A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3–6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.V.G. Sunnichan and R. Kumria contributed equally to this investigation     

Frequency and Diversity of Fungi Colonizing Tissues of Upland Cotton

A study of the mycoflora of upland cotton in Alabama was conducted throughout the 2000 and 2001 growing seasons. Plants were sampled at seedling, first bloom, full bloom, and maturity stages of development. Thirty-seven genera representing 58 species of fungi were isolated, including 9 species of Fusarium. Fusarium oxysporum, F. solani, and F. equiseti were the most common members of this genus occurring at all four sampling stages in both years. Eight species accounted for 67% of the total fungi isolated during the two-year study. Alternaria alternata was the most common fungus encountered, accounting for 19 and 10% of the total fungi isolated in 2000 and 2001, respectively. Twenty species of fungi are reported for the first time colonizing upland cotton tissues.     

中国30个陆地棉主栽品种的SSR指纹图谱

利用SSR分子标记技术,对中国不同生态棉区曾经或正在种植的主要来源于岱字棉、斯字棉、福字棉、乌干达棉的30个陆地棉主栽品种进行了DNA指纹分析。从1 803对SSR引物中筛选到重复性好、多态性丰富的20对核心引物。这些引物分属棉花15条染色体,共检测到116个等位基因,平均每对引物5.8个;PIC值范围为0.384~0.900,平均为0.716;MI值范围为1.152~9.000,平均为4.374。30个品种中有4个品种具有各自的特异引物,可将其与其它26个品种区分开,其它26个品种可利用至少2对引物组合进行区分。为更方便、准确地鉴定各品种,构建了30个品种20对核心引物的十进制数字指纹代码。该研究为陆地棉的品种鉴定和纯度检测、新品种权益保护以及标准DNA指纹库构建提供了重要依据。     

High Frequency Somatic Embryogenesis in Cotton

A highly reproducible system for efficient somatic embryogenesis was developed to regenerate plantlets from cotton (Gossypium hirsutum L.) cultivars (Nazilli M-503 and Nazilli 143). Shoot apices, hypocotyls and nodes of 10-d-old seedlings were used as explants. High frequency (100 %) embryogenic calli was initiated from all tested explants on Murashige and Skoog (1962) (MS) media supplemented with 1 g dm^–3 polyvinylpyrrolidone (PVP), 1 mg dm^–3 6-benzylaminopurine (BAP), 0.5 mg dm^–3 kinetin for Nazilli M-503 and 1 g dm^–3 PVP, 2 mg dm^–3 BAP, 0.5 mg dm^–3 kinetin for Nazilli-143. Globular stage somatic embryos were produced 4 months after transfer to hormone-free MS medium supplemented with 1 g dm^–3 PVP. Subsequent subculture of globular embryos every 3 weeks on hormone-free MS medium led to the development of torpedo and cotyledonary stage embryos with the frequency of 75 and 83.2 % from hypocotyl explants of Nazilli M-503 and Nazilli-143, respectively. Afterwards, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination and development into plantlets. The highest germination frequency (42.9 %) for Nazilli M-503 somatic embryos were obtained on hormone-free MS medium after 5 months with hypocotyl explants, whereas germination frequencies of Nazilli-143 embryos from hypocotyl, node and apex explants varied between 22 – 30 %.     

The rate of cellulose increase is highly related to cotton fibre strength and is significantly determined by its genetic background and boll period temperature

This experiment was conducted to study the relationship between the increase in cellulose content in developing cotton bolls and their final cotton fibre strength. The rate of cellulose increase over time was estimated using logistical regression, and the logistic equation parameters were then used to compare different cotton cultivars in different temperature environments. The increase in cellulose content followed a typical “S” curve, with the boll period time divided into slow-fast-slow stages. In different cultivars, the final fibre strength was closely related to the characters of the fast cellulose content increasing stage, negatively related to the maximal cellulose increasing rate (P < 0.05), and positively related to the duration of the fast cellulose content increasing stage (P < 0.01). In the same cultivar, low temperature reduced the maximal cellulose increasing rate and prolonged the duration of the fast cellulose increasing stage. The results indicate that, in diverse genetic background, long-lasting and tempered cellulose growth during the rapid cellulose increasing stage is of significant benefit to high strength fibre development. For closely related cotton cultivars, decreasing the maximal cellulose increasing rate and the termination of rapid cellulose increasing stage reduced fibre strength that often occurs when temperatures are low.     

