Regeneration Frequency Research Articles

In vitro step up NaCl treatment method as inducer of salinity tolerance in Basmati rice varieties

Background: It has been recorded that salt negatively impacts rice crop's seedling and reproductive stages. Globally, soil salinity is 2nd most important abiotic factor, profoundly impacting rice productivity. The purpose of current research is to persuade tolerance for salinity in salt sensitive varieties of Basmati rice [Basmati-385 (Bas-385), Super Basmati (Sup-Bas) and Basmati-2000 (Bas-2000)] by use of in vitro step-up NaCl treatment method.Methods: The in vitro step up NaCl treatment method was used to produce salt-tolerant calli from salt-sensitive Basmati rice varieties. The regeneration of these salt-tolerant calli in a salt-free medium led to the M1 generation. Subsequently, M2 and M3 generations were cultivated and garnered under salt stress conditions.Results: The highest callus induction percentage was achieved for all Basmati rice varieties by using MS medium supplemented with 2.0 mg/L 2,4-D. These calli underwent in vitro step up NaCl treatments, with the maximum tolerance level to NaCl being 1.0% across all varieties. Bas-385 and Bas-2000 exhibited regeneration frequencies of 75% and 85% respectively when cultured in MS medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA, while Sup-Bas displayed a regeneration frequency of 79% in MS medium comprising 3.0 mg/L BAP with 0.5 mg/L NAA. In subsequent M2 and M3 generations, a decline was observed in all selected agronomic and morphological features in all varieties under salinity stress in comparison to parent plants.Conclusion: This research validates the induction of salt tolerance in salt susceptible varieties of Basmati rice by in vitro step up NaCl treatment technique.Keywords: In vitro selection; Salt tolerance; Basmati rice; Step up NaCl treatment; Callogenesis; Plant regeneration

Study on the Establishment of Efficient Leaf Regeneration System in ‘Yuluxiang’ Pear

The ‘Yuluxiang’ pear is a key cultivated variety in China, celebrated for its high quality. However, it exhibits a low leaf regeneration frequency of only 35.0%, which hinders its transgenic breeding process. To establish an efficient regeneration system, we utilized tissue culture seedlings of the ‘Yuluxiang’ pear and investigated various factors influencing leaf regeneration: plant growth regulators, natural organic materials, leaf wounding and positioning methods, duration of dark culture, ages and lines of plantlets, as well as culture containers. Our results indicated that the optimal medium for leaf regeneration consisted of MS supplemented with 6-BA (6-Benzyl Aminopurine) 1.5 mg/L, NAA (α-Naphthalene acetic acid) 0.4 mg/L along with 10% (v/v) coconut water. Suitable wounding involved ensuring no damage to leaves while placing the abaxial side facing down on the medium; the ideal duration for dark culture was determined to be 21 days; optimal plantlet age was found to be 20 days; both plantlet line 1 and line 2 demonstrated effectiveness; triangle bottles were identified as appropriate culture containers. In summary, we successfully established an efficient leaf regeneration system for the ‘Yuluxiang’ pear that achieved a maximum regeneration frequency of 96.70% with an average bud number of 5.15 per explant. This system also proved effective for the ‘Qiuyue’ pear, yielding a regeneration frequency of 88.89% and an average bud number of 3.44 per explant. After investigating the germination methods of 200 leaves from this screened leaf regeneration system of ‘Yuluxiang’, it was found that there were both direct and indirect regeneration methods, and the germination rate of direct and indirect regeneration was 68.00% and 70.50%, respectively. Therefore, this study also laid a solid foundation for the future genetic transformation of ‘Yuluxiang’ pear.

Sonication-assisted Rhizobium radiobacter-mediated genetic transformation of Indian Lotus (Nelumbo nucifera Gaertn.).

