Analysis of tissue culture-derived variation in pea (Pisum sativum L.)-preliminary results

Summary The possibility of producing agronomically-useful somaclones via organogenesis and somatic embryogenesis from callus cultures of pea (Pisum sativum L.) was studied. Organogenic calli were induced from immature leaflets on MSB medium with NAA and BAP. Embryogenic calli were derived either from immature zygotic embryos (using 2,4-D) or from shoot apices (using picloram) of aseptically-germinated seedlings.The seed progenies (T₁ to T₃-generation) of primary regenerants were grown in field conditions and their phenotypic variation was evaluated and compared with control, non-tissue culture-derived plant material. In addition, electrophoretic analyses of selected isoenzyme systems and total proteins have been done. The results do not show dramatic changes in qualitative and quantitative traits. The evaluation of at least two future generations (T₄, T₅) is planned.Abbreviations BAP 6-benzylaminopurine - IBA indole-3-butyric acid - MSB medium (mineral salts after Murashige & Skoog, 1962, vitamins after Gamborg et al., 1968) - NAA -naphthalene-acetic acid, picloram-4-amino-3,5,6-trichloro picolinic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - ORG organogenesis - SE somatic embryogenesis     

Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz)

An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 1037antibiotic resistant callus lines, of which 526 showed GUS expression. Of the 241 callus lines that were transferred to maturation medium 219formed somatic embryos. Thirty-seven of the 38 lines that were transferred to germination medium produced plants. GUS-positive plants could be obtained from 31 lines; in 14 of those lines 100% of the produced plants were GUS-positive, the remaining 17 lines yielded GUS-positive plants at an average of 72%. The transgenic nature of these plants was confirmed by Southern blot analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Introduction of Transgenes to Control Blackheart in Pineapple (Ananas Comosus L.) cv. Smooth Cayenne by Microprojectile Bombardment

Summary A transformation technique for the introduction of transgenes to control blackheart by particle bombardment has been developed for pineapple cv. Smooth Cayenne. Leaf callus cultures capable of high frequency organogenesis with a short regeneration time were used as explant material. Gus and gfp reporter genes were used to observe and determine transient and stable expression. The ppo gene, isolated from pineapple, was introduced to control blackheart. Co-transformation occurred with constructs containing the nptII gene conferring geneticin resistance. We have recovered 15 independent transgenic gus and gfp lines each from 8 separate experiments and 22 ppo lines from 11 experiments. Gus, gfp, ppo and nptII positive plants have been regenerated, which have been shown by Southern blot analysis to be stable transgenics containing multiple copies of the introduced genes. These results show that biolistic gene delivery in pineapple can be successfully achieved at an acceptable efficiency of 0.21–1.5% for genetic improvement of ’Smooth Cayenne’, the industry standard throughout the world.     

Selection of a highly-regenerative genotype of white clover (Trifolium repens L.) and plant regeneration from protoplasts derived from this genotype

Summary Callus cultures were induced from hypocotyl sections of 24 varieties of white clover (Trifolium repens L.). The calli did not show any significant difference of growth among the varieties. After the calli has been transferred to three regeneration media, green-spot formation was observed on calli derived from some seedlings. Remarkable intra- and intervarietal variations in the emergence of green spots and some trends between the origin of varieties and the frequency of green spots were observed. In most cases, the green spots turned brown without showing further differentiation, and only two genotypes formed shoots. A callus from a seedling of the Swedish variety Undrom has sustained high levels of plant regeneration throughout 24 months of culture. Protoplasts derived from this selected genotype were divided into cell colonies. 8P (Kao, 1977) medium containing 0.5 mg/1 2,4-D and 0.5 mg/1 kinetin was the most suitable medium for inducing divisions in protoplasts. When subcultured into solid B5 medium, the colonies produced calli, which when transferred to a regeneration medium, formed shoots. This genotype is expected to a useful subject for genetic engineering of white clover.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2ip 6-, -dimethylallylamino purine - NAA -naphthaleneacetic acid     

Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants

Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for -glucuronidase (GUS).Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T₀). This chimeric plant passed the transferred nptII gene to its T₁ progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T₀ spikes and 15 T₁ offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T₂ and T₃ plants were produced by isolating embryos from green grains of transgenic T₁ and T₂ plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T₂ offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin ^R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.     

