μM GA3 Research Articles

Development of a Feasible and Efficient In Vitro Rescue Protocol for Immature Prunus spp. Embryos

The major factors affecting the in vitro immature embryo rescue efficiencies from Prunus persica or P. armeniaca accessions have been identified, along with improving the feasibility. Variations in the woody plant medium (WPM) were used depending on the embryo size. Embryos less than 5 mm long were cultured in WPM supplemented with 1 μM BAP and 1 μM GA3, while embryos bigger than 5 mm long were cultured in hormone-free medium, with or without vermiculite. The environmental in vitro culture conditions consisted of three phases: a (I) stratification at 4 °C during a 3- to 5-month-long period in the dark, followed by (II) growth of germinated embryos at 14 °C for a 4-week-long period, with 12 h light a day, which favors plantlet development, and finally, (III) growth at 24 °C, with 16 h light a day, until the plantlets were acclimatized in the greenhouse. The germination of smaller embryos, at the end of phase I, ranged from 82.2% to 22.1% for apricots and flat peaches, respectively, whereas for bigger embryos, the germination varied from 97.3% to 53.2% for the same species. The embryo germination for peaches and nectarines ranged from 40.1% to 30.3% for smaller embryos, and from 91.9% to 63.0% for bigger embryos. Endo- and epiphytic contamination, affecting from 7.4% to 52.9% of cultured embryos, depending on the fruit type and conservation conditions, and the capacity to acclimate to soil conditions, ranging from 50.4% to 93.2%, were the two most important factors influencing the protocol’s efficiency and feasibility. Considering the overall efficiencies, expressed as hardened plants transferred to field plots over clean uncontaminated embryo, the values ranged from 55.8% for nectarines, 54.0% for peaches, 45.6% for apricots, and 23.3% for flat fruits. The addition of vermiculite to the culture medium significantly improved the plantlet development, avoiding subculture to fresh medium when an extension of phase III was required before acclimatization. Compared to laboratory glassware, the use of food glass containers with air-permeable sealing film, along with vermiculite-containing medium, significantly reduced the costs when handling the large number of embryos required for breeding programs.

Physiological response of microalga Dunaliella parva when treated with MeJA, GA3.

DpAP2 is a transcription factor regulating carotenoid biosynthesis pathway. It was speculated that MeJA significantly decreased expression of DpAP2 gene, then the decreasing DpAP2 expression significantly inhibited expression of some key enzyme genes such as PSY, PDS and GGPS in carotenoid biosynthesis pathway. In contrast, it was speculated that GA3 significantly increased expression of DpAP2 gene, then the increasing DpAP2 expression significantly increased expression of some key enzyme genes such as PDS and GGPS in carotenoid biosynthesis pathway. To increase the content of carotenoid, we evaluated the effect of DpAP2 overexpression on carotenoid accumulation in D. parva. Transgenic D. parva showed a higher carotenoid content (3.18 mg/g DW) compared with control group (2.13 mg/g DW) at 9 d. The dosage effects of exogenous hormones MeJA and GA3 were found in D. parva cells treated with different concentrations of MeJA (10, 20, 50, 100 μM) and GA3 (10, 20, 50, 100 μM). The high concentrations of MeJA (10-100 μM) inhibited the accumulation of carotenoid, and the relative expression of DpAP2, PSY, PDS and GGPS decreased significantly. On the contrary, the relative expression of DpAP2, PDS and GGPS increased significantly when D. parva was treated with 10, 20, 50 and 100 μM GA3, which promoted the biosynthesis of carotenoid. Therefore, we inferred that there was a hierarchical regulation from hormone, transcription factor, key enzyme gene to carotenoid accumulation in carotenoid biosynthesis. Carotenoid biosynthesis was enhanced by DpAP2 overexpression (1.4930 fold of control) and exogenous substances such as GA3 (1.5889 fold of control), which laid a foundation for massive accumulation of carotenoids in microalgae. In the future, further studies were required to demonstrate the complex regulatory network.

