Regeneration Of Cotton Research Articles

In vitro regeneration of cotton (Gossypium hirsutum L.) cultivar KC3 with controlled phenolic secretion by using Kappaphycus alvarezii sulfated polysaccharide extract and plant growth regulators

In vitro regeneration of cotton (Gossypium hirsutum L.) cultivar KC3 with controlled phenolic secretion by using Kappaphycus alvarezii sulfated polysaccharide extract and plant growth regulators

Chromosome-level genome assembly and annotation of a potential model organism Gossypium arboreum ZB-1

Recent advancements in plant regeneration and synthetic polyploid creation have been documented in Gossypium arboreum ZB-1. These developments make ZB-1 a potential model within the Gossypium genus for investigating gene function and polyploidy. This work generated the sequence and annotation of the ZB-1 genome. The contig-level genome was constructed using the PacBio high-fidelity reads, encompassing 81 contigs with an N50 length of 112.12 Mb. The Hi-C data assisted the construction of the chromosome-level genome, which consists of 13 pseudo-chromosomes and 39 un-anchored contigs, with a total length of about 1.67 Gb. Repetitive sequences accounted for about 69.7% of the genome in length. Based on ab initio and evidence-based prediction, we have identified 48,021 protein-coding genes in the ZB-1 genome. Comparative genomics analysis revealed conserved gene content and arrangement between ZB-1 and G. arboreum SXY1. The single nucleotide polymorphism occurrence rate between ZB-1 and SXY1 was about 0.54 per 1,000 nucleotides. This study enriched the genomic resources for further exploration into cotton regeneration and polyploidy mechanisms.

The Study of Exogenous Auxin and Cytokinins in Embryogenesis and Fiber Genes Expression during In Vitro Regeneration of Cotton (Gossypium hirsutum L.)

The efficiency of regeneration media, based on the morpho-cytochemical response and embryogenesis gene expression of widely cultivated cotton in Indonesia (Gossypium hirsutum L.), needs to be studied further. This study's objectives are to identify the effect of 2,4-D and IBA hormones on the morphology and induction of cotton callus; to determine the effect of regeneration media on the morphology, cytochemical, and gene expression of cotton plant during embryogenesis. This study was conducted by inducing cotton callus on MS0 medium, MS Basal + 0.1 mg/L 2,4-D and MS Basal + 0.1 mg/L IBA. The best callus induction results were regenerated on the regeneration media, namely M1 (MS Vitamin + 0.01 ppm 2,4-D + 0.3 ppm IBA + 0.5 ppm Kinetin), M2 (MS Vitamin + 0.01 ppm 2,4-D + 0.5 ppm IBA + 0.5 ppm Kinetin), M3 (MS Vitamin + 0.00 ppm 2,4-D + 0.3 ppm IBA + 0.5 ppm Kinetin), and M4 (MS Vitamin + 0, 00 ppm 2,4- D + 0.5 ppm IBA + 0.5 ppm Kinetin). The embryogenesis gene expression was observed. The best morphological results for callus induction were obtained on MS Basal + 0.1 mg/L 2,4-D medium with a compact and white callus, indicates high embryogenic and regeneration ability. The highest percentage of regenerated callus parameters was found in M2 media and further confirmed by cytochemical characterization. Embryogenesis genes expressed were GhSERK1, GhSERK2, LEC, GhWUS, and GhWOX11. Other fiber-related genes GhMYB25-like, GhHD-1, and F3H were also expressed during the embryogenesis.

