Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice

Summary Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.Florida Agricultural Experiment Station Journal Series No. 8807. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable     

Growth and regenerability of long-term suspension cultures of the U.S. rice cultivar Mercury

Suspension cultures of the U.S. rice cultivar Mercury have been maintained in modified General Medium for more than 3 years. These suspensions have continued to have high and relatively stable regeneration rates. Two different explants, immature panicles and seeds, were compared during the development of these embryogenic suspensions. Initial formation of secondary embryogenic callus from immature panicles on induction medium was greater than that from seeds. Suspensions of these two cell lines, however, did not differ morphologically and maintained similar regeneration rates. After 5 months in culture the rates of regeneration began to decline. The suspensions were plated onto regeneration medium without growth regulators for 2 weeks and then embryogenic cells were manually selected and used to develop secondary suspensions. Through this simple rejuvenation procedure, the suspensions retained high and stable regeneration rates. Variability in suspension growth, however, was observed during the culture period. Slower growth occurred at weeks 13, 15, 27, and 29 and was associated with a decrease in regeneration rates. Reproductive fertility of regenerated plants remained high for 3.5 years but then declined.Abbreviations CH casein (acid hydrolysate) - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige & Skoog basal medium - SE standard error     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

An improved technique for culture of rice panicles

An improved technique for long-term culture of rice caryopses is necessary for physiological and genetic studies. Panicles of three rice (Oryza sativa) cultivars `Lemont', `Gummo-byeu' and `Hwasung-byeu' were cultured in liquid media with combinations of light versus dark, panicle position, nitrogen level (5-40 mM), and sucrose level (29–351 mM). Grain growth was increased when panicles were positioned horizontally, partially submerged in the media, owing to greater media contact and apoplastic uptake as observed by fluorescent dyes. The optimal media assimilate supply included 175 mM sucrose and 5 mM nitrogen. Grain fill occurred for up to four weeks; grain dry weight reached 80% of that on intact plants, with 50% germination. This technique should allow for future physiological studies with rice or other panicle-bearing species.     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

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     

Accessing genes in the tertiary gene pool of rice by direct introduction of total DNA fromZizania palustris (wild rice)

Transfer of useful genes from wild relatives of crop plants has relied upon successful conventional crossing or the availability of the cloned gene. Co-bombardment of rice callus with total genomic DNA from wild rice (Zizania palustris) and a plasmid containing a gene confirming hygromycin resistance allowed recovery under selection of transgenic plants with grain characteristics from wild rice. Amplified Fragment Length Polymorphism (AFLP) analysis suggested that a significant amount of DNA fromZizania was introduced by this procedure. One plant had 16 of a possible 122Zizania specific AFLP markers detected with the primers used. This approach may have potential for introgression of genes from wild relatives in other cases where highly efficient transformation methods are available.     

Genotypic effects on the callus formation from different explants of rice,Oryza sativa L.

A total of 108 rice varieties were examined for their tissue culture responses. Callus tissues were initiated from the seed, radicle, coleoptile and anther explants. Our results indicated that genotypes differed in the ability to develop vigorously growing callus. The callus growth responses in seed, radicle and coleoptile cultures were intercorrelated, but were not correlated with that in anther culture.     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

A comparison of methods for callus culture and plant regeneration of RD25 rice (Oryza sativa L.) in two laboratories

While methodology is transferable from one laboratory to another, an exact transfer does not usually occur and even a nearly exact transfer of methods does not always result in repeatable data. Researchers should not expect that an effort to duplicate a published procedure will necessarily lead to identical results.In attempting to transfer rice tissue culture methods between laboratories in Fort Collins, Colorado, USA and Bangkok, Thailand, we discovered that a combination of the methods of each laboratory produced the best results in term of callus productions and plant regeneration. In the experiments reported here, the type of culture vessel used and the geographical location were also important variables.Supported by the USAID/Cooperative Agreement No DAN-4137-A-00-4053-00.     

PFGE analysis of the rice genome: estimation of fragment sizes,organization of repetitive sequences and relationships between genetic and physical distances

Pulsed-field gel electrophoresis (PFGE) has been applied to analyze the rice nuclear genome. Probing 56 RFLP probes selected from the 12 rice chromosomes to PFGE blots of nine rare-cutting restriction enzymes revealed that there are relatively high numbers of rare-cutting restriction sites in the rice genome. The average sizes of restriction fragments detected by single-copy probes are smaller than 200 kb for all of the rare-cutting restriction enzymes examined. Sizes of fragments detected by repetitive probes are variable, depending on the probes analyzed. By using PFGE, a tandemly repeated sequence, Os48, was found to be tightly linked to telomeric tandem repeats but not physically linked to r5s genes with which sequence homology had been observed. Relationships between genetic and physical distances have been established for three different chromosomal segments. In these regions 1 cm corresponds to ca. 260 kb on average. Analysis of a cluster of RFLP markers on chromosome 3 revealed that genetically clustered RFLP markers are also physically closely linked, suggesting that clustering of genetic markers may result in part from uneven distribution of single-copy sequences.     

High frequency plant regeneration from rice protoplasts by novel nurse culture methods

Summary Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.     

