<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting

Background

Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T₁ progeny.

Results

An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T₁ seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T₁ progeny displayed Mendelian inheritance.

Conclusions

This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.

     

Molecular markers for late blight resistance breeding of potato: an update

Late blight is the most devastating disease of the potato crop that can be effectively managed by growing resistant cultivars. Introgression of resistance (R) genes/quantitative trait loci (QTLs) from the Solanum germplasm into common potato is one of the plausible approaches to breed resistant cultivars. Although the conventional method of breeding will continue to play a primary role in potato improvement, molecular marker technology is becoming one of its integral components. To achieve rapid success, from the past to recent years, several R genes/QTLs that originated from wild/cultivated Solanum species were mapped on the potato genome and a few genes were cloned using molecular approaches. As a result, molecular markers closely linked to resistance genes or QTLs offer a quicker potato breeding option through marker‐assisted selection (MAS). However, limited progress has been achieved so far through MAS in potato breeding. In near future, new resistance genes/QTLs are expected to be discovered from wild Solanum gene pools and linked molecular markers would be available for MAS. This article presents an update on the development of molecular markers linked to late blight resistance genes or QTLs by utilization of Solanum species for MAS in potato.     

High level of resistance to Sclerotinia sclerotiorum in introgression lines derived from hybridization between wild crucifers and the crop Brassica species B. napus and B. juncea

Sclerotinia rot caused by the fungus Sclerotinia sclerotiorum is one of the most serious and damaging diseases of oilseed rape and there is keen worldwide interest to identify Brassica genotypes with resistance to this pathogen. Complete resistance against this pathogen has not been reported in the field, with only partial resistance being observed in some Brassica genotypes. Introgression lines were developed following hybridization of three wild crucifers (viz. Erucastrum cardaminoides, Diplotaxis tenuisiliqua and E. abyssinicum) with B. napus or B. juncea. Their resistance responses were characterized by using a stem inoculation test. Seed of 54 lines of B. napus and B. juncea obtained from Australia, India and China through an Australian Centre for International Agricultural Research (ACIAR) collaboration programme were used as susceptible check comparisons. Introgression lines derived from E. cardaminoides, D. tenuisiliqua and E. abyssinicum had much higher levels (P < 0.001) of resistance compared with the ACIAR germplasm. Median values of stem lesion length of introgression lines derived from the wild species were 1.2, 1.7 and 2.0 cm, respectively, as compared with the ACIAR germplasm where the median value for stem lesion length was 8.7 cm. This is the first report of high levels of resistance against S. sclerotiorum in introgression lines derived from E. cardaminoides, D. tenuisiliqua and E. abyssinicum. The novel sources of resistance identified in this study are a highly valuable resource that can be used in oilseed Brassica breeding programmes to enhance resistance in future B. napus and B. juncea cultivars against Sclerotinia stem rot.     

Induction of novel variants through physical and chemical mutagenesis in Barbeton daisy (Gerbera jamesonii Hook.)

Summary

Gerbera (Gerbera jamesonii) is an attractive ornamental flower of high economic importance. The present investigation was aimed at generating novel flower colour variants in the gerbera cultivar ‘Harley’ through physical and chemical mutagenesis. In vitro-raised shoot cultures of gerbera, established from petiole explants, were exposed to different doses of γ-rays (1.5, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, or 30.0 Gy) using a Cobalt-60 source emitting 2.51 kGy h^–1. To induce mutations through chemical mutagenesis, different concentrations of ethylmethane sulphonate [EMS; 0.1, 0.2, 0.5, 0.8, or 1.0% (v/v)] were administered for 10 min or for 20 min. The LD50 values calculated for shoot survival and the induction of mutations were approx. 6.5 Gy for γ-rays and 0.65% (v/v) EMS for 10 min, or ≤ 0.1% (v/v) EMS for 20 min. Investigations revealed a negative correlation between mutagen dose and plant survival, both in vitro and after acclimatisation. Morphological variants showing changes in leaf shape, leaf size, scape length, flower diameter, and flower colour were obtained. Significantly, early flowering was induced in all mutated plants compared to non-mutated plants.The high frequencies of colour variants obtained using Bγ-rays, or the application of EMS to in vitro-raised shoot cultures could be an effective way to improve gerbera cultivars.     

