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.     

Physiological Effects of 1-Methylcyclopropene on Well-Watered and Water-Stressed Cotton Plants

The current study investigated the effect of 1-methylcyclopropene (1-MCP), an ethylene inhibiting compound, in alleviating the detrimental effect of drought on cotton plants. The experiment was conducted in a growth chamber in 2006 and 2007. Treatments consisted of (T1) an untreated control well-watered, (T2) 1-MCP at 10 g ai/ha well-watered, (T3) an untreated control water-stressed, and (T4) 1-MCP at 10 g ai/ha water-stressed. Water-stress treatment consisted of withholding water from the pots until stomatal closure. The water-stress regime and the 1-MCP treatments were imposed at the pinhead-square stage, approximately 4 weeks after planting. Water-stressed plants treated with 1-MCP had a higher stomatal resistance, less negative water potential, higher activity of antioxidant enzymes, and better maintenance of membrane integrity. The greatest effects on stomatal resistance were observed at 5 days after treatment initiation, in which water-stressed 1-MCP-treated plants exhibited stomatal resistance of 0.079 m² s mmol⁻¹, whereas water-stressed untreated plants exhibited only 0.047 m² s mmol⁻¹. There was no significant effect of 1-MCP on water-use efficiency, transpiration, and dry matter production. These results indicated that application of 1-MCP to water-stressed cotton may have the potential to lower levels of stress in treated plants.     

Cloning and characterization of a symbiosis-related gene from an ectomycorrhizal fungus Laccaria bicolor

An in vitro system for a Laccaria bicolor×Pinus resinosa interaction was used to identify and clone a symbiosis-regulated gene from L. bicolor employing the mRNA differential display technique (DDRT–PCR). The DDRT–PCR identified several cDNAs that are differentially expressed as early as 6 h into the interaction. One such cDNA was used to screen a L. bicolor cDNA library enriched for mRNAs expressed during early interaction with red pine seedlings. Characterization of a cDNA clone, PF6.2, showed that it contained a 1551 bp insert coding for a protein of 433 amino acids. Sequence analysis of the PF6.2 cDNA revealed the presence of several evolving repeats in the protein. To confirm this, the gene corresponding to PF6.2 was isolated and sequenced. The PF6.2 gene consisted of seven exons interrupted by six relatively small introns. Although the amino-acid sequence of the PF6.2 did not show significant overall similarity to any previously characterized proteins, of several direct repeats it contained a feature similar to other proteins involved in signal transduction through protein–protein interaction. Northern analysis showed that the PF6.2 mRNA was detectable in the fungus 6 h after interaction and continued to be expressed in established ectomycorrhizas, suggesting that it plays an important role in the formation and maintenance of the symbiosis.     

Leaf Wilting Movement Can Protect Water-Stressed Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) Plants Against Photoinhibition of Photosynthesis and Maintain Carbon Assimilation in the Field

Under severe water stress, leaf wilting is quite general in higher plants. This passive movement can reduce the energy load on a leaf. This paper reports an experimental test of the hypothesis that leaf wilting movement has a protective function that mitigates against photoinhibition of photosynthesis in the field. The experiments exposed cotton (Gossypium hirsutum L.) to two water regimes: water-stressed and well-watered. Leaf wilting movement occurred in water-stressed plants as the water potential decreased to −4.1 MPa, reducing light interception but maintaining comparable quantum yields of photosystem II (PS II; Yield for short) and the proportion of total PS II centers that were open (qP). Predrawn F _v/F _m (potential quantum yield of PS II) as an indicator of overnight recovery of PS II from photoinhibition was higher than or similar to that in well-watered plants. Compared with water-stressed cotton leaves for which wilting movement was permitted, water-stressed cotton leaves restrained from such movement had significantly increased leaf temperature and instantaneous CO₂ assimilation rates in the short term, but reduced Yield, qP, and F _v/F _m. In the long term, predrawn F _v/F _m and CO₂ assimilation capacity were reduced in water-stressed leaves restrained from wilting movement. These results suggest that, under water stress, leaf wilting movement could reduce the incident light on leaves and their heat load, alleviate damage to the photosynthetic apparatus due to photoinhibition, and maintain considerable carbon assimilation capacity in the long term despite a partial loss of instantaneous carbon assimilation in the short term.     

Water-deficit stress effects on pistil biochemistry and leaf physiology in cotton (Gossypium hirsutum,L.)

Flowering in cotton (Gossypium hirsutum L.) is a sensitive stage to water-deficit stress, but the effects on metabolism are not well understood. The objective of this study was to monitor gas exchange responses of cotton plants under conditions of limited water supply and evaluate the effects on the carbohydrate concentrations and glutathione reductase levels in the cotton flower. Growth chamber experiments were conducted in 2008 and 2009, with normal day/night conditions of 32/24 °C and optimum quantities of Hoagland's nutrient solution applied until flowering. Treatments were imposed at flowering and consisted of control (Control), where optimum quantities of water were applied, and water stress (WS) where 50% of optimum quantity of water was supplied. Water-deficit stress resulted in a significant decrease in leaf stomatal conductance. Leaf photosynthetic and respiration rates were similarly decreased compared to the control. Ovary and style water potential of water-stressed leaves were significantly higher compared to the water potential of water stressed leaves, indicating that cotton flowers are fairly resistant to changes in the water status of the plant. However, carbohydrate concentrations of water-stressed pistils (ovary and style) were significantly increased compared to the control and a similar pattern was observed in the levels of glutathione reductase of water-stressed pistils. In conclusion, water-deficit stress during flowering resulted in significant decreases in leaf gas exchange functions as well as leaf water potential. Cotton pistils appeared to be less sensitive since they were able to maintain water potential similar to the control under limited water supply and increase glutathione reductase levels. However, pistil carbohydrate metabolism was significantly affected resulting in accumulation of both hexose and sucrose indicating a perturbation in sucrose cleaving and hexose utilizing enzymes that could potentially have as a consequence a decrease in fertilization and seed set efficiency.     

Cloning and expression of <Emphasis Type="Italic">GhTM6</Emphasis>, a gene that encodes a B-class MADS-box protein in <Emphasis Type="Italic">Gossypium hirsutum</Emphasis>

A full-length cDNA designated GhTM6, which encodes an organ differentiation-related B-class MADS-box protein, was isolated from Upland cotton (Gossipium hirsutum) by screening a normalized fulllength cDNA library and using a RT-PCR strategy. The translated sequence analysis indicated that the polypeptide contained MADS-box and K domains and had a classic TM6 motif, i.e., the paleoAP3 in the C-terminal region. The phylogenetic analysis showed that GhTM6 is closest to CeTM6, MaTM6, BuTM6, and PhTM6. Quantitative RT-PCR analysis showed that the GhTM6 gene was expressed at high levels in all tissues examined, such as those from squares, flowers, petals, stamens, and carpels under normal growth conditions. GhTM6 was expressed at high levels before floral initiation and declined thereafter. Furthermore, six stamens were seen in the transgenic tobacco flower as compared to five stamens in a wild-type flower. The results indicated that GhTM6 did not exhibit the full B-function spectrum, because it is only involved in the determination of stamen organ identity. However, its function in cotton will need to be examined in transgenic cotton plants.