Shoot Induction Medium Research Articles

Efficient Plantlet Regeneration from Branches in Mangifera indica L.

Mango (Mangifera indica L.) is one of the most significant tropical and subtropical fruit species, with high ecological and economic value. However, research on the in vitro culture of mangoes is relatively weak, so establishing an efficient and stable mango plant regeneration system is of great significance. In this study, a preliminary mango regeneration system was established with Mangifera indica L. cv. Keitt from young branches as the starting explants. The results showed that the optimal plant growth regulator (PGR) formula for direct adventitious shoot induction on the branches was 1 mg/L 6-benzylaminopurine (6-BA) + 0.1 mg/L a-naphthaleneacetic acid (NAA), with an adventitious shoot induction rate of 73.63% and an average of 6.76 adventitious shoots. The optimal basal medium for adventitious shoot induction was wood plant medium (WPM), with an adventitious shoot induction rate of 63.87% and an average of 5.21 adventitious shoots. The optimal culture medium for adventitious shoot elongation was WPM + 1 mg/L 6-BA + 0.5 mg/L NAA, with an adventitious shoot elongation rate of 89.33% and an average length of 5.17 cm. The optimal formula for the induction of mango rooting was Douglas fir cotyledon revised medium (DCR) + 3 mg/L indole-3-butyric acid (IBA), with a maximum rooting rate of 66.13% and an average rooting quantity of 6.43. The genetic fidelity of the in vitro-regenerated plants was evaluated using inter-simple sequence repeat (ISSR) molecular markers. There was no difference between the in vitro-regenerated plants and the parent plant. This study provides an efficient and stable propagation system for Mangifera indica L., laying the foundation for its rapid propagation and genetic improvement.

In vitro mass propagation of Dendrocalamus asper (Giant bamboo) through direct organogenesis

Dendrocalamus asper (giant bamboo) is a clumping type of bamboo belonging to the Poaceae family. Due to its economic and environmental value, demand for this species has increased tremendously. Conventional propagation methods have limitations due to low seed viability and the lack of healthy clumps. Therefore, an in vitro mass propagation protocol was developed to provide healthy plants for large-scale plantations. Seeds were used as the explant and they were surface sterilized and cultured on MS medium free of growth regulators. Nodal segments taken from in vitro germinated seedlings were used for shoot initiation. The best medium for shoot induction (MS medium supplemented with 0.0–2.5 mg/L BAP), best medium for multiple shoot induction (MS medium supplemented with 0.0–5.0 mg/L BAP), effect of shoot cluster size (shoot clusters containing 1–4 shoots) and effect of physical state of the medium (semisolid and liquid media) on multiple shoot induction were determined using shoots per node, mean shoot length and mean number of leaves per shoot after 6 weeks of incubation. Elongated shoots were transferred into a rooting medium and the best medium for root induction (MS medium supplemented with 0.0–5.0 mg/L IBA and IAA) and effect of cluster size (shoot clusters containing 1–4 shoots) on rooting were determined using the number of roots and root length after 6 weeks of incubation. All the cultures were maintained under a 16-hour photoperiod. Well-developed plantlets were transferred to coir pellets and after four weeks transferred into different potting mixtures containing different combinations of sand, compost and coir dust. Unless otherwise mentioned there were at least twenty replicates in all treatments. The highest mean number of shoots per node (16.87±0.52), mean shoot length (4.12±0.27 cm) and mean number of leaves per shoot (4.80±0.33) were observed in the MS medium supplemented with 1.0 mg/L BAP. The MS medium supplemented with 2.0 mg/L BAP was the best for multiple shoot induction, shoot cluster with 3 shoots was the best cluster size and the liquid medium had a better effect on shoot multiplication. The half-strength MS medium supplemented with 2.0 mg/L IBA was the best for in vitro root induction with the highest mean number of roots (7.15±0.77) and a mean root length of 10.79±1.11 cm. Shoot clusters with 3 shoots was the best cluster size for root induction. A sand: compost: coir dust (1:1:1) mixture was the best potting mixture giving 100% survival. These findings provide a reliable micropropagation protocol for D. asper, which holds great promise for meeting the growing demand for bamboo resources and promoting sustainable bamboo cultivation.

