Pathogenesis-Related Proteins and Their Transgenic Expression for Developing Disease-Resistant Crops: Strategies Progress and Challenges

Various pathogenic microorganisms (such as fungi, bacteria, viruses and nematodes) affect plant viability and productivity. However, plants combat these pathogens by inducing their defense mechanism to sustain their fitness. The aggregation of pathogenesis-related (PR) proteins in response to invading pathogens is a crucial component of a plant’s self-defense mechanism. PR proteins induce innate resistance in plants through fungal cell wall disintegration, membrane permeabilization, transcriptional suppression, and ribosome inactivation. Earlier studies have demonstrated their crucial role in determining resistance against phytopathogens, making them a promising candidate for developing disease-resistant crop varieties. Plant genetic engineering is a potential approach for developing disease-resistant transgenic crops by employing several PR genes (thaumatin, osmotin-like proteins, chitinases, glucanases, defensins, thionins, oxalate oxidase, oxalate oxidases like proteins/germin-like proteins and LTPs). Furthermore, the overexpression of PR proteins enhances the resistance against phytopathogens. As a result, this chapter gives an overview of PR proteins, including their classification, functional characterization, signaling pathways, mode of action and role in defense against various phytopathogens. It also highlights genetic engineering advances in utilizing these genes singly or synergistically against various phytopathogens to impart disease resistance. Various challenges faced with the products of transgenic technology and synergistic expression of different groups of PR proteins were also discussed

Desiccation of callus enhances somatic embryogenesis and subsequent shoot regeneration in sugarcane

332-334Callus cultures were established in three commercial sugarcane varieties, viz., CoJ 64, CoJ 83 and CoJ 86, from spindle leaf segments on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4 mg/L) and 6-benzylaminopurine (BAP; 0.5 mg/L). The calli were sub-cultured on MS+2,4-D (2 mg/L)+BAP (0.5 mg/L)+agar (0.8% w/v) medium (control) and MS+2,4-D (2 mg/L)+BAP (0.5 mg/L)+agar (1.6% w/v) medium (treatment) to study the effect of desiccation caused by double agar on somatic embryogenesis. Per cent somatic embryogenesis observed in treatment-calli of sugarcane varieties CoJ 64, CoJ 83 and CoJ 86 was 90, 90.63 and 89.66, respectively; while in control-calli the corresponding figures were 66.67, 64.52 and 63.33. Likewise, shoot regeneration from desiccated calli on MS+BAP (0.5 mg/L) medium was also higher over non-desiccated control, i.e., 84.27, 86.52 and 83.13% as compared to 54.35, 56.25 and 50.38%, respectively in CoJ 64, CoJ 83 and CoJ 86. Thus, this fairly simple double agar medium provided an alternative method for improving somatic embryogenesis and, hence, regeneration frequency of sugarcane callus

Transformation of tomato using biolistic gun for transient expression of the <img src='/image/spc_char/beta.gif'>-glucuronidase gene

363-369 We report for the first time, conditions for the biolistic transformation of tomato for the introduction of -glucuronidase gene (gusA) into different explants viz., shoot tips, hypocotyls and cotyledons of genotype IPA-3. The respective per cent plant regeneration in different explants was 95.16, 79.30 and 90.14% on MS media supplemented with BAP (2.0 mg L-1) and Kn (1.0 mg L-1). The influence of physical parameters of particle gun on rates of transient GUS expression have been investigated viz., quantity of DNA, distance between the microcarrier launch assembly and target tissues; and the biological factors associated with the target tissues i.e., effect of osmoticum (mannitol), pre-bombardment culture period and post-bombardment culture period. Maximum GUS expression of 25.00, 33.00 and 22.20% was respectively recorded when 10-d-old shoot tips, hypocotyls and cotyledons were bombarded with 18 L of DNA suspension. A firing distance of 7.5 cm was found to be most suitable recording 34.12, 36.56 and 22.69% transient GUS expression in shoot tips, hypocotyls and cotyledons, respectively. Addition of osmoticum into the culture medium reduced per cent GUS expression significantly in all the explants even at 1 molar concentration. Pre-culture of explants prior to bombardment also deduced the transformation efficiency, however post bombardment culture period of one day resulted in maximum GUS expression in all the explants. </smarttagtype

