Screening of cultivars for tissue culture response and establishment of genetic transformation in a high-yielding and disease-resistant cultivar of Theobroma cacao

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao. Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T0 plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.

In vitro Regeneration of Clematis Plants in the Nikita Botanical Garden via Somatic Embryogenesis and Organogenesis

The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars ‘Alpinist,’ ‘Ay-Nor,’ ‘Bal Tsvetov,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Kosmicheskaya Melodiya,’ ‘Lesnaya Opera,’ ‘Madame Julia Correvon,’ ‘Nevesta,’ ‘Nikitsky Rosovyi,’ ‘Nikolay Rubtsov,’ ‘Serenada Kryma,’ and ‘Vechniy Zov’ were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3–0.4% Cl2 (15 min), and 1% thimerosal (10 min), 1-cm long segments with a single node were introduced to an in vitro culture. The explants were established on the basal MS medium supplemented with BAP (2.20–8.90 μM) and 0.049 μM NAA, or TDZ (3.0; 6.0, and 9.0 μM) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 μM BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 μmol m–2 s–1, at a temperature of 24 ± 1°C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 μM BAP, or 6.0 μM TDZ were optimal cytokinin concentrations for micropropagation. The explants of ‘Alpinist,’ ‘Ay-Nor,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Nevesta,’ and ‘Serenada Kryma’ cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 μmol m–2 s–1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 μM BAP with 0.09 μM IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in ‘Crystal Fountain’ (100%), ‘Crimson Star’ (100%), ‘Nevesta’ (97%), and ‘Ay-Nor’ (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.

PRIMARY AND SECONDARY SOMATIC EMBRYOGENESIS OF CACAO: THE EFFECT OF EXPLANT TYPES AND PLANT GROWTH REGULATORS

<p class="abstrakinggris">The success of cacao somatic embryogenesis is affected by many factors, including the basal salt medium, the genotype, the explant type, and the concentration and composition of plant growth regulators (PGRs). The study aimed to evaluate the effects of PGRs composition on the primary somatic embryo (PSE) response and the effect of explant type and PGRs composition used in inducing PSE on the secondary somatic embryogenesis (SSE) response. PSEs were induced from basal petal and staminoid explants of MCC 01 and MCC 02 clones on DKW medium containing 2,4-D 2 mg l^-1+ kinetin 0.5 mg l^-1 or 2,4-D 2 mg l^-1 + kinetin 0.125 – 0.250 mg l^-1 + thidiazuron (TDZ) 2.5 – 5 µg l^-1 or 2,4-D 2 mg l^-1 + TDZ 10 µg l^-1. Genotype, explant type, and PGR composition dependently affected PSE response. The best PSE response was obtained from staminoid explant of MCC 02 clone on medium containing 2,4-D 2 mg l^-1 + kinetin 0.5 mg l^-1 (20%, 9 embryos). The explant type and PGR composition used in inducing PSEs affect the SSE response. The highest SSE response of MCC 01 clone was obtained from petal explant with medium containing 2,4-D 2 mg l^-1 + kinetin 0.5 mg l^-1. The formation of SSEs could increase the multiplication rate of MCC 01 clone by 7 times.</p>

Long-Term Maintainable Somatic Embryogenesis System in Alfalfa (Medicago sativa) Using Leaf Explants: Embryogenic Sustainability Approach