Optimizing callus induction and indirect organogenesis in non-dormant corm explants of Gloriosa superba (L.) via media priming

IntroductionGloriosa superba L. is an endangered ornamental plant of significant medicinal, commercial, and cultural value. This study establishes an efficient protocol for rapid in vitro propagation of this plant species through callus-mediated organogenesis using non-dormant corm explants.MethodsPlant tissue culture techniques were employed to facilitate the in vitro regeneration process of Gloriosa superba L. This encompassed various stages, including the acquisition of plant material, surface sterilization, formulation of growth media, and the execution of callogenesis, shooting, rooting, and acclimatization experiments.Results and DiscussionInvestigation into the impact of plant growth regulators on callogenesis unveiled diverse callus morphologies, dependent on regulator type and concentration, with the NAA and KN combination emerging as the most effective for callus induction. Specifically, the callus induction medium supplemented with 1.5 mg L−1 NAA, 0.5 mg L−1 KN, and 10 mg L−1 casein hydrolysate (CH) achieved remarkable results, boasting an 81.25% callus induction rate and a substantial callus biomass fresh weight of 333 mg. Furthermore, the combination of BAP and NAA facilitated optimal shoot primordia induction and shooting in callus derived from non-dormant corm explants. The shoot induction medium, enriched with 2.0 mg L−1 BAP, 0.5 mg L−1 NAA, 5 mg L−1 CH, and 20% (v/v) coconut water (CW), resulted in an impressive 83.33% shoot primordia formation rate and an average of 6.86 shoot primordia per callus. For root induction, the medium supplemented with 1.0 mg L−1 IBA displayed a high root induction rate of 81.25% and robust rooting (+++). Successful acclimatization of in vitro plantlets in controlled environments and shade net houses culminated in thriving growth, and upon transplantation into a garden soil blend (garden soil : sand : vermiculite, 2:1:1, v/v), with direct sunlight exposure, the plantlets demonstrated commendable development and form, boasting a 69% survival rate at the seven-week mark. These findings offer a robust foundation for scalable and sustainable propagation strategies, ensuring the conservation and utilization of this valuable species.

Development of transgenic Brassica napus plants with AtATM3 gene to enhance cadmium and lead tolerance

Four popular genotypes of Brassica napus, cv. BARI Sarisha-8, BARI Sarisha-13, Binasarisha-4, and Binasarisha-8 were subjected to cadmium (Cd) and lead (Pb) stress to identify a genotype with enhanced tolerance of Cd and Pb. The goal was to use the selected genotype for genetic engineering, thereby improving traits related to heavy metal tolerance and accumulation to remediate polluted agricultural soils. The genotype, BARI Sarisha-8 with superior tolerance to both Cd and Pb stresses, was chosen for genetic engineering with Arabidopsis thaliana ABC transporter of the mitochondrion 3 (AtATM3) gene. The transformation process involved coculturing the cotyledon explants with Agrobacterium strain GV3101 carrying a binary vector containing the hygromycin phosphotransferase (HPT) gene as a selectable marker and the AtATM3 gene. Transformation efficiency was significantly enhanced with a two-day co-cultivation period on shoot induction medium composed of MS medium, α-naphthaleneacetic acid (0.5 mg/L), and 6-benzyladenine (3 mg/L), supplemented with acetosyringone (20 mg/L), along with a four-day delay in exposing the explants to the selective agent hygromycin. Hygromycin-resistant shoots were obtained by employing a three-step selection process. The refined protocol resulted in a transformation efficiency of 15.38%. Polymerase chain reaction (PCR) analysis confirmed the integration of AtATM3 and HPT genes into the host genome in all recombinant plants. Transgenic B. napus plants expressing the AtATM3 gene exhibited notable improvements in tolerance, demonstrating a 1.4- to 1.7fold increase in Cd tolerance and a 1.3- to 1.5-fold increase in Pb tolerance compared to the wild type (nontransformed) under both Cd and Pb stress conditions.

Plant Regeneration via Organogenesis in Jerusalem Artichokes and Comparative Analysis of Endogenous Hormones and Antioxidant Enzymes in Typical and Atypical Shoots.

