Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a destructive soil-borne disease leading to huge yield loss.  We previously reported that Klebsiella variicola FH-1 could degrade atrazine herbicides, and the vegetative growth of atrazine-sensitive crops (i.e., soybean) was significantly increased in the FH-1-treated soil.  Interestingly, we found that FH-1 could promote soybean growth and induce resistance to S. sclerotiorum.  In our study, strain FH-1 could grow in a nitrogen-free environment, dissolve inorganic phosphorus and potassium, and produce indoleacetic acid and a siderophore.  The results of pot experiments showed that K. variicola FH-1 promoted soybean plant development, substantially improving plant height, fresh weight, and root length, and induced resistance against S. sclerotiorum infection in soybean leaves.  The area under the disease progression curve (AUDPC) for treatment with strain FH-1 was significantly lower than the control and was reduced by up to 42.2% within 48 h (P<0.001).  Moreover, strain FH-1 rcovered the activities of catalase, superoxide dismutase, peroxidase, phenylalanine ammonia lyase, and polyphenol oxidase, which are involved in plant protection, and reduced malondialdehyde accumulation in the leaves.  The mechanism of induction of resistance appeared to be primarily resulted from the enhancement of transcript levels of PR10, PR12, AOS, CHS, and PDF1.2 genes.  The colonization of FH-1 on soybean root, determined using CLSM and SEM, revealed that FH-1 colonized soybean root surfaces, root hairs, and exodermis to form biofilms.  In summary, K. variicola FH-1 exhibited the biological control potential by inducing resistance in soybean against S. sclerotiorum infection, providing new suggestions for green prevention and control.

Upland cotton (Gossypium hirsutum L.) is the most important natural textile fiber crop worldwide. Plant height (PH) is a significant component of plant architecture, strongly influencing crop cultivation patterns, overall yield, and economic coefficient. However, cotton genes regulating plant height have not been fully identified. Previously, an HD-Zip gene (GhHB12) was isolated and characterized in cotton, which regulates the abiotic and biotic stress responses and the growth and development processes. In this study, we showed that GhHB12 was induced by auxin. Moreover, overexpression of GhHB12 induces the expression of HY5, ATH1, and HAT4, represses the spatial-temporal distribution, polar transport, and signaling of auxin, alters the expression of genes involved in cell wall expansion, and restrains the plant height in cotton. These results suggest a role of GhHB12 in regulating cotton plant height, which could be achieved by affecting the auxin signaling and cell wall expansion.

In Bacillus thuringenesis (Bt) transgenic cotton, the cotton boll has the lowest insecticidal protein content when compared to the other organs.  The present study investigated the effects of amino acid spray application at the peak flowering stage on the cotton boll Bt toxin concentration and yield formation.  Boll protein synthesis and carbohydrate conversion were also studied to reveal the fundamental mechanism.  Three treatments (i.e., CK, the untreated control; LA1, five amino acids; LA2, 21 amino acids) were applied to two Bt cultivars of G. hirsutum (i.e., the hybrid Sikang 3 and the conventional Sikang 1) in the cotton-growing seasons during 2017 and 2018.  Amino acid spray application at the peak flowering stage resulted in an increase of 5.2–16.4% in the boll Bt protein concentration and an increase of 5.5–11.3% in the seed cotton yield, but there was no difference between the two amino acid treatments.  In addition, amino acid applications led to increases in the amino acid content, soluble protein content, glutamate pyruvate transaminase (GPT) activity, glutamate oxaloacetate transaminase (GOT) activity, glucose content, fructose content and soluble acid invertase (SAI) activity.  This study also found that Bt protein content, enhanced boll number and the weight of opened bolls were closely related to carbon and nitrogen metabolism.  The Bt protein content had significant linear positive correlations with amino acid and soluble protein contents.  Enhanced boll number had significant linear positive correlations with the GPT and GOT activities from 15–25 days after flowering (DAF).  The weight of opened bolls from 55–65 DAF had a significant linear positive correlation with the SAI activity.  These results indicate that the enhancement of boll protein synthesis and carbohydrate conversion by amino acid application resulted in a simultaneous increase in the boll Bt protein concentration and cotton lint yield.

