Somatic Embryogenesis in Vitis for Genome Editing: Optimization of Protocols for Recalcitrant Genotypes

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have proven to be very recalcitrant to regeneration via somatic embryogenesis. In this work, the development of a protocol for improved production of embryogenic calluses is described. Two sterilization protocols were tested: (a) a lower active chlorine concentration for a longer time (LS); and (b) a higher chlorine concentration for a shorter time (HS), in combination with the absence or presence of citric acid in the growing substrate in the first growth media. The embryogenic calluses formation in Chardonnay, a cv. with a high embryogenic response, was significantly higher in presence of citric acid in the initial growing substrate regardless of the sterilization protocol. In Aglianico, a cv. with a lower embryogenic response, no significant differences were observed. Instead, in a recalcitrant cv. as Italia, we obtained a 13-fold increase in embryogenic calluses formation performing sterilization of flowers with the HS protocol compared to LS.

Optimized somatic embryogenesis and plant regeneration in elite Argentinian sugarcane (Saccharum spp.) cultivars

Background Biotechnological breeding of elite sugarcane cultivars is currently limited because of the difficulty of regenerating plants by tissue culture. Here, we report that commercially elite sugarcane genotypes, which are adapted to Argentinian agro-ecological conditions, are capable of being regenerated via indirect somatic embryogenesis. Leaf rolls of five elite genotypes were cultured following two callus induction protocols using different concentrations of 2,4-D as the growth regulator. Embryogenic calluses were regenerated under light conditions. Regenerated plants were subsequently acclimatized in the greenhouse under two acclimatization procedures before being transplanted to the field. Results Four of the five genotypes were able to form somatic embryos following the two induction protocols. The variables related to embryogenic callus production were influenced by the interaction between genotype and culture conditions. For plant regeneration, the embryogenic calluses were further cultured on an IBA-supplemented medium, where we observed a high genotype dependence. Calluses from the four cultivars regenerated a good number of plants. With the procedures described here, we obtained more than 90% of well-acclimatized plants both in the greenhouse and in the field. Conclusions This protocol provides a simple way to regenerate sugarcane plants through indirect somatic embryogenesis. Also, the results confirm that tissue culture ability is highly genotype-dependent in sugarcane. Our findings suggest that these elite cultivars could be good candidates for biotechnological breeding.

High-Efficiency Somatic Embryogenesis from Seedlings of Koelreuteria paniculata Laxm.

Research Highlights: In the current study, we established a method for plant regeneration via somatic embryogenesis (SE) in Koelreuteria paniculata Laxm. for the first time. Background and Objectives: K. paniculata is an important ornamental and medicinal plant in China. However, the plant has difficulty with asexual reproduction, which imposes a limitation on large-scale propagation. Materials and Methods: Embryogenic calluses were induced from stems of aseptic seedlings on induction media. The effects of different media types and concentrations of N6-benzyladenine (BA), α-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on callus induction were examined. Embryogenic calluses were then transferred to Driver-Kuniyuki Walnut (DKW) media containing NAA (0.1–0.2 mg L−1) or 2,4-D (0.5–2.0 mg L−1) to develop somatic embryos. Cotyledon embryos were cultured on DKW media containing NAA (0.1–0.2 mg L−1) until maturation, and were then transferred to 1/2 DKW medium supplemented with 1.0 mg L−1 indole-3-butyric acid (IBA) to produce complete plants. The effects of IBA and NAA on rhizogenesis were then examined by clonal culture. Results: The maximum callus induction frequency (80.25%) was obtained on DKW medium supplemented by 0.5 mg L−1 BA, 0.25 mg L−1 NAA, and 1.5 mg L−1 2,4-D. NAA had a more pronounced effect on somatic embryo growth than did 2,4-D, with a maximum SE frequency (54.75%) observed with 0.1 mg L−1 NAA added to DKW medium. For clonal culture, the highest rooting rate (52%) was observed on 1/4 DKW medium containing 1.5 mg L−1 IBA. Histology studies confirmed the presence of embryogenic calluses and somatic embryos in different stages. Conclusions: This protocol provides a novel method for large-scale propagation of K. paniculata, and creates opportunities for genetic engineering in this species.

Morphohistological development of the somatic embryo of Typha domingensis

Background. The sustainable methods of propagation for Typha domingensis through somatic embryogenesis can help to mitigate its current condition of ecological marginalisation and overexploitation. Then, the hypothesis established that the variation of the concentration of auxin and light conditions in sequential stages of culture generate different morphogenetic routes that can be monitoring by morphohistological markers. Methods. Murashige and Skoog medium at half ionic strength, 3% sucrose and 0.1% ascorbic acid were used in the induction, proliferation and embryogenic maturation. Induction started with aseptic germinates cultured in 0.5 mg L-1 of 2,4-dichlorophenoxyacetic. Four concentrations of 0 to 2 mg L-1 of 2,4-dichlorophenoxyacetic, that generated four embryogenic lines, were evaluated in darkness. Maturation of the somatic embryo took place, in each embryogenic line, without auxin and under light and dark conditions. Results. The yellow and brown callus, as well as oblong and scutellar somatic embryos were recorded in the methodological sequence. The embryogenic differentiation was described with histological analysis. The induced cultures produced both somatic embryos in a small proportion. The percentages of the yellow callus on the explant and of suspended cells in the embryogenic proliferation were greater with the three concentrations of 2,4-dichlorophenoxyacetic. While, the brown callus predominated without auxin. The somatic embryo developed under light and dark conditions, and presented globular, oblong, scutellar and sparsely coleoptilar stages. Discussion. The combined effect of auxin concentrations and light-dark conditions generated conditions that favoured the development of embryogenic calluses and somatic embryos (globular, oblong, scutellar and coleoptilar) in an asynchronous process with respect to the stages of embryogenic induction, proliferation, and maturation. Indeed, differentiation and cellular organization of this process were compatible with descriptors of the embryogenic stages recorded by other aquatic and terrestrial monocotyledons.