The Jerusalem artichoke (Helianthus tuberosus) is a tuberous plant with considerable nutrient and bioactive compounds. The optimization of the in vitro clonal propagation protocol is critical for large-scale reproduction and biotechnological applications of Jerusalem artichoke production. In this work, in vitro plant regeneration from the stem nodes of the Jerusalem artichoke via direct organogenesis is presented. In the shoot induction stage, the stem segments produced more shoots with vigorous growth on MS medium containing 0.5 mg/L 6-benzylaminopurine (6-BA). The concentrations of 6-BA and gibberellic acid (GA3) were both optimized at 0.5 mg/L for shoot multiplication, and the combination of 0.05 mg/L indole-3-butyric acid (IBA) and 0.05 mg/L 1-naphthylacetic acid (NAA) was the most responsive for root induction, yielding the largest number of roots. The regenerated plantlets were successfully hardened at a 96% survival rate and vigorously grew in the field. The genetic stability of the regenerated plants was confirmed by flow cytometry and simple sequence repeat (SSR) analysis. However, 17.3% of shoots on the optimum shoot induction medium had withered leaves and excessive callus (atypical shoots), which greatly reduced the induction efficiency. Enzyme activity in the typical and atypical shoots was compared. The atypical shoots had significantly higher levels of endogenous indole-3-acetic acid (IAA) and abscisic acid (ABA), as well as increased activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), whereas the content of 6-BA, zeatin (ZT), and GA3 was significantly reduced. The activity of the three enzymes was positively correlated with the content of IAA and ABA, while being negatively correlated with that of 6-BA, ZT, and GA3. The results suggest that the poor growth of the atypical shoots might be closely related to the significant accumulation of endogenous IAA and ABA, thus significantly increasing antioxidant enzyme activity.

Influence of High Concentrations of Copper Sulfate on In Vitro Adventitious Organogenesis of Cucumis sativus L.

Copper (Cu) is an essential plant micronutrient. This report is the first to assess the effects of high copper sulfate (CuSO4) levels on in vitro callus and shoot formation of cucumber. Four-day-old cotyledon explants from the inbred line Wisconsin 2843 and the commercial cultivars Marketer and Negrito were used. Murashige and Skoog (MS)-derived callus and shoot induction medium containing 0.5 mg L−1 indole-3-acetic acid (IAA) and 2.5 mg L−1 6-benzylaminopurine (BAP) was supplemented with CuSO4 (0.2–5 mg L−1). The response on callus-derived shoots showed that the optimal concentration of CuSO4 was 8- to 200-fold greater than in standard MS medium. Shoot frequency (SF) and shoot number (SN) were assessed, and Marketer > Negrito > Wisconsin 2843, where 1, 0.2 and 5 mg L−1 CuSO4 produced the highest results, respectively. SF and SN increased 6- and 10-fold in Wisconsin 2843 and twice in the other cultivars. All explants formed calluses, and in two of the three cultivars, callus extension was significantly affected by CuSO4 application. SN showed a strong relationship with CuSO4 levels and no association with callus extension. The results show that specific CuSO4 concentrations higher than in standard MS medium increase adventitious cucumber shoot organogenesis.

An improved in-vitro regeneration protocol using scutellum of mature and immature embryos of wheat

Generally, conventional practices take a long time to improve agronomic traits in common wheat (Triticum aestivum L.). However, biotechnological tools provide the best opportunity to enhance crop potential within a limited time. A simple and repeatable protocol for wheat regeneration is the priority for transforming the genetic makeup of elite wheat cultivars. Among 10 types of callus induction media (CIM), CIM4 with supplemented Murashige and Skoog (MS) media+ dicamba (2 mg/l) has 98.22 and 97.33% callus induction efficiency (CIE) in immature and mature scutellum, respectively. Precocious germination, considered a key barrier during callus induction, was fully controlled by excising the embryonic axis. Among 29 different shoot induction media (SIM) assessed in the study, SIM 29 containing zeatin 5 mg/l, 6-benzylaminopurine (BAP) 5 mg/l, 2,4-dichlorophenoxyacetic acid (2,4-D) 0.25 mg/l,1-naphthaleneacetic acid (NAA) 0.25 mg/l, and copper sulfate (CuSO4) 20 mg/l was found most responsive for shoot induction. The combined effect of cytokinin and CuSO4 resulted in an improved wheat regeneration response in two popular Indian wheat varieties, namely HD2967 and HD3171. Rooting was observed at 100% using IAA and IBA @ 0.2 mg/l concentration in root induction media (RIM). The whole in-vitro regeneration protocol from embryo to transfer in the greenhouse took 8 to 9 weeks. The in-vitro regeneration protocol developed in the present study could further be utilized to improve the genetic makeup of elite wheat cultivars utilizing genetic engineering tools.

