Yichun Nie

Isolation and Characterization of Genes Associated to Cotton Somatic Embryogenesis by Suppression Subtractive Hybridization and Macroarray

Somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway and is a notable illustration of cell totipotency. To identify genes involved in SE, subtractive polymerase chain reaction (PCR) was performed to generate transcripts highly enriched for SE-related genes, using cDNA prepared from a mixture of embryogenic callus and preglobular somatic embryos, as the tester, and cDNA from nonembryogenic callus, as the driver. After differential screening and subsequent confirmation by reverse Northern blot analysis, a total of 671 differentially expressed cDNA fragments were identified, and 242 unigenes significantly up-regulated during cotton SE were recovered, as confirmed by Northern blot and reverse-transcription PCR analysis of representative cases, including most previously published SE-related genes in plants. In total, more than half had not been identified previously as SE-related genes, including dominant crucial genes involved in transcription, posttranscription, and transportation, and about one-third had not been reported previously to GenBank or were expected to be unknown, or newly identified genes. We used cDNA arrays to further investigate the expression patterns of these genes in differentiating gradient culture, ranging from proembryogenic masses to somatic embryos at every stage. The cDNA collection is composed of a broad repertoire of SE genes which is an important resource for understanding the genetic interactions underlying SE signaling and regulation. Our results suggested that a complicated and concerted mechanism involving multiple cellular pathways is responsible for cotton SE. This report represents a systematic and comprehensive analysis of genes involved in the process of somatic embryogenesis.

Factors affecting transformation efficiency of embryogenic callus of Upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens

A reliable and high-efficiency system of transforming embryogenic callus (EC) mediated by Agrobacterium tumefaciens was developed in cotton. Various aspects of transformation were examined in efforts to improve the efficiency of producing transformants. LBA4404 and C58C3, harboring the pΔgusBin19 plasmid containing neomycin phosphortransferase II (npt-II) gene as a selection marker, were used for transformation. The effects of Agrobacterium strains, acetosyringone (AS), co-cultivation temperature, co-cultivation duration, Agrobacterium concentration and physiological status of EC on transformation efficiency were evaluated. Strain LBA4404 proved significantly better than C58C3. Agrobacterium at a concentration of 0.5 × 108 cells ml−1 (OD600=0.5) improved the efficiency of transformation. Relatively low co-cultivation temperature (19 °C) and short co-cultivation duration (48 h) were optimal for developing a highly efficient method of transforming EC. Concentration of AS at 50 mg l−1 during co-cultivation significantly increased transformation efficiency. EC growing 15 days after subculture was the best physiological status for transformation. An overall scheme for producing transgenic cotton is presented, through which an average transformation rate of 15% was obtained.

Overexpression of Rice NAC Gene SNAC1 Improves Drought and Salt Tolerance by Enhancing Root Development and Reducing Transpiration Rate in Transgenic Cotton

The SNAC1 gene belongs to the stress-related NAC superfamily of transcription factors. It was identified from rice and overexpressed in cotton cultivar YZ1 by Agrobacterium tumefaciens-mediated transformation. SNAC1-overexpressing cotton plants showed more vigorous growth, especially in terms of root development, than the wild-type plants in the presence of 250 mM NaCl under hydroponic growth conditions. The content of proline was enhanced but the MDA content was decreased in the transgenic cotton seedlings under drought and salt treatments compared to the wild-type. Furthermore, SNAC1-overexpressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in the greenhouse. The performances of the SNAC1-overexpressing lines under drought and salt stress were significantly better than those of the wild-type in terms of the boll number. During the drought and salt treatments, the transpiration rate of transgenic plants significantly decreased in comparison to the wild-type, but the photosynthesis rate maintained the same at the flowering stage in the transgenic plants. These results suggested that overexpression of SNAC1 improve more tolerance to drought and salt in cotton through enhanced root development and reduced transpiration rates.

Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8–10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.

