To optimize protocol for obtaining hairy roots of L. with high antiviral activities, factors such as four strain types of (A4, ATCC15834, R-1601, 8196), two explant types, namely cotyledonous and primary leaves of seedlings, and different cultivation durations (30 and 90 d) were studied. The formation of hairy roots was observed after 2 to 4 wk of incubation, depending on the type of explant and the strain of used. The maximum transformation frequency (85.7%) was observed in the cotyledons genetically modified with the strain ATCC15834. The transgenic nature of hairy roots was revealed by PCR with primers to the oncogenes and . The absence of contamination of the culture by was confirmed by primers to the and genes. Phytochemical analysis showed that accumulation of individual metabolites in the line samples exceeded their levels in the native roots. Catechin content in the cultures of long-term cultivation (90 d) was found 1.4 to 2.2 times higher than the same samples of short cultivation (30 d) and 4.8 to 10.8 times higher in comparison with the native roots. The most productive in terms of catechin level were hairy roots of long-term cultivation obtained during the transformation of primary leaves of seedlings with ATCC15834 strain. These data were consistent with the highest antiviral activities against influenza viruses of A (H5N1) and A (H3N2) subtypes with neutralization indexes 6.5 to 6.75 log10, and selectivity index values were in the range 15.4 to 16.4.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine () differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.

The medicinal plant L. is a rich source of hyoscyamine and scopolamine, the tropane alkaloids. The use of hairy root cultures has focused significant attention on production of important metabolites such as stable tropane alkaloid production. Elicitation is an effective approach to induce secondary metabolite biosynthetic pathways. Hairy roots were derived from cotyledon explants inoculated with and elicited by iron oxide nanoparticles (FeNPs) at different concentrations (0, 450, 900, 1800, and 3600 mg L) for different exposure times (24, 48, and 72 h). The highest hairy root fresh and dry weights were found in the medium supplemented with 900 mg L FeNPs. Antioxidant enzyme activity was significantly increased in induced hairy roots compared to non-transgenic roots. The highest hyoscyamine and scopolamine production (about fivefold increase over the control) was achieved with 900 and 450 mg L FeNPs at 24 and 48 h of exposure time, respectively. This is the first report of the effect of FeNP elicitor on hairy root cultures of a medicinal plant. We suggest that FeNPs could be an effective elicitor in hairy root cultures in order to increase tropane alkaloid production.

Enset ( (Welw.) Cheesman) is an economically important staple food crop in Ethiopia, especially in the southern and southwestern regions. It is called "false banana" due to its resemblance to banana, but inability to produce any edible fruit. The crop is clonally propagated using field-grown suckers. This study reports the development of a robust regeneration technique to propagate large numbers of plantlets using corm discs containing intercalary meristematic tissues. Hundreds of shoot buds were induced from corm discs of enset cultivar 'Bedadeti' cultured on Murashige and Skoog (MS) medium supplemented with 1.5 mg L 2,4-dichlorophenoxyacetic acid, 0.216 mg L zeatin, and 2 g L activated charcoal. The shoot buds were regenerated into complete plantlets when transferred onto MS medium supplemented with 1 mg L 6-benzylaminopurine and 2 g L activated charcoal. More than 100 plantlets were generated in 4 mo from corm discs isolated from a single mother plantlet. Well-rooted plantlets were acclimatized in soil with 100% success, and did not show any apparent phenotypic abnormalities under glasshouse conditions. This efficient regeneration system could be very useful for the rapid multiplication of clean pathogen-free planting material.

-mediated plant transformation is typically conducted by inoculating plant tissues with an suspension containing approximately 10-10 bacteria mL, followed by a 2-3-d co-culture period. Use of longer co-culture periods could potentially increase transformation efficiencies by allowing more time for to interact with plant cells, but bacterial overgrowth is likely to occur, leading to severe tissue browning and reduced transformation and regeneration. Low bacterial inoculum levels were therefore evaluated as a means to reduce the negative outcomes associated with long co-culture. The use of low inoculum bacterial suspensions (approximately 6 × 10 bacteria mL) followed by long co-culture (15 d) led to the production of an average of three transformed sunflower shoots per explant while the use of high inoculum (approximately 6 × 10 bacteria mL) followed by short co-culture (3 d) led to no transformed shoots. Low inoculum and long co-culture acted synergistically, and both were required for the improvement of sunflower transformation. Gene expression analysis via qRT-PCR showed that genes related to plant defense response were generally expressed at lower levels in the explants treated with low inoculum than those treated with high inoculum during 15 d of co-culture, suggesting that low inoculum reduced the induction of plant defense responses. The use of low inoculum with long co-culture (LI/LC) led to large increases in sunflower transformation efficiency. This method has great potential for improving transformation efficiencies and expanding the types of target tissues amenable for transformation of different plant species.

