Expression of the CCCH-tandem zinc finger protein gene OsTZF5 under a stress-inducible promoter mitigates the effect of drought stress on rice grain yield under field conditions.

Increasing drought resistance without sacrificing grain yield remains an ongoing challenge in crop improvement. In this study, we report that Oryza sativa CCCH-tandem zinc finger protein 5 (OsTZF5) can confer drought resistance and increase grain yield in transgenic rice plants. Expression of OsTZF5 was induced by abscisic acid, dehydration and cold stress. Upon stress, OsTZF5-GFP localized to the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF5 under the constitutive maize ubiquitin promoter exhibited improved survival under drought but also growth retardation. By introducing OsTZF5 behind the stress-responsive OsNAC6 promoter in two commercial upland cultivars, Curinga and NERICA4, we obtained transgenic plants that showed no growth retardation. Moreover, these plants exhibited significantly increased grain yield compared to non-transgenic cultivars in different confined field drought environments. Physiological analysis indicated that OsTZF5 promoted both drought tolerance and drought avoidance. Collectively, our results provide strong evidence that OsTZF5 is a useful biotechnological tool to minimize yield losses in rice grown under drought conditions.

Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field.

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non-transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.

Modulation of Tropane Alkaloids' Biosynthesis and Gene Expression by Methyl Jasmonate in Datura stramonium L.: A Comparative Analysis of Scopolamine, Atropine, and Hyoscyamine Accumulation.

Scopolamine and atropine are two medicinal alkaloids derived from Datura stramonium L. with anticholinergic properties. This study explored how methyl jasmonate (MJ), a plant growth regulator, affects the biosynthesis and accumulation of these alkaloids in different plant tissues. The expression levels of putrescine N-methyltransferase (PMT), tropinone reductase I (TR1), and hyoscyamine 6ß-hydroxylase (h6h), three critical enzymes in the biosynthetic pathway, were also analyzed. The results indicated that MJ at 150 µM increased the production of scopolamine and atropine in both leaves and roots, while MJ at 300 µM had an adverse effect. Furthermore, MJ enhanced the expression of PMT, TR1, and h6h genes in the roots, the primary site of alkaloid synthesis, but not in the leaves, the primary site of alkaloid storage. These results imply that MJ can be applied to regulate the biosynthesis and accumulation of scopolamine and atropine in D. stramonium, thereby improving their production efficiency.

Calcium-Dependent Protein Kinase 5 (OsCPK5) Overexpression in Upland Rice (Oryza sativa L.) under Water Deficit.

Water deficit significantly affects global crop growth and productivity, particularly in water-limited environments, such as upland rice cultivation, reducing grain yield. Plants activate various defense mechanisms during water deficit, involving numerous genes and complex metabolic pathways. Exploring homologous genes that are linked to enhanced drought tolerance through the use of genomic data from model organisms can aid in the functional validation of target species. We evaluated the upland rice OsCPK5 gene, an A. thaliana AtCPK6 homolog, by overexpressing it in the BRSMG Curinga cultivar. Transformants were assessed using a semi-automated phenotyping platform under two irrigation conditions: regular watering, and water deficit applied 79 days after seeding, lasting 14 days, followed by irrigation at 80% field capacity. The physiological data and leaf samples were collected at reproductive stages R3, R6, and R8. The genetically modified (GM) plants consistently exhibited higher OsCPK5 gene expression levels across stages, peaking during grain filling, and displayed reduced stomatal conductance and photosynthetic rate and increased water-use efficiency compared to non-GM (NGM) plants under drought. The GM plants also exhibited a higher filled grain percentage under both irrigation conditions. Their drought susceptibility index was 0.9 times lower than that of NGM plants, and they maintained a higher chlorophyll a/b index, indicating sustained photosynthesis. The NGM plants under water deficit exhibited more leaf senescence, while the OsCPK5-overexpressing plants retained their green leaves. Overall, OsCPK5 overexpression induced diverse drought tolerance mechanisms, indicating the potential for future development of more drought-tolerant rice cultivars.

Stipagrostis pennata (Trin.) De Winter Artificial Seed Production and Seedlings Multiplication in Temporary Immersion Bioreactors

This study was conducted to develop the protocol for artificial seed production of Stipagrostis pennata (Trin.) De Winter via somatic embryo encapsulation as well as test a temporary bioreactor system for germination and seedling growth. Embryogenic calli were encapsulated using sodium alginate and calcium chloride and then sowed in the Murashige and Skoog (MS) germination medium in in vitro cultures. The experiments were conducted as a factorial based on a completely randomized design with three replications. The treatments include three concentrations of sodium alginate (1.5%, 2.5%, and 3.5%), two ion exchange times (20 and 30 min), and two artificial seed germination media (hormone-free MS and MS supplemented with zeatin riboside and L-proline). Germination percentage and number of days needed until the beginning of germination were studied. The highest percentage of artificial seed germination was obtained when 2.5% sodium alginate was used for 30 min (ion exchange time) and when the seeds were placed on the MS germination medium supplemented with zeatin riboside and L-proline. The results of the analysis of variance in the temporary immersion bioreactor system showed that the main effects observed on the seedling growth were associated with different growth hormones in culture media and the number of feeding cycles. Experimental results also indicated that the total protein analyses of zygotic seedlings and seedlings originating from the synthetic seeds showed no statistically significant differences between these samples.

