploid Timothy (Phleum pratense L.) via Anther Culture

Pollen embryos were obtained from eight genotypes and viable green plants were regenerated from four genotypes in an anther-culture experiment with 165 genotypes of Phleum pratense L. Formation of proembryos inside the cultured anthers during the first 10—12 days was significantly influenced by genotypes and by the type of nutrient media. Primary embryos developed into multiple secondary embryos before regeneration of plants. Among a total of 62 plants regenerated, only 13 were albinos. Of the green regenerants, 11 were triploid while 35 were hexaploid when DNA-content was measured by flow cytometry. Eight plants with a triploid DNA-content did possess the triploid chromosome number of 21. Triploid and hexaploid regenerants from two different parents showed simplified isozyme (GPI and PGD) banding patterns relative to that of their parents.     

Callus Culture-Derived Somaclonal Variation in the Tropical Pasture Legume Stylosanthes guianensis (Aubl.) Sw.

Plants were regenerated from leaf-derived callus cultures of five genotypes of Stylosanthes guianensis. There were substantial genotypic differences in morphogenetic potential, ranging from 95–100 % of cv.‘Graham' calh to less than 1% of cv,‘Oxley' calh forming shoots. A total of 1024 regenerants (SC₁) were planted out, with 28.1 % surviving to set seed. Variant characters including albinism, reduced fertility, dwarfism and leaf morphology were observed. Variation in selfed progeny (SQ₂) of 138 SC₁ plants was assessed in a replicated field trial involving 2484 SO₂ individuals. Variant phenotypes were observed in 55 (39.9 %) SC₂ families. Many families were segregating for albinism, altered morphology or reduced fertility. Thirty families had lower yields than their parent and earlier and later flowering variants were found. Eight tetraploid families were identified. These were characterized by more erect habit, larger leaves, stems, flowers and seeds, reduced fertility and yield and a tendency to flower later.     

A comparison of somaclonal variation in rice plants derived and not derived from protoplasts

To clarify the effect of the type of in vitro culture on the generation of somaclonal variation, protoplast-derived rice plants were compared with rice plants derived from suspension culture or primary calli (not derived from protoplasts). Regenerated plants showing polyploid-like phenotypes appeared at a higher frequency (33–70%) in plants derived from protoplasts than in those not derived from protoplasts (3–6%). In the first progeny (R₁) generation of all regenerated plants, 120 of 368 lines (33%) segregated plants with mutated characters such as albino, dwarf and sterile. In quantitative traits, 62 (21%) and 144 (50%) of 290 Rj lines showed shorter culm and lower seed-fertility, respectively, compared with the control line derived from the selfed seeds of the original cultivar. The frequency of the mutant-possessing R₁ lines among lines derived from protoplasts was not significantly different from those not derived from protoplasts. These results indicate that isolation and culture of protoplasts does not enhance genetic changes other than cytogenetical changes.     

Evidence for phenotypic assortative mating for flower colour in periwinkle

Two experiments were conducted with periwinkle, Catharanthus roseus, to determine the extent of natural outcrossing. Three white-flowered, fully self-fertile, monogenic recessive mutants, namely, dwarf, wavy leaf margin and curved leaf were used, together with their parental white-flowered variety, ‘Nirmal’, and a normal pink-flowered variety, PS-1. The extent of total outcrossing ranged from 43.4 to 79.3% among mutants. Outcrossing between white × white-flowered plants ranged from 28.3 to 65.3% and was two to four times greater than that between white × pink-flowered plants in the three mutants. The extent of out-crossing between white × pink-flowered plants was similar “02.2-15.0%” in all mutants and also similar to that in the normal white-flowered variety,‘Nirmal’(00.4%), where white × white flower out-crossing could not be estimated. There were no large differences in the number of seeds per fruit, percentage fruit set and germination percentage of seeds obtained from self, white × white and white × pink flower crosses made in the glasshouse. There were also no significant differences in the number of flowers produced by the genotypes used in the study. The observed higher frequency of white × white flower matings compared with white × pink flower matings appeared to be due to the constancy of flower colour exhibited by the butterfly pollinators Pachliopta hector and Catopsilia pyranthae during their flower visits. Observations made on the occurrence of natural self-pollination revealed that automatic self-pollination did not occur in periwinkle.     

