Obtaining Insect-resistant Cotton by Transformation with Agrobacterium

ABSTRACT
A plant expression vector, pBinLK, carrying two insecticidal genes: pea lectin gene and soybean Kunitz trypsin inhibitor gene, were constructed and successfully transferred into 4 upland cotton (Gossypium hirsutum L.) cultivars, Xinluzao-1, Xinluzhong-2, Jihe-321 and Liao-9 via Agrobacterium-mediated transformation. Hypocotyl segments from aseptic seedlings were used as transgene recipients. After co-cultivation, kanamycin-resistant calli were screened, and somatic embryos and regenerated plants were obtained using various media. Transgenic cotton plants with two insecticidal genes were confirmed by ELISA, PCR and PCR Southern analysis. Bioassays demonstrated that the transgenic plants had significant resistance to larvae of cotton bollworm (Helicoverpa armigera Hübner).

Back to Table of contents

Response to In Vitro Regeneration of Immature Zygotic Embryos in Cotton (Gossypium spp)

ABSTRACT
The in vitro culture response of immature zygotic embryos (IZE), from six Gossypium hirsutum and one G. barbadense variety, was studied. Explants were collected from plants grown in the field and the greenhouse. Bolls were collected 12, 14 and 16 days after pollination (DAP) from the field and 10, 12 and 14 DAP from the greenhouse. The young embryos were cultured in vitro in SH and BT supplemented with 2,4-D and kinetin. All genotypes responded well to callogenesis in spite of the environmental conditions where they were grown. Callus formation (%) was higher when the bolls were collected 16 DAP for embryos collected from the field and 14 DAP for embryos collected from the greenhouse. The callus developed was tested for its embryogenic and organogenic potential in several media without positive results. During the callus induction period, it was observed that in some explants the epidermal cells from the upper cotyledon and the hypocotyl were diversified into globular or heart shape embryoids. For their further development, they were transferred to a modified MS medium. Some of the young embryoids formed roots and leaves but they never developed into regular plantlets.

Back to Table of contents

Random Amplified Polymorphic DNA Studies in Cotton

ABSTRACT
Polymerase chain reaction (PCR)-based random amplified polymorphic DNA (RAPD) analysis can to be an important tool in the hands of plant breeders. Among its many applications, RAPD analysis can be used to measure genetic diversity and for DNA fingerprinting. In this investigation, 22 genotypes, representing cultivated Gossypium hirsutum, G. barbadense and G. arboreum with diverse morphological traits and one wild species G. thurberi, were subjected to RAPD analysis using 20 random 10-mer primers. DNA was extracted and quantified by established methods for RAPD analysis and the PCR conditions were standardized. Eleven primers produced 128 amplification products of which 119 (92.97%) were polymorphic between at least two of the entries. The size of the amplification products ranged between 201 and 2888 bp. Specific markers were observed for each of the four species. A set of five primers distinguished all 23 genotypes. Five cultivar-specific markers were also observed. A dendrogram constructed from the RAPD data classified all the genotypes into two broad clusters of wild and cultivated species. The cultivated species were further classified into G. arboreum, G. hirsutum and G. barbadense clusters. The wild species (G. thurberi), which fell away from the cultivated species, was about 18 percent similar to them. The similarity of the G. arboreum and G. hirsutum clusters was 47 percent. Gossypium thurberi appeared to be more closely related to G. barbadense than to the other two cultivated species.

Back to Table of contents

Genetics and Combining Ability of Leaf Curl Virus Resistance in Cotton (Gossypium hirsutum L.)

ABSTRACT
The cotton leaf curl virus (CLCV) disease caused by Gemini viruses has resulted in failure of the cotton crop in many countries. The losses caused by this disease range from 20 to 80 percent depending upon the stage of viral infection by virulent cotton white fly (Bemisia tabaci). The only permanent approach of combating this menace is through resistant varieties and hybrids. An understanding of the genetics and combining ability of CLCV resistance is a pre-requisite for breeding cultivars resistant to this disease. The present investigation was undertaken for that purpose. The experimental material consisted of 90 crosses involving resistant and susceptible parents and segregating generations. The experiments were conducted at Punjab Agricultural University, Ludhiana under artificial conditions and at the Cotton Research Station, Abohar, under disease epiphytotic conditions. The CLCV resistance was controlled by more than two genes along with inter-allelic interactions. Good general combining parents included LH 1134, IL 85, IL 89 and IL 99 as females and F 54 as male parent for virus resistance. PIL 43 x PIL 8 (LHH 144), LH 1832 x F 54, LH 1818 x F 54, and LH 1556 x R 27 were good specific combining crosses for CLCV resistance. The resistant crosses involved either one or both resistant/tolerant parents. The breeding approaches for CLCV resistance are discussed.

