Huamin Si

Phosphomannose-isomerase (pmi) gene as a selectable marker for rice transformation via Agrobacterium

A new selectable marker system was adapted for use in Agrobacterium-mediated transformation of rice (Oryza sativa L.). This selection system utilizes the pmi gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. Only transformed cells were capable of utilizing mannose as a carbon source. Transgenic rice plants regenerated from selected transformed immature embryo-derived calli on media containing various concentrations of mannose. The highest transformation frequency of 6.0% was obtained when a combination of 25 g/l mannose and 5 g/l sucrose was used. Molecular and genetic analyses showed that the plants contained the pmi gene and the gene was transmitted to the progeny in Mendelian fashion. The results indicated that the mannose selection system, which was devoid of the disadvantages of antibiotic or herbicide selection, could be used for rice Agrobacterium-mediated immature embryo transformation.

The Effect of the Crosstalk between Photoperiod and Temperature on the Heading-Date in Rice

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions.

Characterization and fine mapping of a novel rice narrow leaf mutant nal9.

A narrow leaf mutant was isolated from transgenic rice (Oryza sativa L.) lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 (narrow leaf 9). The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F₂ generations of a cross between the nal9 mutant and Longtepu were used for mapping. With 24 F₂ mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent Clp protease proteolytic subunit gene (ClpP) was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the ClpP cDNA driven by the 35S promoter. Accordingly, the ClpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work.

Genetic analysis and identifi- cation of a large leaf angles (lla) mutant in rice

Mutants are essential genetic materials to elucidation of biological functions of genes involved. Characterization and isolation of genes in mutants is one of the research tasks in functional genomic era. T-DNA insertional mutagenesis has provided an efficient way to identify genes in plant species, in which the mutated genes could be rapidly isolated once the mutant was confirmed by T-DNA insertion. About 40% of 620 cloned Arabidopsis genes with mutant phenotypes were determined by T-DNA tagging. In recent years, a large number of T-DNA insertional rice mutant pools were established. Moreover, three genes were successfully isolated from rice. Leaf angles, the angles between main culms and leaves, are one of the important features of the plant types. Investigation on the mechanism of leaf angle formation will probably provide the basic knowledge of plant breeding on plant architectures. Several QTLs of leaf and flag leaf angles in rice were identified, which would provide the useful materials for manipulating these QTLs in a marker-assisted selection program. However, the major genes, which control leaf angles, have not been reported yet and very little is known about the mechanism of leaf angle formation. Based on T-DNA inserted rice (Oryza sativa L. subsp. Japonica cv. Zhonghua11) mutant pool, which contains about 10000 insertional mutant lines, a large leaf angles (lla) mutant T429 was found in T1 lines. At seeding stage, the leaf angles of lla mutant were larger than those of the wild type. In addition, the mutant seedlings showed semi-dwarf. At heading stage, the abnormal phenotypes of lla mutants were observed in plant height, leaf angel, leaf blade length and leaf blade width. The plant of lla was shorter than the wild type (Fig. 1). The length and width of the lla mutant leaves were shorter and wider than those of the wild type (unpublished data). Under natural long day conditions, the heading date of lla was about two weeks later than that of the wild type. Under winter natural short day conditions in greenhouse, the heading date of lla was about one week later than that of the wild type while the plant height and leaf angles remained significantly different from the control. This indicated that the mutant phenotype was slightly influenced by environmental conditions such as photoperiod and temperature. Different concentrations of GA3 were used to treat the plants at both seeding and heading stages; the results showed that the plant heights of wild type and the lla mutant were all sensitive to exogenous GA3 at different concentrations. But plant heights and leaf angles of GA3 treated mutant plants differed significantly from the corresponding wild types. At the same time, leaf angles of GA3 treated wild type and lla did not show any visible differences from the corresponding untreated plants.

Classification of environmentally induced genetic male sterile lines of rice based on their fertility responses to photoperiod and temperature

The use of environmentally induced genetic male sterile (EGMS) rice could alter the development of hybrid rice from a three-line system to a two-line system. It is critical for the utilization of EGMS rice to determine which are the main environmental factors influencing fertility changes. Fertility responses to photoperiod (P) and temperature (T) were studied in 101 EGMS rice lines under nine controlled regimes combining three photoperiods (15·0, 14·0 and 12·5 h)x three temperatures (30·1, 24·1 and 23·1 °C). According to the variance analysis of seed-setting data, 96% of the total EGMS lines studied could be divided into three types as follows: (1) photoperiod-sensitive genetic male sterility (PGMS) characterized statistically by significant ( P 0·05) P and P × T interaction effects but by a non-significant T effect on fertility, (2) thermosensitive genetic male sterility (TGMS) by a significant T effect, a non-significant P effect and by either a significant or a non-significant P × T interaction effect on fertility, and (3) photo-thermosensitive genetic male sterility (P-TGMS) by only a significant P × T interaction effect on fertility. Among the japonica EGMS lines studied, PGMS, TGMS and P-TGMS accounted for 32·3, 9·7 and 51·6%, respectively. However, among the indica EGMS lines, no PGMS lines were detected and most of them were TGMS or P-TGMS (61·4 and 35·7%, respectively). The results indicate that the selection of indica PGMS lines of rice might be very difficult. The availability of different types of EGMS rice in two-line system hybrid rice is evaluated and the selection of an ideal model of response to photoperiod and temperature for indica EGMS is discussed.