Increase of in vitro regeneration in Malus domestica by the application of phosphatase inhibitors

The effect of the phosphatase inhibitors sodium fluoride and sodium orthovanadaate on adventitious shoot regeneration from primary leaf explants was tested in eight genotypes of apple Malus domestica Borkh. Ten different concentrations between 0.5 and 100μM of each substance were added to a modified Murashige and Skoog regeneration medium containing 0.9 μM TDZ and 0.5 μM IBA. In general, all the con-centrations tested were able to promote shoot formation, although distinct maxima could be observed depending upon the genotype and the inhibitor. Shoot yield was influenced mostly by the genotype and by the concentration of the inhibitor, as well as the interaction between these two factors. In six of the eight genotypes the total number of regenerated shoots increased significantly compared with the control. This was achieved by an increase of the number of regenerating explants as well as the number of shoots per regenerating explant Comparing these results with those of former publications, application of the two phosphatase inhibitors resulted in an improved regeneration response in the corresponding genotypes. This method may also contribute to the understanding of in vitro regeneration in plants.     

The influence of auxin type on the array of somaclonal variants generated from somatic embryogenesis of eggplant, Solanum melongena L.

Somaclonal variation is a possible source of variation in plant breeding. To apply this approach to eggplant breeding, somaclonal variations were observed among plants regenerated through somatic embryogenesis induced by 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Variations including leaf shape, plant height, flower number per cluster, fruit shape, anther number per flower and pollen fertility were compared among ? 300 plants (R₀). Although these variations were confirmed among plants regenerated using both auxins, the frequencies of somaclonal variations in leaf shape, plant height, fruit shape and pollen fertility in the NAA experiment were higher than those in the 2,4-D experiment. Variations in flower number and anther number were also confirmed among plants from both experiments, although no significant differences in their frequency were observed. Subsequently, the inheritance of variations (leaf shape, fruit shape, and flower number) observed in R₀ was investigated from generation to generation (R₁). Variations in leaf shape and fruit shape were inherited while those in flower number were not. From these results we concluded that, although a high frequency of somaclonal variations were observed among plants from both methods, embryogenesis with NAA was more efficient than 2,4-D in eggplant.     

Protoplast technology for the breeding of top-fruit trees (Prunus, Pyrus, Malus, Rubus) and woody ornamentals

Summary Until recently, temperate fruit trees and woody ornamentals have been regarded as recalcitrant to biotechnological breeding approaches based on protoplasts. This however should no longer be the case, as procedures are now available, not only for the regeneration of complete plants from protoplasts of various tissues of such species, but also for the exploitation of protoplast technology for their genetic manipulation. This paper will examine the recent advances and state of the art in this domain, with particular attention to the use of protoplast technology as a novel tool in the breeding of rosaceous top-fruit tree species and woody ornamentals. Problems and their solutions within the context of regenerating plants from isolated protoplasts of stone (Prunus spp.), pome (Pyrus spp., Malus spp.) and small (Rubus spp.) fruits, and of several shrubby ornamental genotypes (Lonicera spp., Weigela spp., Forsythia spp., Cotoneaster spp.) will be addressed. Interspecific (Prunus spinosa + Prunus cerasifera) and intergeneric (Forsythia spp. + Syringa spp.) somatic hybridization within this group of species, as well as the use of protoplasts for host/pathogen interaction studies (Pyrus/Erwinia amylovora) will also be discussed.     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Ploidy analysis in water yam, Dioscorea alata L. germplasm

Poor reproductive development in yams (Dioscorea spp.) has often been attributed to the polyploid nature of the crop. In this study, flow cytometry was used to determine the ploidy level of 53 accessions of Dioscorea alata, mostly from West African countries, Chad and Puerto Rico. Nuclei were isolated from young leaf material and stained with DAPI(4,6-diamidino-2-phenylindole). The nuclear genome size (2C) was measured as an indicator of the ploidy level. Dioscorea rotundata genotypes with known ploidy levels were used as standards. The results showed that the majority of plants were hexaploid (84.9%) with a smaller percentage of tetraploids (15.1%). A higher number of male plants were hexaploid than tetraploids. This is at variance with earlier findings, which reported that hexaploid male plants are rare. Higher ploidy levels were not directly related to sparse or erratic flowering as previously reported as profuse flowering occurred in some male hexaploid accessions. These findings have important implications for yam breeding in relation to yam genetic resources. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production of fertile transgenic Brassica napus by Agrobacterium-mediated transformation of protoplasts