Mapping QTLs that control the performance of rice tissue culture and evaluation of derived near-isogenic lines

Quantitative trait loci (QTLs) that control the performance of tissue culture in rice were detected by using 116 RFLP markers and 183 BC₁F₃ lines derived from two varieties, Koshihikari and Kasalath. With time, the seed callus of Koshihikari tends to turn brown and stop growing, while that of Kasalath remains yellowish-white and proliferates continuously. The performance of tissue culture in the induction of calli from seed, the subculture of induced calli, and shoot regeneration were evaluated by five indices: induced-callus weight, induced-callus color, subcultured-callus volume, subcultured-callus color, and regeneration rate. Through callus induction and subculture, eight putative QTLs (P < 0.001) were located on chromosomes 1, 4, and 9. Among these QTLs, five Kasalath alleles and three Koshihikari alleles improved tissue culture performance. No QTL for regeneration was found. Among all the QTLs, qSv1 explained the largest phenotypic variance, 33%, in subcultured-callus volume. In induced-callus color, two detected QTLs accounted for 36.4% of the total phenotypic variance; this was the highest score among the five indices used to evaluate the performance of tissue culture. Three near-isogenic lines for QTLs, located in two regions on chromosome 1, were developed to evaluate their tissue culture performance. The Kasalath alleles in qSv1 and qSc1-1 improved callus color through callus induction and subculture, and increased the subcultured-callus volume and the fresh weight of regenerated calli, including shoots, roots, and differentiated structures. In qSc1-2, the Kasalath allele improved callus color through induction and subculture. These results verified the presence of QTLs for the volume and color of subcultured callus on chromosome 1, qSv1, qSc1-1, and qSc1-2.     

Improved isolated microspore culture efficiency in medium with maltose and optimized growth regulator combination in japonica rice (Oryza sativa)

The influence of maltose and growth regulators on microspore culture response was investigated in japonica rice. High frequency of callus induction of isolated microspores was obtained with liquid medium containing MS salts, 100 mg l^–1 myo-inositol, 1 mg l^–1 thiamine-HCl, 500 mg l^–1 glutamine, 60 g l^–1 maltose, and several growth regulators. The effect of maltose on promoting callus formation was associated with keeping a high proportion of swollen microspores after 5 day preculture and increasing the microspore division rate on the 3rd day after culture initiation. No significant effect of maltose in place of sucrose on plantlet regeneration was seen in regeneration medium. Among the growth regulators tested, the combination of auxin 2,4-dichlorophenoxyacetic acid (1 mg l^–1), naphthaleneacetic acid (1 mg l^–1), and cytokinin (6-benzyl-aminopurine 1 mg l^–1) in the medium proved to be much better for callus formation than in the other media, and the percentage of callusing microspores of that medium reached 0.86%. Indole-3-acetic acid (0.5 mg l^–1) and kinetin (2 mg l^–1) in regeneration medium were beneficial for green plantlet differentiation. The results also showed that the frequencies of microspores initial division, callus formation and green plant regeneration varied among genotypes no matter what kind of growth regulator and sugar were used. Xiushui 117 was the best variety for callusing followed by 02428 & Taipei 309. Taipei 309 showed a good ability for green plantlet regeneration.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - 6-BA 6-benzylaminopurine - KT kinetin - IAA indole-3 acetic acid     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Growth and respiration of rice (Oryza sativa L.) Cells in suspension culture

Studies on the growth and respiration of batch suspension cultures of rice (Oryza sativa L.) in a reference medium containing Murashige-Skoog salts, 2% (w/v) sucrose and yeast extract are reported. It was found that the yeast extract contributed 70% of the phosphate in this medium, and that the cells grew equally well in continued subculture in a defined medium which contained 6 mM phosphate and 3% (w/v) sucrose and the remaining Murashige-Skoog salts. Cell clumps (up to 1.5 mm diameter) were prevalent in the initial cultures in the reference medium. In such cultures the critical O₂ pressure of cell respiration was high (125 M), and ethanol accumulated. When cell clumps were routinely removed during several weekly subcultures on the defined medium cultures were obtained in which no clumps were present, the critical O₂ pressures was decreased to 40 M and no ethanol accumulated.This work was supported by grant PCM-84-03542 from the U.S. National Science Foundation.     

Molecular mapping of rice chromosomes

Summary We report the construction of an RFLP genetic map of rice (Oryza sativa) chromosomes. The map is comprised of 135 loci corresponding to clones selected from a PstI genomic library. This molecular map covers 1,389 cM of the rice genome and exceeds the current classical maps by more than 20%. The map was generated from F₂ segregation data (50 individuals) from a cross between an indica and javanica rice cultivar. Primary trisomics were used to assign linkage groups to each of the 12 rice chromosomes. Seventy-eight percent of the clones assayed revealed RFLPs between the two parental cultivars, indicating that rice contains a significant amount of RFLP variation. Strong correlations between size of hybridizing restriction fragments and level of polymorphism indicate that a significant proportion of the RFLPs in rice are generated by insertions/delections. This conclusion is supported by the occurrence of null alleles for some clones (presumably created by insertion or deletion events). One clone, RG229, hybridized to sequences in both the indica and javanica genomes, which have apparently transposed since the divergence of the two cultivars from their last common ancestor, providing evidence for sequence movement in rice. As a by product of this mapping project, we have discovered that rice DNA is less C-methylated than tomato or maize DNA. Our results also suggest the notion that a large fraction of the rice genome (approximately 50%) is single copy.