The Heat Stress Transcription Factor LlHsfA4 Enhanced Basic Thermotolerance through Regulating ROS Metabolism in Lilies (Lilium Longiflorum)

Heat stress severely affects the annual agricultural production. Heat stress transcription factors (HSFs) represent a critical regulatory juncture in the heat stress response (HSR) of plants. The HsfA1-dependent pathway has been explored well, but the regulatory mechanism of the HsfA1-independent pathway is still under-investigated. In the present research, HsfA4, an important gene of the HsfA1-independent pathway, was isolated from lilies (Lilium longiflorum) using the RACE method, which encodes 435 amino acids. LlHsfA4 contains a typical domain of HSFs and belongs to the HSF A4 family, according to homology comparisons and phylogenetic analysis. LlHsfA4 was mainly expressed in leaves and was induced by heat stress and H2O2 using qRT-PCR and GUS staining in transgenic Arabidopsis. LlHsfA4 had transactivation activity and was located in the nucleus and cytoplasm through a yeast one hybrid system and through transient expression in lily protoplasts. Over expressing LlHsfA4 in Arabidopsis enhanced its basic thermotolerance, but acquired thermotolerance was not achieved. Further research found that heat stress could increase H2O2 content in lily leaves and reduced H2O2 accumulation in transgenic plants, which was consistent with the up-regulation of HSR downstream genes such as Heat stress proteins (HSPs), Galactinol synthase1 (GolS1), WRKY DNA binding protein 30 (WRKY30), Zinc finger of Arabidopsis thaliana 6 (ZAT6) and the ROS-scavenging enzyme Ascorbate peroxidase 2 (APX2). In conclusion, these results indicate that LlHsfA4 plays important roles in heat stress response through regulating the ROS metabolism in lilies.

Chromosome characterization and physical mapping of 18S rDNA in Lilium longiflorum originated interspecific hybrids using combined genomic and fluorescent in situ hybridization

This study was aimed at differentiating parental genomes, examining intergenomic composition, and mapping mitotic metaphase chromosomes by localizing parental and 18S rDNA probes in seven interspecific hybrid progenies that originated from Lilium longiflorum. Since in situ hybridization has not been previously used in lily breeding, flow cytometry was used in conjunction with genomic and fluorescent in situ hybridization to determine the genomic contribution of each parent to the interspecific progenies. A significant variation was observed in the DNA content, chromosome length, and 18S loci in F1 as compared to the female and male parents. L. longiflorum showed nearly two times higher DNA content than the male parents and L. longiflorum × Asiatic progenies, but eight times higher than L. longiflorum × L. hansonii. Genomic in situ hybridization results revealed that both female and male parents contributed an equal number of chromosomes to their interspecific F1 offspring. Fluorescent in situ hybridization mapping revealed that 18S rDNA had 8, 6 and 7 loci in L. longiflorum parents, i.e., White heaven, Bright tower, and White tower, respectively, whereas each Asiatic cultivar and L. hansonii used as male showed 8 and 12 loci respectively. Interspecific progenies showed 8 and 7 loci in LA, and 10–11 in LM hybrids. These cytogenetic results implied equal genetic and chromosomal contribution from both parents to their intergenomic progenies. Therefore, this combined (Schwarzacher et al., 1992)cytogenetic method has the potential to be an affordable and time-saving approach in lily breeding that could determine the status of hybrids and their genomic origin while achieving physical mapping and detecting genes in different genomes.

​Effect of Different Media on In vitro Rooting in Different Cultivar of Lilium longiflorum Cv. Elite, Brunello, Cordelia

Background: Lilium (Lilium longiflorum Thunb.) belongs to the family Liliaceae and is a native of Northern Hemisphere (up to South Canada and Siberia). Conventionally Lilium can easily be propagated by sexual and asexual methods of propagation but these prevalent methods are not capable of meeting the increasing demand in domestic and global market. Generally, Lilium is propagated through bulbs but, limited number of bulbs per plant, long dormancy period of bulbs which again results into non-availability of planting material throughout the year. Keeping in view the above facts, the present study was undertaken with the following objective: “To standardize the cost effective protocol for micro propagation of lilium to produce disease free and true to type plants at a faster rate”. Methods: The present investigation was carried out in the Tissue Culture Laboratory of the Centre for Research and Application in Plant Tissue Culture. The experiment was laid out in a C.R.D. (Factorial) with three replications. In vitro raised bulblets were separated out and were transferred on to the root regeneration media. Different levels of NAA were used in MS media for the rooting of in vitro raised bulblets and percent rooting of plantlet is recorded. Result: It was interesting to note that the media LR-3 (MS + NAA 1.0 mg/l) is most efficient for rooting in all type of cultivars. All the three cultivars used responded very poor on media LR-1 (MS basal).

