L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis)

Successful Agrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant’s recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stable Agrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regeneration media. Four-day-old in vitro derived cotyledon explants were infected with A. tumefaciens strain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to an Agrobacterium suspension (OD600 of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO3 combination exhibited the highest β-glucuronidase (GUS) expression (94%) and explant regeneration efficiency (76%). After 3 days, the cotyledon explants were subjected to three selection cycles with gradually increasing hygromycin B concentrations (10 to 12 mg/L). The incorporation and expression of hptII in T0 transformed plants were verified by polymerase chain reaction and Southern blot analyses. These transgenic plants (T0) were fertile and morphologically normal. Using the present protocol, a successful transformation efficiency of 14% was achieved, and this protocol can be applied for genome editing and functional studies to improve Chinese cabbage traits.

Thiol Compounds, Pre-Conditioning and Orientation of Explants – Important Factors Affecting Regeneration from Cotyledons of Legume Crop Sesbania Aculeata

Sesbania aculeata is a multipurpose legume crop grown primarily for green manuring in the rice-based cropping system. Besides this, it is an industrial crop and is also used as food in many parts of the world. The present work reports for optimization of various parameters (growth medium, plant growth regulators, pre-conditioning, orientation of explant, and presence of thiol compounds) affecting in vitro regeneration using mature cotyledon explants. The 5-day-old mature cotyledon explants excised from seedlings grown on Murashige and Skoog (MS) salts and Gamborg (B5) vitamins medium containing 15 μM 6-benzylaminopurine were cultured with its adaxial side facing on medium containing 2.5 μM 6-benzylaminopurine and 50 mg/L thiourea and produced multiple shoots (7 ‒ 8) in 100% cultures within 28 days. Healthy shoots were rooted on half-strength Murashige and Skoog (MS) salts and full-strength vitamins medium augmented with 2.5 μM indole-3-butyric acid.

Screening of Suitable Plant Regeneration Protocols for Several Capsicum spp. through Direct Organogenesis

Peppers (Capsicum spp.) are recalcitrant to in vitro culture regeneration, making the application of in vitro-based breeding strategies difficult. We evaluated the impact of different combinations of auxins, cytokinins and micronutrients on the induction of direct organogenesis in cotyledon and hypocotyl explants of C. annuum, C. baccatum and C. chinense. We found variation in the regeneration response among species and type of explant. In this way, the average numbers of shoots per cotyledon and hypocotyl explant were, respectively, 1.44 and 0.28 for C. annuum, 4.17 and 3.20 for C. baccatum and 0.08 and 0.00 for C. chinense. Out of the six media, the best overall results were obtained with the medium Pep1, which contained 5 mg/L BAP (6-benzylaminopurine), 0.5 mg/L IAA (indole-3-acetic acid) and 0.47 mg/L CuSO4, followed by a subculture in the same medium supplemented with 10 mg/L AgNO3 (medium Pep1.2). The best result for the Pep1 + Pep1.2 medium was obtained for C. baccatum using cotyledon explants, with 8.87 shoots per explant. The explants grown in medium Pep1 + Pep1.2 were the ones with greener tissue, while overall the hypocotyl explants were greener than the cotyledon explants. Our results indicate that there is wide variation among Capsicum species in terms of regeneration. Our results suggest that the synergistic effect of copper and silver resulted in a higher regeneration rate of Capsicum explants. Explants with shoots were transferred to different media for elongation, rooting and acclimatization. Although acclimatized plantlets were obtained for C. baccatum and C. chinense, an improvement in these latter stages would be desirable for a high throughput regeneration pipeline. This work contributes to the improvement of Capsicum regeneration protocols using specific combinations of medium, explant and genotype, reaching the levels of efficiency required for genetic transformation and of gene editing technologies for other crops.

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

The Effect of Sodium Butyrate on Adventitious Shoot Formation Varies among the Plant Species and the Explant Types

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.