Highly efficient oil–water separation of superhydrophobic cellulose II aerogel based on dissolution and regeneration of cotton in lithium bromide system

In recent years, the frequent occurrence of oil spills and the excessive discharge of oily wastewater have caused serious damage to the environment. Therefore, the development of oil/water separation materials has attracted widespread attention. Here, we have successfully synthesized cellulose aerogels (CAs) based on a green and simple LiBr system and modified the CAs with methyltrimethoxysilane (MTMS) for the fabrication of hydrophobized cellulose II aerogels (HCAs). The prepared HCAs have low density (0.021–0.039 g/cm3), high porosity (97.6–98.7 %) and hierarchical porous structure. The water contact angle of the HCAs was 150°±5°, exhibiting the excellent hydrophobic and lipophilic properties of the HCAs. Oil/water separation experiments have shown that HCAs has outstanding separation efficiency of over 96 % for a variety of oil/water mixtures. Furthermore, the HCAs also exhibited excellent reusability for long-term use. HCAs are promising for a wide range of practical applications in oil–water separation, including oil/chemical spills and industrial effluent discharge, owing to the green and facile preparation process, excellent separation efficiency and superior reusability.

A Stable Agrobacterium rhizogenes-Mediated Transformation of Cotton (Gossypium hirsutum L.) and Plant Regeneration From Transformed Hairy Root via Embryogenesis.

Genetic transformation is a powerful tool to study gene function, secondary metabolism pathways, and molecular breeding in crops. Cotton (Gossypium hirsutum L.) is one of the most important economic crops in the world. Current cotton transformation methods take at least seven to culture and are labor-intensive and limited to some cultivars. In this study, we first time achieved plantlet regeneration of cotton via embryogenesis from transformed hairy roots. We inoculated the cotyledon explants of a commercial cultivar Zhongmian-24 with Agrobacterium rhizogenes strain AR1193, harboring a binary vector pBI-35S::GFP that contained the NPT II (neomycin phosphotransferase) gene and the GFP (green fluorescent protein) gene as a fluorescent marker in the T-DNA region. 82.6% explants produced adventitious roots, of which 53% showed GFP expression after transformation. 82% of transformed hairy roots produced embryonic calli, 12% of which regenerated into stable transformed cotton plants after 7 months of culture. The integration of GFP in the transformed cotton genomes were confirmed by PCR (Polymerase chain reaction) and Southern blot analysis as well as the stable expression of GFP were also detected by semi-quantitative RT-PCR analysis. The resultant transformed plantlets were phenotypically, thus avoiding Ri syndrome. Here we report a stable and reproducible method for A. rhizogenes-mediated transformation of cotton using cotyledon as explants, which provides a useful and reliable platform for gene function analysis of cotton.

Dynamic Transcriptome Analysis Reveals Uncharacterized Complex Regulatory Pathway Underlying Genotype-Recalcitrant Somatic Embryogenesis Transdifferentiation in Cotton.

As a notable illustration of totipotency and plant regeneration, somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway, the key step for genetic engineering. Investigations examining the totipotency process are of great fundamental and practical importance in crop biotechnology. However, high-frequency regeneration of cotton via SE has been limited due to genotype-dependent response. The molecular basis deciphering SE genotype recalcitrance remains largely unexplored in cotton. In the current study, to comprehensively investigate the dynamic transcriptional profiling and gene regulatory patterns involved in SE process, a genome-wide RNA sequencing analysis was performed in two cotton genotypes with distinct embryogenic abilities, the highly embryogenic genotype Yuzao 1 (YZ) and the recalcitrant genotype Lumian 1 (LM). Three typical developmental staged cultures of early SE—hypocotyls (HY), nonembryogenic calli (NEC) and primary embryogenic calli (PEC)—were selected to establish the transcriptional profiles. Our data revealed that a total of 62,562 transcripts were present amongst different developmental stages in the two genotypes. Of these, 18,394 and 26,514 differentially expressed genes (DEGs) were identified during callus dedifferentiation (NEC-VS-HY) and embryogenic transdifferentiation (PEC-VS-NEC), respectively in the recalcitrant genotype, 21,842 and 22,343 DEGs in the highly embryogenic genotype. Furthermore, DEGs were clustered into six expression patterns during cotton SE process in the two genotypes. Moreover, functional enrichment analysis revealed that DEGs were significantly enriched in fatty acid, tryptophan and pyruvate metabolism in the highly embryogenic genotype and in DNA conformation change otherwise in the recalcitrant genotype. In addition, critical SE-associated expressed transcription factors, as well as alternative splicing events, were notably and preferentially activated during embryogenic transdifferentiation in the highly embryogenic genotype compared with the recalcitrant genotype. Taken together, by systematically comparing two genotypes with distinct embryogenic abilities, the findings in our study revealed a comprehensive overview of the dynamic gene regulatory patterns and uncharacterized complex regulatory pathways during cotton SE genotype-dependent response. Our work provides insights into the molecular basis and important gene resources for understanding the underlying genotype recalcitrance during SE process and plant regeneration, thereby holding great promise for accelerating the application of biotechnology to cotton for improving its breeding efficiency.