GbTCP, a cotton TCP transcription factor, confers fibre elongation and root hair development by a complex regulating system

As the most important natural raw material for textile industry, cotton fibres are an excellent model for studying single-cell development. Although expression profiling and functional genomics have provided some data, the mechanism of fibre development is still not well known. A class I TCP transcription factor (designated GbTCP), encoding 344 amino acids, was isolated from the normalized cDNA library of sea-island cotton fibre (from –2 to 25 days post anthesis). GbTCP was preferentially expressed in the elongating cotton fibre from 5 to 15 days post anthesis. Some expression was also observed in stems, apical buds, and petals. RNAi silencing of GbTCP produced shorter fibre, a reduced lint percentage, and a lower fibre quality than the wild-type plants. Overexpression of GbTCP enhanced root hair initiation and elongation in Arabidopsis and regulated branching. Solexa sequencing and Affymetrix GeneChip analysis indicated that GbTCP positively regulates the level of jasmonic acid (JA) and, as a result, activates downstream genes (reactive oxygen species, calcium signalling, ethylene biosynthesis and response, and several NAC and WRKY transcription factors) necessary for elongation of fibres and root hairs. JA content analysis in cotton also confirmed that GbTCP has a profound effect on JA biosynthesis. In vitro ovule culture showed that an appropriate concentration of JA promoted fibre elongation. The results suggest that GbTCP is an important transcription factor for fibre and root hair development by regulating JA biosynthesis and response and other pathways, including reactive oxygen species, calcium channel and ethylene signalling.

FACTORS AFFECTING SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM A RANGE OF RECALCITRANT GENOTYPES OF CHINESE COTTONS (GOSSYPIUM HIRSUTUM L)

SummaryA new protocol has been developed for the highly efficient somatic embryogenesis and plant regeneration of 10 recalcitrant Chinese cotton cultivars. Calluses and embryogenic calluses were induced on MSB1 medium containing the optimal combination of indolebutyric acid (IBA; 2.46 μM) and kinetin (KT; 2.32 μM). Up to 86.7% of embryogenic calluses differentiated into globular somatic embryos 2 mo. after culture on MSB2 medium containing double KNO3 and free of growth regultors. Up to 38.3% of the somatic embryos were converted into complete plants in 8 wk on MSB3 medium with l-asparagine (Asn)/l-glutamine (Gln) (7.6/13.6 mM). The plants were successfully transferred to soil and grew to maturity. With the protocol described here, we have obtained hundreds of regenerating plantlets from 10 recalcitrant cultivars, which is important for the application of tissue culture to cotton breeding and biotechnology.

Genetic Diversity of Asian Cotton (Gossypium arboreum L.) in China Evaluated by Microsatellite Analysis

Asian cotton (Gossypium arboreum L.) was once widely cultivated in China. It has also been a valuable source of genetic variation in modern cotton improvement. In this study, the genetic diversity of selected G. arboreum accessions collected from different regions of China was evaluated by microsatellite (simple sequence repeats, SSRs) analysis. Of the 358 microsatellite markers analyzed, 74 primer pairs detected 165 polymorphic DNA fragments among 39 G. arboreum accessions examined. Twelve accessions could be fingerprinted with one or more SSR markers. With the exception of two accessions, DaZiJie and DaZiMian, genetic similarity coefficients among all accessions ranged from 0.58 to 0.87 suggesting high level of genetic variation in the G. arboreum collections. The UPGMA dendrogram constructed from genetic similarity coefficients revealed positive correlation between cluster groupings and geographic distances. In addition, comparison of the microsatellite amplification profiles of the diploid G. arboreum and tetraploid Gossypium hirsutum L. found that size distribution of amplified products in G. arboreum was dispersive and that of G. hirsutum was relatively concentrated. The information on the genetic diversity and SSR fingerprinting from this study is useful for developing mapping populations for constructing diploid cotton genetic linkage map and tagging economically important traits.