This study aimed to develop a reliable and efficient genetic transformation method for the ornamental Indian Lotus (Nelumbo nucifera Gaertn.) using the sonication-assisted Rhizobium radiobacter-mediated transformation technique. To conduct the transformation, shoot apical meristem explants were infected with Rhizobium radiobacter (synonym Agrobacterium tumefaciens) strain LBA 4404 containing a binary vector pBI121 that harbours the GUS reporter gene (uidA) and kanamycin resistance gene nptII for plant selection. To improve the transformation efficiency, we optimized parameters such as bacterial cell density, sonication duration, infection time, co-cultivation duration, acetosyringone concentration, cefotaxime, and kanamycin concentrations. Sonication treatment at 42kHz for 90s recorded the highest transformation efficiency. The selection of regenerated plantlets was performed on a kanamycin-supplemented selection medium. The putative transformants showed GUS expression in the leaves and petioles. The presence of the GUS gene was also confirmed in the putative transformants through PCR, with the appearance of the expected amplicon size of 520bp. The presence of nptII was confirmed by PCR in the putatively transformed plants with an amplicon size of 530bp. The maximum regeneration frequency obtained was 72.66%, and the highest transformation efficiency achieved was 9.0% in the Indian Lotus.

Optimization of Shoot Regeneration and Agrobacterium-mediated Transformation in Strawberry (Fragaria ×ananassa)

Efficient methods of plant transformation and tissue culture are essential for the genetic engineering and advanced molecular breeding of plants, but neither is well established in cultivated octoploid strawberry (Fragaria ×ananassa). In the present study, a method for shoot regeneration was established and optimized for two genetically different strawberry cultivars, Sweet Sensation® Florida 127 (FL127) and Florida Brilliance (FB). Runner segments at the tip, node, and petiole obtained from greenhouse-grown plants were used as explants for comparisons of shoot regeneration rate. ‘FL127’ showed the highest frequency of shoot regeneration in the optimized condition, whereas ‘FB’ showed the best response to a lower concentration of N6-benzyladenine (BA) (0.01 mg/L) in the same media type. The average conversion frequencies of somatic embryos into shoot regenerations from the runner tips (RTs) were 42.8% in ‘FL127’ and 56.9% in ‘FB’, respectively. Using these optimized tissue culture conditions, Agrobacterium-mediated CRISPR/Cas9 gene editing was conducted to examine the efficiency of transformation and target gene editing for the phytoene desaturase, FaPDS, in the cultivar, FL127. A total of 234 explants inoculated with Agrobacterium harboring Cas9-FaPDS resulted in an 80.3% callus induction efficiency, with 13.3% of regenerated plants exhibiting partial or complete albino phenotypes. Amplicon sequencing of edited progeny showed mutations (substitutions, insertions, and deletions) at the guide RNA (gRNA) target sites or flanking regions of all FaPDS homoeologous copies. Our results provide effective tissue culture and transformation methods for strawberry functional genomics research and gene editing guided cultivar improvement.

Identification of quantitative trait loci for in vitro plant regeneration from leaf microexplants in cucumber (Cucumis sativus L.).

Plant regeneration in tissue cultures is crucial for the application of biotechnological methods to plant breeding. However, the genetic basis of in vitro plant regeneration is not fully understood. For cucumber, regeneration protocols from different types of explants have been reported, but thus far, the molecular basis of regeneration from cotyledon explants has only been studied. The aim of this work was to identify quantitative trait loci (QTLs) for in vitro plant regeneration from cucumber leaf microexplants. Plant regeneration was evaluated using a population of recombinant inbred lines (RILs) developed from a cross between line B10, characterized by high regeneration efficiency, and the low regeneration efficiency line Gy14. All RILs were scored for frequency of callus formation, organogenesis, and shoot regeneration. RILs with regeneration efficiencies higher than that of line B10 have been observed. QTLs for the frequency of organogenesis and shoot regeneration were identified. All the QTLs were mapped on cucumber chromosome 6, explaining 11.9 to 20% of the phenotypic variance. The major-effect QTL for organogenesis or6.1 was located on the upper arm of chromosome 6. The QTLs for shoot regeneration frequency, sr6.1A and sr6.1B, were located on the lower arm of chromosome 6. Analysis of the genomic region corresponding to these QTLs combined with gene expression profiling revealed that CsARF6 and CsWOX9 are gene candidates underlying these QTLs. This study is a step toward identifying the genes controlling the ability of cucumber plant regeneration from leaf explants.

Somatic embryogenesis and shoot organogenesis in peanut cv. 'Georgia-12Y' and successful transfer to the soil.