Embryogenic Capabilities of Cotton through Tissue Culture and Expression Analysis of SERK3 for Regeneration Potential

Plant tissue culture technique was used to develop a regeneration procedure in local Pakistani cotton varieties. Three cotton varieties Coker 312, CEMB66 and Klean cotton were cultured on MS media supplemented with vitamins and growth regulators. The results showed that the regeneration potential of Coker 312 was higher, CEMB 66 moderate while Klean cotton lacks the cotton regeneration potential. The molecular analysis identified the Somatic Embryogenesis Receptor Kinase3 (SERK3) gene and its expression analysis confirmed that it plays role for embryo formation through tissue culture. Its low expression resulted in calli to die rather than forming embryos. Therefore, SERK3 gene is considered important for callus induction and its development into primary embryogenic and embryogenic stage. Further investigations will be helpful to understand the mechanism of regeneration potential of cotton through tissue culture.

Target-mimicry based miRNA167-diminution ameliorates cotton somatic embryogenesis via transcriptional biases of auxin signaling associated miRNAs and genes

In vitro regeneration of cotton seems to be highly genotype dependent and typically ensues through the extraordinarily complex mechanism of somatic embryogenesis (SE). During the acquisition of embryogenic potential by the somatic cells, 21–24 nucleotides long regulatory microRNAs, especially miRNA167 plays a crucial role in the regulation of auxin signaling by degrading or inhibiting target mRNA molecules. To reveal the mechanism of regulation of target genes and the influence of key SE-associated miRNAs on the acquisition of embryogenic potential by the somatic cells, we employed target-mimicry approach of miR167 diminution for loss-of-function analysis encompassing callogenesis and in vitro embryogenesis stages of a fully-regenerating line of Coker 310 cultivar. Results demonstrated that miR167-diminution led to enhanced callogenesis and increased production of somatic embryos in the mimic-transformed events. In the MIM167-transformed calli, ARF6/ARF8, auxin-responsive GH3, auxin transporter Aux1, and auxin influx/efflux carrier LAX3, PIN1 and PIN2 genes showed a greater shift than control/non-transformed events, and suggested that miR167 diminution led to enhanced cellular auxin-signaling. Moreover, auxin-signaling associated microRNAs-miR160, miR393, miR166, miR156 and miR157 were differentially expressed in MIM167 mimic events indicating that diminution of miR167 also influenced the expression of these miRNAs. Evidently, miR166-LEC2 pair supplemented auxin signaling associated miR167-ARF6/ARF8 module that consequently may have resulted into substantial phenotypic transformations during embryonic transitions of cotton cultures. Coordinated and fine-scale expression-network of auxin signaling-associated miRNAs and genes in miR167-target mimic transgenic calli of cotton, directed the incessant production of somatic embryos.

Cotton Hairy Root Culture as an Alternative Tool for Cotton Functional Genomics.