Temporary immersion bioreactor system for propagation by somatic embryogenesis of hybrid larch (Larix × eurolepis Henry).

Somatic embryogenesis (SE) has high potential for large-scale clonal propagation of conifers. Different types of bioreactor cultures have been tested for the conifer SE process where the temporary immersion bioreactors (TIBs) have proved to be useful across the different developmental steps of the SE process. In the present study the use of TIBs was tested for hybrid larch (Larix × eurolepis Henry). The results showed two-fold increases in both fresh weight (FW) of pro-embryogenic masses (PEMs) and yield of cotyledonary embryos in the TIBs compared to solid medium in plates. For the germination phase, the highest number of roots per plant, the root length and height of plants were also obtained in the TIBs. The results show that the TIB system can be successfully used to support scale up of plant production in all steps of the SE process from proliferation to germination of hybrid larch (Larix × eurolepis Henry).

Protocol development for somatic embryogenesis, SSR markers and genetic modification of Stipagrostis pennata (Trin.) De Winter.

BACKGROUND: Stipagrostis pennata (Trin.) De Winter is an important species for fixing sand in shifting and semi-fixed sandy lands, for grazing, and potentially as a source of lignocellulose fibres for pulp and paper industry. The seeds have low viability, which limits uses for revegetation. Somatic embryogenesis offers an alternative method for obtaining large numbers of plants from limited seed sources. RESULTS: A protocol for plant regeneration from somatic embryos of S. pennata was developed. Somatic embryogenesis was induced on Murashige & Skoog (MS) medium supplemented with 3 mg·L-1 2,4-D subsequently shoots were induced on MS medium and supplemented with 5 mg·L-1 zeatin riboside. The highest shoots induction was obtained when embryogenic callus derived from mature embryos (96%) in combination with MS filter-sterilized medium was used from Khuzestan location. The genetic stability of regenerated plants was analysed using ten simple sequence repeats (SSR) markers from S. pennata which showed no somaclonal variation in regenerated plants from somatic embryos of S. pennata. The regenerated plants of S. pennata showed genetic stability without any somaclonal variation for the four pairs of primers that gave the expected amplicon sizes. This data seems very reliable as three of the PCR products belonged to the coding region of the genome. Furthermore, stable expression of GUS was obtained after Agrobacterium-mediated transformation using a super binary vector carried by a bacterial strain LBA4404. CONCLUSION: To our knowledge, the current work is the first attempt to develop an in vitro protocol for somatic embryogenesis including the SSR marker analyses of regenerated plants, and Agrobacterium-mediated transformation of S. pennata that can be used for its large-scale production for commercial purposes.

Refinement of a clearing protocol to study crassinucellate ovules of the sugar beet (Beta vulgaris L., Amaranthaceae).

BACKGROUND: Clearing methods allow relatively quick processing of plant material and examination of cellular structures by rendering tissues and organs translucent. They have been adapted for plant embryology, primarily to study ovule development, megasporogenesis, megagametogenesis and embryogenesis. Such clearing methods overcome several disadvantages of the conventional embedding-sectioning techniques that are arduous and time-consuming. Although numerous protocols with different clearing solutions have been described, there have been no reports to date proposing a reliable method to clear the crassinucellate ovules of the sugar beet (Beta vulgaris L.), an economically important crop. Therefore, this study aims to find a suitable approach to improve the tissue transparency of sugar beet ovules at different developmental stages. RESULTS: We established a methyl salicylate-based protocol that significantly improved the transparency of the B. vulgaris ovule structures, which allowed us to observe the megagameto- and embryogenesis of that species. This was achieved by (1) chemical softening of the tissues; (2) vacuum pump-assisted infiltration step; (3) shaking-assisted incubation with clearing mixtures; and (4) manual removal of the chemically softened seed coat. CONCLUSIONS: The effectiveness of our method is due to the strategy combining various approaches at different stages of the procedure aiming at increasing the accessibility of the internal ovule structures to the clearing solution. The results of this study may be applied in sugar beet breeding programs, and it will provide a basis for further investigation of numerous aspects of the species' embryology. Moreover, that unique approach may be easily adapted to other species developing crassinucellate ovules.

Overexpression of a phospholipase (OsPLDα1) for drought tolerance in upland rice (Oryza sativa L.).