Intergeneric symmetric and asymmetric somatic hybridization in Festuca and Lolium

Summary Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10–500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.Species-specific sequences from F. arundinacea and L. multiflorum being dispersed and evenly-represented in the corresponding genomes were isolated and used for the molecular characterization of the nuclear make-up of the intergeneric, somatic Festulolium plants recovered. The irradiation of Italian ryegrass protoplasts with 250 Gy X-rays prior to fusogenic treatment favoured the unidirectional elimination of most or part of the donor chromosomes. Irradiation of L. multiflorum protoplasts with 500 Gy produced highly asymmetric (over 80% donor genome elimination) nuclear hybrids and clones showing a complete loss of donor chromosomes.The RFLP analysis of the organellar composition in symmetric and asymmetric tall fescue (+) Italian ryegrass regenerants confirmed their somatic hybrid character and revealed a bias towards recipient-type organelles when extensive donor nuclear genome elimination had occurred.Approaches aimed at improving persistence of ryegrasses based on asymmetric somatic hybridization with largely sexually-incompatible grass species (F. rubra and Alopecurus pratensis), and at transferring the cytoplasmic male sterility trait by intra- and inter-specific hybridization in L. multiflorum and L. perenne, have been undertaken.Abbreviations cpDNA chloroplast DNA - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxy-acetic acid - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Plant regeneration from callus culture in potato

Summary A procedure for plant regeneration from callus culture of potato, Solanum tuberosum L. is described. Calli were induced from 1–2 mm long shoot apices of potato cultivars Cara and A25/19 on half-strength Murashige and Skoog's medium (half-MS) supplemented with 3.2 mg IAA (indole-3-acetic acid), 1.0 mg kinetin (6-furfurylamino)purine], and 0.5 mg 2,4-D [2,4-dichlorophenoxy)acetic acid]/1. Sixty percent explants produced nodular calli on this medium within 30 days. Calli differentiated into shoot-primordia when subcultured on half-MS medium supplemented with 0.5 mg 2,4-D and 1.0 mg zeatin [6-(4-hydroxy-3-methybut-2 enylamino)amino purine]/1. Differentiated calli on half-MS medium without growth hormones produced complete plantlets which were cloned on the same medium and transferred into soil.     

Biolistic introduction of a synthetic Bt gene into elite maize

Summary A synthetic Bt gene encoding a truncated version of the CryIA(b) protein derived from Bacillus thuringiensis was successfully introduced into elite maize using microprojectile bombardment of immature embryos. The method used to initiate and identify transformation events is described. We describe the detailed parameters used for the Biolistics device as well as the plasmids used for the transformations. The plasmids contained the synthetic Bt gene driven by either the 35S CaMV promoter or a combination of two tissue-specific promoters, leaf and pollen, derived from maize. Specific conditions for the culture of Type I callus from immature embryos, the phosphinothricin (PPT) selection protocol, and the regeneration of plants are discussed. T0 and T1 plants were initially identified using the pH-dependent chlorophenol red test and/or the histochemical -glucuronidase (GUS) assay. PCR and Southern data confirm the presence of the 35S CaMV promoter and the synthetic Bt gene.     

Genotypic and exogenous factors affecting shoot regeneration from anther callus of linseed (Linum usitatissimum L.)

Summary The objective of this study was to investigate factors affecting the regeneration capacity of linseed anther culture. Four different environmental conditions in a phytotron were tested with regard to their effects on anther donor plants of cv. Hella. Anther response and shoot regeneration from anther callus was maximal when donor plants were grown in a 16 hrs-day at 14°C day/8°C night temperature. Anthers of four linseed genotypes were cultured on different media. Maximum shoot regeneration was achieved when the induced calli were transferred onto a modified N6 medium containing zeatin (1 mg l^-1). Most of the calli regenerated shoots in the second subculture on regeneration media. Shoots were rooted on modified B5 or MS media containing NAA (0.1 mg l^-1). Cytological examinations of incubated anthers and root tips of regenerated plants indicated that the anther calli were derived from microspores.Abbreviations B5 Gamborg's (1975) medium - BAP 6-benzylaminopurine - 2,4D dichlorophenoxyacetic acid - N6 Chu's (1978) medium - NAA -naphthaleneacetic acid - MS Murashige & Skoog's (1962) medium - ZEA zeatin     

Improved frost tolerance and winter survival in winter barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by <Emphasis Type="Italic">in vitro</Emphasis> selection of proline overaccumulating lines