Although well-accepted as the ultimate method for cotton functional genomics, Agrobacterium tumefaciens-mediated cotton transformation is not widely used for functional analyses of cotton genes and their promoters since regeneration of cotton in tissue culture is lengthy and labor intensive. In certain cases, A. rhizogenes-induced hairy root culture has been a suitable molecular tool for functional analyses of genes and promoters for plants that are difficult to regenerate by A. tumefaciens-mediated transformation. Similarly, A. rhizogenes-induced hairy root cultures are an alternative tool for cotton functional genomics. In this chapter, the advantages and disadvantages of using A. rhizogenes-induced cotton hairy root culture over A. tumefaciens-mediated cotton transformation are discussed. The procedures for transformation, generation, selection, and molecular analyses of transgenic cotton hairy roots are introduced by describing the functional analysis of a cotton promoter in cotton hairy roots generated by A. rhizogenes-mediated transformation.

Characterization of GhSERK2 and its expression associated with somatic embryogenesis and hormones level in Upland cotton

Somatic embryogenesis (SE) is one of the most important steps during regeneration of cotton, but the molecular mechanism of SE remains unclear. SOMATIC EMBRYOGENSIS RECEPTOR KINASE (SERK) gene is known to function in SE. A homolog GhSERK2 (accession number: JF430801) was cloned from Upland cotton and characterized for its functions in SE. GhSERK2 expressed in different tissues and showed higher expression level in floral organs than vegetative ones with the highest levels in ovule and anther. GhSERK2 expressed during SE with a high level at globular embryos stage. Upon treatment with indole-3-butytic acid (IBA), the transcription level of GhSERK2 was induced and promoted SE subsequently. A 2-day treatment of 2,4-dichlorophenoxyacetic acid (2,4-D) induced the expression of GhSERK2, but treatments of 2,4-D for longer periods sharply inhibited the GhSERK2 transcription level of embryogenic callus (EC). The levels of hormones, including 3-indoleacetic acid (IAA), abscisic acid (ABA), and brassinosteroid (BR), were increased in the initial calli induced from the over-expression of GhSERK2 cotton. Our results indicated that GhSERK2 expression was associated with induction of SE and closely related to hormone levels during tissue culture in Upland cotton, and the gene might play an important role in regeneration of cotton.

English

The need for alternative strategies to obtain transgenic cotton via apical shoot was necessitated due to the recalcitrance of cotton regeneration from somatic embryogenesis, this has greatly slowed down the development of transgenic cottons. To this effect, an optimized regeneration system from apical shoot was developed for three varieties of cotton. Ninety-five percent seed surface sterility was observed in seed germination using a combination of hydrogen perioxide and Clorox as sterilizing medium. Highest shoot elongation rate was achieved on MS supplemented with 2.5 mg/L BAP + 0.1% (w/v) AC, rapid shoot growth occurred with kinetin supplemented media. Rooting efficiency of the three improved cultivars of cotton (Gossypium hirsutum), Samcot 9,11 and 13 were optimized using the optimum medium for rooting of difficult-to-root in vitro regenerated shoots of cotton which consist of MS basal salts and modified MS vitamins, supplemented with 3% sucrose, 0.2 mg/L IBA, without activated charcoal. In the end, an improved regeneration protocol with rooting efficiency up to 47% and regeneration rate up to 87% by combining rooting induction, indole acetic acid (IAA) shock and graft technique was developed.   Key words: Allium sativum, cotton tissue culture, transgenic plant, optimized regeneration of cotton.

English

In vitro regeneration of cotton (Gossypium spp.) has been a subject of intense research for the last two decades because of the commercial value of the crop. A study was conducted to assess the callusing potential of two local cotton genotype varieties viz., MCU-5 and SVPR-2 and two exotic genotypes, Coker 310 and 312. High significant difference was observed between media composition, genotype and explant types used for callus induction. Among the 24 media composition screened for callusing, the medium CIM3 (MS + 0.1 mg/l 2,4-D + 0.5 mg/l kinetin) exhibited successful and faster induction of calli. Callus initiation was found to be quicker from hypocotyls than cotyledons. Calli developed from hypocotyl explants were large, rough and friable, while cotyledon-derived calli were friable and medium sized. Significantly higher callus induction frequencies were observed in hypocotyl (97.3%) than cotyledon (95.8%) explants which were collected from 7 day-old seedlings irrespective of genotype. Explants collected from younger (4 day-old) and older (14 day-old) plants showed poor and low callus induction frequencies.   Key words: Callus, cotyledon, Gossypium hirsutum, hypocotyl, media composition.