Plant Regeneration in Amorphophallus muelleri Blume. through Organogenic

Porang (Amorphophallus muelleri Blume.) usually used to functional food raw materials, caused its high glucomant content. Limitation of conventional propagation both vegetative and generative affected to decreasing of porang production to fulfil market demand. To solve the problem, plant tissue culture technique was chosen to rapid propagate bulbils of porang. So, the aim of this research to determine the combination and concentration of Plant growth Regulators (PGRs), such as 6-Benzylaminopurine (BAP), Naphthaleneacetic Acid (NAA), and Thidiazuron (TDZ) for quality of callus, also growth of callus and shoots for rapid propagation of porang, cause PGRs are one of factors that affect the success of in vitro propagation. Experimental design of this research was completely randomized designed with statistical analysis using analysis of variance (Anova) one way. Difference combination and concentration of cytokinin and auxin as manipulation variables, which several parameters growth of callus, shoots, and roots was observed to indicate the rapid organogenic of porang. The optimal shoot induction medium was Murashige and Skoog (MS) media with 5.0 mg.L-1 BAP and 0.2 mg.L-1 NAA on which number of shoots and shoot height reached 15±1.8 and (7.2±1.86) cm. After shoot formation stage, the proper media for rooting stage was determined, which was Murashige and Skoog (MS) media with 1.0 mg.L-1 NAA resulted the rooting rate reached 100% and roots appear earlier. Optimal combination and concentration of PGRs on micropropagation of Amorphophallus muelleri Blume. from bulbils explant should be known to increase the production of porang, which can fulfil the market demand.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain.

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the β-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

In Vitro Propagation and Histochemical Analysis of Launaea sarmentosa (Willd.) Kuntze

Launaea sarmentosa (Willd.) Kuntze., an edible perennial herb, is critically considered an endangered species due to its habitat destruction. In vitro L. sarmentosa propagation was therefore conducted to preserve this plant. Two types of explants including leaf and stolon were applied to induce the multiple shoots by culturing on Murashige and Skoog (MS) medium supplemented with BA (0, 1, and 2 mg/L) and 2,4-D (0, 1, 2, and 4 mg/L). The optimal shoot induction medium for both explants was MS medium containing 2 mg/l BA (presented 100 % shoot induction). Roots were induced by various types and concentrations of auxins [1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA)]. The highest percentage of rooting (70.00 ± 11.05 %) was obtained from stolon-derived shoots on MS medium supplemented with 0.1 mg/l IAA. One-hundred percent survivability was presented after transforming plantlets into a mixture of soil, sand, and peat moss (1:1:1). Starch, protein, and lipid were accumulated in leaves detected by histochemical technique. The obtained information will be a useful tool for mass propagation of L. sarmentosa and to obtain basic knowledge for plant accumulating substances. HIGHLIGHTS This paper highlights 2 types of sarmentosa explant including leaves and stolons have great potential to produce multiple shoots after inoculation in MS medium adding BA. All regenerated plants get success for acclimatization and transplantation to the greenhouse. In vitro plantlets of L. sarmentosa leaves accumulated fat, protein and carbohydrate which might be related with precursor of medicinal properties GRAPHICAL ABSTRACT

Influence of sodium salicylate on adventitious organogenesis of a commercial cucumber cultivar

Cucumber is an important vegetable crop belonging to the Cucurbitaceae family. An optimized regeneration system is required to address different biotechnological approaches in this species, such as large-scale multiplication, as well as genome editing and genetic transformation techniques. The aim of this study was to investigate the effect of different concentrations of the phenolic compound sodium salicylate (NaSA), a salicylic acid derivative, on in vitro callus and shoot regeneration of a commercial cultivar of cucumber. Four-day-old cotyledon explants from the slicing cultivar 'Marketer' were employed. Murashige and Skoog (MS)-derived shoot induction medium containing 0.5 mg L-1 indole-3-acetic acid (IAA) and 2.5 mg L-1 6-benzylaminopurine (BAP) was supplemented with NaSA. Frequency and shoot number were enhanced by 1.5-fold with NaSA at 0.1 µM. Higher salicylate levels led to increased callus formation and decreased shoot regeneration. The application of sodium salicylate at a specific concentration showed a positive trend in in vitro adventitious organogenesis of a commercial cucumber cultivar. Some probable mechanisms that may underlie the beneficial effects of salicylic acid/salicylates on in vitro regeneration were also discussed.