Plant chlorophyll biosynthesis and chloroplast development are two complex processes that are regulated by exogenous and endogenous factors.  In this study, we identified OsDXR, a gene encoding a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice.  OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process.  OsDXR was highly expressed in rice leaves, and subcellular localization indicated that OsDXR is a chloroplast protein.  Many genes involved in chlorophyll biosynthesis and chloroplast development were differentially expressed in the OsDXR knock-out lines compared to the wild type.  Moreover, we found that the RNA editing efficiencies of ndhA-1019 and rpl2-1 were significantly reduced in the OsDXR knock-out lines.  Furthermore, OsDXR interacted with the RNA editing factor OsMORF1 in a yeast two-hybrid screen and bimolecular fluorescence complementation assay.  Finally, disruption of the plastidial 2-C-methyl-derythritol-4-phosphate pathway resulted in defects in chloroplast development and the RNA editing of chloroplast genes.

Soybean yield has been increased through high planting density, but investigating plant height and petiole traits to select for compact architecture, lodging resistance, and high yield varieties is an underexplored avenue to improve yield.  We compared the relationship between yield-related traits, lodging resistance, and petiole-associated phenotypes in the short petiole germplasm M657 with three control accessions over 2017-2018 in four locations of the Huang-Huai region.  The results showed M657 exhibited stable and high tolerance to high planting density and resistance to lodging, especially at the highest density (8×10⁵ plants ha^-1).  Regression analysis showed that shorter petiole length was significantly associated with increased lodging resistance.  Yield analysis showed that M657 achieved higher yields under higher densities, especially in the north Huang-Huai region.  There are markedly different responses to intra- and inter-row spacing designs among varieties in both lodging and yield related to location and density.  Lodging was positively correlated with planting density, plant height, petiole length, and number of effective branches, and negatively correlated with stem diameter, seed number per plant, and seed weight per plant.  The yield of soybean was increased by appropriately increasing planting density on the basis of current soybean varieties in the Huang-Huai region.  This study provides a valuable new germplasm resource for introgression of compact architecture traits amenable to high yield in high density planting systems and establishes a high-yield model of soybean in the Huang-Huai region.

Phenotypic screening of soybean germplasm suitable for high planting density is currently the most viable strategy to increase yield.  Previous studies have shown that soybean varieties with dwarf features and a short petiole often exhibit a compact plant architecture which could improve yield through increased planting density, although previously reported short petiole accessions were ultimately not usable for breeding in practice.  Here, we established a method to assess petiole length and identified an elite mutant line, M657, that exhibits high photosynthetic efficiency.  The agronomic traits of M657 were evaluated under field conditions, and appeared to be stable for short petiole across seven locations in northern, Huang–Huai, and southern China from 2017 to 2018.  Compared with the Jihuang 13 wild type, the mutant M657 was shorter in both petiole length and plant height, exhibited lower total area of leaf, seed weight per plant and 100-seed weight, but had an increased number of effective branches and the growth period was prolonged by 2–7 days.  Using M657 as a parental line for crosses with four other elite lines, we obtained four lines with desirable plant architecture and yield traits, thus demonstrating the feasibility of adopting M657 in breeding programs for soybean cultivars of high density and high yield.

