Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH4NO3 and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6-7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.

The Aluminum (Al) and proton (H ) ions are major acid soil stress factors deleteriously affecting plant root growth and crop yield. In our preliminary studies, cotton seedlings (Gossypium hirsutum L.) displayed very sensitive phenotypes to Al and H rhizotoxicities. Based on previous Arabidopsis results, we aimed to characterize the Al stress responsive Sensitive to Proton rhizotoxicity 1 (GhSTOP1) transcription system in cotton by RNAi mediated down-regulation. With the help of seed embryo apex explants, we developed transgenic cotton plants overexpressing a GhSTOP1-RNAi cassette with NPTII selection. Kanamycin tolerant T1 seedlings were further considered for Al and H stress tolerance studies. Down-regulation of the GhSTOP1 displayed increased sensitivity to Al and proton rhizotoxicities and the root growth was significantly reduced in RNAi-lines. The expression profile of GhALMT1 (Aluminum-activated Malate Transporter 1), GhMATE (Multidrug and Toxic Compound Extrusion), GhALS3 (Aluminum Sensitive 3) and the key genes involved in the GABA shunt were downregulated in the transgenic RNAi lines. Additionally, the lateral root initiation process was delayed and the expression of GhNAC1 which is involved in lateral root initiation was also suppressed in transgenic lines. Besides, overexpression of GhSTOP1 in Arabidopsis accelerated root growth and AtMATE and AtALMT1 expression under Al stress conditions. These analyses indicate that the GhSTOP1 is necessary for the expression of several genes which are necessary for acid soil tolerance mechanisms and lateral root initiation. This article is protected by copyright. All rights reserved.

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 T 0 plant seeds. Polymerase chain reaction and southern blotting analysis were used to confirm the integration of Chi II transgene in the T 0 plants genome of putative transgenics. Quantitiave and qualitative (SDS-PAGE) analyses were also carried out to confirm the expression of chitinase enzyme in T 0 plants. Further, randomly selected transgenic plants (T 0 ) 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 1×10 5 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.

An in vitro propagation system was developed for castor-bean (Ricinus communis L. cv. TMV 6) through cotyledon derived callus cultures. The impact of different concentrations of auxins, cytokinins, additives, amino acids and sugars were evaluated for callus induction and shoot proliferation. Green compact nodular organogenic callus was obtained on the medium fortified with Murashige and Skoog (MS) salts, B5 vitamins, 2.0 mg dm -3 6-benzyladenine and 0.8 mg dm -3 α-naphthalene acetic acid (NAA). Multiple shoot proliferation from the callus cultures was achieved on the medium with MS salts, B5 vitamins, 2.5 mg dm -3 thidiazuron (TDZ), 0.4 mg dm -3 NAA and 15 mg dm -3 glutamine. During multiple shoot induction the phenolic secretion was controlled by the addition of 15 mg dm -3 polyvinylpyrolidone. The proliferated shoots were elongated on the medium comprising MS salts, B5 vitamins, 1.5 mg dm -3 TDZ and 0.3 mg dm -3 gibberellic acid. The elongated shoots were rooted on the medium containing MS salts, B5 vitamins, 0.3 mg dm -3 indole-3-butyric acid and 0.6 mg dm -3 silver nitrate. After root induction, the plants were hardened in earthen pots containing sand, soil and vermiculite.