Back to Table of contents

Development of Transgenic Insect-protected Cotton Plants

ABSTRACT
An artificially synthesized Bacillus thuringiensis insecticidal protein gene, co-constructed with the GUS reporter gene (Bt/GUS), was transferred into fertilized ovaries of the elite cotton (Gossypium hirsutum L.) cultivars Simian 3 and Zhongmiansuo 12 by the pollen tube pathway. Transgenic cotton plants were recovered from the seeds in the treated bolls. Histochemical analysis for GUS activity indicated that the gene was expressed in the transgenic R1 plants of the two recipient cultivars. The presence of the Bt gene in the GUS-positive R1 plants was confirmed by PCR and the same results were obtained in the R2 plant populations. This indicated stable integration of the Bt gene into the recipients and its inheritance from R1 to R2 generations. Resistance to the cotton bollworm (Helicoverpa armigera Hübner) was identified in these transgenic plants. In R1, five plants highly toxic to the insect were found: S545, S591, S636, and S1001 from “Simian 3+Bt/GUS”, and Zh1109 from “Zhongmiansuo 12+Bt/GUS”, with larva mortality up to 91.6%, 93.8%, 92.3%, 85.7% and 75.0%, respectively. Insect-resistant R5 strains were derived from the R1 transgenic insect-resistant plants via selfing and breeding, showing the maintenance of the transgene and insect-resistance and the practical potential in cotton production.

Back to Table of contents

Induction of Multiple Shoot Differentiation and Increasing Transformation Frequency in Shoot Tips of Egyptian Cotton

ABSTRACT
Multiple shoot differentiation and regeneration from cotton (Gossypium barbadense L.) shoot tips of aseptically-grown seedlings was developed on Murashige and Skoog media supplemented with 100 mg/L myo-inositol, 1.0 mg/L thiamin-HCL, 2% glucose, 1X Gamborg vitamin, 100 mg/L citric acid, 100 mg/L ascorbic acid, 0.1 mg/L kinetin, various levels of BA and TDZ, and 0.2% phytagel. Multiple shoot differentiation and regeneration was genotype independent when tested on four Egyptian cottons, Giza 45 and Giza 87 as extra long staple varieties, and Giza 85 and Giza 89 as long staple varieties. Plant regeneration required a specific concentration of BA and TDZ depending on the origin of the meristems. The frequency of explants forming multiple shoots ranged from 40 % to 65 % among the genotypes and the number of shoots ranged from 2 to 15 per responding explant. More than 80% of the shoots could be rooted. The frequency of genotype-independent transformation of Egyptian cotton varieties by Agrobacterium was increased by microprojectile wounding of the apical meristem. Bombardment was with tungsten (0.5 m) particles and four rupture disk pressures, 650 psi, 900 psi, 1300 psi and 2000 psi. Increasing rupture disk pressure produced a higher transformation frequency by A. tumefaciens but decreased the percentage of recovered plantlets. Regenerated plants were phenotypically normal and all of the mature plants initiated flowers and set variable R1 seeds.

Back to Table of contents

Transformation of Egyptian Cotton Tissue (Gossypium barbadense) Using Agrobacterium tumefaciens

ABSTRACT
Callus induction from hypocotyl and cotyledonary explants of Egyptian cotton (Gossypium barbadense), extra-long staple (Giza 45, Giza 77) and long staple (Giza 85), were evaluated for in vitro callus induction and maintenance utilising two media formulations (G2) and (CI). Giza 85 produced significantly better callus than Giza 45 and Giza 77. However, induction of callus was genotype dependent and highly variable, not only among varieties, but also among explants of the same variety. Attempts to control tissue contamination and decay of calli were successful. Explants from three varieties were tested for regeneration using G2 and CI media containing different levels of kanamycin to test for tolerance level in callus tissue. Callus induction and growth were inhibited at 25 mg/L kanamycin. Cotyledon and hypocotyl tissues of the three varieties were transformed successfully, and transformation efficiency varied by variety and type of explant. Cotyledon and hypocotyl explants from 12-day-old aseptically germinated seedlings were inoculated with a non-oncogenic Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid vector pBI121 with a chimeric nopaline synthase (NOS) promoter driving the neomycin phosphotransferase (NPT II) gene and a CaMV35S promoter driving the -glucuronidase (GUS) gene. After three days co-cultivation, explants were placed on callus induction medium containing 25 mg/L kanamycin for selection of transformed tissue. The presence of (NPT II) in crude cellular extracts from embryogenic callus tissue was detected by ELISA test. Expression of the GUS gene was confirmed histochemically followed by dot blot analyses to screen for DNA integration using an Xba I- EcoR1 fragment containing GUS gene coding sequences as probe.