A Genetic and Metabolic Analysis Revealed that Cotton Fiber Cell Development Was Retarded by Flavonoid Naringenin

Flavonoids and flavenoid synthesis affect white cotton fiber development. The cotton (Gossypium spp.) fiber is a unique elongated cell that is useful for investigating cell differentiation. Previous studies have demonstrated the importance of factors such as sugar metabolism, the cytoskeleton, and hormones, which are commonly known to be involved in plant cell development, while the secondary metabolites have been less regarded. By mining public data and comparing analyses of fiber from two cotton species (Gossypium hirsutum and Gossypium barbadense), we found that the flavonoid metabolism is active in early fiber cell development. Different flavonoids exhibited distinct effects on fiber development during ovule culture; among them, naringenin (NAR) could significantly retard fiber development. NAR is a substrate of flavanone 3-hydroxylase (F3H), and silencing the F3H gene significantly increased the NAR content of fiber cells. Fiber development was suppressed following F3H silencing, but the overexpression of F3H caused no obvious effects. Significant retardation of fiber growth was observed after the introduction of the F3H-RNA interference segment into the high-flavonoid brown fiber G. hirsutum T586 line by cross. A greater accumulation of NAR as well as much shorter fibers were also observed in the BC1 generation plants. These results suggest that NAR is negatively associated with fiber development and that the metabolism mediated by F3H is important in fiber development, thus highlighting that flavonoid metabolism represents a novel pathway with the potential for cotton fiber improvement.

Mapping QTLs of traits contributing to yield and analysis of genetic effects in tetraploid cotton

Fiber yield and yield components – including lint index (LI), seed index (SI), lint yield (LY), seed cotton yield (SCY) and number of seeds per boll (NSPB) – were investigated on the farm of Huazhong Agricultural University in a population of 69 F2 individuals and corresponding F2:3 families derived from a cross between high-fiber-yield Gossypium hirsutum CV Handan 208 and a low-fiber-yield Gossypium barbadense CV Pima 90. On the basis of the genetic map constructed previously from the same population by Lin et al. (Plant Breed., 2005), quantitative trait locus (QTL) analysis was performed with the software QTL Cartographer V2.0 using composite interval mapping method (LOD ≥ 3.0). A total of 21 QTLs were identified, which were located in 15 linkage groups. The number of QTLs per trait ranged from one to seven. Of these QTLs detected, one affecting LI explained 24.3% of phenotypic variation (PV), five influencing SI explained 16.15–39.21% of PV, seven controlling LY explained 13.01–28.35% of PV, and two controlling SCY explained 22.76 and 39.97% of PV, respectively. Simultaneously, the detected six QTLs for NSPB were located on five linkage groups, which individually explained 28.01–38.32% of the total phenotypic variation. The results would give breeders further insight into the genetic basis of fiber yield.

GbPDF1 Is Involved in Cotton Fiber Initiation via the Core cis-Element HDZIP2ATATHB2

Cotton (Gossypium spp.) fiber cells are seed trichomes derived from the epidermal layer of the cotton seed coat. The molecular components responsible for regulating fiber cell differentiation have not been fully elucidated. A cotton PROTODERMAL FACTOR1 gene (GbPDF1) was found to be expressed preferentially during fiber initiation and early elongation, with highest accumulation in fiber cells 5 d post anthesis. PDF1 silencing caused retardation of fiber initiation and produced shorter fibers and lower lint percentage compared with the wild type, indicating that the gene is required for cotton fiber development. Further analysis showed that a higher accumulation of hydrogen peroxide occurred in the RNA interference transgenic cotton lines. Meanwhile, the expression of several genes related to ethylene and pectin synthesis or sugar transport during cotton fiber growth was found to be significantly reduced in the PDF1-suppressed cotton. Three proteins interacting with GbPDF1 in yeast and in planta might involve cellular signaling or metabolism. GbPDF1 promoter::GUS constructs in transgenic cotton were predominantly expressed in the epidermis of ovules and developing fibers. Progressive deletions of the GbPDF1 promoter showed that a 236-bp promoter fragment was sufficient for basal GbPDF1 transcription in cotton. Mutation of putative regulatory sequences showed that HDZIP2ATATHB2, an element within the fragment, was essential for PGbPDF1-1 expression. The binding activity between this cis-element and nuclear extracts from fiber-bearing cotton ovules at 5 d post anthesis was specific. We conclude that GbPDF1 plays a critical role together with interaction partners in hydrogen peroxide homeostasis and steady biosynthesis of ethylene and pectin during fiber development via the core cis-element HDZIP2ATATHB2.