This work aimed to evaluate the drought tolerance of transformed plants of the cultivar BRSMG Curinga that overexpress the rice phospholipase D α1 (OsPLDα1) gene. The productivity of independent transformation event plants of the OsPLDα1 gene was evaluated in an experiment where 19 days of water deficit were applied at the reproductive stage, a very strict growing condition for upland rice. The non-genetically modified cultivar (NGM) under drought treatment reduced productivity by 89% compared with that under irrigated treatment, whereas transformed plants (PLDα1_E2) reduced productivity by only 41%. After the drought treatment, the PLDα1_E2 plants productivity was five times greater than that of the NGM plant. Moreover, no adverse effects on growth and development of the transgenic plants were observed. Seven days after the resumption of irrigation, PLDα1_E2 plants had higher stomatal conductance, greater photosynthetic rate, and transpiration rate than did NGM plants, as well as a higher expression level of the OsPLDα1 gene. A delay in the senescence process was observed in these PLDα1_E2 plants, and this was determined for the recovery of photosynthesis, with greater expression of the Rubisco and lower expression of the SOD. This finding was suggestive of decreased oxidative stress, probably due to gas exchange by the partial closure of the stomata of these transformed plants, which prevented the formation of reactive oxygen species. OsPLDα1 gene overexpression resulted in a reduction in production loss under severe water deficit and revealed a possibility for the development of upland rice cultivars that are more tolerant to extreme drought conditions.

Ensifer-mediated transformation: an efficient non-Agrobacterium protocol for the genetic modification of rice.

While Agrobacterium-mediated transformation (AMT) remains the most widely used technique for gene transfer in plants, interest exists for the use of non-Agrobacterium gene delivery systems due to freedom-to-operate issues that remain with AMT across several jurisdictions. In addition, the plant pathogenic mode of action of Agrobacterium tumefaciens significantly increases the costs to passage engineered cultivars through the regulatory process. Ensifer adhaerens (OV14) is a soil-related bacterium with the proven ability to genetically modify the model plant A. thaliana and the staple crop S. tuberosum (Wendt et al., Trans Res 21:567-578, 2012). While previous work was relevant for dicotyledonous species, in this study, the efficacy of Ensifer adhaerens (OV14)-mediated transformation (EMT) was determined on two japonica rice varieties, Curinga and Nipponbare, and the recalcitrant indica variety, IR64. The results indicated that E. adhaerens (OV14) exhibits infection efficiencies ranging between 50-70 %, 90-100 % and 90-95 % for Curinga, Nipponbare and IR64 respectively. Curinga and Nipponbare plants transformed with E. adhaerens (OV14) and A. tumefaciens (LBA4404 and EHA105) were regenerated achieving transformation efficiencies of 16 % and 26-32 % for Curinga and 7 and 4 % for Nipponbare respectively. Separately, the transformation of IR64 was only recorded via EMT (transformation efficiency ~1 %). Integration analyses conducted on 24 transgenic rice lines illustrated that T-DNA insertion occurred randomly throughout the rice genome for EMT (and AMT), with similar integration patterns in the rice genomic DNA observed for both bacterial species.

Current issues in cereal crop biodiversity.

The exploration, conservation, and use of agricultural biodiversity are essential components of efficient transdisciplinary research for a sustainable agriculture and food sector. Most recent advances on plant biotechnology and crop genomics must be complemented with a holistic management of plant genetic resources. Plant breeding programs aimed at improving agricultural productivity and food security can benefit from the systematic exploitation and conservation of genetic diversity to meet the demands of a growing population facing climate change. The genetic diversity of staple small grains, including rice, maize, wheat, millets, and more recently quinoa, have been surveyed to encourage utilization and prioritization of areas for germplasm conservation. Geographic information system technologies and spatial analysis are now being used as powerful tools to elucidate genetic and ecological patterns in the distribution of cultivated and wild species to establish coherent programs for the management of plant genetic resources for food and agriculture.

Induction of parthenocarpy in tomato via specific expression of the rolB gene in the ovary.

The molecular signals for the development of the ovary into fruit following ovule fertilization are not clear. However, in many species, including tomato ( Lycopersicon esculentum Mill.), auxins and auxin transport inhibitors can substitute for fertilization as activators of fruit set, suggesting that this plant hormone plays a key role in this process. In agreement, transgenes for auxin biosynthesis expressed under ovary- or ovule-specific promoters were shown earlier to enable parthenocarpic (i.e. seedless) fruit development. In the present study, we tested an alternative approach for the induction of parthenocarpy that is based on ovary-specific expression of the Agrobacterium rhizogenes-derived gene rolB. This gene was chosen because rolB transgenic plants manifest several syndromes characteristic of auxin treatment. Tomato plants transformed with a chimeric construct containing the rolB gene fused to the ovary- and young-fruit-specific promoter TPRP-F1 developed parthenocarpic fruits. Fruit size and morphology, including jelly fill in the locules of the seedless fruits, were comparable to those of seeded fruits of the parental line. Although it is not known whether ROLB signals for the same cassette of genes involved in fertilization-dependent fruit development, it clearly activates a battery of genes that enable successful completion of seedless fruit development in tomato.