Profile Distribution of Carbon Fractions Under Long‐term Rice‐wheat and Maize‐wheat Production in Alfisols and Inceptisols of Northwest India

The knowledge of profile distribution of soil organic carbon (SOC) in long‐term agricultural systems could help to store atmospheric carbon in the soil. We investigated profile distribution of easily oxidisable Walkley–Black SOC pool (SOC_WB) under long‐term rice‐wheat (R‐W) and maize‐wheat (M‐W) cropping systems under soils of different pedogenesis. The soil samples were collected from the characteristic genetic horizons and analysed for carbon fractions. The SOC_WB was the highest in soils under R‐W systems in both Alfisols and Inceptisols. The SOC_WB stock in the deeper profile horizons under R‐W system was significantly (p < 0·05) higher than that under M‐W system especially in Typic Hapludalfs. Long‐term R‐W system could store on average 3·55 Mg ha⁻¹ more SOC_WB than M‐W system in the Ap horizon. The SOC_WB stock in the Ap horizon of all pedons was significantly (p < 0·05) higher in Alfisols than that in Inceptisols. About 60–90% of the total profile SOC_WB stock was contributed by B‐horizon because of its greater extent. Considering the whole profile, clay was negatively correlated with SOC fractions; however, the SOC fractions were closely related to each other. This study reveals that the distribution of SOC_WB is different in long‐term R‐W and M‐W systems not only in surface but also in the deeper horizons and the magnitude of the variation is influenced by the specific pedogenic processes. This indicates the significance of profile SOC_WB stock instead of topsoil SOC_WB stock in quantifying carbon retention potential of the long‐term management practices. Copyright © 2014 John Wiley & Sons, Ltd.     

Genetic diversity of maize inbred lines in relation to downy mildew

A major emphasis in maize breeding in Asian countries has been the improvement for resistance to downy mildew, a serious disease that causes significant yield losses. A total of 102 inbred lines, including lines from Asian breeding programs, Mexico, USA and Germany, were analyzed with 76 SSR markers to measure diversity and investigate the effect of selection for downy mildew resistance. A mean polymorphism information content of 0.59, with a range of 0.14 to 0.83, was observed. Diversity at the gene level showed an average of 5.4 alleles per locus and a range of two to 16 alleles per locus, with a total of 409 alleles. About half of the alleles in the Asian lines had frequencies of 0.10 or less, and only 2% had frequencies > 0.80, indicating the presence of many alleles, and thus a high level of diversity. Some of the high-frequency alleles were in chromosomal regions associated with disease resistance. However, the frequencies of alleles in three SSR loci that are linked to a QTL for resistance to downy mildews in Asia were not significantly different in the subtropical/tropical Asian lines as compared to all the lines in the study. Lines from the US, Germany, and China, comprised three clusters of temperate maize(GS = 0.31), while those from India, Indonesia, Philippines, Thailand, Vietnam and CIMMYT comprised seven indistinct clusters of subtropical and subtropical maize (GS = 0.29). We conclude that maize breeding activity in Asia has not caused a decline in the overall amount of diversity in the region. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of multi-nutrient extractants for determination of available P,K, and micronutrient cations in soil

Sixty soil samples (0–0.15 m depth) collected randomly from different districts of Himachal Pradesh were used to evaluate ammonium bicarbonate-diethylenetriaminepenta acetic acid (AB-DTPA) and acid ammonium acetate-ethylene diamine tetra acetic acid (AAAc-EDTA) over standard methods for P, K, and micronutrient cations. Among Olsen P, Bray P1, AB-DTPA P, and AAAc-EDTA P, available P (kg ha^?1) varied from 7.8 to 44.3, 9.5 to 61.1, 6.5 to 38.8, 10.5 to 52.1; available K (kg ha^?1) among NH4OAc, AB-DTPA, and AAAc-EDTA ranged from 103.6 to 372.3, 86.6 to 364.9, and 74.6 to 362.5. In case of micronutrients cations, AB-DTPA in general, extracted more amounts as compared to DTPA alone. AAAc-EDTA correlated highly with the Olsen P. AB-DTPA-K and AAAc-EDTA-K correlated well with standard method (NH4OAc) however; AAAc-EDTA had higher correlation with AB-DTPA for K extractability. Among methods for micronutrients highest significant correlations were observed between AB-DTPA and DTPA for Cu, Fe, and Zn.