Morphohistological development of the somatic embryo of Typha domingensis

Background. The sustainable methods of propagation for Typha domingensis through somatic embryogenesis can help to mitigate its current condition of ecological marginalisation and overexploitation. Then, the hypothesis established that the variation of the concentration of auxin and light conditions in sequential stages of culture generate different morphogenetic routes that can be monitoring by morphohistological markers. Methods. Murashige and Skoog medium at half ionic strength, 3% sucrose and 0.1% ascorbic acid were used in the induction, proliferation and embryogenic maturation. Induction started with aseptic germinates cultured in 0.5 mg L-1 of 2,4-dichlorophenoxyacetic. Four concentrations of 0 to 2 mg L-1 of 2,4-dichlorophenoxyacetic, that generated four embryogenic lines, were evaluated in darkness. Maturation of the somatic embryo took place, in each embryogenic line, without auxin and under light and dark conditions. Results. The yellow and brown callus, as well as oblong and scutellar somatic embryos were recorded in the methodological sequence. The embryogenic differentiation was described with histological analysis. The induced cultures produced both somatic embryos in a small proportion. The percentages of the yellow callus on the explant and of suspended cells in the embryogenic proliferation were greater with the three concentrations of 2,4-dichlorophenoxyacetic. While, the brown callus predominated without auxin. The somatic embryo developed under light and dark conditions, and presented globular, oblong, scutellar and sparsely coleoptilar stages. Discussion. The combined effect of auxin concentrations and light-dark conditions generated conditions that favoured the development of embryogenic calluses and somatic embryos (globular, oblong, scutellar and coleoptilar) in an asynchronous process with respect to the stages of embryogenic induction, proliferation, and maturation. Indeed, differentiation and cellular organization of this process were compatible with descriptors of the embryogenic stages recorded by other aquatic and terrestrial monocotyledons.

Embryo Germination Development of Coffea arabica L.at Various Media Composition, Subcultures Stages and Embryo Size

Most reliable and efficient protocol for Coffea arabica L. of Sigararutang variety plant regeneration was established using embryoid as an early explant from the induction of embryogenic callus phase. A completely randomized designs with 5 replications was designed to accomplish 20 protocols of embryo germina-tion methods with different steps of subculture, size of embryo and germination medium. The embryogenic calluses from the flush leave explant were induced embryoid on a half-strength MS medium fortified with a half-strength combina-tion vitamin of 1.8 mg/L nicotinic acid, 10.1 mg/L thiamin HCl and 3.1 mg/L pyri-doxine, 50 mg/L myo inositol, 33 mg/L L.cistein, 1 mg/L Kinetin, 0.1 mg/L NAA, 20 gr/L sucrose, 2.4 gr/L gelrite and pH 5.5.The result showed that Protocol 17 was the most effective, with 59,2% of rooted cotyledons, 4.04 cm of length of roots, 1.68 cm of length of hypocotyl, 20.8% of opened cotyledons and 100% of cotyledonary embryo at the end of 8 weeks which used the B medium, large embryos and twice phase of subculture from liquid medium to solid medium. The Protocol 17 is stable protocol from low to high value. Protocol 8 is the steady protocol from high to low value. Protocols 17 and 8 are the highest and lowest ranking, respectively, for each parameter. Protocol 17 is the most suitable for the germination embryo somatic.

The effect of media composition on multiplication of grape rootstocks in vitro

The current demand for in vitro cultures of grape rootstocks, not only for mass production of plants, but also for genetic engineering is evident. The study on micropropagation of grape rootstock genotypes namely Kober 5BB, Kober 125AA and Teleki 5C was performed. The aim of the study was to develop an optimized protocol to obtain large quantity of plant material. Protocol is based on regeneration via organogenesis, considering that grape embryogenic calluses are laborious to establish and the genotype of the regenerated plants can be altered. Using of Driver and Kuniyuki Walnut media for the establishing of proliferating cultures gave better results than Murashige Skoog media in case of all used rootstocks. Subsequent cultivation on modified Murashige Skoog media with 1-naphtalene acetic acid and increased concentration of cytokynin was characterized by multiplication of cultures and formation of clusters with high multiplication capability. The clusters obtained from rootstock genotypes were suitable for mass propagation as well as for genetic transformation due to their high ability of regeneration.