Embryogenic calli derived from anther cultures of the two-rowed winter barley cultivar Igri were plated on solid L3 medium containing the proline analogue hydroxyproline (Hyp), 10–20 mmol l^–1. Exposure to Hyp caused severe degeneration of most of the calli. Hyp resistant calli, distinguishable by their lighter colour and higher growth rate, and control calli not exposed to Hyp were plated on L3 regeneration medium. From 22,500 anthers exposed to Hyp 46 Hyp resistant regenerates were obtained, which were transferred to soil. After cultivation for 5–10 weeks at normal growth conditions they were cold hardened at 2 C under short day conditions together with control regenerates. Frost tolerance assays with segments of fully grown leaves of unhardened and cold hardened plants revealed that Hyp resistant regenerants were significantly more frost tolerant than the control regenerants. Improved frost tolerance was found also in the progenies R₁ to R₉, and genotypic segregation in the R₁ generation in a 1:2:1 ratio was indicated. Increased proline content was observed in the R₂ generation and in subsequent generations and was significantly (P 0.001) correlated with increased frost tolerance in the Hyp lines. Comparative studies of R₉ progenies from homozygous R₂ plants with the wild type Igri under field conditions in winter at three locations in Europe as well as crossing experiments confirmed the heritable improvement of frost tolerance and winter survival, respectively, in the Hyp lines. The results support the hypothesis that proline accumulation in cold acclimated winter barley plants is causally related to the acquisition of frost tolerance. Moreover, the described biotechnological procedure may be applicable in breeding programs for improved winter hardiness and possibly also for other stress tolerances.     

Selection and characterization of a 5-methyltryptophan resistant mutant in Zea mays L.

Summary A maize plant resistant to 5-methyltrytophan (5MT) was selected from M₂ seeds (Zea mays L. Danggin, inbred line) originating from ears treated with ethylmethane sulfonate (0.2%) at 6 hr after self-pollination. Genetic analysis of the progeny of plants selected from a medium containing 50 ppm 5MT showed that 5MT resistance was inherited as a single dominant nuclear gene. This resistance was also expressed in callus and seedling. Analysis of the free amino acids in kernels and calli showed that homozygous resistant plants (MR1) contained higher levels of total free amino acids than sensitive plants and calli. In particular, the their kernels the levels of tryptophan, threonine and serine were, respectively, 4.5, 5.9 and 6.3 times higher than those of the sensitive plants. From the results, it may be expected that mutants resistant to amino acid anologs will be useful not only for studying amino acid biosynthesis but also for improving the nutritional quality of maize.Abbreviations EMS Ethylmethane sulfonate - 5MT 5-methyltryptophan - 2,4-D 2-4-dichlorophenoxyacetic acid     

An assessment of morphogenic and transformation efficiency in a range of varieties of potato (Solanum tuberosum L.)

Summary To provide a truly genotype-independent transformation system, it is necessary to be able to transform a wide range of potato genotypes. The ability to regenerate shoots in vitro was determined for 34 potato varieties using tuber disc explants. Following a culture regime used extensively in previous studies with the variety Desiree, half of the varieties could be regenerated from tuber discs and half could not. From a sample of varieties that could be regenerated from tuber discs, all but one variety gave transgenic plants. Twelve varieties were evaluated for the capacity to regenerate shoots from leaf and internode explants excised from in vitro grown plants. All of the varieties tested regenerated adventitious shoots. Leaf and internode explants from 5 varieties were subsequently used for transformation, and transgenic plants were produced from two potato varieties that did not give transgenic plants from tuber disc explants. Some varieties could not be transformed by either method, and will require modification of the in vitro regeneration and transformation system to be successful.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion

Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.     

Plant regeneration from immature inflorescence callus cultures of wheat,rye and triticale

Summary Callus cultures were initiated from inflorescence explants of wheat, rye and triticale on MS medium supplemented with 2 mgl^-1 2,4-D+5% CW or 2 mgl^-1 2,4-D+0.5 mgl^-1 BA. On transfer of the cultures to medium supplemented with 15% CW+0.2 mgl^-1 NAA or 1 mgl^-1 BA+0.1 mgl^-1 IAA, shoot buds and embryoids were produced. Full fledged plantlets obtained on MS medium supplemented with NAA were transferred to the field. Cytological analysis showed the plants to be diploid. However, the regenerated plantlets were shorter, produced fewer tillers and had lower fertility compared to the control.Abbreviations BA Benzyladenine - CW coconut water - IAA indoleacetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Screening wild Lycopersicon species for resistance to powdery mildew (Oidium lycoperiscum)