The cotton direct seeding after wheat (rape) harvested is under trial and would be the future direction at the Yangtze River Valley region of China.  The objective of this study was to quantify the effects of branch and stem architecture on cotton yield and identify the optimal cotton architecture to compensate the yield loss due to the reduction of individual production capacity under high planting density in the direst seeding after wheat harvested cropping system.  The characteristics of the stem and branch architecture and the relationships between architecture of the stem and branch with yield formation were studied on eight short season cotton cultivars during 2015 and 2016 cotton growth seasons.  Based on the two years results, three cultivars with different architectures of stem and branch were selected to investigate the effect of mepiquat chloride (MC) application on the architecture of the stem and branch, boll retention, and the yield in 2017.  Significant differences were observed on plant height, all fruiting nodes to branches ratio (NBR) in the cotton plant, and the curvature of the fruiting branch (CFB) among the studied cultivars.  There were three types of stem and fruiting branch structures: Zhong425 with stable and suitable plant height and NBR (about 90 cm and 2.5, respectively), high CFB (more than 10.0), and high boll retention speed and seed cotton yield; Siyang 822 with excessive plant height and NBR, low CFB, and low boll retention speed and seed cotton yield; and other studied cultivars with unstable structure of stem and branch, boll retention speed, and seed cotton yield across years.  And MC application could promote the appropriate plant height and NBR and high CFB and thus resulted in high boll retention speed and the yield.  The results suggested that the suitable plant height and NBR (about 90 cm and 2.5 respectively), and high CFB (more than 10.0), which was related to both genotype and cultural practice, could promote the higher boll retention speed and seed cotton yield.

This study was conducted to investigate the effects of alternating high temperature on Cry1Ac protein content on Bt cotton cultivars Sikang 1 (SK-1, a conventional cultivar) and Sikang 3 (SK-3, a hybrid cultivar). In 2011 and 2012, cotton plants were subjected to high temperature treatments ranging from 32 to 40°C in climate chambers to investigate the effects of high temperature on boll shell insecticidal protein expression. The experiments showed that significant decline of the boll shell insecticidal protein was detected at temperatures higher than 38°C after 24 h. Based on the results, the cotton plants were treated with the threshold temperature of 38°C from 6:00 a.m. to 6:00 p.m. followed by a normal temperature of 27°C during the remaining night hours (DH/NN) in 2012 and 2013. These treatments were conducted at peak boll growth stage for both cultivars in study periods of 0, 4, 7, and 10 d. Temperature treatment of 32°C from 6:00 a.m. to 6:00 p.m. and 27°C in the remaining hours was set as control. The results showed that, compared with the control, after the DH/NN stress treatment applied for 7 d, the boll shell Cry1Ac protein content level was significantly decreased by 19.1 and 17.5% for SK-1 and by 15.3 and 13.7% for SK-3 in 2012 and 2013, respectively. Further analysis of nitrogen metabolic physiology under DH/NN showed that the soluble protein content and the glutamic pyruvic transaminase (GPT) activities decreased slightly after 4 d, and then decreased sharply after 7 d. The free amino acid content and the protease content increased sharply after 7 d. The changes in SK-1 were greater than those in SK-3. These results suggest that under DH/NN stress, boll shell Cry1Ac protein content decline was delayed. Reduced protein synthesis and increased protein degradation in the boll shell decreased protein content, including Bt protein, which may reduce resistance to the cotton bollworm.

Cotton cultivars with brown (Xiangcaimian 2), green (Wanmian 39) and white (Sumian 9) fiber were investigated to study fiber developmental characteristics of natural-colored cotton and the effect of hormones on fiber quality at different stages after anthesis. Fiber lengths of both natural-colored cottons were lower than the white-fibered control, with brown-fibered cotton longer than green. Fiber strength, micronaire and maturation of natural-colored cotton were also lower than the control. The shorter fiber of the green cultivar was due to slower growth during 10 to 30 days post-anthesis (DPA). Likewise, the lower fiber strength, micronaire and maturation of natured-colored cotton were also due to slower growth during this pivotal stage. Indole-3-acetic acid (IAA) content at 10 DPA, and abscisic acid (ABA) content at 30 to 40 DPA were lower in the fibers of the natural-colored than that of the white-fibered cotton. After applying 20 mg L^–1 gibberellic acid (GA3), the IAA content at 20 DPA in the brown and green-fibered cottons increased by 51.07 and 64.33%, fiber ABA content increased by 38.96 and 24.40%, and fiber length increased by 8.13 and 13.96%, respectively. Fiber strength, micronaire and maturation were also enhanced at boll opening stage. Those results suggest that the level of endogenous hormones affect fiber quality. Application of external hormones can increase hormone content in natural-colored cotton fiber, improving its quality.