Temporal and Spatial Expression Analysis of Shoot-Regeneration Regulatory Genes during the Adventitious Shoot Formation in Hypocotyl and Cotyledon Explants of Tomato (CV. Micro-Tom)

Enhancing the competence for plant regeneration in tissue culture studies is an important issue not only for efficient genetic transformation of commercial crops but also for the reproducibility of scientific reports. In this study, we investigated optimization of several tissue culture conditions including plant growth regulators, types and ages of explants, culture densities, and plant position in order to improve the competence of adventitious shoot formation of the tomato (Solanum lycopersicum cv. Micro-Tom). In addition, we examined the differential expression of D-type cyclin (CYCD3-1) and several shoot regeneration regulatory genes from hypocotyl and cotyledon explants of tomato during shoot organogenesis. A treatment of 1 mg L−1 Zeatin and 0.1 mg L−1 Indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium containing 3% sucrose was optimal for adventitious shoot formation from hypocotyl and cotyledon explants. The younger explants exhibited more shoot formation regardless of explant types. Additionally, those closest to the shoot apical meristem produced more shoots compared to the other regions in the hypocotyl and the cotyledon explants. Gene expression of CYCD3-1, SHOOT MERISTEMLESS (STM), and cytokinin dependent WUSCHEL (WUS) was significantly higher in younger explants than in older ones. Furthermore, an increase in CYCD3-1, STM, and WUS expression was evident at the distal part of hypocotyls and the proximal part of cotyledons compared to other regions. These differential gene expression profiles exhibited good agreement with the results of shoot formation obtained from diverse explants of tomato. These results suggest that temporal and spatial gene expression of shoot regeneration regulatory genes plays an important role in enhancing the competence and the reproducibility of adventitious shoot formation from tomato explants.

In-vitro Callus Induction and Regeneration of Brinjal (Solanum melongena L.) through Cotyledon

Brinjal is one of the most popular, nutritional and vegetable crops in the world. It plays a vital role in the national economy as a cash crop. Tissue culture techniques used for in-vitro plant regeneration through cotyledon explants of eggplant (Solanum melongena L.) with different combinations of plant growth hormones BAP (4.44, 6.66, 8.88, 11.10 and 13.32µM) and IAA (0.57, 1.14 and 1.71µM) used for in-vitro regeneration of brinjal. The cotyledon explants used in this study, the highest callus induction found on BAP 8.88 µM and IAA 1.14 µM. The callus induction occurred after 15days from initiation, shoot induction occurred after 30 days from initiation and shoot elongation was carried out on the same medium, shoot elongation occurred after 45 days from initiation. MS hormone-free medium found best for root regeneration, the elongated shoots were selected and transferred to a test tube containing MS hormone-free rooting medium and the elongated shoots produce roots after 15 days. Then the rooted plantlets were transferred to poly-cup with a pre-sterilized mixture of coco peat for primary hardening under poly-tunnel for 10days. Subsequently, there generated plantlets acclimatized under the greenhouse. Then, hardened plants transferred to the open field for further development. This plant regeneration method can be useful for the production of the disease-free plant.

In vitro callus and shoot regeneration in Enterolobium cyclocarpum (Jacq.) Grised. - a fast timber yielding species

This study was conducted to investigate the in vitro callus induction and rapid shoot regeneration potential in Enterolobium cyclocarpum, a plant native to central Mexico but widely introduced into Africa. The leaf, stem and nodal explants of E. cyclocarpum were cultured on full strength Murashige and Skoog (MS) medium supplemented with different concentrations of Cytokinins - Benzyladenine (BA) and/or Kinetin and Auxins - Naphthalene acetic acid (NAA) and/or 2,4-Dichlorophenoxylacetic acid (2,4-D) each alone and in combination. The leaf explants did not respond to these treatments. The Nodal explants were best for caulogenesis, while the explant responses were in the order- nodal > stem > cotyledon for callogenesis in MS medium supplemented with BA and/or Kin combined with NAA and/or 2,4-D. The varied combinations induced white compact callus. The highest callus production was observed on MS medium supplemented with 2.7 µM NAA + 2.2 µM BA and 5.4 µM NAA alone. Nodal and cotyledon explants developed callus and multiple shoots on MS supplemented with a combination of cytokinin (BA and/or Kin.) and auxin (NAA and/or 2,4-D). The maximum number of 3.98 ± 0.37 and 2.1±0.11 shoots/explants were recorded for nodal and cotyledon explants on MS medium supplemented with a combination of 8.8 µM BA+2.7 µM NAA and 2.2µM BA+2.7 µM NAA respectively. On the basal medium, 10% of the excised shoots rooted successfully. Thus, this in vitro method can be exploited for conservation and mass propagation of this fast timber yielding tree and also utilized for embryogenesis studies.