Effect of growth regulators on in vitro morphogenic response of tomato

526-530A series of experiments were conducted to explore the in vitro morphogenic response of tomato genotypes viz., Castle Rock, Punjab Upma, VFN-8 and IPA-3, under different concentrations and combinations of growth regulators. The analysis of variance at 5% level of significance indicated that the differences among different genotypes, hormonal regimes as well as their interactions were statistically significant. The MS medium supplemented with BAP @ 3.0 mg L⁻¹ and IAA 2.5 mg L⁻¹ was optimum for callus induction, plant regeneration and number of shoots per explant. The maximum per cent callus induction and plant regeneration in the genotypes Castle Rock, Punjab Upma, VFN-8 and IPA-3. was 81.23, 76.69, 68.13 and 65.12%; and 46.91, 48.02, 57.14 and 60.23%, respectively, The respective average number of shoots per culture were 7.03, 6.92, 8.19 and 9.19. At higher and lower levels of hormones, a considerable decline was recorded in per cent callus induction, plant regeneration and number of shoots per explant. The best rooting was found to be in the ½ MS medium supplemented with 0.2 mg L⁻¹ IBA. Among the four soil mixtures studied viz., vermiculite, perlite, coco-peat and mixture of three (vermiculite, perlite and cocopeat in the ratio of 1:1:1), maximum plantlet survival rate was recorded in the mixture of three for genotypes Castle Rock and Punjab Upma and in vermiculite for genotype VFN-8

Standardization of different parameters for ‘particle gun’ mediated genetic transformation of sugarcane (Saccharum officinarum L.)

31-34Three different parameters, viz. quantity of DNA, size of target tissue and distance between microcarrier launch assembly and target tissue (firing distance), for ‘particle gun’ (Bio-Rad) mediated transformation were optimized using embryogenic calli of sugarcane (target tissue), gusA (reporter gene) and hpt (hygromycin resistance gene). Of 12, 18 and 24 µL of DNA (gusA and hpt) suspension used per two bombardments, highest (33.33%) GUS expression was observed with 18 µL of suspension carrying 1.88 µg DNA. Bombardments made into 2-5 mm calli exhibited 55.55% GUS expression, while no expression was obtained in 7.5-12 mm calli after 7 h of their incubation in X-gluc. Out of different firing distances, viz. 2.5, 5.0, 7.5 and 10.0 cm, maximum intensity of GUS expression was recorded when calli were bombarded at a distance of 5.0 cm from the microcarrier launch assembly in the ‘particle gun’

Red rot resistant transgenic sugarcane developed through expression of β-1,3-glucanase gene.

Sugarcane (Saccharum spp.) is a commercially important crop, vulnerable to fungal disease red rot caused by Colletotrichum falcatum Went. The pathogen attacks sucrose accumulating parenchyma cells of cane stalk leading to severe losses in cane yield and sugar recovery. We report development of red rot resistant transgenic sugarcane through expression of β-1,3-glucanase gene from Trichoderma spp. The transgene integration and its expression were confirmed by quantitative reverse transcription-PCR in first clonal generation raised from T0 plants revealing up to 4.4-fold higher expression, in comparison to non-transgenic sugarcane. Bioassay of transgenic plants with two virulent C. falcatum pathotypes, Cf 08 and Cf 09 causing red rot disease demonstrated that some plants were resistant to Cf 08 and moderately resistant to Cf 09. The electron micrographs of sucrose storing stalk parenchyma cells from these plants displayed characteristic sucrose-filled cells inhibiting Cf 08 hyphae and lysis of Cf 09 hyphae; in contrast, the cells of susceptible plants were sucrose depleted and prone to both the pathotypes. The transgene expression was up-regulated (up to 2.0-fold in leaves and 5.0-fold in roots) after infection, as compared to before infection in resistant plants. The transgene was successfully transmitted to second clonal generation raised from resistant transgenic plants. β-1,3-glucanase protein structural model revealed that active sites Glutamate 628 and Aspartate 569 of the catalytic domain acted as proton donor and nucleophile having role in cleaving β-1,3-glycosidic bonds and pathogen hyphal lysis

Structural model showing protein-ligand interaction.

<p>β-1,3-glucanase protein (GenBank Accession No. AIC32930) docked with D-glucose.</p