     Heterosis is an important biological phenomenon, and it has been used to increase grain yield, quality and resistance to abiotic and biotic stresses in many crops. However, the genetic mechanism of heterosis remains unclear up to now. In this study, a set of 184 chromosome segment substitution lines (CSSLs) population, which derived from two inbred lines lx9801 (the recurrent parent) and Chang 72 (the donor parent), were used as basal material to construct two test populations with the inbred lines Zheng 58 and Xun 9058. The two test populations were evaluated in two locations over two years, and the heterotic loci for plant height and ear height were identified by comparing the performance of each test hybrid with the corresponding CK at P<0.05 significant level using one-way ANOVA analysis and Duncan’s multiple comparisons. There were 24 and 29 different heterotic loci (HL) identified for plant height and ear height in the two populations at two locations over two years. Three HL (hlPH4a, hlPH7c, hlPH1b) for plant height and three (hlEH1d, hlEH6b, hlEH1b) for ear height were identified in the CSSLs×Zheng 58 and CSSLs×Xun 9058 populations as contributing highly to heterosis performance of plant height and ear height across four environments. Among the 29 HL identified for ear height, 12 HL (41.4%) shared the same chromosomal region associated with the HL (50.0%) identified for plant height in the same test population and environment.

Soil aggregate stability and organic carbon (OC) are regarded as effective indicators of soil structure and quality. A longterm field experiment was established in 2006 to examine the influence of tillage systems on soil aggregation and OC in a sandy loam soil in the Huang-Huai-Hai Plain of China. The study involved eight treatments: plowing every year with (TS) and without residue (T), plowing every 2 years with (2TS) and without residue (2T), plowing every 4 years with (4TS) and without residue (4T), and no plowing with (NTS) and without residue (NT). In 2013, soil samples were collected at depths of 0–5, 5–10 and 10–20 cm, and separated into three aggregate-size classes: macroaggregates (>250 μm), microaggregates (53–250 μm) and the silt+clay fraction (<53 μm) using wet sieving method. Soil parameters measured were water-stable aggregates, geometric mean diameter (GMD), mean weight diameter (MWD) and OC concentrations in different aggregate- size fractions and in bulk soil. The tillage treatments significantly (P<0.05) influenced soil aggregate stability and OC distribution. Higher MWD and GMD were observed in 2TS, 4TS and NTS as compared to T. With increasing soil depth, the amount of macroaggregates and MWD and GMD values were increased, while the proportions of microaggregates and the silt+clay fraction were declined. The OC concentrations in different aggregate fractions at all soil depths followed the order of macroaggregates>microaggregates>silt+clay fraction. In the 0–5 cm soil layer, concentrations of macroaggregateassociated OC in 2TS, 4TS and NTS were 14, 56 and 83% higher than for T, whereas T had the greatest concentration of OC associated with the silt+clay fraction in the 10–20 cm layer. Soil OC concentrations under 4TS and NTS were significantly higher (P<0.05) than that of T in the 0–10 cm layer. Residue retention promoted formation of macroaggregates, increased macroaggregate-associated OC concentrations and thus increased total soil OC stock. The macroaggregate-associated OC was positively correlated (R2=0.96) with soil OC concentration, while the silt+clay fraction-associated OC was negatively correlated (R2=0.82) with soil OC concentration. The concentration of soil OC was positively correlated with MWD (R2=0.94) and GMD (R2=0.92). We concluded that increasing tillage intensity led to a loss of carbon (C)-rich macroaggregates and an increase of C-depleted silt+clay fraction. The conservation tillage system, especially NTS and 4TS, increased soil aggregate stability and promoted OC accumulation in macroaggregates, provided the potential to improve soil C sequestration and soil structure in the Huang-Huai-Hai Plain of China.