Optimizing pollen germination and subcellular dynamics in pollen tube of Torreya grandis

Torreya grandis, a dioecious Taxaceae species of significant economic value in southeast China, presents challenges for natural pollination due to asynchronous maturation of its sex organs and low pollen vitality. In order to enhance fertilization success through artificial pollination of T. grandis, this study investigated the optimal conditions for in vitro pollen germination and pollen tube growth of T. grandis. The optimal in vitro growth medium was found to contain 29 mM sucrose, 0.8 mM H3BO3, 0.72 mM CaCl2, and 0.32 mM MgSO4, supplemented with 4 μM NAA, 2 μM GA3, and 5 μM 2,4-D at pH=5.6. Under these conditions, we achieved a maximum pollen germination ratio of 69.99 ± 5.17 % and a pollen tube length of 34.38 ± 6.04 µm after 6 days germination at 28°C. FM4–64 dye and Mitotracker Red staining revealed highly dynamics of vesicles and mitochondria during germination, which were accumulated at the tip of pollen tube and exhibited biphasic movement patterns. The total number, motion rate, and movement velocity of vesicles as well as mitochondria showed an initially increase followed by a gradual decrease pattern. The presence of sucrose in the medium significantly increased the dynamics and metabolic activity of both vesicles and mitochondria, which may relate with higher pollen germination ratio and faster pollen tube growth compared to sucrose-depleted conditions. Thus, these findings shed light on the physiological characteristics of Torreya pollen germination and provide scientific information for improving Torreya fruit yield through artificial pollination.

Interacting effects of phytohormones and fruit pruning on the morpho-physiological and biochemical attributes of bell pepper

Several factors, such as pruning and phytohormones, have demonstrated an influence on both the quantity and quality in the bell pepper. A factorial experiment using a completely randomized design was conducted on the Lumos yellow bell in a greenhouse. Treatments were the fruit pruning (0, 10, and 30%) and foliar application of phytohormones auxin (AUX) and gibberellic acid (GA3) at concentrations of 10 µM AUX, 10 µM GA3, 10 µM AUX + 10 µM GA3+, and 20 µM AUX + 10 µM GA3 along with controls. The plants were sprayed with phytohormones in four growth stages (1: flowering stage when 50% of the flowers were on the plant, 2: fruiting stage when 50% of the fruits were the size of peas, 3: fruit growth stage when 50% of the fruits had reached 50% of their growth, and 4: ripening stage when 50% of the fruits were at color break). The results of the present investigation showed that pruning rate of 30% yielded the highest flesh thickness and vitamin C content, decreased seed count and hastened fruit ripening. The use of GA3 along with AUX has been observed to augment diverse fruit quality characteristics. According to the results, the application of 10% pruning in combination with 20 µM AUX and 10 µM GA3 demonstrated the most significant levels of carotenoids, chlorophyll, and fruit length. The experimental group subjected to the combined treatment of 30% pruning and 10 µM AUX + 10 µM GA3 showed the most noteworthy levels of vitamin C, fruit weight, and fruit thickness. The groups that received the 10 µM GA3 and 20 µM AUX + 10 µM GA3 treatments exhibited the most favorable fruit flavor. According to the research results, the implementation of hormonal treatments 10 µM AUX and 10 µM AUX + 10 µM GA3 in combination with a 30% pruning strategy resulted in the most advantageous yield of bell peppers.

Exploring somatic embryogenesis in Ajuga multiflora Bunge: Profiling lipophilic metabolites via HPLC, GC-FID, and GCMS– analysis

