Molecular analysis of plants regenerated from embryogenic cultures of hybrid sugarcane cultivars (Saccharum spp.)

Summary The genomic stability of tissue culture regenerants of sugarcane (Saccharum spp. hybrids, cvs CP721210, CP68-1067 and B43-62) was analyzed by DNA restriction fragment length polymorphism (RFLP). Plants regenerated from calli, cell suspensions, cryopreserved cell suspensions and protoplasts were used. Total DNA isolated from 19 different sources was digested with EcoRI, HindIII, BamHI, BamHI, EcoRI and PstI and probed with six known maize mitochondrial genes (coxI, coxII, atpA, atp6, atp9 and rrn18-rrn5), three random maize mitochondrial cosmid clones, two random maize chloroplast cosmid clones and a wheat Nor locus clone. Hybridization patterns indicated that the variation observed was minor and appeared only in the secondcycle regenerants. No differences were observed among the three cultivars and the regenerants from calli, suspension culture, cryopreserved suspension culture and protoplasts. Mitochondrial DNA (mtDNA) isolated from CP72-1210 plants and its embryogenic cell suspensions, and bulk samples from all CP72-1210 regenerants pooled together were digested with EcoRI, HindIII, PstI, BamHI and SalI and probed with three recombinationally active wheat mtDNA clones, K, K3 and X2. No variation in the mtDNA restriction patterns was observed between the CP72-1210 plants and its regenerants. However, restriction pattern variation was observed only from EcoRI digestion, and hybridization patterns of K3, K and X2 revealed minor variations in the mtDNA of cell suspensions when compared with the DNA of the CP72-1210 plant. Except for a qualitative variation detected by the X2 probe and minor stoichiometric variations detected by the K3 probe, sugarcane DNAs were found to be stable after plant regeneration.Florida Agriculture Experiment Station Journal Series No. R-02703     

Mitochondrial DNA variation in long-term tissue cultured rice lines

Summary The effects of long-term tissue culture on mitochondrial DNAs were examined using rice (Oryza sativa) cell suspension cultures. Mitochondrial DNAs were isolated from P. I. 353705 (an indica subspecies of rice similar to Asam 5), its anther-culture-derived line BL2 (an 8-year-old cell suspension culture), and five other cell lines (A1, A7, A11, A13, and A23), also derived from BL2 and independently selected for resistance to the lysine analog, S-(2-amino)-ethyl-L-cysteine. Mitochondrial DNAs of the rice lines were digested with ten restriction endonucleases (BamHI, BglII, EcoRI, EcoRV, HindIII, PstI, PvuII, SalI, SmaI, and XhoI), electrophoresed, and transferred to nylon membranes. Southern blots were hybridized with one rice and five maize probes containing mitochondrial genes. The restriction patterns of ten Southern blots and hybridization patterns of 60 endonuclease/probe combinations were analyzed. DNAs from all sources produced unique restriction patterns when digested with HindIII or BglII; with the other endonucleases an array of similarities and differences was observed. Lines BL2 and A11 showed unique patterns with all restriction endonucleases tested. No hybridization pattern differences were observed among the lines when probes containing apt9 and atpA were used. However, extensive hybridization pattern differences were observed with coxI, coxII, rrn18-rrn5, and atp6 probes. Both restriction and hybridization patterns revealed variation due to tissue culture effect. Coxll was most efficient in revealing the uniqueness of BL2. Among the analog selected lines A11 was most divergent, and probes rrn18-rrn5 and atp6 were most efficient in revealing its distinctiveness. Unique mitochondrial genomic organizations were found to be associated with long-term tissue culture.Florida Agricultural Experiment Station Journal Series No. R-00213     

Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.     

Is Nor region variability in wheat invariably caused by tissue culture?