Many animal feed grains contain high β-glucan in the cell wall. Pigs do not secret β-glucanase to degrade the β-glucan in their feed. The indigestible β-glucan not only blocks the release of nutrients from the grain cell wall, but also increases the digesta viscosity in the gastrointestinal tract of pigs. Therefore, dietary β-glucan significantly inhibits nutrient digestion and absorption in pigs. Transgenic expression of β-glucanase in the digestive tract of pigs may offer a solution to solve this problem. In the current study, four arti?cial codon-optimized β-glucanases genes was prepared and expressed in porcine cells. Only pBgA and pEgx showed high activity in transfected pig kidney cells. To improve the pH range and pH stability of β-glucanase, the two β-glucanases, pBgA and pEgx, were co-expressed in pig kidney cells and salivary gland cells by Linker A3 or 2A peptide. The resulting dual enzymes of pBgA3pEg and pBg2ApEg showed significantly enlarged pH range and significantly increased pH stability, as compared to parental enzymes. These results provide useful data for future study on increasing the feed digestibility of pigs by transgenic expression of β-glucanase in their salivary glands.

Given the public concern over the safety of genetically modified (GM) technology and products, the article elaborated the safety regulatory and administration on agricultural GMOs in China. China has made a set of laws and regulations of GMO safety management and confirmed competent authorities with clear-cut responsibilities. According to the laws and regulations, GMO products before entering markets have to pass through safety evaluation, get production and processing permission and be labeled correctly. For the importation of GM products, China has set up an import approval system. In addition, China has established technical supportive systems, including safety evaluation specifications, trial specifications and criteria specifications. The existing regulatory system supervises and regulates all activities and work related to agricultural GMOs in China.

Higher boll worm survival rates were detected after high temperature presented during square period in Bt cotton. The objective of this study was to investigate the effects of high temperature level on the Bt efficacy of two different types of Bt cotton cultivars at squaring stage. During the 2011 to 2013 cotton growth seasons, high temperature treatments ranged from 34 to 44°C in climate chambers, and field experiments under high temperature weather with various temperature levels were conducted to investigate the effects of the high temperature level on square Bt protein concentration and nitrogen metabolism. The climate chamber experiments showed that the square insecticidal protein contents reduced after 24 h elevated temperature treatments for both cultivars, whereas significant declines of the square insecticidal protein contents were detected at temperature >38°C, and only slightly numerical reductions were observed when temperature below 38°C. Similar high temperature responses were also observed at the two field experimental sites in 2013. Correspondingly, high temperature below 38°C seems have little effect on the square amino acid concentrations, soluble protein contents, glutamic- pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) activities as well as protease and peptidase activities; however, when the temperature was above 38°C, reduced soluble protein contents, enhanced amino acid concentrations, decreased GPT and GOT activities, bolstered protease and peptidase activities in square were detected. In general, the higher the temperature is (>38°C), the larger the changes for the above compound contents and key enzymes activities of the square protein cycle. The findings indicated that the unstable insect resistance of the square was related to high temperature level during square stage.

A rational plant population is an important attribute to high yield of cotton, because it can provide a beneficial micro environment within the canopy for plant growth and development as well as yield formation. A 2-yr field experiment was conducted to determine the optimal plant density based on cotton yield in relation to the canopy micro environment (canopy temperature, relative humidity and light transmittance). Six plant densities (1.2-5.7 plants m-2) were arranged with a completely randomized block design. The highest cotton yield (1 507 kg ha-1) was obtained at 3.0 plants m-2 due to more bolls per unit ground area (79 bolls m-2), while the lowest yield (1 091 kg ha-1) was obtained at 1.2 plants m-2. Under the moderate plant density (3.0 plants m-2), there was a lower mean daily temperature (MDT, 27.1°C) attributing to medium daily minimum temperature (Tmin, 21.9°C) and the lowest daily maximum temperature (Tmax, 35.8°C), a moderate mean canopy light transmittance of 0.51, and lower mean daily relative humidity (MRH) of 79.7% from June to October. The results suggest that 3.0 plants m-2 would be the optimal plant density because it provides a better canopy micro environment.

