Comparative assessment of multi-trait plant growth-promoting endophytes associated with cultivated and wild Oryza germplasm of Assam, India.

This paper presents a comparative study of endophytic bacteria from cultivated (Oryza sativa) and wild rice (Oryza rufipogon) plants and their functional traits related to plant growth promotion. A total of 70 bacterial isolates were characterized by both biochemical and molecular identification methods. Taxonomic classification showed dominance of three major phyla, viz, Firmicutes (57.1%), Actinobacteria (20.0%) and Proteobacteria (22.8%). Screening for in vitro plant growth-promoting activities revealed a hitherto unreported endophytic bacterium from wild rice germplasm, Microbacterium laevaniformans RS0111 with highest indole acetic acid (28.39 ± 1.39 µg/ml) and gibberellic acid (67.23 ± 1.83 µg/ml) producing efficiency. Few other endophytic isolates from cultivated rice germplasm such as Bacillus tequilensis RHS01 showed highest phosphate solubilizing activity (81.70 ± 1.98 µg/ml), while Microbacterium testaceum MKLS01 and Microbacterium enclense MI03 L05 showed highest potassium (53.42 ± 0.75 µg/ml) and zinc solubilizing activity (157.50%). Fictibacillus aquaticus LP20 05 produced highest siderophore (64.8%). In vivo evaluation of plant growth-promoting efficiencies of the isolates showed that Microbacterium laevaniformans RS0111, Microbacterium testaceum MKLS01 and Bacillus tequilensis RHS 01 could increase rice grain yield by 3.4-fold when compared to the control group. This study indicates the potentiality of rice endophytes isolates as an effective bioinoculants.

Assessment of efficacy of mutagenesis of gamma-irradiation in plant height and days to maturity through expression analysis in rice.

Introduction of semi-dwarfism and early maturity in rice cultivars is important to achieve improved plant architecture, lodging resistance and high yield. Gamma rays induced mutations are routinely used to achieve these traits. We report the development of a semi-dwarf, early maturing and high-yielding mutant of rice cultivar 'Improved White Ponni', a popular cosmopolitan variety in south India preferred for its superior grain quality traits. Through gamma rays induced mutagenesis, several mutants were developed and subjected to selection up to six generations (M6) until the superior mutants were stabilized. In the M6 generation, significant reduction in days to flowering (up to 11.81% reduction) and plant height (up to 40% reduction) combined with an increase in single plant yield (up to 45.73% increase) was observed in the mutant population. The cooking quality traits viz., linear elongation ratio, breadthwise expansion ratio, gel consistency and gelatinization temperature of the mutants were similar to the parent variety Improved White Ponni. The genetic characterization with SSR markers showed variability between the semi-dwarf-early mutants and the Improved White Ponni. Gibberellin responsiveness study and quantitative real-time PCR showed a faulty gibberellin pathway and epistatic control between the genes such as OsKOL4 and OsBRD2 causing semi-dwarfism in a mutant. These mutants have potential as new rice varieties and can be used as new sources of semi-dwarfism and earliness for improving high grain quality rice varieties.

Assessment of germination, phytochemicals, and transcriptional responses to ethephon priming in soybean [Glycine max (L.) Merrill].

The present work aims to dissect the underlying signaling pathways associated with soybean [Glycine max (L.) Merrill] seed hormo-priming with ethephon (Eth). Our results demonstrated that soybean germination improved significantly upon Eth priming (Ethp). Phytohormone quantification shows relative enhanced endogenous gibberellin A4 (GA4) levels concomitant with impaired biogenesis and signaling of auxin, viz., indole acetic acid (IAA) and abscisic acid (ABA). Phytochemical analysis revealed relative reduced levels of individual and total raffinose family oligosaccharide (RFO) components, starch, soluble sugars, and sucrose concomitant with enhanced levels of reducing sugars, glucose, cellular ATP, and acetyl-CoA pools. Secondary metabolite analysis revealed the activation of the mevalonate (MVA) pathway with a concomitant suppression of the plastidal 2-methyl-d-erythritol-4-phosphate/1-deoxy-d-xylulose-5-phosphate (MEP/DOX) and phenylpropanoid pathways, substantiated by relative reduced levels of total phenolics, tannins, and proanthocyanidin. Ethp also enhances the in vitro antioxidative activity (viz., 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and ferric reducing antioxidant power (FRAP)) and endogenous antioxidants levels (viz., flavonoids, isoflavones, ß-carotene, vitamin C, and vitamin E). Further quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed transcriptional pattern of representative genes in agreement with these metabolic alterations.

CRISPR-Based Assessment of Gene Specialization in the Gibberellin Metabolic Pathway in Rice.