Back to Table of contents

Utilization of Cotton Haploids in Basic and Applied Research

ABSTRACT
Systematic cytological work in the genus Gossypium L. resulted in isolation of haploids (2n=2x=26) in tetraploid (2n=4x=26) G. hirsutum, G. barbadense and their F2 interspecific crosses and monoploid (n=x=13) cultivated Asiatic (2n=2x=26) cotton. The haploids and monoploids were studied for differences in their morphological characters at haploid and diploid levels. On the basis of cytological observation in haploids and monoploids, a new concept of the basic chromosome number (x=7) in the genus and the polyploid nature of Gossypium spp. (2n=2x=26) is proposed. The haploids were found to be a shorter and easier means for securing desirable interspecific genetic combinations. Most interspecific hybridization reported in Gossypium so far are at the tetraploid and diploid levels. The success in transfer of characters in such crosses is limited by sterility in F1s. Development of fixed lines from these interspecific gene transfer becomes a long-term program with a low success probability. The use of G. hirsutum haploids (2n=2x=26), AhDh, for hybridization with different wild Gossypium species is a new and shorter approach in this regard

Back to Table of contents

Development of an Efficient Regeneration and Transformation System for Commercially Grown South African Cotton Cultivars

ABSTRACT
While establishing a system for genetic transformation of South African cotton cultivars, a method of regeneration and transformation of the shoot apical meristem of seedlings was developed. Shoot apices from young cotton seedlings (Gossypium hirsutum cvs Sabie, Jassid, Palala and 107/1) were placed onto Murashige and Skoog (MS) basal medium (pH 5.8) containing MS vitamins, 2% sucrose, 0.7% agar and 0.1 mg/L kinetin. Within two weeks of culture, the shoot meristems differentiated into multiple green shoots and spontaneous root regeneration occurred two months later. Plantlets were placed in soil and hardened off in the growth chamber. Four months later the plants had grown to maturity and cotton bolls had formed. This regeneration protocol demonstrated a successful technique for the recovery of cotton plants from tissue culture using the shoot apical meristem. Shoot apical meristems of seedlings were used for transformation and were co-cultivated with Agrobacterium tumefaciens containing the phosphinothricin acetyltransferase (pat) and the neomycin phosphotransferase (npt II) genes. After co-cultivation, selection was carried out on regeneration medium containing 100 mg/L kanamycin and 1 mg/L glufosinate ammonium (PPT). Control explants were completely inhibited from growing on the selection medium, whereas transformed explants gave rise to multiple shoots resistant to PPT and kanamycin after four weeks. The integration of the pat gene in the transformed plant DNA was confirmed by polymerase chain reaction

Back to Table of contents

Haploid Induction in Cotton: A Future Perspective

ABSTRACT
The value of haploids in genetic analysis and plant breeding has long been known. This is more so in cotton, since the production of double haploids in large numbers could open the way for the development of partial interspecific hybrids between Gossypium hirsutum and G. barbadense. For the induction of haploids in cotton three approaches were applied, namely: anther culture, induction of parthenogenesis after pollination of cotton flowers with alien pollen, and semigamy. In vitro culture of anthers originated from 10 cotton varieties and a number of intraspecific and interspecific cotton hybrids resulted only in the production of callus with a number of haploid cells and very few embryoids. Haploid and double haploid plants were obtained in a relatively small number after pollination of a semigametic line with pollen from interspecific cotton hybrids. Pollination of cotton flowers with pollen from related species (Hibiscus syriacus, Abelmoschus esculentus, Malva sylvestris, Datura stramonium, Hibiscus cannabinus) and in vitro culture of young ovules resulted in the production of abnormal plantlets when A. esculentus and D. stramonium were the pollinators and of mature cotton plants when the pollinator was H. cannabinus.

Back to Table of contents