A protocol for Agrobacterium tumefaciens‐mediated transformation of Brassica napus mesophyll protoplasts is described. A strain with a neomycin phosphotransferase (nptII) gene and a KCS gene under control of a napin promoter was used at co‐cultivation. Transformed protoplasts were regenerated to fertile and morphologically normal transgenic plants. Transformants were confirmed by PCR of the nptII gene and NAP/KCS expression cassette, and Southern blot analysis. Seeds of the transformants showed a changed fatty acid profile: two transformants had a higher erucic acid level and differed significantly from that of B. napus. Genetic analysis of the progeny revealed that the kanamycin resistance introduced was inherited in a Mendelian fashion.     

Genetic analysis for haploid-regenerations responses of the hexaploid-wheat anther cultures

Genetic variability in response to anther culture was investigated in 49 winter hexaploid wheats, comprising 33 pure lines (F₁₀) derived from a composite cross programme and their 16 parental genotypes. All genotypes were grown in a randomized block design with three replications in a controlled greenhouse. The number of embryoids and total plant regeneration per 100 anthers, as well as the numbers of green and albino plants regenerated per 100 embryoids, were measured. Significant genetic variability was observed among the 49 genotypes for all the traits studied. All traits showed high heritability. Among the genotypes compared, DC²30N and 1BPT-40 gave the best results for the production of embryoids and IBPT-78 had the highest value for the production of green plants. The genotype IBPT-34 developed a large number of albino plants, and it should be useful as a parent in studies to determine the genetic control of albino plants in wheat.     

The induction of tetraploidy in chicory (Cichorium intybus L. var. Magdebourg) by protoplast fusion

Summary Fusions of chicory leaf protoplasts were realized in order to obtain a higher number of tetraploid plants compared to the traditional doubling techniques with colchicine. Thanks to the fusion technique, using a mixture of PEG, DMSO and a solution of CaCl₂ (pH 10.5), 25% tetraploid (2n=36) plants were obtained. Among the 167 regenerated plants, only one aneuploid of a near tetraploid level (2n=33) was identified.     

Improvement of somatic embryogenesis and plant regeneration from durum wheat (Triticum turgidum var. durum Desf.) scutellum and inflorescence cultures

Scutellum and inflorescence explants of four genotypes of durum wheat(Triticum turgidum var. durum Desf.) were used to define culture conditions to obtain high frequencies of embryogenesis and plant regeneration in vitro. Under all conditions tested, scutellum cultures gave higher frequencies of embryogenesis and plant regeneration than inflorescence cultures. Two different auxins, 2,4-D(2,4-dichlorophenoxyacetic acid) and picloram(4-amino-3,5,6-trichloropicolinic acid), were compared for their effect on scutellum and inflorescence explant response in vitro. Picloram was found to significantly increase the frequency of plant regeneration from both explants. When cultures were grown on regeneration medium containing zeatin for two three-week passages, the frequency of plant regeneration increased by between 20–30% compared with cultures exposed to hormones for a single three-week passage. Finally, the addition of 1 mg/l 6-BAP (6-benzyl aminopurine) to the plantlet growth medium was found to enhance tiller production in regenerants. The optimized culture conditions were applicable to the four genotypes tested and frequencies of plant regeneration varied between 97% to 100% for scutellum cultures (2 mg/l picloram in induction medium) and between45% and 80% for inflorescence cultures (4 mg/l picloram in induction medium). The number of plants regenerated per explant was improved over previous procedures, with means of 34 plants per scutellum, and 16 plants per inflorescence explant. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production and molecular characterization of Citrus intergeneric somatic hybrids between red tangerine and citrange