Transgenic Plants of Colonial Bentgrass from Embryogenic Callus via Agrobacterium-mediated Transformation

Colonial bentgrass (Agrostis tenuis Sibth. Fl. Oxen.) is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable and repeatable approach in transforming the grass using Agrobacterium (strain LBA4404), in which -glucuronidase (gus) gene was used as a reporter and hygromycin phosphotransferase (hpt) gene as a selectable marker. This vector was effective in transforming 7-week-old calluses derived from mature seeds cultured on MS medium supplemented with 2,4-D. A two-step solid medium selection with increasing hygromycin concentration (from 50 to 70 mg l^–1) was used to obtain resistant calluses. Hundreds of transgenic plants have been produced from several independent transformed calluses. The presence of functional -glucuronidase (GUS) was detected in hygromycin-resistant calluses, young leaves and roots of transgenic plants. The transgenic plants collected from greenhouse showed strong resistance to 50 mg l^–1 hygromycin solution. Four putative transgenic plants and one control plant were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the hpt gene were clearly shown in transgenic plants.     

Cotton shoot plays a major role in mediating senescence induced by potassium deficiency

The objective of this study was to determine the roles of shoot and root in the regulation of premature leaf senescence induced by potassium (K) deficiency in cotton (Gossypium hirsutum L.). Two contrasting cultivars (CCRI41, more sensitive to K deficiency; and SCRC22, a less sensitive cultivar) were selected for self- and reciprocal-grafting, using standard grafting (one scion/one rootstock), Y grafting (two scions/one rootstock) and inverted Y grafting (one scion/two rootstocks) at the seedling stage. Standard grafting was studied in the field in 2007 and 2008. There were no obvious differences in senescence between CCRI41 and SCRC22 scions while supplied with sufficient K. However, SCRC22 scions showed significantly greater K content, SPAD values (chlorophyll content), soluble protein content and net photosynthetic rates than CCRI41 scions while grown in K deficient solution or soil, regardless of rootstock cultivars, grafting types, growth stage and growth conditions. Also, SCRC22 scions had greater yield and less variation in boll weight either between upper- and lower sympodials, or between proximal and distal fruit positions from the main stem in the field under K deficiency, probably owing to reduced leaf senescence. Although the effect of rootstocks on leaf senescence under K deficiency was significant in some cases, the scion cultivars explained the highest percentage of variations within grafting treatments. The shoot-to-root feedback signal(s), rather than high shoot demand for K nutrition, was involved in the shoot regulation of premature senescence in cotton plants, achieved possibly by altering root K uptake.     

First evidence of trans-resveratrol production in cell suspension cultures of cotton (Gossypium hirsutum L.)

For the first time, trans-resveratrol, a stilbene, has been identified in cotton cell suspensions. Cell suspensions of Coker 312, a cultivar which produces embryogenic structures, acccumulate trans-resveratrol contrary to those of cultivar R405-2000, which do not. This stilbene may be a good phenolic marker for induction of somatic embryogenesis in cotton.     

Effects of brassinosteroid on cotton regeneration via somatic embryogenesis

Brassinolide (BR), which is the most biologically active brassinosteroid, was used to examine the potential effect of hormone on cotton somatic embryogenesis. Ten-day-old cotton (Gossypium hirsutum L., cv. Cooker) seedlings were used for explant source and hypocotyls were removed and cultured on MS basal medium with B₅ vitamins supplemented with 1 mg/L 6-benzylaminopurine + 0.5 mg/L kinetin for callus induction. After one month proliferating calli pieces were collected and cultured on MS basal medium containing various concentrations of BR (0.1, 0.5, 1.0 μM) with their controls. BR treatments were negatively effective on the fresh weight of calli when compared to control. Differential somatic embryogenesis maturation rates due to BR treatment were observed. Somatic embryogenesis was stimulated especially for transition to cotyledonary phase at 0.5 mg/L BR. Histological preparations from embryogenic calli and somatic embryos at different stages of development revealed the spontaneous polyploidisation during early somatic embryogenesis on BR-treated calli. Present results suggest that BR negatively effected calli growth, however, had a stimulating role in maturation of somatic embryos.     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid     