Summary In a previous study we observed extensive Nor region variability in tissue-culture derived plants of only one out of three tested wheat cultivars. This finding prompted us to further question whether or not this variability was invariably caused by in vitro culture. In the present study, the upper halves of spikes from four source plants of the inbred cultivar ND7532 were removed 12 days after anthesis. The immature embryos from these halves were cultured and regenerated into plants. The lower halves of the same spikes were retained on the plants to obtain mature caryopses. DNA was extracted from seedlings, cut with TaqI endonuclease, run on agarose gels, and the respective Southern blots were probed with the plasmid pTA71 to reveal the Nor region patterns. The sexual progeny of regenerants from three out of four source plants derived from the immature embryos provided Nor region patterns which were exactly identical to the patterns obtained from seedlings which germinated from the caryopses matured on the respective source spikes. The regenerants from the fourth source plant provided variable Nor region patterns. Analyses of the Nor region patterns of 21 individual seedlings germinated from caryopses of this source plant showed that 18 had a three-fragment pattern (consisting of 3.0, 2.7 and 1.9 kb fragments) while three seedlings lacked one (2.7 of 1.9 kb) fragment. Furthermore, the next sexual progeny of the regenerants which had a three-fragment pattern further segregated into three- and two-fragment patterns.These results, in conjunction with previous reports on Nor region variability among tissue-culture derived plants, suggest that this variability is not invariably related to in vitro culture.     

Efficient regeneration system from wheat leaf base segments

Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 1 mg dm⁻³ naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm⁻³ kinetin (6.3 plantlets per segment).     

Cell culture induced introgression of Thinopyrum intermedium chromatin into common wheat

To introduce useful genes from the wild species Thinopyrum intermedium into cultivated wheat, a wheat-Th. intermedium disomic addition line (TAI27) was used as source material for tissue culture. TAI27 contains, beside the 42 wheat chromosomes, a pair of smaller chromosomes that is cytologically discernible. Based on restriction fragment length polymorphism (RFLP) analysis, this chromosome pair was determined to be a recombinant one, comprising segments with homoeology to at least two chromosome groups of wheat, i.e., group 2 and 7. Sixty-eight green plants were regenerated from six month-old embryogenic calli initiated from immature embryos of TAI27. Four of the plants were found to have only 42 cytologically normal-looking chromosomes. Southern blot analysis using a Th. intermedium-enriched repetitive probe showed that one of the plants had hybridization fragments specific to Th. intermedium, indicating introgression of chromatin during the cell culture process. This revised version was published online in June 2006 with corrections to the Cover Date.     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

Comprehensive molecular characterization of tissue-culture-derived Hordeum marinum plants

Summary Scuttelar calli of Hordeum marinum readily and efficiently regenerate functional plants. In order to assess genetic variability among the regenerants we employed multiple analytic tools, which included molecular and biochemical assays. Total DNA extract from regenerated plants was digested with at least two restriction enzymes and hybridized to four nuclear and six mitochondrial coding sequences, in addition to one nuclear and three mitochondrial noncoding probes. SDS-PAGE analyses of hordein extracted from seeds of regenerated plants and activity assays of -amylase were also performed. The nuclear and mitochondrial genomes of 50 regenerated plants demonstrated relative stability when assessed with coding sequences and by biochemical analyses. However, the mitochondrial noncoding probes revealed one qualitative somaclonal variant characterized by a loss of a hybridizing fragment. Moreover, changes in the methylation patterns of the rRNA genes and the nontranscribed spacer were revealed in another regenerated plant. The albino plant regenerated was characterized by a loss of three chloroplast DNA BamHI fragments.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

Biochemical and molecular analysis of plants derived from embryogenic tissue cultures of napier grass (Pennisetum purpureum K. Schum)

Summary We have investigated the extent of biochemical and molecular variation in 63 plants of napier grass (Pennisetum purpureum K. Schum.) regenerated from 3- to 24-week-old embryogenic callus cultures. The calli were derived from cultured basal segments of young leaves and immature inflorescences obtained from a single fieldgrown donor plant. The entire population was analyzed for the activity of 14 isozyme systems, but no qualitative variation was found at any of the loci examined. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in a representative sample of regenerated plants. Our results confirm earlier reports of the genetic uniformity of plants derived from somatic embryos and highlight their value both for clonal propagation and for genetic transformation.     