An efficient regeneration system was established through somatic embryogenesis and shoot organogenesis using mature embryos explants of peanut cultivar 'Georgia-12Y'. The role of plant growth regulator combinations was investigated for embryogenic callus and somatic embryo induction. Results showed that Murashige and Skoog (MS) medium supplemented with 20 μM picloram (4-amino 3, 5, 6-trichloropicolinic acid), casein hydrolysate (0.2 g/L), sucrose (30 g/L) and sorbitol (10 g/L) supported callus induction in dark and higher number of somatic embryos in light. No somatic embryos were induced at 0.1 μM to 10.0 μM of 2,4-Dichloro phenoxy acetic acid (2,4-D) and picloram individually. The highest regeneration frequency of 90% was recorded on 40 μM 2,4-D + casein hydrolysate (0.2 g/L), sucrose (30 g/L) and sorbitol (10 g/L). The plantlets regenerated via somatic embryogenesis did not exhibit any morphological abnormalities. Double staining with acetocarmine and Evans blue distinguished between embryogenic and non-embryogenic callus. Histological observations confirmed distinct developmental stages of somatic embryos. On the other hand, highest number of shoots were induced in response to MS + 15 μM thidiazuron (TDZ) among various treatments tested. Incubation of shoots on plant growth regulator free MS medium induced in-vitro flowering after 12 weeks under light conditions. The induction of embryogenic and morphogenic callus and production of fertile peanut plants using manipulations of various plant growth regulators is reported on peanut cultivar 'Georgia- 12Y'.

In vitro Multiplication of Cymbidium finlaysonianum (Lindl.) from Leaf Segments

The current investigation is envisioned to conserve one of the floriferous species i.e. Cymbidium finlaysonianum through in vitro leaf segment culture. The Cymbidiums are highly ornamental and have huge commercial demand, hence their germplasm needs to be conserved. Leaf segments (1.0 cm size) procured from 28 weeks of sterilize cultures were utilized as explants. These were inoculated in Murashige and Skoog medium (1962) enriched with auxin and cytokinin at 1 mgl-1. The highest regeneration frequency i.e. 80% was observed in 6-benzylaminopurine (BAP), followed by 60% regeneration growth in a-naphthalene acetic acid (NAA). Early development of plantlets was observed within 14 weeks of culturing in MS+BAP medium. This study demonstrates an effective use of in vitro multiplication methodology for Cymbidium finlaysonianum, which can be used for large-scale multiplication and conservation of this valuable orchid species before it goes extinct. The use of leaf segments as explants offers a promising approach for orchid breeding and genetic improvement programs. . KEYWORDS :6-benzylaminopurine (BAP), Cymbidium finlaysonianum, Germplasm, Leaf segment

Efficient Regeneration of Transgenic Rice from Embryogenic Callus via Agrobacterium-Mediated Transformation: A Case Study Using GFP and Apple MdFT1 Genes

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.

Evaluating the effect of cupric sulphate on morphogenic structure induction and plant regeneration in spring barley anther culture in vitro

Aim. Investigation was aimed to ascertain possibility to increase the efficiency of spring barley haploid production in anther culture in vitro by addition of cupric sulphate to the solution for cut tiller cold pretreatment and by increase of cupric sulphate concentrations in the media for morphogenic structure induction and plant regeneration. Methods. Cut tillers were cold pretreated for 5 days at 4ºC in water and in the solutions contained 5 µM and 10 µM of cupric sulphate. Isolated anthers were cultivated on agar solidified nutrient medium containing N6 macro-and MS micronutrients, physiologically active substances, maltose (9.0 %) and on the media with increased (10 µM) content of cupric sulphate. Results. No effect of cupric sulphate addition on cut tiller viability and morphogenesis in anther culture in vitro was found. The lowest plant regeneration frequency was obtained when cupric sulphate was added both to inductive and regenerative media. Сonclusions. Addition of cupric sulphate to the pretreatment solution as well as increase of this microelement concentration (up to 10 µM) in the nutrient media can’t be considered as the methodological approach suitable to enhance the efficiency of spring barley haploid production in anther culture in vitro.