The Effect of Colchicine on Profiles and Contents of Withanolides of Ciplukan (Physalis angulata) In vitro

This study aimed to determine the effect of in vitro polyploid induction using colchicine on the content of withanolides in Physalis angulata shoots. Polyploidy induction used two types of tissue, namely seeds from in vivo ripe fruit and in vitro cotyledonary nodes. Each tissue type was soaked in 0.1% colchicine for 0, 24, 48 and 72 hours. Seeds that had been soaked in colchicine were germinated in vitro and the cotyledonary nodes of seedling were used as explants for shoot induction. Meanwhile, the cotyledonary nodes that had been soaked in colchicine were immediately cultured on shoot induction medium. Media for shoot induction was MS + BAP 2 mg/L + IAA 0.05 mg/L. The shoots derived from multiplication were used for the analysis of withanolides content using the HPLC method. The results showed that a total of 38 withanolides compounds were detected in the retention time 21.6 to 36.8 minutes. Soaked the seeds in colchicine solution was more effective in increasing the withanolides content than the cotyledonary node tissues. All exposure time in colchicine only affected the intensity but had no effect on the profile of the withanolides.

In vitro propagation of Ocimum sanctum L., Ocimum canum Sims., and Ocimum tenuiflorum L., and evaluation of antioxidant, MMP-9 down regulation of eugenol and camphor

• Eugenol and camphor could be suitable candidates for preventing diseases caused by oxidative stress and free radicals. • Eugenol and camphor are effective MMP-9 inhibitors under in vitro . • Cytokinins are most suitable for micropropagation. Medicinal plants have long been used as a source of drugs. Ocimum species contain a diverse range of distillate that are abundant in phenolics, as well as a variety of other organic ingredients such as phytoconstituents. The present study was undertaken to standardize the medium for shoot and callus induction using nodal and leaf explants of Ocimum sanctum, Ocimum canum and Ocimum tenuiflorum and to perform RAPD analysis for identification of somaclonal variants. To evaluate the free radical scavenging activity of eugenol and camphor and to test MMP-9 inhibition of eugenol and camphor using gelatin zymography. Zeatin (6.8 µM) was observed to be suitable for the induction of shoots among the cytokinins evaluated in Ocimum sanctum and Ocimum canum . BA (6.6 µM) was shown to be more effective for shoot induction in Ocimum tenuiflorum . Supplemented B5 medium with 2,4-D (6.7µM and 4.5 µM) and B5 medium supplemented with NAA (8.5 µM) were reported to be favorable for callus induction in Ocimum sanctum and Ocimum canum respectively. The polymorphism was discovered in in vitro produced plants of Ocimum sanctum, Ocimum canum , and Ocimum tenuiflorum using the primers OPK4 and OPK10. Antioxidant activity of eugenol and camphor has been evaluated using various in vitro antioxidant assays. This study clearly indicates that eugenol is a potent free radical scavenging agent when compared with camphor. Eugenol and camphor were used to alleviate inflammation caused by lipopolysaccharide-induced overexpression of matrix metalloproteinase-9. Both eugenol and camphor are effective MMP-9 inhibitors in vitro , according to the results of this investigation. RT-PCR analysis clearly indicates the down regulation of over expressed MMP-9.

Ectopic expression of WOX5 promotes cytokinin signaling and de novo shoot regeneration.