Induction of triploid Citrus plants from endosperm calli in vitro

Summary Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12–14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis.Florida Agricultural Experiment Station Journal Series No. R-00627     

Protoplast electrofusion between common wheat (Triticum aestivum L.) and Italian ryegrass (Lolium multiflorum Lam.) and regeneration of mature cybrids

Summary This study describes the development of electrofusion techniques using the ‘donor-recipient’ model for the production of cybrids between common cultivated winter wheat (Triticum aestivum L.) cv. Jinghua No. 1 and a phylogenetically remote, sexually incompatible grass species, Italian ryegrass (Lolium multiflorum Lam.), which belong to two different subtribes: Triticinae and Loliinae. Wheat protoplasts were metabolically inactivated by iodoacetamide before fusion, while protoplasts of Italian ryegrass were X-ray irradiated before protoplast isolation. The suspension cells were directly used to optizmize the inactivation parameters. By exploring the minimum irreversible membrane breakdown strength, the electrofusion parameters were optimized just a few minutes before electrofusion began. A total of 108 green plantlets were obtained, and about half of the green plants uncontrollably necrotized. Among all green plants, 14 were rooted normally and transplanted in growth chamber or field and developed to maturity. All these transplanted plants were male sterile with smaller and off-white anthers. Seeds were obtained by crossing with Jinghua No. 1. Three transplanted regenerants possessed the characteristics of glume facing the rachis, which was the taxonomic characteristic distinguishing the two subtribes of Triticinae and Lolliinae. Although Southern blot hybridization analysis of 33 randomly selected regenerants using a wheat ribosomal DNA probe (pHA71) did not find any differences to wheat, analysis using two mitochondrial probes B342 (cox l), 490 (Pro II) and one chloroplastidic probe pHve H5 revealed that 31 plants were ‘true cybrids’ by showing ryegrass-specific band(s) or new band(s). It also showed that the mitochondria and chloroplasts were not coexistent as the restriction fragment length polymorphism band of Italian ryegrass was not detected by the mitochondrial probes 7 (26s), B342 (cox I), pHJ2-7-1 (cox II), B30 (atp9), and the chloroplast probe pHvc P5. To regenerate the cybrids, the regeneration capacity of the recipient (wheat) was crucial in this study.     

Embryogenesis and Regeneration of Green Plantlets from Wheat (Triticum aestivum) Leaf Base

A short-term regeneration system from leaf-base-derived callus of wheat (Triticum aestivum L.) was developed. Embryogenic callus formation and shoot regeneration were achieved from the first basal segments of 3–4-day-old seedlings. Callus formation frequency as well as plantlet regeneration frequency was dependent on the composition of basal medium and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). MS medium with 2,4-D 4.5–9.0 mol l^–1 was optimal for the culture of wheat leaf base. Effects of different combinations of plant growth regulators, which were added in either callus induction medium or shoot regeneration medium, were tested. Adding of BAP in callus induction medium shortened the time of shoot emergence but could not improve the producing of embryogenic calli and green plantlets. Optimal ratio of 2,4-D, BAP and NAA gave similar regeneration frequency to control. Existence of cytokinins in regeneration medium had no effect on increasing the regeneration frequency. The regenerants could grow to normal, fertile plants after they were transferred into soil.     