The present study aims to assess the effect of different concentrations of auxins and combinations of plant growth regulators (PGRs), specifically auxin and cytokinin, on somatic embryogenesis in Ajuga multiflora. We also aim to compare the lipophilic metabolite profiles, such as carotenoids (CAs), fatty acids (FAs), phytosterols (PSs), and tocopherols (TPs), in globular somatic embryos (SEs), germinated SEs, and shoots of SEs derived plantlets. This assessment is carried out utilizing high performance liquid chromatography, gas chromatography-flame-ionization detection, and gas chromatography–mass spectrometry techniques. The results reveal that the application of 2,4-dichlorophenoxyacetic acid (2,4-D) had the most significant impact on somatic embryogenesis, inducing both SE initiation (34.4%) and the number of SEs (3.5) per explant. Among the explants used, leaf explants were found to be the most effective for SE induction (SEI). Additionally, the study explores the combined effect of 2,4-D and cytokinins (N6-benzyladenine, N6-BA, or 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, TDZ) on somatic embryogenesis, highlighting optimal conditions for SEI percentages and the number of SEs per explant. The maximum SE induction rate (100%) and the number of SEs (45.9) per leaf explant were obtained on an SEI medium with 30 µM of 2,4-D and 2.3 µM of TDZ. The maturation and conversion of SEs were also examined, emphasizing the role of GA3 in achieving the highest conversion percentage (94.0 ± 2.7 at 0.8 µM GA3). Furthermore, the study details the biochemical composition during various stages of somatic embryogenesis, including CAs, TPs, PSs, and FAs. Globular SEs displayed comparatively lower concentrations of CAs than their germinated counterparts and shoots from SE-derived plantlets, while the total PSs content remained relatively consistent across all stages. Conversely, fatty acids tended to accumulate more in globular SEs, with the exception of α-Linolenic acid. These findings provide valuable insights into the optimization of somatic embryogenesis protocols in A. multiflora, with implications for plant tissue culture, regeneration and lipophilic metabolites production.

Influence of FeSO4.7H2O, Indole-3-Butyric Acid and Different Nutrient Medium on In Vitro Sapling Propagation and Micrografting of Walnut (Juglans Regia L.)

Walnuts are considered a functional food and play a significant role worldwide in people's regular diets. The cultivation and trade of walnut rootstocks and saplings are crucial agricultural activities globally and in Türkiye. Rootstocks are used for grafting and propagating different walnut varieties, and they are essential for tree development, fruit yield, product quantity, and most importantly, their ability to adapt to various ecological conditions and resist diseases and pests. Fruit growing and production of fruit rootstocks of deep-rooted plants in horticulture is given high importance in many countries. However, poor rooting and slow-growing sapling prevent establishing high-yield clonal production. Especially walnut sapling propagation is more complicated and time-consuming than other woody plants. Our research aims to grow high-quality saplings by using biotechnological techniques. This study used modifications to the current in vitro basal medium and evaluated the effects on in vitro rooting and micrografting of walnut species. The optimal medium for shoots induction (2.93±0.90) of the plant was determined as 4 number medium (Murashige and Skoog (MS) nutrient medium with 5.4 µM NAA + 6.9 µM TDZ + 0.6 µM GA3 and solidified with 0.7% agar). In trials to develop sapling, the 10.33 and 6 rooted (2.5cm root length) shoots were obtained from MS nutrient medium with 14.7 and 19.6 µM of IBA and 348.42 µM of FeSO4. 7H2O, and two micrografted plants in MS medium with 19.6 µM of IBA and 348.42 µM of FeSO4.7H2O survived. After three months in the growth chamber, six saplings were ready for transfer to the field. Our findings suggested that FeSO4.7H2O and high dose IBA treatment in MS medium is efficient for in vitro rooting and obtaining in vitro micrografted saplings.

Role of calcium signaling in cadmium stress mitigation by indol-3-acetic acid and gibberellin in chickpea seedlings.