Expression of Mannose-Binding Insecticidal Lectin Gene in Transgenic Cotton (<i>Gossypium</i>) Plant

Cotton ( Gossypium spp ) is an important world crop. Despite the efforts made through traditional breeding methods, cotton breeders still faced with many problems, i.e., narrow genetic base, inability to use alien genes and difficulty in breaking gene linkages. Breeders attempted genetic transformations analyses tools to overcome these problems with very little success, hence the need for transgenic intervention. In this report, an optimized cotton regeneration system from shoot apices used to transform cotton wit insecticidal lectin gene from Allium sativum . Cotton regeneration system was observed to be genotype independent with a regeneration rate of 85% obtained within 16 weeks. The age of explants and the size of isolated tips have a significant effect on shoot tip elongation. The elongation rates of the three varieties were not significantly different from each other (p=0.1573). It was observed that Samcot 9 had the highest rooting efficiency (47%), and Samcot 13 had the least rooting efficiency (36%). Though the difference in rooting efficiency was not significant in the three varieties (P=0.08) Transgenic cotton plants were obtained via Agrobacterium -mediated transformation using shoot apices as explants. Transformation rate was 1.3% using LBA 4404 with β-glucuronidase (GUS) gene. The mean number of GUS positive apices was 67% higher when acetosyringone was included in the medium. Agrobacterium concentration and co-cultivation time have a significant effect on transient GUS expression. The highest GUS positive number was observed at OD 600 0.6 and co-cultivation for 3 days. Putative transgenic plants were confirmed by leaf GUS assay, kanamycin leaf test and molecular analysis of putative young leaves.

Transgenic Indian Cotton (Gossypium hirsutum) Harboring Rice Chitinase Gene (Chi II) Confers Resistance to Two Fungal Pathogens

Problem statement: The present investigation described a simple and reproducible protocol for transgenic cotton regeneration and characterization of chitinase (Chi II) gene expression against two different fungal pathogens in cotton. Approach: Transgenic cotton (Gossypium hirsutum cv. SVPR2) plants were produced by pCambia-bar-Chi II (13.8 kb) under the control of the CaMV 35S promoter, harbored in the strain LBA 4404 Agrobacterium tumefaciens by using shoot tip explants. Results: Finally, from the 10 experiments, 21.8% of transformation frequency was recorded. Segregation ratio of 3:1 was recorded in the T0 plant seeds. Polymerase chain reaction and southern blotting analysis were used to confirm the integration of Chi II transgene in the T0 plants genome of putative transgenics. Quantitiave and qualitative (SDS-PAGE) analyses were also carried out to confirm the expression of chitinase enzyme in T0 plants. Further, randomly selected transgenic plants (T0) were analyzed for disease tolerance by evaluating them with spores of Fusarium oxysporum and Alternaria macrospora. All the selected PCR positive plants showed enhanced disease resistance against Fusarium wilt. The plants selected randomly showed an enhanced survival rate compared with the control when they were grown in earthen pots inoculated with 1×105 spores 100-1 g of soil mixture. Another four randomly selected plantlets were sprayed with spores of Alternaria macrospora in order to test their tolerance to Alternaria leaf spot disease. After 20 days of culture, the number of lesions per leaf and the lesion length per leaf spot in non-transferred leaves increased. In the case of transgenic plantlets, lesion formation was completely absent. Conclusion: The disease resistance against Fusarium wilt and Alternaria leaf spot in cotton strains would serve as good breeding materials for producing fungal disease resistant cotton varieties.