WOX5 has a potential in activating cytokinin signaling and shoot regeneration, in addition to its role in pluripotency acquisition. Thus, overexpression of WOX5 maximizes plant regeneration capacity during tissue culture. In vitro plant regeneration involves two steps: callus formation and de novo shoot organogenesis. The WUSCHEL-RELATED HOMEOBOX 5 (WOX5) homeodomain transcription factor is known to be mainly expressed during incubation on callus-inducing medium (CIM) and involved in pluripotency acquisition in callus, but whether WOX5 also affects de novo shoot regeneration on cytokinin-rich shoot-inducing medium (SIM) remains unknown. Based on the recent finding that WOX5 promotes cytokinin signaling, we hypothesized that ectopic expression of WOX5 beyond CIM would further enhance overall plant regeneration capacity, because intense cytokinin signaling is particularly required for shoot regeneration on SIM. Here, we found that overexpression of the WOX5 gene on SIM drastically promoted de novo shoot regeneration from callus with the repression of type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, negative regulators of cytokinin signaling. The enhanced shoot regeneration phenotypes were indeed dependent on cytokinin signaling, which were partially suppressed in the progeny derived from crossing WOX5-overexpressing plants with cytokinin-insensitive 35S:ARR7 plants. The function of WOX5 in enhancing cytokinin-dependent shoot regeneration is evolutionarily conserved, as conditional overexpression of OsWOX5 on SIM profoundly enhanced shoot regeneration in rice callus. Overall, our results provide the technical advance that maximizes in vitro plant regeneration by constitutively expressing WOX5, which unequivocally promotes both callus pluripotency and de novo shoot regeneration.

Agrobacterium-mediated genetic transformation of the most widely cultivated superior clone Eucalyptus urophylla × E. grandis DH32-29 in Southern China.

Eucalyptus, as an economically important species for wood and paper industries, remains a challenge to genetic improvement by transgenic technology owing to the deficiency of a highly efficient and stable genetic transformation system, especially in cultivated superior clones. Eucalyptus urophylla × E. grandis clone DH32-29 is most widely planted in southern China, but it is relatively recalcitrant to adventitious bud regeneration, which blocks the establishment of a genetic transformation system. Here, an efficient adventitious bud regeneration and transformation system of Eucalyptus was established using E. urophylla × E. grandis DH32-29 as material. The in vitro leaves from microshoots that were subcultured for 20-25 days were immersed into liquid Woody Plant Medium supplemented with 0.02 mg·L-1 α-naphthaleneacetic acid (NAA) and 0.24 mg·L-1 forchlorfenuron [callus-inducing medium (CIM)]. After 15 days, explants were transferred to a medium containing 0.10 mg·L-1 NAA and 0.50 mg·L-1 6-benzyladenine (shoot-inducing medium, SIM) for adventitious bud induction. The highest regeneration efficiency of adventitious buds was 76.5%. Moreover, an Agrobacterium tumefaciens-mediated genetic transformation system was optimized. The leaves were precultured for 7 days and infected for 30 min with A. tumefaciens strain EHA105 grown to a bacterial density of 0.3 (OD600). After 72 h of cocultivation in the dark, leaves were transferred to CIM supplemented with 100 mg·L-1 cefotaxime (Cef), 100 mg·L-1 timentin, and 15 mg·L-1 kanamycin (Kan) for 15 days to induce calluses. Then, the explants were transferred to SIM supplemented with the same concentration of antibiotics, and the fresh medium was replaced every 15 days until resistant adventitious buds appeared. After inducing roots in root-inducing medium supplemented with 200 mg·L-1 Cef and 75 mg·L-1 Kan, completely transgenic plants were obtained. Using the aforementioned method, the transformation frequency can reach 1.9%. This provides a powerful approach for genetic improvement of E. urophylla × E. grandis DH32-29 and gene function analysis in Eucalyptus.

Histidine kinase inhibitors impair shoot regeneration in Arabidopsis thaliana via cytokinin signaling and SAM patterning determinants.