Given the adverse impacts of heavy metals on plant development and physiological processes, the present research investigated the protective role of indole-3-acetic acid (IAA) and gibberellic acid (GA3) against cadmium (Cd)-induced injury in chickpea seedlings. Therefore, seeds germinated for 6 days in a medium containing 200 μM Cd alone or combined with 10 μM GA3 or 10 μM IAA. Both GA3 and IAA mitigated Cd-imposed growth delays in roots and shoots (80% and 50% increase in root and shoot length, respectively). This beneficial effect was accompanied by a significant reduction in Cd2+ accumulation in both roots (74% for IAA and 38% for GA3) and shoots (68% and 35%, respectively). Furthermore, these phytohormones restored the cellular redox state by reducing the activity of NADPH oxidase and downregulating the transcription level of RbohF and RbohD genes. Likewise, hydrogen peroxide contents were reduced by GA3 and IAA supply. Additionally, GA3 and IAA countered the Cd-induced reduction in total phenols, flavonoids, and reducing sugars in both roots and shoots. The exogenous effectors enhanced the activities of catalase, ascorbate peroxidase, and thioredoxin, as well as the corresponding gene expressions. Interestingly, adding GA3 and IAA to the Cd-contaminated germination media corrected the level of calcium (Ca2+) ion within seedling tissues. This effect coincided with the upregulation of key genes associated with stress sensing and signal transduction, including auxin-binding protein (ABP19a), mitogen-activated protein kinase (MAPK2), calcium-dependent protein kinase (CDPK1), and calmodulin (CaM). Overall, the current results suggest that GA3 and IAA sustain the Ca2+ signaling pathway, resulting in metal phytotoxicity relief. Amendment of agricultural soils contaminated with heavy metals with GA3 or IAA could represent an effective practice to improve crop yield.

Ex vitro rooting of micro-propagated intergeneric papaya through phloroglucinol

Micropropagation techniques of papaya have been optimized Worldwide. However, standard rapid multiplication technique of papaya at commercial level with economically viable is still lacking. Since establishment of in vitro culture pretty easy at the same time in vitro rooting of papaya is still difficult. However, in vitro rooting is costly and the roots initiated in vitro do not have lateral branches and root hairs, making them difficult to acclimatize. Hence ex vitro rooting induction of micro-propagated plantlets and acclimatization were conducted. In vitro cultures were established using shoot tips from seedlings of selected advanced intergeneric hybrid. They were inoculated onto Murashige and Skoog (MS) medium + 3% sucrose + 1 µM BAP + 1 µM GA3 + 0.1 µM IAA + 0.25% phytagel until plantlets were obtained. For ex vitro rooting, plantlets were cultured with various concentrations of phloroglucinol (PG) to investigate the appropriate condition of rooting. Direct rooting (82%) of papaya micro-cutting into potting medium (perlite + vermiculite + coco peat; in the ratio of 1:1:2) subsequent dipping into phloroglucinol was more effective than in vitro rooting and led to amended survival percentage (82%) of micro-cutting during acclimatization. PG at 1000 µM was extra effectual than IBA in encouraging root development. Sachet method was found to be the best appropriate for ex vitro rooting and hardening of papaya with single-step acclimatization.

Gibberellins as a novel mutagen for inducing 2n gametes in plants.

The plant hormone gibberellin (GA) regulates many physiological processes, such as cell differentiation, cell elongation, seed germination, and the response to abiotic stress. Here, we found that injecting male flower buds with exogenous gibberellic acid (GA3) caused defects in meiotic cytokinesis by interfering with radial microtubule array formation resulting in meiotic restitution and 2n pollen production in Populus. A protocol for inducing 2n pollen in Populus with GA3 was established by investigating the effects of the dominant meiotic stage, GA3 concentration, and injection time. The dominant meiotic stage (F = 41.882, P < 0.001) and GA3 injection time (F = 172.466, P < 0.001) had significant effects on the frequency of induced 2n pollen. However, the GA3 concentration (F = 1.391, P = 0.253) did not have a significant effect on the frequency of induced 2n pollen. The highest frequency of GA3-induced 2n pollen (21.37%) was observed when the dominant meiotic stage of the pollen mother cells was prophase II and seven injections of 10 μM GA3 were given. Eighteen triploids were generated from GA3-induced 2n pollen. Thus, GA3 can be exploited as a novel mutagen to induce flowering plants to generate diploid male gametes. Our findings provide some new insight into the function of GAs in plants.

Regulation of dormancy break and germination of safflower seeds: the role of GA3, light and cold temperatures.