PERFORMANCE EVALUATION AND VARIABILITY ANALYSIS FOR MAJOR GROWTH AND FLOWERING TRAITS OF Lilium longiflorum THUNB. GENOTYPES

The performance evaluation and variability estimation are some of the most important basic steps in any breeding scheme and are usually taken as a pre-breeding task. This experiment was carried out to evaluate the performance and estimate the variability for major growth and flowering traits of Sixteen Lilium longiflorum Thunb genotypes in a randomized complete block design (RCBD) with three replications. The estimated analysis of variance (ANOVA) demonstrated prevailing significant variability for studied ten growth and flowering traits of all 16 genotypes. The experiment was conducted to quantify the genetic variability, heritability, and genetic advance as a percentage of the mean (GAM), for understanding the breeding potentiality of L. longiflorum genotypes based on their performance for growth and flowering traits, so that pre-breeding scheme could be executed properly. The estimated genotypic coefficient of variation (GCV) and phenotypic coefficient of variation (PCV) value, along with heritability estimate and GAM, revealed that almost all studied traits besides flower diameter were controlled by additive gene action. For these traits, improvement would be possible through the selection after cross-breeding among these genotypes. The choice of genotypes for the hybridization scheme could be made based on the mean performance of those genotypes for the particular traits of the breeding scheme.

Hormonal endogenous changes in response to the exogenous 6-benzylaminopurine application in pre- and post-harvesting lilium flower stalks

Phyto-hormones play a key role in regulating plant responses to stress. Cytokines are a type of phyto-hormones involved in the regulation of many important biological processes related to growth, development, and response to environmental variables. The exogenous application of cytokines increases the possibility of delaying senescence; however, this is a physiological process, and, under certain conditions, degradation processes may be triggered. The effect of 6-bencilaminopurine application and the endogenous hormonal changes involved in lilies floral stalks after their cutting were studied. In order to improve vase life and quality of Lilium longiflorum ‘Brindisi’ flower stalks, they were sprayed with 6-BAP, at a concentration of 300 ppm at pre-harvest, post-harvest, and pre- and post-harvest stages. After that, they were compared to non-sprayed control stalks. The application of 6-BAP caused endogenous hormonal changes in abscisic acid and cytokinin levels, and the most effective treatment was pre-harvest spraying. This treatment proved to be an appropriate method to improve the stalk tolerance to post-harvest stress as it delayed the appearance of senescence symptoms and reduced the speed of chlorophyll degradation with differences of up to 10% with respect to untreated stalks. In addition, the opening of flowers was delayed by up to 2 days, although there were no significant differences in total vase life.

Impact of Manganese on Pollen Germination and Tube Growth in Lily

In vitro culture of Lilium longiflorum pollen grains was carried out to determine the role of manganese in pollen germination and pollen tube growth. Pollen germination was adversely affected by the presence of manganese (>10 −8 M), whereas low concentrations (10 −12 –10 −10 M) stimulated the process. Manganese caused morphological anomalies during tube growth, characterized by irregular pollen tube thickening and swollen tips. The main effect was the anomalous cell wall formation at the tip, in which the presence of several organelles reduced the number of secretory vesicles. A loose network of fibrillar material and spherical aggregates, mostly in the tip region, was detected, and this material was progressively loosened into the surrounding medium. As a response to potential toxicity, the excess manganese was isolated in vacuoles, which formed an internal barrier against penetration of manganese to the tip area. Elevated manganese concentrations might affect plant reproduction, resulting in anomalies in gamete development. Consequently, the loss in genetic diversity and decreased fruit set ultimately lower yield.

3D mechanical characterization of single cells and small organisms using acoustic manipulation and force microscopy

Quantitative micromechanical characterization of single cells and multicellular tissues or organisms is of fundamental importance to the study of cellular growth, morphogenesis, and cell-cell interactions. However, due to limited manipulation capabilities at the microscale, systems used for mechanical characterizations struggle to provide complete three-dimensional coverage of individual specimens. Here, we combine an acoustically driven manipulation device with a micro-force sensor to freely rotate biological samples and quantify mechanical properties at multiple regions of interest within a specimen. The versatility of this tool is demonstrated through the analysis of single Lilium longiflorum pollen grains, in combination with numerical simulations, and individual Caenorhabditis elegans nematodes. It reveals local variations in apparent stiffness for single specimens, providing previously inaccessible information and datasets on mechanical properties that serve as the basis for biophysical modelling and allow deeper insights into the biomechanics of these living systems.

Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.