Proline Accumulation,Photosynthetic Abilities and Growth Characters of Sugarcane （Saccharum officinarum L.） Plantlets in Response to Iso-Osmotic Salt and Water-Deficit Stress

The aim of this study was to investigate the biochemical, physiological and morphological responses of sugarcane to iso- osmotic salt and water-deficit stress. Disease-free sugarcane plantlets derived from meristem cuttings were photo- autotrophically grown in MS media and subsequently exposed to -0.23 （control）, -0.67 or -1.20 MPa iso-osmotic NaCl （salt stress） or mannitol （water-deficit stress）. Chlorophyll a （Chl a）, chlorophyll b （Chl b）, total carotenoids （Cx＋c）, maximum quantum yield of PSII （Fv/Fm）, photon yield of PSII （ΦPSII）, stomatal conductance （Gs） and transpiration rate （E） in the stressed plantlets were significantly reduced when compared to those of plantlets of the control group （without mannitol or NaCl）, leading to net-photosynthetic rate （Pn） and growth reduction with positive correlation. In addition, physiological changes and growth parameters of plantlets in the salt stress conditions were more sharply reduced than those in waterdeficit stress conditions. On the other hand, the proline content and non-photochemical quenching （NPQ） in the leaves of stressed plantlets increased significantly, especially in response to iso-osmotic salt stress. The chlorophyll pigments in iso-osmotic stressed leaves were significantly degraded （r^2 = 0.93）, related to low water oxidation （r^2 = 0.87）, low net- photosynthetic rate （r^2 = 0.81）, and growth reduction （r^2 = 0.97）. The multivariate biochemical, physiological and growth parameters in the present study should be further used to develop salt, or drought, tolerance indices in sugarcane breeding programs.     

Study on DNA Cytosine Methylation of Cotton （Gossypium hirsutum L.） Genome and Its Implication for Salt Tolerance

To study the relations between DNA methylation and abiotic stress responses in cotton （Gossypium hirsutum L.）,the methylation-sensitive amplified polymorphism （MSAP） method was used to investigate the differences in methylation level and the change of cytosine methylation patterns under salt （NaCl） stress in two different salt-tolerant cotton lines.The results showed that the number of the cytosine methylation of CCGG sites in high salt-tolerant cotton line was less than that in low salt-tolerant line.Under salt stress,extensive cytosine methylation alterations including hypermethylation and demethylation as well as the potential conversion of methylation types occurred in the salt-treated cotton line compared with the corresponding control.Interestingly,salt stress-induced demethylation loci that occurred in high salt- tolerant cotton line were greater than those in low salt-tolerant cotton line,however,salt stress-induced hypermethylation loci in the high salt-tolerant cotton line were less than those in low salttolerant cotton line.It suggested that the demethylation positively contributed to salt tolerance and the hypermethylation had negative effect on salt tolerance in cotton.     

Analysis on Salt Tolerance of Transgenic Maize（Zea mays L. ） Crosses with BcWRKY I Gene in Seedling Stage

为了明确外源基因提高玉米耐盐性的效果,以6份转BcWRKYI基因的玉米杂交组合及其对应的6份非转基因对照组合为试材,对其发芽率、耐盐级别、株高、干重、脯氨酸含量等7个指标进行分析。结果表明：耐盐级别、发芽率、干重、叶绿素舍量和相对电导率的变化均达到显著水平,株高的变化达到极显著水平,脯氨酸含量转基因与非转基因组合间差异不显著。     

In vitro Plant Regeneration from the Mature Tissue of Navel Orange （Citrus sinensis L. Osbeck） by Direct Organogenesis

An efficient in vitro regeneration system by direct organogenesis from mature nodal and internodal stem segments of Newhall navel orange (Citrus sinensis L. Osbeck) was developed. Illuminating conditions together with plant growth regulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation is beneficial for the adventitious bud regeneration. The highest regeneration frequency (85.2%) and bud formation efficiency (3.7 per responsive internodal stem segment) were obtained in the media supplemented with 1.0 mg L^-1 BAP and 0.5 mg L^-1 NAA. ABA at 0.2 mg L^-1 positively affected the bud formation efficiency, which amounted to 8.5 buds per internodal segment in the presence of BAP at 1.0 mg L^-1 The adventitious shoots successfully rooted and were transferred to the soil.     

Advanced Backcross QTL Analysis for the Whole Plant Growth Duration Salt Tolerance in Rice（Oryza sativa L.）

Salinity is a major factor limiting rice yield in coastal areas of Asia. To facilitate breeding salt tolerant rice varieties, the wholeplant growth duration salt tolerance（ST） was genetically dissected by phenotyping two sets of BC2F5 introgression lines（ILs） for four yield traits under severe natural salt stress and non-stress filed conditions using SSR markers and the methods of advanced backcross QTL（AB-QTL） analysis and selective introgression. Many QTLs affecting four yield traits under salt stress and nonstress conditions were identified, most（〉90%） of which were clustered in 13 genomic regions of the rice genome and involved in complex epistasis. Most QTLs affecting yield traits were differentially expressed under salt stress and non-stress conditions. Our results suggested that genetics complementarily provides an adequate explanation for the hidden genetic diversity for ST observed in both IL populations. Some promising Huanghuazhan（HHZ） ILs with favorable donor alleles at multiple QTLs and significantly improved yield traits under salt stress and non-stress conditions were identified, providing excellent materials and relevant genetic information for improving rice ST by marker-assisted selection（MAS） or genome selection.     