The safflower crop is considered a great alternative for crop rotation since drought tolerance and low production cost are attractive for its choice. However, safflower seeds show dormancy soon after dispersal from the mother plant, making it difficult to successfully establish plants using newly harvested seeds. The influence of temperature, gibberellin and light/dark on dormancy break of safflower seeds during storage were investigated. In a completely randomized design, freshly harvested seeds or stored for 100 and 200 days (paper bag, 20 °C/ 60% UR) were treated with GA3 (0 and 100 µM), at 4, 10 and 25 °C, in the presence and absence of light, during the germination test. Seeds were evaluated for germination percentage, germination speed and seedling survival after 21 days. The temperature of 10 °C, in combination with GA3 (0/100 µM), or light/dark, provided the highest seed germination results, for freshly-harvested seeds and stored seeds. Collectively, these observations indicate that dormancy was not affected by gibberellic acid (100 µM GA3) and the germination results at 21 days were significantly higher, in relation to the use of GA3, under light or dark. Recently harvested seeds could efficiently germinate at 10 °C in the dark, while seeds dry-stored at 20 °C had decreased germination percentages.

Overexpression of gibberellin 2-oxidase 4 from tall fescue affected plant height, tillering and drought tolerance in rice

Gibberellin (GA) is an important phytohormone that regulates plant growth and development. GA-2-oxidases (GA2oxs) have been found to affect plant height, tillering and stress tolerance by inactivating endogenous bioactive GAs. The present study aimed to isolate GA2oxs genes from tall fescue (Festuca arundinacea), analyze their expression level under abiotic stresses (10% PEG6000 induced drought stress, 150 mM NaCl induced salt stress, heat stress at 38 °C, and cold stress at 4 °C) and search for valuable genes for improving stress tolerance. Among eight FaGA2ox genes, FaGA2ox4 was found to represent a highest expression level at 24 h of PEG-induced drought stress and was chosen for further analysis. Compared with wildtype plants, transgenic rice plants overexpressing FaGA2ox4 exhibited a dwarf and high-tillering phenotype and the dwarf phenotype was partially compensated by foliar application of 10 μM GA3. In addition, transgenic plants showed improved drought stress tolerance, as manifested by higher leaf relative water content and lower electrolyte leakage than wildtype. Further qRT-PCR analysis demonstrated that the enhanced drought stress tolerance may be associated with decreased expression levels of genes involved in chlorophyll degradation (OsSGRL) and GA synthesis (OsGA3ox1) as well as increased expression of ABA-synthesizing gene (OsNCED3) in transgenic plants overexpressing FaGA2ox4.

The identification of suitable internal reference genes in quinoa seeds subjected to abscisic acid and gibberellin treatment

Abstract: Quinoa has been recognized as the sole “comprehensive nutritional crop”; however, it is susceptible to pre-harvest sprouting (PHS). While quantitative reverse transcription polymerase chain reaction (RT-qPCR) has been extensively employed for gene expression level detection, the selection of suitable reference genes is imperative to ensure precise gene expression quantification across diverse conditions. This study aims to identify stable reference genes in quinoa seeds under ABA and GA, in order to provide a basis for subsequent research on PHS. Seeds were subjected to different concentrations of ABA and GA (10 μM, 50 μM, 100 μM, and 200 μM). The most suitable treatment concentration was determined based on seed viability. Here, MON1, GAPDH, EIF3, EF1α, ACT, TUB1, and TUB6 were selected as candidate genes. The suitability of these reference genes under different conditions was assessed using various methods including Ct values, geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder. Based on the results obtained from the hormone experiments, it was observed that the application of 100 μM ABA and 200 μM GA yielded the most advantageous outcomes. Additionally, the most appropriate reference genes for different treatments are ACT and TUB1 (H2O treatment), EIF3 and MON1 (ABA, GA treatment and also for the combined data set of the three groups). However, GAPDH exhibited the least stability across all treatments. In summary, ACT is recommended as the reference gene for natural quinoa germination, while EIF3 and MON1 should be used for ABA and GA treatments.