Since the late 1980s powdery mildew, designated Oidium lycopersicum, frequently invaded the tomato crop in Western Europe. All commercial cultivars are susceptible. To screen for resistance in wild species a reliable and efficient disease test was developed. Young plants with two to three true leaves are inoculated at high relative humidity by spraying with a freshly prepared suspension of 2×10⁴ conidia, ml^–1. Symptoms are periodically evaluated according to a scale based on the percentage of leaf area with mycelium.One hundred and twenty seven accessions, representing eight wild Lycopersicon species, were screened for resistance to O. lycopersicum. A large variation in resistance was found between species. L. hirsutum was the most resistant species; L. pennellii was moderately resistant; species of the subgeneric group of L. esculentum and of the peruvianum-complex were all susceptible. L. parviflorum was classified separately due to a large variation between accessions. Except for this species, a low variation was found between accessions within species. High levels of resistance were observed in four accessions of L. hirsutum, in one of L. parviflorum and in one of L. peruvianum. This resistance is characterized by a very low disease incidence and a strongly restricted mycelium growth and lack of sporulation.     

Callus induction and plant regeneration from anther and inflorescence culture of Sorghum

Summary Twenty-five inbred lines, including grain and forage types from the USA and China, two hybrids, one Sorghum almum, and one Parasorghum (S. versicolor) were tested for their response to anther culture. Three nutrient media were effective in inducing anther calli from six cultivars (Xin White, TX 403-TSB, DDY Sommer Milo, TX 2779, Brawley, and Spur Federal) and one was effective for plant regeneration for one cultivar, Xin White. Averaged over media, callus induction frequency (number of calli per 100 anthers) was highest in cultivars Xin White and TX 403-TSB (6.7 and 3.9%, respectively). The means of cultivars for media C17-2 and Ms-t-z-2, 4.3 and 3.2%, respectively, were superior to that for medium 85D3-2 (0.1%). Expressed as an average of the six cultivars and three media the mean calli induction frequency was 2.6%; however, differential responses of genotype and medium were noted. Among the 10 regeneration media tested, medium MS-d-4 containing Murashige and Skoog basal components plus 2.0 mg/l indole-3-acetic acid (IAA) and 2.5 mg/l kinetin was the most effective for plant regeneration. Numbers of albino plants and calli developing only roots increased directly with callus-induction time, whereas the frequency of plant regeneration decreased. Regenerated plants had varied numbers of chromosomes in root tip cells: 10, 15, 20, 40, and 60. The 29 regenerated plants that reached maturity, however, were highly fertile and contained only 10 bivalents in pollen mother cells. Normal chromosome number and behavior for the regenerated plants suggest that induced calli originated from cells other than microspores. However, spontaneous chromosome doubling in microspore-derived haploids may occur. The appearance of albinos also implies that haploids may have been produced from anther culture.Joint contribution of the Dept. of Agronomy and USDA-ARS, Kansas Agricultural Experiment Station, Manhattan, KS 66506-5501, USA. Contribution no. 88-566-J.     

In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. II. Culture filtrate technique and inheritance of Fusarium-resistance in the somaclones

Summary Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R₃. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R₀) than the non-selected ones. Among 18 R₁ lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R₃ lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R₃ selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.     

Interspecific hybrids of Cucumis metuliferus × C. anguria obtained through embryo culture and somatic embryogenesis

Summary Interspecific crosses between Cucumis metuliferus Naud. and C. anguria L. were obtained through embryo culture. Embryos in the rabbit-ear to advanced fluke-shaped stages were rescued 34–99 days after pollination. Plants were obtained through direct embryo culture, and through somatic embryogenesis from immature embryos. For direct embryo culture, fluke-shaped embryos were stored in sterile water in darkness for three days at 25C prior to transfer on Murashige and Skoog (MS) culture medium plus 1.0 M 6-benzylamino-purine. Multiple plants were obtained from single embryos through somatic embryogenesis of rabbit-ear stages on MS plus 10 M indole-3-acetic acid and 5 M 6-benzylamino-purine. Evidence of hybridization included leaf shape intermediate between the two parents, penduncle shape prior to fertilization which resembled the male parent, low pollen viability and isoelectric focussing of protein bands for acid phosphatase of leaf extracts.     

High frequency somatic embryogenesis with a pampeana-derived genotype of alfalfa (Medicago sativa L.)

Summary Somatic embryos of genotype R11 of the alfalfa variety Pampeana were produced from embryogenic calli derived from leaf sections. They were induced by an auxin shock and its development was attempted on six different media. The best condition for somatic embryo production was inducing callus on MS medium plus 10 M 2,4-D and 4,6 M KIN and transferring them, after the auxin shock, to MS with 10–20 mM NH₄ ⁺ and 30 mM proline. More than 500 somatic embryos per plate were produced. Embryos were grown to plants on MS or half strength MS media and all regenerated plants resembled the original R11 genotype. This technique could be useful in alfalfa Pampeana improvement using genetic modification.