Establishment and Optimization of the Regeneration System of Mature Embryos of Maize （Zea mays L.）

A reliable system was developed for regeneration from mature embryos derived from callus of four maize inbred lines （Liao 7980, Dan 9818, Dan 340, and Dan 5026）. The protocol was mainly based on a series of experiments involving the composition of culture medium. We found that 9 pM 2,4-dichlorophenoxyacetic acid in MS medium was optimum for the induction of callus. The induction frequency of primary calli was over 85% for four inbred lines tested. The addition of L- proline （12 mM） in subculture medium significantly promoted the formation of embryogenic callus but it did not significantly enhance growth rate of callus. Efficient shoot regeneration was obtained on regeneration medium containing 2.22 μM 6- benzylaminopurine in combinations with 4.64 μM Kinetin. Regenerated shoots were rooted on half-strength MS medium containing 2.85 μM indole-3-butyric acid. This plant regeneration system provides a foundation for genetic transformation of maize.     

Identification of QTLs for Starch Content in Sweetpotato （Ipomoea batatas （L.） Lam.）

Sweetpotato （Ipomoea batatas （L.） Lam.） breeding is challenging due to its genetic complexity. In the present study, interval mapping （IM） and multiple quantitative trait locus （QTL） model （MQM） analysis were used to identify QTLs for starch content with a mapping population consisting of 202 F1 individuals of a cross between Xushu 18, a cultivar susceptible to stem nematodes, with high yield and moderate starch, and Xu 781, which is resistant to stem nematodes, has low yield and high starch content. Six QTLs for starch content were mapped on six linkage groups of the Xu 781 map, explaining 9.1-38.8% of the variation. Especially, one of them, DMFN 4, accounted for 38.8% of starch content variation, which is the QTL that explains the highest phenotypic variation detected to date in sweetpotato. All of the six QTLs had a positive effect on the variation of the starch content, which indicated the inheritance derived from the parent Xu 781. Two QTLs for starch content were detected on two linkage groups of the Xushu 18 map, explaining 14.3 and 16.1% of the variation, respectively. They had a negative effect on the variation, indicating the inheritance derived from Xu 781. Seven of eight QTLs were co-localized with a single marker. This is the first report on the development of QTLs co-localized with a single marker in sweetpotato. These QTLs and their co-localized markers may be used in marker-assisted breeding for the starch content of sweetpotato.     

Haploid Induction via In vitro Gynogenesis in Tomato （Solanum lycopersicum L.）

In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely because the integument grew faster than embryo sacs did and hindered the fiarther development of embryo sacs. Therefore, subsequent callus morphogenesis might be originated from the integument. Thousands ofcalli from the two tomato varieties were obtained. Five diploid plants were regenerated after 15 months of subculturing. To eliminate the hindering effect of integument on embryo sac cells, the protoplasts of embryo sacs were prepared and cultured. After 48 hours of culture, the protoplasts of embryo sacs doubled in size and gradually formed clusters of cells. These results suggested that gynogenesis might be a potential way for haploid induction in tomato.     

Early Identification of Stable Transformation Events by Combined Use of Antibiotic Selection and Vital Detection of Green Fluorescent Protein （GFP） in Carrot （Daucus carota L.） Callus

Genetic transformation is a useful technique to complement conventional breeding in crop improvement. Although carrot has been a model organism for in vitro embryogenesis study, genetic transformation of carrot is still lengthy and labor intensive. An efficient transformation and detection system is desirable. Direct infection of Agrobacterium to carrot calli has provided an easy way for carrot genetic transformation. To improve the efficiency of antibiotic selection in this method, we report the combined use of an improved green-fluorescent protein, referred to as smGFP, to establish a versatile selection method for carrot callus transformation system. By combining antibiotic selection with the bright fluorescence observed in the callus tissue, we were able to easily identify stable transformants in early stage of the transformation process. In addition to the GFP expression of the callus cells, the transgenic nature of callus cells was confirmed with Southern and Western analysis. We found we can link the simplicity of carrot-callus-cell transformation, early detection of stable transformants with antibiotic selection, visualization of GFP fluorescence, and molecular analysis （Southern and Western） of callus tissue （non-photosynthetic tissue） to provide a more efficient way in identifying stable transformants at early stage of carrot transformation.     