Development of a Robust In vitro Regeneration Protocol in Cassava (Manihot esculenta C.) Using Axillary Bud Explants for Cryopreservation

Cassava is an important crop as it is the source of energy in the diet for the most of the tropical countries. Field gene bank is the only viable option for the maintenance of cassava germplasm. Cryopreservation offers the long-term conservation of vegetatively propagates crops without losing its viability through an effective in vitro regeneration system. In vitro regeneration response in YTP-1 and Sree Athulya genotypes of cassava was standardized through optimization of various factors influencing organogenesis. Among the different media tested, highest shoot induction frequencies of 81.4% and 77.8% were recorded in medium supplemented with 0.5 mg/l BAP + 0.2 mg/l NAA (CMSI 2) for YTP 1 and Sree Athulya respectively in the present study. 3 to 5 multiple shoots from a single shoot apex obtained from 70-80% of explants within 50-60 days. Highest regeneration frequencies of 56.6%and 46.6% were recorded in medium supplemented with 1 mg/l IAA (CRM 2) for YTP1 and Sree Athulya. As a result, an easy in vitro regeneration method that is genotype independent was created and may be effectively used to preserve cassava germplasm in the cryogen bank.

A single-use electrochemical biosensor system for ultrasensitive detection of Aflatoxin B1 in rice, corn, milk, peanut, chili pepper samples

Aflatoxin B1, a common food contaminant in peanuts and corn and a genotoxic carcinogen in humans poses a significant risk for hepatocellular carcinoma, making its detection crucial; this study aims to develop a label-free electrochemical biosensor using a disposable indium tin oxide polyethylene terephthalate (ITO-PET) electrode modified with 3-Aminopropyltriethoxysilane for detecting Aflatoxin B1 in real food samples. Initially, optimization steps for the proposed biosensor were conducted using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. Characterization steps such as storage capacity, regeneration, and single frequency impedance (SFI) were completed for the proposed disposable biosensor after the optimization steps. The electrochemical biosensor, based on AFB1, exhibited excellent repeatability and reproducibility. It had a broad dynamic detection range from 0.1 fg/mL to 500 fg/mL, with low limits of detection (LOD) and quantitation (LOQ) at 0.19 fg/mL and 0.65 fg/mL, respectively. Finally, the proposed AFB1-based biosensor system was applied to real food samples (rice, chili pepper, milk, corn, and peanuts) for testing and validation.

Comparison Between Maize Inbred and Hybrid Lines for Their Amenability to Agrobacterium Mediated Transformation and In Vitro Plant Regeneration

The field of genetic engineering and molecular breeding utilizes maize immature embryos for transformation studies. However, there are hinderances such as the unavailability of immature embryos throughout the year and their low transformation frequencies which make them unsuitable for use at a commercial level. Among cereals, maize has prime importance due to its productivity, industrial products, animal feed and fodder. Regeneration capability in maize transformation studies varies due to differences in genetic makeup and explant source. In this study, we evaluate mature embryos as explants for in vitro plant regeneration. Six varieties including 3 hybrid lines (Pioneer 3025, SG 2002 and Neelam) and 3 inbred lines (NCML 107, CML 161 and FBF 3368) were screened for Agrobacterium mediated transformation amenability using mature maize embryos. The two inbred lines NCML 107 and CML 161 performed best showing 100% callus induction frequency, 93.33% and 86.66% transient GUS expression, and 43.75% and 13.3% regeneration frequency, respectively. Using split seed as an explant, better regeneration was observed in NCML 107 (14.86%) and CML 161 (7.5%) as compared to mature embryos, NCML 107 (10%) and CML 161 (2%). Here we report successful plant regeneration (regeneration medium supplemented with Kinetin + BAP) using split seeds as an explant. Our results demonstrate the capability of two elite maize inbred lines (NCML 107 & CML 161) for their amenability to Agrobacterium mediated transformation and better tissue culture response; hence can be selected in maize transformation studies for improving crop varieties for enhanced nutritional content and better adaptation.