Alfalfa (Medicago sativa) is one of the most important forage legume crops because of its mass production and high feeding value. It originated in Asia and is one of the most ancient plants cultivated throughout the world as a fodder. Despite the well-studied somatic embryogenesis of alfalfa, there is a lack of a long-term maintainable somatic embryogenic system. Every time an embryogenic callus culture must be started from new explants, which is laborious, costly and time consuming. In addition to this, endogenous microorganisms present in ex vitro explants of alfalfa can often cause contamination, reducing the efficiency of callus culture. An attempt was made to establish long-term continuous somatic embryogenesis system in alfalfa using cultivar Regen-SY. Nine somatic embryogenesis pathways were studied and evaluated for embryo yield, plant conversion rate and embryogenic sustainability. Somatic embryos passed through the same stages (globular, heart-shaped, torpedo and cotyledonary) as characteristic of the zygotic embryo and secondary somatic embryogenesis was also observed. B5H-B5 system showed the highest embryo yield and plant conversion rate whereas SH4K-BOi2Y system demonstrated the highest embryogenic sustainability and maintained the embryogenic potential even after six subculture cycles. Scanning electron microscopy was applied to study the morphology of the somatic embryos and secondary somatic embryogenesis. Therefore, long-term maintainable somatic embryogenesis system protocol was developed through this study, which will help to enhance and accelerate the alfalfa biotechnology research.

Primary and secondary somatic embryogenesis in Jatropha curcas L. From leaf transverse thin cell layers

An efficient method for plant regeneration in Jatropha curcas L. via primary and secondary somatic embryogenesis culture from ex vitro leaves of 6-month-old plants was presented in this study. Leaves were cut into transverse thin cell layers (tTCLs) and cultured on MS medium supplemented with kinetin (KIN) at 0.5, 1.0, 1.5, and 2.0 mg/l in combination with indole-3-butyric acid (IBA) at 0.1, 0.5, and 1.0 mg/l or 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0, 1.5 and 2.0 mg/l . The highest embryogenic callus formation rate (89.3%) was obtained on medium supplemented with 1.0 mg/l KIN and 1.5 mg/l 2,4-D. The calli were selected for the study of primary somatic embryogenesis on MS medium containing 2,4-D (0.01, 0.03, 0.05, and 0.07 mg/l) or KIN (0.5, 1.0, 1.5, and 2.0 mg/l). The highest primary somatic embryos formation rate (76.67%) was achieved on MS medium supplemented with 1.0 mg/l KIN. The primary embryos were cultured on medium supplemented with KIN (0.1, 0.5, 1.0, 1.5, and 2.0 mg/l) combined with 0.2 mg/l indole-3-butyric acid (IBA) or 0.05 mg/l 2,4-D. The combination of 1.5 mg/l KIN and 0.05 mg/l 2,4-D was suitable for secondary embryos formation. Embryos proliferated rapidly, and the highest number of secondary embryos (77.5 embryos) wasobtained from a single primary embryos inoculated. Results also showed that the addition of proline (0.75 g/l) or spermidine (0.15 mM) to the culture medium increased the number of secondary embryos considerably. The fully developed plantlets exhibiting healthy roots and shoots were obtained when somatic embryos were sub-cultured onto B5 medium containing 1.5 mg/l IBA.

Morpho-anatomical characterization of secondary somatic embryogenesis in Azadirachta indica (Meliaceae)

Background and Aims: Meliaceae species are extremely recalcitrant during germination and in vitro processes. Therefore, this research focuses on characterization and optimization of a highly efficient system by secondary somatic embryogenesis in Azadirachta indica, which is an important step for enhancing secondary metabolite production and regeneration in recalcitrant species.Material and Methods: Leaf and cotyledon sections were induced in MS medium supplemented with benzylaminopurine (BAP) alone, or combined with 1-naphthaleneacetic acid (NAA) and, abscisic acid (BA) with thidiazuron (TDZ).Key results: Azadirachta indica developed primary somatic embryos with BAP. Shoot and root formation occurred at low concentrations of BAP, while somatic embryogenesis was favored under high levels of BAP or TDZ. Primary and secondary somatic embryos were evidenced continuously and asynchronously. The highest amount of somatic embryos was obtained with cytokinins. However, the concentration might be significant to differentiate between primary and secondary embryos. Moreover, the auxins are key for inducing histodifferentiation in embryos. Shoot induction occurred after transfer of the embryos to hormone-free MS medium. The shoots were rooted in MS1/2.Conclusions: The secondary somatic embryos were distinguished and characterized during the whole process and the efficient system was established with cotyledon sections at short term, which offers several advantages such as the production of metabolites.