Transgene integration and organization in Cotton (Gossypium hirsutum L.) genome

While genetically modified upland cotton (Gossypium hirsutum L.) varieties are ranked among the most successful genetically modified organisms (GMO), there is little knowledge on transgene integration in the cotton genome, partly because of the difficulty in obtaining large numbers of transgenic plants. In this study, we analyzed 139 independently derived T0 transgenic cotton plants transformed by Agrobacterium tumefaciens strain AGL1 carrying a binary plasmid pPZP-GFP. It was found by PCR that as many as 31% of the plants had integration of vector backbone sequences. Of the 110 plants with good genomic Southern blot results, 37% had integration of a single T-DNA, 24% had two T-DNA copies and 39% had three or more copies. Multiple copies of the T-DNA existed either as repeats in complex loci or unlinked loci. Our further analysis of two T1 populations showed that segregants with a single T-DNA and no vector sequence could be obtained from T0 plants having multiple T-DNA copies and vector sequence. Out of the 57 T-DNA/T-DNA junctions cloned from complex loci, 27 had canonical T-DNA tandem repeats, the rest (30) had deletions to T-DNAs or had inclusion of vector sequences. Overlapping micro-homology was present for most of the T-DNA/T-DNA junctions (38/57). Right border (RB) ends of the T-DNA were precise while most left border (LB) ends (64%) had truncations to internal border sequences. Sequencing of collinear vector integration outside LB in 33 plants gave evidence that collinear vector sequence was determined in agrobacterium culture. Among the 130 plants with characterized flanking sequences, 12% had the transgene integrated into coding sequences, 12% into repetitive sequences, 7% into rDNAs. Interestingly, 7% had the transgene integrated into chloroplast derived sequences. Nucleotide sequence comparison of target sites in cotton genome before and after T-DNA integration revealed overlapping microhomology between target sites and the T-DNA (8/8), deletions to cotton genome in most cases studied (7/8) and some also had filler sequences (3/8). This information on T-DNA integration in cotton will facilitate functional genomic studies and further crop improvement.     

Establishment of molecular markers and linkage groups in two F2 populations of Upland cotton

Two F₂ populations of cotton (Gossypium hirsutum L.) from the crosses of HS46 x MARCABUCAG8US-1-88 (MAR) and HS46 x Pee Dee 5363 (PD5363) were characterized for restriction fragment length polymorphisms (RFLPs) using DNA probes. Seventy-three probe/enzyme combinations were used in the HS46 x MAR population analysis, which resulted in 42 informative polymorphic fragments. These 42 moleclar markers represented 26 polymorphic loci, which consisted of 15 codominant and 11 dominant (+/-) genotypes. Chi-square analyses of these loci fit expected genotypic ratios of 121 and 31, respectively An analysis of these loci with the MAPMAKER program resulted in the establishment of four linkage groups A, B, C, and D with 4,2,2, and 2 loci, respectively, as well as 16 unlinked loci. Six probe-enzyme combinations were assayed on the HS46 x PD5363 population, which resulted in 11 informative polymorphic fragments. These 11 fragments represented 6 polymorphic loci, 1 dominant (+/-) and 5 codominant genotypes. The MAPMAKER analysis of these loci yielded 2 linked loci. Thus, a total of 53 polymorphic fragments and 32 polymorphic loci, representing five linkage groups, were identified among the two families.Contribution of the USDA-ARS in cooperation with the Miss Agric For Exp Stn.     