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.

Reactions of some phosphorus compounds with cellulose dissolved in aqueous alkaline solution

AbstractDissolution of cellulose in aqueous hydroxide offers a procedure for regeneration of cotton or wood celluloses. The aqueous NaOH system for dissolving cellulose seems to have considerable potential for fiber production and also as a medium for preparing cellulose derivatives. Reactions with phosphoric and phosphorous acids, phenyl‐ and phenoxy phosphonic dichlorides, and an amido phosphite oligomer on aqueous dissolved cellulose are described. The resulting products are characterized by analytical and spectral methods (infrared and X‐ray diffraction) to determine phosphorus and nitrogen content, substitution degree, and polymerization degree. Particular attention was given to the effects produced by chemical modification on the crystalline pattern of microcrystalline cellulose. Some remarks are also made about the thermooxidative behavior of phosphorylated cellulose. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 327–333, 2003

Development of a highly regenerable elite Acala cotton (Gossypium hirsutum cv. Maxxa): a step towards genotype-independent regeneration

A step towards genotype-independent regeneration of cotton (Gossypium hirsutum L.) was achieved by selection for regeneration potential (RG) in commercial seed of elite cultivars. A callus induction medium (MCIM) empirically determined for the cultivar `Maxxa' paved the way for RG selection among individual genotypic variants within a cultivar. MCIM consists of a basal Murashige-Skoog medium, supplemented with a unique combination of two synthetic auxins. Hypocotyl explants of `Coker 312', `Maxxa' and `Riata' seedlings cultured on MCIM successfully produced a high quality, friable callus as defined by its color, texture, size, and organization. Based on the number of fertile plants regenerated on a per seedling basis, RG was estimated as 17.4%, 44.4% and 80% in Acala cotton cultivars `Maxxa', `Ultima', and `Riata', respectively. The high RG of the cultivar Riata, a Round-up Ready® transgenic cultivar in a Maxxa genetic background, is likely due to additional RG alleles introgressed from the transgenic parent. Genotypic differences between cultivars for RG was reflected by the need for supplemental kinetin to efficiently regenerate `Ultima' plantlets via somatic embryogenesis. RG selection pressure through two cycles of selection resulted in development of advanced highly regenerable `Max-R' lines in an elite genetic background with immediate potential as suitable germplasm for breeding and biotechnology applications. Based on the results presented here, strategies for genotype-independent transformation and regeneration of cotton are proposed that integrate selection and introgression of regeneration potential in improvement programs.

Adaptation of Cotton Shoot Apex Culture to Agrobacterium-Mediated Transformation

A protocol is presented for rapid genotype-independent transformation and regeneration of cotton (Gossypium spp.) from shoots isolated from germinating seedlings. Isolated shoots are inoculated with a super-virulent strain of Agrobacterium tumefaciens, subjected to a mild antibiotic selection, and directly regenerated as shoots in vitro. Shoots do not dedifferentiate and mutation rates are low. Rooted shoots can be obtained within 6–10 weeks of isolation and inoculation depending on the cotton cultivar.

In vitro proliferation of shoots and regeneration of cotton

Shoot proliferation from different explants of several Indian cultivars of cotton was studied in culture. Cotyledonary nodes taken along with the shoot apex of seedlings produced multiple shoots on modified MS nutrient agar supplemented with cytokinins. 6-Benzyladenine was most effective in inducing growth of multiple shoots. Explants of several genotypes formed organogenic masses that differentiated to secondary shoots on repeated subculture. The isolated shoots were rooted on basal medium supplemented with naphthaleneacetic acid and were transferred to soil after acclimatization.

Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.)

Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0°C/min). A cooling rate of 0.5°C/min provided a satisfactory recovery rate (30%) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of 1°C/min. Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soil.