Reversible protein phosphorylation is a post-translational modification involved in virtually all plant processes, as it mediates protein activity and signal transduction. Here, we probe dynamic protein phosphorylation during de novo shoot organogenesis in Arabidopsis thaliana. We find that application of three kinase inhibitors in various time intervals has different effects on root explants. Short exposures to the putative histidine (His) kinase inhibitor TCSA during the initial days on shoot induction medium (SIM) are detrimental for regeneration in seven natural accessions. Investigation of cytokinin signaling mutants, as well as reporter lines for hormone responses and shoot markers, suggests that TCSA impedes cytokinin signal transduction via AHK3, AHK4, AHP3, and AHP5. A mass spectrometry-based phosphoproteome analysis further reveals profound deregulation of Ser/Thr/Tyr phosphoproteins regulating protein modification, transcription, vesicle trafficking, organ morphogenesis, and cation transport. Among TCSA-responsive factors are prior candidates with a role in shoot apical meristem patterning, such as AGO1, BAM1, PLL5, FIP37, TOP1ALPHA, and RBR1, as well as proteins involved in polar auxin transport (e.g., PIN1) and brassinosteroid signaling (e.g., BIN2). Putative novel regeneration determinants regulated by TCSA include RD2, AT1G52780, PVA11, and AVT1C, while NAIP2, OPS, ARR1, QKY, and aquaporins exhibit differential phospholevels on control SIM. LC-MS/MS data are available via ProteomeXchange with identifier PXD030754.

Enhancement of shoot regeneration by treatment with inhibitors of auxin biosynthesis and transport during callus induction in tissue culture of Arabidopsis thaliana.

In two-step culture systems for efficient shoot regeneration, explants are first cultured on auxin-rich callus-inducing medium (CIM), where cells are activated to proliferate and form calli containing root-apical meristem (RAM)-type stem cells and stem cell niche, and then cultured on cytokinin-rich shoot-inducing medium (SIM), where stem cells and stem cell niche of the shoot apical meristem (SAM) are established eventually leading to shoot regeneration. In the present study, we examined the effects of inhibitors of auxin biosynthesis and polar transport in the two-step shoot regeneration culture of Arabidopsis and found that, when they were applied during CIM culture, although callus growth was repressed, shoot regeneration in the subsequent SIM culture was significantly increased. The regeneration-stimulating effect of the auxin biosynthesis inhibitor was not linked with the reduction in the endogenous indole-3-acetic acid (IAA) level. Expression of the auxin-responsive reporter indicated that auxin response was more uniform and even stronger in the explants cultured on CIM with the inhibitors than in the control explants. These results suggested that the shoot regeneration competence of calli was enhanced somehow by the perturbation of the endogenous auxin dynamics, which we discuss in terms of the transformability between RAM and SAM stem cell niches.

In vitro culture initiation and regeneration of two highly productive clones of poplar

In vitro shoot regeneration is a fast and reliable method for propagation of valuable tree genotypes and important step in genetic manipulations. This study aimed to establish an optimal in vitro culture initiation and regeneration protocol for highly productive poplar clones ‘Novoberlinska-3’ (Populus pyramidalis × P. laurifolia) and ‘Volosystoplidna’ (P. trichocarpa). In vitro culture was initiated either on MS (IM-MS) or WPM (IM-WPM) medium both containing BAP (0.2 mg.L-1) and IBA (0.1 mg.L-1). Results demonstrated that the best initiation medium for the clone ‘Volosystoplidna’ was IM-WPM on which 93.3 % of explants survived, while explants from ‘Novoberlinska-3’ better survived on IM-MS. After establishing both genotypes into aseptic culture, regeneration experiments were started using two types of explants, leaf and petioles, planted on callus induction medium containing 2iP (1.02 mg.L-1) and NAA (1.86 mg.L-1). Furthermore, microshoots were placed on shoot induction medium supplemented with 0.04 mg.L-1 thidiazuron, and then transferred on shoot elongation medium with 0.2 mg.L-1 BAP. Regeneration protocol showed better performance of poplar ‘Novoberlinska-3’, but it was also efficient for ‘Volosystoplidna’. Plant regeneration from leaf explants in the clone ‘Novoberlinska-3’ showed the highest regeneration percentage (92.3 %) and the number of shoots per explant (3.1), which significantly exceeded other explants. Our results showed a significant difference between the survivability of two clones during culture initiation. The differences in regeneration rates between the clones as well as leaf and petiole explants were also determined. Obtained aseptic cultures of highly productive poplar hybrids will be used in our further studies, especially for genetic transformation.

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and β-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.

Knockout of Tobacco Homologs of Arabidopsis Multi-Antibiotic Resistance 1 Gene Confers a Limited Resistance to Aminoglycoside Antibiotics.

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the Arabidopsis Multi-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacum MAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs–knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants’ sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.