Indirect in vitro Regeneration in Four Varieties of Potato (Solanum tuberosum L.) from Internodal Segments and Leaf Explants

Internodal segments and leaf explants from four varieties of potato (Solanum tuberosum L.) cultivated in Bangladesh, namely, Asterix, Diamant, Granola and Lady Rosseta were utilized for indirect in vitro regeneration of shoots through callus culture. The best responses regarding the reaeration of shoots were obtained when the explants were cultured on MS medium supplemented with 4.0 mg/l BAP and 1.0 mg/l IAA and 0.5 mg/l GA3. The highest frequency of multiple shoot regeneration was recorded from internodal segments rather than from the leaf explants. Hormone free MS medium was found to be the most effective for healthy root induction from the in vitro raised excised shoots. Following adequate root induction, the in vitro regenerated plantlets were successfully transplanted and established to soil for further growth and development of tubers. Plant Tissue Cult. & Biotech. 34(1): 83-92, 2024 (June)

Clonal Propagation of Turmeric (Curcuma longa) and Confirmation of Genetic Fidelity of the Micropropagated Shoots by RAPD Markers

Turmeric (Curcuma longa) is used as a spice and has excellent medicinal value. However, the field multiplication rate of turmeric plants is not satisfactory. In vitro methods could be a feasible alternative to vegetative propagation in the field. This study was aimed at developing a suitable protocol for the clonal propagation of turmeric confirming the genetic fidelity of the in vitro regenerated plantlets. The explants of shoot bud (full, half, and quarter), root, rhizome, and leaf were cultivated on MS medium with BAP, NAA, and Kinetin. Cultured shoot buds showed a higher regeneration frequency and shoots per explant than those obtained from other explants. The highest shoot regeneration was 72.49% from shoot buds, followed by rhizome (68.76%) and the lowest was 53.93 % from ¼ shoot buds. There were significant effects of growth regulator combinations and concentrations in shoot regeneration from shoot bud explant. The highest shoot regeneration per shoot bud explant was 4.42 with BAP at 20.0 mg/l and NAA at 0.5 mg/l at 24 weeks. The best rooting response with 9.75 roots per explant was reported with 1.0 mg/l BAP with 1.0 mg/l NAA. The rooted plants were successfully transferred to pots for their acclimatization. Both the micropropagated shoots and their mother plant produced monomorphic bands with the six RAPD markers that confirm the genetic fidelity of micropropagated clones. These results showed that in vitro raised plantlets of turmeric had no risk of somaclonal variations and were found to be true-to-type in nature over the culture period. The protocol developed through this investigation has the potential application for the rapid multiplication of turmeric plants. Plant Tissue Cult. & Biotech. 34(1): 55-69, 2024 (June)

Direct shoot regeneration from cotyledonary node of Uraria picta (Jacq.) Desv. ex DC., an important plant of dashmula drugs, and assessment of genetic fidelity, metabolic profiling, and anti-diabetic activity

Uraria picta root is used in the polyherbal product ‘Dashmula’. Its exploitation for formulation preparation has depleted its availability, leading to medicine adulteration. Direct shoot regeneration from the cotyledonary node holds promise as a source of raw material. This study aimed to develop a regeneration protocol for U. picta and validate its genetic and metabolic fidelity. The seeds of U. picta showed low germination rates, prolonged dormancy, and poor viability. Root exploitation in the wild poses a threat to its availability in nature. Seedling’s derived cotyledonary nodes cultured on B5 medium supplemented with BAP (0.5–3 mg L−1), Kinetin (0.5–3 mg L−1), Thidiazuron (0.01–1 mg L−1), and meta-Topolin (0.1–4 mg L1). To address hyperhydricity in regenerated shoots, cotyledonary nodes were cultured on high-agar concentration media. Microshoots were exposed to IBA solution (50–800 mg L−1) pulse treatment for rooting. Tissue-cultured plants genetic fidelity was assessed using ISSR and SCoT markers, while metabolic fidelity was studied with HRMS. The chlorophyll content, antioxidant, and antidiabetic activity of micropropagated plants were evaluated. The highest shoot regeneration frequency, with a maximum of 6.57±0.278 shoots per explant, was achieved using 2 mg L−1meta-Topolin. The shoots were elongated, had expanded leaves, and were hyperhydrated. BAP (2 mg L−1) induced a maximum of 9.83±0.333 shoot buds per explant. BAP caused explant browning, profuse callus formation, dwarfing, and hyperhydric shoots. Hyperhydricity was alleviated with a higher agar concentration (1 %). IBA (400 mg L−1) induced a maximum of 2.18±0.090 roots per shoot and a root length of 9.23±0.033 cm. Tissue-cultured and mother plants exhibited clonal fidelity, similar metabolite and chlorophyll content, strong antioxidant activity, and equal efficacy for inhibiting α-amylase and α-glucosidase. This method can propagate elite clones of U. picta and offer its improvement via genetic transformation.