Micropropagation of Hoya carnosa, H. kerrii, H. parasitica, and H. longifolia using tray-based floating and stationary hydroponic systems

Hoya is a wild ornamental plant with medicinal properties that have been extensively exploited from its natural habitat, causing a reduction at an alarming rate. The current study is aiming to fabricate an affordable and simple-to-use hydroponic system for the conservation and mass production of four Hoya species (H. carnosa, H. kerrii, H. parasitica, and H. longifolia). Tray-based floating and stationary hydroponic systems consist of Hoya plants (cuttings) inserted in a thermocol sheet and suspended in MS nutrient solution. Cuttings derived from mature plants were subjected to full MS (Murashige and Skoog), 1/2 MS, 1/3 MS, and 1/4 MS nutrient media for growth optimization. Subsequently, the ½ MS nutrient medium with explants was further augmented with different concentrations (0.5, 1.5, 2.5, 3.5, and 4.5 µM) of auxins like 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), indole acetic acid (IAA) and IBA (2.5 µM) optimized for rooting. A combination of 1/2 MS + IBA (2.5 μM) proved to be the optimal medium for rooting in Hoya species when compared to all other treatments.Further, improvements in root number were recorded with augmentation of 25, 50, 100, 150, and 200 µM of Zinc Sulphate (ZnSO4). Half MS nutrient solution with 2.5 µM IBA and 100 μM ZnSO4 resulted a maximum of 15 roots in H. carnosa, 20 in H. parasitica, 38 in H. kerrii, and 19 in H. longifolia. The effect of 5.0 µM GA3 (Gibberellic acid) was also evaluated on bud break in rooted plants. Foliar spray of GA3 on acclimatized plants subsequently reduced the bud break time, and the highest reduction of time was observed from 21 d to 7 d in H. parasitica. Transfer of hydroponically raised plantlets was under stress during in vitro to field conditions. Stress markers (MDA, SOD, and CAT) for defense and photosynthetic pigments for better survival of hydroponically grown plants were monitored during acclimatization. MDA content was stable at 60 to 70 d while SOD and CAT activity was stabilized and influenced upon its peak on the same days under the poly-house condition in the propagated Hoya species. Photosynthetic pigments were gradually increased, and maximum values were recorded at 70 d of acclimatization. The present study proposed a reliable protocol for the conservation and mass propagation of most of the Hoya species.

Tissue Culture and Regeneration of Three Rose Cultivars

Methods of in vitro regeneration protocols were developed for three elite rose cultivars, Chewnicebell (Oso Easy Italian Ice®), Bucbi (Carefree Beauty™), and Cheweyesup (Ringo All-Star™). We evaluated the effects of different types and concentrations of auxins [dichlorophenoxyacetic acid (2,4-D) and trichlorophenoxyacetic acid (2,4,5-T)], carbohydrates [sucrose, glucose, and fructose], and cytokinins [thidiazuron (TDZ) and 6-bezylaminopurine (BAP)] on callus induction and regeneration from leaf explants. The greatest amount of regenerative callus was obtained on media containing 10 µM 2,4-D and 30 g·L−1 sucrose for Italian Ice® (40%), 10 µM 2,4-D and 60 g·L−1 glucose for Carefree Beauty™ (24%), and 5 µM 2,4,5-T and 30 g·L−1 sucrose for Ringo All-Star™ (32%). The greatest regeneration occurred when callus was transferred to media consisting of 1/2 MS media supplemented with 2.9 µM GA3 and 5 µM TDZ for Italian Ice® and Ringo All-Star™, and with 2.9 µM GA3 and 20 µM TDZ for Carefree Beauty™. Plantlets regenerated from callus were cultured on maintenance media and successfully transferred ex vitro. This study highlights the genotype-specific responses among rose cultivars and provides the first reports of in vitro regeneration for Italian Ice® and Ringo All-Star™.