Somatic hybridization has been an effective and successful technique for plant improvement. In this paper, embryogenic callus protoplasts of red tangerine (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from citrange (C. sinensis × P. trifoliata, a Chinese local strain) in an effort to produce complementary tetraploid citrus rootstocks. Regenerated embryoids grew slowly and were vulnerable to browning. Twelve plants were finally regenerated, rooted and transplanted into a greenhouse. Root‐tip chromosome counting of five randomly‐selected plants revealed most cells were tetraploid (2n = 4x = 36), but aneuploid cells also existed. Flow cytometry analysis further confirmed their tetraploid nature. Nuclear simple sequence repeat (SSR) analysis verified their hybridity. Further mitochondrial genome analysis by restriction fragment length polymorphism and cleaved amplified polymorphic sequence revealed their mtDNA banding pattern was identical to that of red tangerine, the embryogenic callus parent; while their chloroplast DNA inheritance was random as revealed by chloroplast SSR analysis, in addition to cpDNA co‐existence detected in one plant. Cytological and molecular analysis indicated that somatic hybrid plants between red tangerine and citrange had been successfully obtained.     

Progress made in FEC transformation of cassava

In cassava friable embryogenic callus (FEC) has been used to obtain transgenic plants using particle bombardment, electroporation, and Agrobacterium tumefaciens. FEC cultures have been obtained in 6 of the10 tested genotypes. In all genotypes FEC could be regenerated into plants,however the efficiency differed between the genotypes. Almost all plants regenerated from 6 months old FEC cultures of TMS604444, Adira 4,Thai 5 and M7 were morphological similar to control plants. However, in R60 and R90 a large number of plants were not identical to control plants. Older FEC lines of TMS60444 have a reduced ability to regenerate plants and the plants show somaclonal variation. Somaclonal variation is observed in the same extend in transgenic and non-transgenic plants. The origin of this variation is both genetic and epigenetic. Luciferase based selection is less efficient in producing transgenic lines than chemical selection. Furthermore Agrobacterium tumefaciens mediated transformation is much more efficient than particle bombardment with respect to the production of transgenic lines. A tentative model is introduced which best describes the effect of different selection regimes on the time period required to produce transgenic plants. Kanamycin and stringent luciferase selection required a shorter period of time than selection based on hygromycin, phosphinothricin or non-stringent luciferase. However, a more significant reduction of time was obtained if young instead of old FEC lines of genotype TMS60444 were used for genetic modification. In accordance to the model these young FEC lines of TMS60444 produced transgenic plants within 4 months with both Agrobacterium tumefaciens combined with kanamycin selection and particle bombardment combined with stringent luciferase selection. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variation in Crown and Root Organic Reserves Among Lucerne Genotypes of Different Morphology and Flower Colour

Previous evidence indicates that differences in the concentration of underground organic reserves can drive the survival and growing ability of lucerne under cold and defoliation stresses. In order to provide the selection process with further information on compounds that may influence plant performance under grazing, we assessed variations in cold‐season concentrations of nitrogen and carbon reserves on genotypes that had been identified for morphological features that possibly enhance grazing tolerance. The selected genotypes encompassed distinct morphological patterns (defined as ‘models’) and different taxa within the Medicago sativa complex, as shown by different flower colours. Crown concentrations of reserves were determined on 90 genotypes, whereas root concentrations were measured on a subsample of 15 genotypes. Wide intergenotypic variation was observed for all reserve substances. Comparisons among models and among flower colour classes highlighted the high concentrations of crown carbohydrates and root and crown‐soluble proteins of the model coded as ‘D1’, characterized by prostrate, rhizomatous habit and long dormancy, which largely corresponded to plants with yellow or variegated flowers, typical of ssp. falcata and × varia, respectively. There was a strong ‘flower colour × storage organ’ interaction for sugar concentration, and the results suggested a preferential compartment of sugars in the roots of purple‐flowered genotypes that belonged to the ssp. sativa. A rank correlation analysis indicated a positive relationship between persistence after two years under grazing of half‐sib progenies deriving from 19 genotypes out of the 90 and crown concentrations of carbohydrates of the 19 mother plants.