Expression of insecticidal protein for transgenic Bacillus thuringiensis (Bt) cotton is unstable and related to nitrogen metabolism. The objective of this study was to investigate the relationship between leaf carbon nitrogen ratio (C/N) and insecticidal efficacy of two Bt cotton cultivars. C/N ratio and Bt protein content were both measured at peak square period and peak boll period respectively under 5-7 d high temperature and different nitrogen fertilizer rates on the Yangzhou University Farm and the Ludong Cotton Farm, China. All plants were grown in field. The results showed that the C/N ratio enhanced slightly and the Bt protein content remained stable at peak square period, but significant increases for the C/N ratio and decreases markedly for the leaf Bt protein concentration were detected at the peak boll period. The similar patterns at the two growth periods were found for the leaf C/N ratio and Bt protein content by different N fertilizer treatments. When nitrogen rate was from 0 to 600 kg ha-1, the C/N ratio was reduced by 0.017 and 0.006 for Sikang 1 and Sikang 3 at peak square period, compared to the 1.350 to 1.143 reduction for Sikang 1 and Sikang 3 at peak boll period, respectively. Correspondingly, the leaf Bt protein contents were bolstered by 2.6-11.8 and 26.9-36.9% at the two different growth periods, respectively. The results suggested that enhanced C/N ratio by high temperature and nitrogen application may result in the reduction of insectiocidal efficacy in Bt cotton, especially in peak boll period.

N fertilization of 300 kg N ha-1 is normally applied to cotton crops in three splits: pre-plant application (PPA, 30%), first bloom application (FBA, 40%) and peak bloom application (PBA, 30%) in the Yangtze River Valley China. However, low fertilizer N plant recovery (NPR) (30-35%) causes problems such as cotton yield stagnation even in higher N rate, low profit margin of cotton production and fertilizer release to the environment. Therefore, it is questioned: Are these three splits the same significance to cotton N uptake and distribution? An outdoor pot trial was conducted with five N rates and 15N labeled urea to determine the recovery and distribution of 15N from different splits in cotton (Gossypium hirsutum L. cv. Huazamian H318) plant. The results showed that, cotton plant absorbed fertilizer 15N during the whole growing period, the majority during flowering for 18-20 d regardless of N rates (150-600 kg ha-1). Fertilizer 15N proportion to the total N accumulated in cotton plant increased with N rates, and it was the highest in reproductive organs (88% averaged across N rates) among all the plant parts. FBA had the highest NPR (70%), the lowest fertilizer N lose (FNL, 19%), and the highest contribution to the fertilizer 15N proportion to the total N (46%) in cotton plant, whereas PPA had the reverse effect. It suggests that FBA should be the most important split for N absorption and yield formation comparatively and allocating more fertilizer N for late application from PPA should improve the benefit from fertilizer.

Telomeres form the ends of eukaryotic chromosomes and serve as protective caps that keep chromosomes structure independency and completeness. The first plant telomere DNA was isolated from Arabidopsis thaliana and was shown to have tandemly repeated sequence 5´-TTTAGGG-3´. The Arabidopsis-type telomere has been found in many plants, but several reports indicate that this sequence is absent in some plants. Up to now, no research has been conducted on the telomere of cotton. In this paper, the Arabidopsis-type telomere sequence was amplified and cloned using the primers designed based on the fragment containing telomere sequence in an Arabidopsis bacterial artificial chromosome (BAC). Fluorescence in situ hybridization (FISH) with cotton metaphase chromosomes using the Arabidopsis-type telomere sequence as probes indicated that the signals were located at all chromosome ends of seven diploid and two tetraploid cotton species with different signal intensities among chromosome complements of different cotton species, even between long and short arms of the same chromosome. To identify the signals of FISH, the genome DNA of Xinhai 7, a cultivar of Gossypium barbadense, digested by BAL-31 nuclease was introduced in this study. The result of BAL-31 digestion indicated that the hybridization signals of FISH represent the outermost DNA sequence of each cotton chromosomes. So we first proved that the telomeric repeats of cotton cross-hybridize with that of Arabidopsis. The results of terminal restriction fragment (TRF) showed significant variation in telomere length among cotton species. The telomere length of cultivated cotton was close to 20 kb and was larger than those of wild cotton species whose telomere length ranged from 6 to 20 kb.