Gibberellin Increased Yield of Sesbania pea Grown under Saline Soils by Improving Antioxidant Enzyme Activities and Photosynthesis

Crop yield is the ultimate manifestation of all physiological changes of crops and external environmental influence. A controlled study was conducted to investigate the effects of exogenous gibberellin on the morphological and physiological characteristics and yield formation of sesbania pea grown in saline soils. Seeds were presoaked with four levels of Gibberellin (GA3) solutions (0, 202.1, 404.2 and 606.3 µM) for 6 h, and then manually direct-sown with a seeding rate of 45 kg ha−1. The morphological parameters (plant height, root length, dry weight), photosynthesis (chlorophyll a and b content), the activities of antioxidant enzymes (superoxide dismutase (SOD); peroxidase (POD); catalase (CAT)), the contents of soluble protein and NSC (non-structural carbohydrates), and seed yield increased with the application of exogenous gibberellin, especially at the level of 404.2 µM GA3. But GA3 had no significant effects on 1000-seed weight. Our study suggested that the appropriate application of exogenous gibberellin could improve the yield of sesbania pea grown in saline soils by increasing photosynthesis and antioxidative defense.

Assessment of somaclonal variation occurrence in micropropagated Mansonia altissimia (A. Chev.) A. Chev. using molecular marker

Plant tissue culture that offers limited space and short time for clonal propagation of plant species can sometimes be discouraging if true to type clones are not generated. This study investigated direct shoot induction and genetic stability of Mansonia altissimia using molecular markers in order to ascertain that the in vitro propagated plantlets were true to type. Nodal cutting was used as explant, and Murashige and Skoog (MS) medium was supplemented with 6-benzyl aminopurine (BAP) (µM) + naphthaleneacetic acid (NAA) (µM), BAP (µM) + Gibberellic acid (GA3) (µM), Kinetin (KIN) (µM) + NAA (µM), KIN (µM) + GA3 (µM), thidiazuron (TDZ) (µM) + NAA (µM), TDZ (µM) + GA3 (µM), for direct micropropagation. PCR amplification of wild plant and direct micropropagation of Mansonia altissimia were done using 15 RAPD and 6 ISSR primers. The highest shoot height (4.77 cm) and number of leaves (5.33) were obtained from 2.5 µM BAP + 3.0 µM GA3. Moreover, the polymerase chain reaction amplification results showed 100% monomorphic and 1.0 level of similarity coefficients, which indicates that there is no occurrence of somaclonal variation.

Cattleya tigrina (Orchidaceae) in vitro regeneration: Main factors for optimal protocorm-like body induction and multiplication, plantlet regeneration, and cytogenetic stability

The induction and subsequent regeneration of protocorm-like bodies (PLB) is the most common morphogenetic pathway for orchid micropropagation. The regeneration of plants from PLB can be a bottleneck for some species due to asynchronicity. Genetic fidelity is another issue, and the cytogenetic stability of regenerated plantlets deserves special attention due to the common occurrence of endopolyploidy in orchids. In the present work, we defined optimal conditions for PLB induction and plant regeneration for Cattleya tigrina, a threatened orchid from the Brazilian Atlantic Rain Forest. We estimated the optimum thidiazuron (TDZ) concentration for PLB induction from leaf explants, while also comparing whole leaves vs thin cell layer (TCL) as explants. For plantlet regeneration from PLB clusters, we investigated the effects of different concentrations of GA3 and sucrose, as well as the dehydration of PLBs and the use of temporary vs continuous immersion systems. Furthermore, the cytogenetic stability of regenerated plantlets was analyzed using flow cytometry, and the differences in stomatal morphology were studied. Exogenous TDZ (25 µM) was the most effective concentration for PLB induction, with 17.5% induction rate. Leaf basal regions, either in whole leaves or in TCL, were the most responsive for PLB induction. The dehydration of PLBs for 120 min increased the fresh weight increment (FWI) in plantlet regeneration, but at the expense of decreased shoot number and increased mortality. Temporary-immersion bioreactors were found to be the most suitable system for PLB multiplication compared to continuous immersion system. Shoot development was challenging due to previously acquired habituation to TDZ. Sucrose and GA3 showed an interactive effect on shoot growth. GA3 improved shoot formation, and maximum shoot production was estimated to occur at 10.3 and 9.7 µM GA3 on media containing 1.5% and 2% sucrose, respectively, resulting in larger number of developed plantlets (∼220) per culture flask. The flow cytometry analysis showed that 26.5% of plants regenerated from PLBs had doubled ploidy levels. The ploidy alterations were reflected in changes on leaf epidermis, such as increased stomatal density and number.