Somatic embryogenesis in cotton (Gossypium) I. Effects of source of explant and hormone regime

Optimal media for induction of somatic embryogenesis from mature and immature tissues ofG. hirsutum L. cv Coker 312 were determined. Explants of three-day-old seedlings form somatic embryos in 100% of cultures when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 0.5 mg/1 kinetin. Mature tissues are more recalcitrant than immature tissues and formed somatic embryos on a limited number of hormone treatments. Stem tissue is most readily induced to form somatic embryos by 2 mg/1 napthaleneacetic acid plus 0.1 mg/1 kinetin, whereas leaf tissue formed embryos best when treated with 0.1 mg/1 2,4-dichlorophenoxyacetic acid plus 1.0 mg/1 (2-isopentyl)-adenine, or 1.0 mg/1 napthaleneacetic acid plus 0.5 mg/1 (2-isopentyl)-adenine.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration

Various aspects of transformation and regeneration processes were examined in efforts to improve the efficiency of production of transgenic cotton (Gossypium hirsutum L.). Green fluorescent protein (GFP) proved to be a valuable tool in elucidating the timing and localization of transient gene expression and in visualizing conversion of transient events to stable transformation events. By day 4 after infection, there was maximal transient activity in the cells at the cut edge of Agrobacterium-infected cotyledon disks. We were able to visualize conversion of some of these events to stable transformation by day 8. The effects of Agrobacterium strains, acetosyringone, and temperature on stable transformation were also evaluated. Strain LBA4404 proved to be significantly better than EHA105. Acetosyringone increased significantly the stable transformation efficiency in cotton. Cocultivation at 21°C, compared to 25°C, consistently resulted in higher transformation frequencies. GFP expression in stably transformed callus was useful in studying the efficiency of selection during early stages of culture. We found that the survival of individual callus lines on selection medium was influenced by their original size and initial transgene expression status. Larger-size calluses and calluses expressing the transgene (GFP) had a higher rate of survival. Survival could be improved by an additional two-week culture on medium high in cytokinin and low in auxin before transfer to a medium to induce embryogenesis. However, this treatment delayed embryogenesis. Various other important aspects of the regeneration process are described and an overall scheme for producing transgenic cotton is presented.     

Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration

Calli of cotton (Gossypium hirsutum L.) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium. Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media. High inorganic salts (MS), low salt (2/3 MS), excess KNO₃, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA₃) and kinetin were tested for their effects on somatic embryo maturation. Long-term embryo proliferation and maturation were best on medium containing MS plus 1.9g/l KNO₃. Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 0.1 mg/l indoleacetic acid (IAA). Plants were recovered from 10.6% of the embryos. When 5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks. Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 0.1 mg/l GA₃. Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite. Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period.     

转OvDREB2B基因陆地棉鉴定及其对水分渗透胁迫的分析

通过花粉管通道技术,以该实验室自育陆地棉品系TH₁和TH₂为材料,将诸葛菜(Orychophragmus vidaceus)抗逆转录因子OvDREB2B基因构建到植物表达载体后,导入棉花基因组,经卡那霉素筛选和分子鉴定表明目的基因已整合到棉花基因组中并表达。将T₁代转基因植株和受体对照在温室中栽培,待植株生长至四叶一心时,用不同渗透势的PEG-6000水溶液进行渗透胁迫处理,分析探讨转基因植株的抗旱效果及其抗旱机理。结果显示:当渗透势为0和0.5 MPa处理时,转基因植株和对照无明显差异;当渗透势为0.8 MPa和1.1 MPa处理时转基因植株较对照抗旱性明显提高。当渗透势为1.1 MPa处理96 h时,对照植株F_v/F_m降至0.2左右,而转基因植株仍正常生长,F_v/F_m值约为0.51,而且初始荧光（F₀）值、净光合速率（P_n）、胞间CO₂浓度（C_i）、蒸腾速率（T_r）等一系列参数转基因植株都明显优于对照,表明DREB2B基因能够提高棉花对水分胁迫的耐受性。     

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km^r) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T₁, T₂, and T₃ progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T₁ AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.