The green-fluorescent protein (gfp) gene was evaluated as a screening marker during cotton (Gossypium hirsutum L.) transforming and plant regeneration. High expression of GFP (green-fluorescent protein) was observed in transgenic cells as early as 42 h after co-culture with Agrobacterium. Most of the stable transformation events were detected in the cells of primary vascular tissue. GFP transient expression could be detected on all the explants after co-culturing for 4 d, however, the highest GFP stable expression was recorded when the explants were co-cultured for 3 d. We believe the transient and stable expression of a foreign gene in genetic transformation were two relative but different events, because high transient expression did not surely lead to high stable transformation. Under the same conditions of in vitro culture, transgenic and non-transgenic calli exhibited different morphological characters on different stages of development. High concentration of plant growth regulators (PGRs) was efficient for somatic embryogenesis of the transgenic calli, which means that the transgenic calli need relatively higher dose of hormone for further growth and somatic embryogenesis than non-transgenic ones. Strong GFP-expression was observed in leaf, stem, petioles, floral tissues, and seedlings of T1 progeny. Segregation ratios of eight transgenic lines were scored for expression of GFP in the T1 progeny that providing further evidence of stable transformation. These results proved that GFP is a powerful reporter gene for protocol optimization, selection, and monitioring in whole transformation events.

The higher survival rates of Helicoverpa amigera larvae were usually observed after adverse climate which was related to extreme temperature (T) and relative humidity (RH) stresses in transgenic Bacillus thuringiensis (Bt) cotton. The unstable resistance of Bt cotton to bollworms has been correlated with the reduced expression of CryIAc d-endotoxin. The objective of this study was to investigate the effects of combined temperature and relative humidity stresses on the leaf CryIAc insecticidal protein expression during critical developmental stages. The study was undertaken on two transgenic cotton cultivars that share same parental background, Sikang 1 (a conventional cultivar) and Sikang 3 (a hybrid cultivar), during the 2007 and 2008 growing seasons at the Yangzhou University Farm, Yangzhou, China. The study was arranged with two factors that consisted of temperature (two levels) and relative humidity (three levels). The six T/RH treatments were 37°C/95%, 37°C/70%, 37°C/50%, 18°C/95%, 18°C/70%, and 18°C/50%. In 2007, the six treatments were imposed to the plants at peak flowering stage for 24 h; in 2008, the six treatments were applied to the plants at peak square, peak flowering, and peak boll stages for 48 h. The results of the study indicated that the leaf insecticidal protein expression in CryIAc was significantly affected by extreme temperature only at peak flowering stage, and by both extreme temperature and relative humidity during boll filling stage. The greatest reductions were observed when the stresses were applied at peak boll stage. In 2008, after 48 h stress treatment, the leaf Bt endotoxin expression reduced by 25.9-36.7 and 23.6-40.5% at peak boll stage, but only by 14.9-26.5 and 12.8-24.0% at peak flowering stage for Sikang 1 and Sikang 3, respectively. The greatest reduction was found under the low temperature combined with low relative humidity condition for both years. It is believed that the temperature and relative humidity stresses may be attributed to the reduced efficacy of Bt cotton in growing conditions in China, where extreme temperatures often increase up to 35-40°C and/or decrease down to 15-20°C, and relative humidity may reach to 85-95% and/or reduce to 40-55% during the cotton growing season.