Effect of Different Treatment on Anther Callus Formation and Browning in Balsam Pear （Mornordica charantia L.）

Brown callus derived from anther limited the application of anther culture in balsam pear. In order to establish a more perfect regeneration system from anther cultuer, the effects of low temperature pretreatment, 2,4-dichlorophenoxyacetic acid （2,4-D）, vitamin C （Vc） and silver nitrate （AgNO3） on callus induction and browning in anther culture of balsam pear （Momordiea charantia L.） were investigated. The results showed that after pretreatment at 4 ℃ for 1 day, callus induction rate was the highest and browning rate was the lowest. Anthers on MS medium supplemented with 2,4-D 0.5 mg/L formed more and better callus. The medium supplemented with Vc or AgNO3 was advantageous to the induction of callus and reduction of browning. When cultured on medium supplemented with 50 mg/L Vc or 5 mg/L AgNO3, callus induction rate was the highest and browning rate was rather low.     

Cytological Observation of Microsporogenesis in Male-Sterile Lines of Chinese Pink （ Dianthus chinensis L.）

This study addressed the differences in microsporogenesis between male sterile and fertile lines of Chinese pink. The microsporogenesis processes of male sterile and fertile lines were histologically examined in squashed pollen grains and in paraffin embedded sections. A stable male-sterile line （H-37B） was obtained following six generations of inbreeding in a self-fertile line, followed by two generations of backcrossing. In the corresponding fertile line, development of the mature pollen grains was followed through the initiation of the sporogenous cell, microsporocyte formation, and the tetrad developmental period. In the male-sterile line, abortion of the developing pollen grains was observed to take place at various stages, namely, sporogenous cell growth, mother cell meiosis, and tetrad transformation to the uninuclear state. The pollen grains of the fertile line were spheroid, turgid, and viable. By contrast, the male-sterile line produced pollen that was irregular in shape, empty, and nonviable. The abortion of the microspore in the male-sterile line appeared to relate to abnormal growth of the tapetum layer.     

Photosynthesis,Growth and Antioxidant Metabolism in Mustard （Brassica juncea L.） Cultivars Differing in Cadmium Tolerance

Two mustard （Brassica juncea L. Czern and Coss.） cultivars, Pusa Jai Kisan and SS2 differing in cadmium （Cd） tolerance were treated with 0, 25 and 50 umol L-1 Cd to study the physiological basis of difference in Cd tolerance. Cultivar SS2 （Cd sensitive） accumulated greater Cd in leaves than Pusa Jai Kisan （Cd tolerant）. Further, SS2 also exhibited higher contents of thiobarbituric acid reactive substances （TBARS） and H2O2 and electrolyte leakage. However, the activities of antioxidant enzymes, catalase （CAT）, ascorbate peroxidase （APX）, dehydroascorbate reduetase （DHAR）, and glutathione reductase （GR） were higher in Pusa Jai Kisan than those in SS2. Contrarily, the activity of superoxide dismutase （SOD） was higher in SS2 than that in Pusa Jai Kisan and was the greatest at 25 umol L-1 Cd. Treatment of 25 umol L-1 Cd induced the maximum activity of enzymes. However, the activity of GR increased up to 50 umol L-1 Cd in both the cultivars. The non-enzymatic antioxidants ascorbate （AsA） and glutathione （GSH） were higher in Pusa Jai Kisan than that in SS2, whereas dehydroascorbate （DHA） and oxidized glutathione （GSSG） were higher in SS2. Photosynthesis and growth were adversely and maximally decreased by 50 umol L-1 Cd treatment in both the cultivars, but SS2 exhibited greater reductions. The protection of photosynthesis and growth and lesser reduction in Pusa Jai Kisan were associated with its capacity to restrict accumulation of Cd in leaves resulting in lower level of TBARS and H2O2 and electrolyte leakage. Moreover, Pusa Jai Kisan exhibited efficient antioxidant metabolism for removal of Cd-induced reactive oxygen species.     

Variation on Wild Sunflower （Helianthus ruderalis L.） for Quantitative and Qualitative Parameters

Our expedition have identified location in the central part of Kosovo where were collected a samples （accessions） at coordinates; Sub-Locality SL-1 （N42°34′36″, E21°07′45″）, altitude 574 m, SL-2 （N42°34′29″, E 21°07′45″altitude 591 m and SL-3 （N42°34′30″, E21°07′46″） on altitude 565 m. The aim of present investigation was carried out to study genetic variability of quantitative and qualitative parameters. The populations from SL-2 were characterized of higher significance on seed oil （23.74%） and protein content （16.74%）. The estimation average value of seed protein and oil content was 15.52% and 23.48% respectively. The variability of plant height ranged from 182.33 cm to as much as 200.89 cm, while distinction＇s between them were ＋ 18.56 cm which was a significant higher at P〈0.05 probability level. The SL-3 showed the higher value of HWK （34.6 g）, head per plant （39.33） and head diameter （9.5 cm） but, was characterized significantly lowest on seed protein content （13.99%）. The all traits included in our investigations showed a higher genetic variability with majority of extreme values. The sunflower populations differed significantly in several parameters measured, they have a different genetic and morphological backgrounds. Results from wild sunflower form indicated notable differences in dispersion of important genes for different traits and showed the potential for using in future for breeding program.