Rapid and Efficient Regeneration of Populus ussuriensis Kom. from Root Explants through Direct De Novo Shoot Organogenesis

Populus ussuriensis is an important tree species with high economic and ecologic values. However, traditional sexual propagation is time-consuming and inefficient, challenging afforestation and wood production using P. ussuriensis, and requires a rapid and efficient regeneration system. The present study established a rapid, efficient, and stable shoot regeneration method from root explants in P. ussuriensis using several plant growth regulators. Most shoot buds (15.2 per explant) were induced at high efficiency under WPM medium supplemented with 221.98 μM 6-BA, 147.61 μM IBA, and 4.54 μM TDZ within two weeks. The shoot buds were further multiplicated and elongated under WPM medium supplemented with 221.98 μM 6-BA, 147.61 μM IBA, and 57.74 μM GA3 for four weeks. The average number and efficiency of elongation of multiplication and elongation for induced shoot buds were 75.2 and 78%, respectively. All the shoots were rooted within a week and none of them showed abnormality in rooting. The time spent for the entire regeneration of this direct shoot organogenesis was seven weeks, much shorter than conventional indirect organogenesis with the callus induction phase, and no abnormal growth was observed. This novel regeneration system will not only promote the massive propagation, but also accelerate the genetic engineering studies for trait improvement of P. ussuriensis species.

In Vitro Cultures and Volatile Organic Compound Production in Chiliadenus montanus (Vhal.) Brullo.

Callus and microshoot cultures were established for Chiliadenus montanus (Vhal.) Brullo. (Asteraceae), a medicinal plant known for producing volatile organic compounds (VOCs). Callus induction was achieved successfully by culturing leaf explants on full-strength Murashige and Skoog medium (MS) supplemented with 2.2 µM 2, 4-dichlorophenoxy acetic acid (2,4-D) and 6.9 µM kinetin (Kin). Successful direct shoot regeneration was achieved using nodal explants cultured onto half-strength MS media supplemented with 1.4 μM Gibberellic Acid (GA3) and 4.4 μM 6-Benzylaminopurine (BAP). Indirect microshoots were successfully regenerated using callus cultured on MS media supplemented with 8.8 μM BAP, 2.2 μM Zeatin, and 1.4 μM GA3 followed by culturing on MS media supplemented with 8.8 μM BAP and 0.5 μM naphthalene acetic acid (NAA). Using wild plant aerial parts, callus and microshoots samples, VOCs were extracted successfully using Headspace Solid-Phase Micro-Extraction (HS-SPME) and analyzed by gas chromatography–mass spectrometry (GC-MS). In wild plant extracts, sesquiterpene hydrocarbons were found to be predominant with the following principal components: Alloaromadendrene (11.92%), trans-Cadina-1(6),4-diene (7.54%), and α-caryophyllene (6.77%). The analysis of in vitro microshoots revealed high levels of oxygenated monoterpenes with cis-Myrtanol (16.62%), and β-Cyclocitral (14.3%) as the main components. Callus extract was dominated by monoterpene hydrocarbons and the main compounds identified were (Z)-β-Ocimene (22.27%), p-Cymene (15.13%), and α-pinene (13.78%). In conclusion, an efficient in vitro production system of VOCs in C. montanus was established that can be used in the future for boosting their production without endangering wild plants.

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill.

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP + 1.4 µM GA3 enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11240-022-02254-z.