Efficient isolated microspore culture protocol for callus induction and plantlet regeneration in japonica rice (Oryza sativa L.)

BackgroundIsolated microspore culture is a useful biotechnological technique applied in modern plant breeding programs as it can produce doubled haploid (DH) plants and accelerate the development of new varieties. Furthermore, as a single-cell culture technique, the isolated microspore culture provides an excellent platform for studying microspore embryogenesis. However, the reports on isolated microspore culture are rather limited in rice due to the low callus induction rate, poor regeneration capability, and high genotypic dependency. The present study developed an effective isolated microspore culture protocol for high-frequency androgenesis in four japonica rice genotypes. Several factors affecting the isolated microspore culture were studied to evaluate their effects on callus induction and plantlet regeneration.ResultsLow-temperature pre-treatment at 4 ℃ for 10–15 days could effectively promote microspore embryogenesis in japonica rice. A simple and efficient method was proposed for identifying the microspore developmental stage. The anthers in yellow-green florets located on the second type of primary branch on the rice panicle were found to be the optimal stage for isolated microspore culture. The most effective induction media for callus induction were IM2 and IM3, depending on the genotype. The optimal concentration of 2, 4-D in the medium for callus induction was 1 mg/L. Callus induction was negatively affected by a high concentration of KT over 1.5 mg/L. The differentiation medium suitable for japonica rice microspore callus comprised 1/2 MS, 2 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 6 g/L agar. The regeneration frequency of the four genotypes ranged from 61–211 green plantlets per 100 mg calli, with Chongxiangjing showing the highest regeneration frequency.ConclusionsThis study presented an efficient protocol for improved callus induction and green plantlet regeneration in japonica rice via isolated microspore culture, which could provide valuable support for rice breeding and genetic research.

Phenotyping in Rye Landrace and Regeneration Frequency Study in Wheat × Rye F1 Hybrid Seeds

The present research aimed to study the phenotyping characterization of a high-altitude Himalayan rye landrace and its crossability with wheat genotypes and investigate the regeneration frequency of wheat × rye F1 hybrid seeds. The study was conducted during the rabi season of 2022-2023 at the Agriculture Research Farm, Department of Plant Breeding and Genetics, Lovely Professional University, Phagwara, Punjab. The phenotypic characterization of the Himalayan rye landrace revealed several adaptive traits that were found suitable for cultivation in the northwest Indo-Gangetic plains. Furthermore, the study investigated the crossability of the Himalayan rye landrace with various wheat genotypes, including 4 genotypes of bread wheat HD2967, UNNAT 343, WH1105, and PBW502 and 4 genotypes of durum wheat PDW291, PDW274, PBW34, and WHD943. Among the germination percentage of all 8 genotypes, 4 in Bread wheat and 4 in Durum wheat was observed. Due to its self-incompatibility, Rye cannot be used to develop inbred lines directly. However, the doubled haploid approach can produce inbred lines in the rye, facilitating the extension of genetic bases and introducing alien chromatin to identify stress tolerances.

Investment in regeneration versus asexual reproduction is resource‐dependent in a freshwater annelid

Abstract The post‐embryonic developmental processes of regeneration and asexual agametic reproduction are widespread and often co‐occur in animals. These traits are of great ecological significance, but their physiological dynamics within species are not well understood. In naid annelids, regeneration and asexual reproduction via fission are evolutionarily related and mechanistically similar yet distinct, making these animals useful systems in which to study resource allocation strategies between the two processes. How asexual reproductive investment varies as a function of somatic investment demands was tested in the naid Pristina leidyi by repeatedly amputating the heads of individual worms, allowing regeneration to proceed, and measuring reproductive output over time. Treatments were replicated under high and low food levels to determine to what extent the investment dynamic between regeneration and fission is affected by the resource pool. Reproductive output was affected by injury and regeneration frequency in a resource‐dependent manner, such that only worms with less food availability exhibited reproductive deficits; injury and regeneration did not affect reproductive output of worms under the high food condition. When reproductive output was decreased, this occurred not through a reduction in offspring quantity but a reduction in offspring quality. In the offspring of experimental animals, body size and fission speed were dependent on parental feeding level and to a lesser and inconsistent extent on parental injury history, but regeneration speed was unaffected by parental treatment. These findings suggest that, in a species capable of both regeneration and asexual reproduction: (1) the resource pool is a key factor mediating the resource investment pattern between regeneration and fission; (2) sacrificing per‐offspring investment rather than fecundity may be an optimal strategy if resources are limiting; (3) regeneration and fission have evolved distinct resource allocation pathways. This work prompts further questions about the physiological dynamics between regeneration and asexual reproduction in animals, such as whether and to what extent these have evolved adaptively, including in response to injury and resource pressures. Read the free Plain Language Summary for this article on the Journal blog.