Increased NaCl-tolerance in wheat (Triticum aestivum L. andT. durum desf.) through in vitro selection

Summary Callus cultures were initiated from immature embryos of oneTriticum aestivum and threeT. durum cultivars. Growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.5, and 0.7%) added to the culture medium during two subsequent subcultures (4 wk each). The growth rate of the calli was determined by the relative fresh weight callus growth (RFWCG). The callus growth of all investigated genotypes was slightly changed in the presence of 0.3 and 0.5% NaCl, but strongly inhibited by 0.7% NaCl. Selected NaCl-tolerant clones were isolated and plants were regenerated on MS-based regeneration medium without NaCl. The regeneration capacity of the selected calli was highly reduced compared to the control. The highest number of regenerants was scored for cv. Gladiator (T. aestivum). All regenerated plants were morphologically normal and many developed to maturity and set seeds. Seedlings from the R₁ generation of selected and control plants were treated with 0.5% NaCl in vivo in liquid cultures for 6 wk. Salt tolerance of the progenies of selected plants appeared in all cultivars, but those derived from calli grown on medium with 0.7% NaCl showed the highest survival rate.T. aestivum showed higher tolerance to NaCl salinity thanT. durum.     

Organellar segregation,rearrangement and recombination in protoplast fusion-derived Brassica oleracea calli

Summary Cauliflower protoplasts were fused to determine the effect of protoplast source and pretreatment on organellar segregation in fusion products. Mitochondrial and chloroplast type were determined for over 250 calli from eight fusions between iodoacetate-treated or -irradiated leaf or hypocotyl protoplasts with fertile or Ogura cytoplasms. Organelles in fusion-derived calli were identified with five mitochondrial probes and one chloroplast probe. Mitochondrial and chloroplast segregation were independent but biased. Most calli had B. oleracea chloroplasts, but more calli had Ogura mitochondria than B. oleracea ones. Neither protoplast source nor pretreatment alone affected organelle segregation. However, iodoacetate treatment of hypocotyl protoplasts reduced their mitochondrial contribution to the fusion products although it did not affect chloroplast segregation. Over half of the calli had mitochondrial genomes distinct from those of either fusion partner; many of these contained the complete mitochondrial genome of one partner along with some mitochondrial DNA from the other. Out of 258 calli, 83 showed evidence of mitochondrial recombination, most commonly by formation of a novel 11-kb PstI fragment near the atp9 region.     

Inheritance of mitochondrial DNA in the conifer Larix

Summary Restriction fragment length polymorphisms between Larix leptolepis and Larix decidua were identified in heterologous hybridization experiments, using wheat mitochondrial DNA probes specific for atp9, coxI, nad3/rps12, and orf25. Analysis of eight individuals of each reciprocal hybrid of these two species revealed that mitochondrial DNA was maternally inherited. Furthermore, sequences homologous to wheat orf25 were also identified in Larix gmelini, Larix siberica, Larix olgensis, and Larix laricina, as well as Ginkgo biloba, Picea mariana, Picea glauca and Pinus contorta.     

Characteristics of the interspecific somatic hybrids <Emphasis Type="Italic">Solanum pinnatisectum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

The morphological, cytological and molecular analyses of the plants regenerated after PEG-induced fusion between mesophyll protoplasts from the dihaploid potato clone H-8105 and the wild tuberous disease-resistant species S. pinnatisectum, were performed. A single fusion experiment yielded 313 calli, although only two calli produced shoots. From the rooted shoots, two stable clones (PT-01-1 and PT-01-2) exhibiting different vigor and habitat, were developed. The plants of PT-01-1 clone grew slowly in vitro, produced tubers after transfer to soil but did not set flowers. In contrast, the plants of the vigorous clone PT-01-2 produced both tubers and flowers after transfer to soil. The flower and tuber morphology of PT-01-1 and PT-01-2 regenerants was intermediate in comparison to the parental species. Cytological analysis revealed that the PT-01-1 clone was pentaploid and the PT-01-2 clone was tetraploid. The molecular (RAPD) analysis confirmed hybridity of both clones. The preliminary tests on late blight resistance of the hybrids showed no differences with a potato parent.