Gibberellin (GA) functions as an essential natural regulator of growth and development in plants. For each step of the GA metabolic pathway, different copy numbers can be found in different species, as is the case with the 13 genes across four enzymatic steps in rice (Oryza sativa). A common view is that such gene duplication creates homologs that buffer organisms against loss-of-function (LOF) mutations. Therefore, knockouts of any single homolog might be expected to have little effect. To test this question, we generated clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) knockouts for these homologs and measured effects on growth and reproduction. Surprisingly, we report here that there is consistently one or more essential gene at each enzymatic step, for which LOF mutation induces death or sterility-suggesting that the GA pathway does not have a redundancy route and that each gene family is essential for GA metabolism. In most of these genes from the same gene family, we observed defects in plant height and infertility, suggesting that the duplicated members retain functions related to GA synthesis or degradation. We identified both subfunctionalization of the three recently diversified homologs OsKO1, OsKO2, and OsKO5 and neofunctionalization in OsKO3 and OsKO4 Thus, although the function of each step is conserved, the evolution of duplicates in that step is diversified. Interestingly, the CRISPR/Cas9 lines at the SD1 locus were typically sterile, whereas the natural sd1 mutants, related to the "Green Revolution" in rice, show normal setting rates. Collectively, our results identify candidates for control of GA production and provide insight into the evolution of four critical gene families in plants.

In vitro seed germination of economically important edible bamboo Dendrocalamus membranaceus Munro.

An in vitro propagation protocol using mature seeds of D. membranaceus was successfully established. Scarcity of seeds in bamboos because of their long flowering periods and irregular seed set resulting in low viability and germination potential, motivated us to undertake the present study. The effects of sterilants, light conditions, exogenous application of plant growth regulators and temperature in overcoming germination barriers in ageing seeds of bamboo were studied. It was found that HgCl2 (0.1%) along with bleach (15%) was more effective in raising aseptic cultures. Dark conditions, high temperatures around 30 degrees C and soaking of seeds in GA3 solution (50 ppm) overnight stimulated high percent of seed germination with corresponding increase in shoot length (2.7 +/- 0.7 mm) and number of sprouts (2.1 +/- 0.7) per explants during culture initiation. 6-benzylaminopurine acted synergistically with kinetin to give optimum germination rate of 70 +/- 13.9% as compared to 63.13% when used individually. For prolonged maintenance of cultures, 2% sucrose was found to be suitable for promoting photomixotrophic micropropagation. Following this procedure, about 65% survival of plantlets could be achieved during hardening. Biochemically seeds consume starchy endosperm for emergence of radicle which is taken as a sign of germination as also evident from the present study. Loss of viability and vigour after a year was confirmed by Tetrazolium chloride test. Micropropagation protocol developed here will ensure regeneration of large number of plants in a relatively short time. Conclusively, in vitro propagation protocol developed in D. membranaceus using mature seeds as an explants is reported for the first time.

Involvement of endogenous gibberellins in potato tuber dormancy and early sprout growth: a critical assessment.

The role of endogenous gibberellins (GAs) in the regulation of potato (Solanum tuberosum) tuber dormancy was examined by determining: 1. changes in endogenous GA levels during natural dormancy progression, 2. the effects of GA biosynthesis inhibitors on tuber dormancy duration and 3. the dormancy status and tuber GA levels in a dwarf mutant of potato. The tubers (cv. Russet Burbank) used in these studies were still completely dormant after 98 days of storage. Between 98 and 134 days of storage, dormancy began to end and tubers exhibited limited (< 2 mm) sprout growth. Tuber dormancy weakened with further storage and tubers exhibited greater rates of sprout growth after 187 days of storage. Tubers stored for 212 days or longer were completely non-dormant and exhibited vigorous sprout growth. Immediately after harvest, the endogenous contents of GA19, GA20, and GA1 were relatively high (0.48-0.62 ng g fresh weight(-1)). The content of these GAs declined between 33 and 93 days of storage. Internal levels of GA19, GA20, and GA, rose slightly between 93 and 135 days of storage reaching levels comparable to those found in highly dormant tubers immediately after harvest. Levels of GA19, GA20, and GA1 continued to increase as sprout growth became more vigorous. Neither GA4 nor GA8 was detected in any tuber sample regardless of dormancy status. Dormant tubers exhibited a time-dependent increase in apparent GA sensitivity. Freshly harvested tubers were completely insensitive to exogenous GAs. As postharvest storage continued, exogenous GAs promoted premature dormancy release with GA1 and GA20 eliciting the greatest response. Injection of up to 5 microg tuber(-1) of kaurene, GA12, GA19 or GA8 had no effect on dormancy release. Sprout growth from non-dormant tubers was also promoted by exogenous GA in the following sequence of activity: GA1 = GA20 > GA19. Kaurene, GA12, and GA8 were inactive. Continuous exposure of developing tubers to inhibitors of GA biosynthesis (AMO-1618, ancymidol, or tetcyclasis) did not extend tuber dormancy but rather hastened dormancy release. Comparison of tuber dormancy and GA1 content in tubers of a wild-type and dwarf mutant of S. tuberosum ssp. andigena revealed a near-identical pattern of dormancy progression in spite of the absence of detectable levels of GA1 in tubers of the dwarf sibling at any time during dormancy progression. Collectively, these results do not support a role for endogenous GA in potato tuber dormancy release but are consistent with a role for GAs in the regulation of subsequent sprout growth.