First Report on Mesophyll Protoplast Isolation and Regeneration System for the Duboisia Species.

The Duboisia species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, employing both conventional breeding methods and advanced biotechnology tools. The primary objective of this research was to establish a highly efficient method for isolating mesophyll protoplasts and facilitating their regeneration, thereby laying a robust foundation for the application of various advanced plant biotechnology tools in the pursuit of genetic enhancement. The mesophyll protoplast isolation process was developed for hybrid D. myoporoides × D. hopwoodii with careful optimisation of the following parameters: leaf strip size; incubation conditions; physical treatment; and enzyme concentration. The optimal parameters were combined in each individual step; the best enzyme concentration was determined to be 2% (w/v) cellulysin and 0.5% (w/v) macerase. Protoplast yield was found to be greatly affected by the enzyme concentrations. The isolated protoplasts were cultured at a density of 0.5 × 105 to best sustain the highest cell division (33.2%) and a microcalli induction frequency of 17.9%. After 40 days of culture in a modified KM8P medium at 25 °C in darkness, visible microcalli were transferred to a solidified Murashige and Skoog (MS) medium with 1 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction under a 16 h photoperiod. After 30 days of culture, compact organogenic calli were transferred into a solid MS medium with 6-benzylaminopurine (BA) alone or thidiazuron (TDZ) alone or in combination with BA or naphthalene acetic acid (NAA) for shoot regeneration. The maximum shoot regeneration frequency (63.3%) was observed in the medium with 1.5 mg L-1 TDZ alone. For the first time, a reliable protoplast isolation and regeneration system from mesophyll cells was established for Duboisia with high protoplast viability, successful microcalli formation, and intact plant regeneration. This innovation will significantly contribute towards the genetic enhancement of the Duboisia species.

Development of regenerated plants of interspecific wheat hybrids using immature embryos as explants

Nowadays particular attention in winter wheat breeding is paid to the introduction of biotechnological methods. The culture of immature embryos in vitro is an alternative way to produce hybrids that often do not develop fully in vivo. The purpose of the current study was to estimate the ability of immature embryos of interspecific winter wheat hybrids for callus induction, morphogenesis, and plant regeneration on artificial nutrient media, as well as to select optimal conditions for obtaining full-fledged plants with all the botanical characteristics of the species. The research material included 5 hybrid combinations of interspecific crosses between durum (Triticum durum DESF.) and common (Triticum aestivum L.) winter wheat. The material was collected during one field season (2022). Immature embryos were used as explants. Culture media were according to Murashige-Skoog (MS), and they differed in the content of hormones and organic substances. There has been found that the hybrid combinations were characterized by a variety of morphogenic responses. The manifestation of callusogenesis was not high enough and averaged 22–32 % depending on the nutrient medium. Rhizogenesis averaged from 1–6.7 %, and ended with the formation of roots. The process of formation of embryoid- and hemmorizogenesis was best demonstrated by the hybrid combination No.1, and on two media. All processes of morphogenesis excepting ‘rhizogenesis’ varied greatly among hybrid combinations. The largest number of regenerated plants was obtained from hybrid combination No.1 (61 pcs., 54.5 %). After vernalization and acclimatization, 38 % of the plants survived from the number of planted embryos. The largest number of heads (79 pcs.) was formed according to hybrid combination No.1, on average 1.46 heads per plant. However, not all heads were fertile. The results of the correlation analysis made it possible to identify indicators that closely correlated with the amount of plant regeneration, namely, the high yield of embryoid- and hemmorizogenic structures ensured a high frequency of plant regeneration (r = 0.769).