The importance of cassava as the fourth largest source of calories in the world requires that contributions of biotechnology to improving this crop, advances and current challenges, be periodically reviewed. Plant biotechnology offers a wide range of opportunities that can help cassava become a better crop for a constantly changing world. We therefore review the state of knowledge on the current use of biotechnology applied to cassava cultivars and its implications for breeding the crop into the future. The history of the development of the first transgenic cassava plant serves as the basis to explore molecular aspects of somatic embryogenesis and friable embryogenic callus production. We analyze complex plant-pathogen interactions to profit from such knowledge to help cassava fight bacterial diseases and look at candidate genes possibly involved in resistance to viruses and whiteflies-the two most important traits of cassava. The review also covers the analyses of main achievements in transgenic-mediated nutritional improvement and mass production of healthy plants by tissue culture and synthetic seeds. Finally, the perspectives of using genome editing and the challenges associated to climate change for further improving the crop are discussed. During the last 30 yr, great advances have been made in cassava using biotechnology, but they need to scale out of the proof of concept to the fields of cassava growers.

The establishment of cocoa embryogenic cell lines in liquid medium starting from high frequency somatic embryogenesis (HFSE) callus is described. The growth kinetics of the cultures during the multiplication and the expression steps conducted in 250 mL Erlenmeyer flasks were described for three genotypes selected for their agronomical traits (EET95, EET96, and EET103). The glucose and dissolved oxygen concentrations and the absorption of Murashige and Skoog medium macronutrients (nitrate, ammonium, potassium, sulfate, calcium, phosphorus, and magnesium) were monitored. The multiplication of the embryogenic calluses in a medium containing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1 mg L, initiated with an inoculation density of 20 g L of callus, was achieved. The growth rate was characterized by two phases, with the second being concomitant with a depletion of phosphorus and magnesium, and a decrease in the embryogenic potential of the callus. The expression of the callus embryogenic capacity was conducted in an auxin-free medium. The embryo production starting from 1 and 5 g L inoculation densities was compared. When placed in the optimal expression conditions in flasks, 1 g of callus produced 1000 to 1500 embryos within 5 to 7 wk. Finally, two paths for improving the plantlet regenerative capacities of cocoa SE produced in liquid medium were identified. Supplementing the expression medium with myo-inositol used as an osmotic agent at a concentration of 50 g L increased the embryo-to-plantlet conversion rate from 13-16% to 40-48%. A 6-wk culture of the embryos on a maturation medium in Petri dishes optimized their subsequent development into plantlets.

Somatic embryogenesis offers many benefits for clonal propagation in large-scale plant production of conifers. A key rate-limiting step is the conversion from early-stage somatic embryos in pro-embryogenic masses (PEMs) to the maturation stage. Immature embryos in PEMs are present at different developmental stages, where some are unable to respond to the maturation treatment, thus limiting yields of mature embryos. Synchronization of early somatic embryo development in PEMs could greatly improve subsequent yields of mature embryos. A temporary immersion bioreactor designed for Norway spruce ( (L.) H.Karst.) was used in this study. Through a specific system for dispersion, connected tissue of PEMs, composed of immature embryos grown in liquid medium in the temporary immersion bioreactors or on solid medium as a control, was dispersed and redistributed in a more uniform spatial arrangement. It was demonstrated that development of mature embryos could be significantly stimulated by dispersion, compared to controls, in both medium types. Synchronization of maturation was evaluated by a statistical approach. The present study shows that the yield of mature embryos from dispersed PEMs was three to five times higher than that from non-dispersed controls in three of four cell lines of Norway spruce tested, both in bioreactors and on solid medium.

(Roxb.) Korth., commonly known as "Kadam," is an endangered and pharmaceutically valued tree of the family Rubiaceae. The numerous medicinal properties are attributed to the various alkaloids of this plant. Poor seedling survival (due to very small size of seeds, approximately 10,000 per gm), overexploitation and habitat destruction are the major constraints in conserving the wild stocks of this species. This paper reports a significant, improved, and repeatable micropropagation protocol of using nodal explants of a mature tree. Nodal explants harvested during spring season from the lopped tree differentiated the maximum number of axillary shoots (5.3 ± 0.82 per node) on full-strength Murashige and Skoog (MS) medium containing 3.0 mg L 6-benzylaminopurine (BAP) and additives (25 mg L each of adenine sulfate, L-arginine, and citric acid and 50 mg L ascorbic acid). Shoots were amplified through (1) recurrent transfer of mother explants and (2) subculturing on fresh nutrient medium. The greatest number of shoots (13.4 ± 1.26) with an average length of 6.2 ± 1.03 cm was produced after 4 wk on MS medium containing 0.5 mg L BAP, 0.25 mg L kinetin (Kin), 0.1 mg L Indole-3-acetic acid (IAA), additives, 100 mg L activated charcoal (AC), and 0.8% (w/v) agar. This is the first report of concurrent rooting and acclimatization (CEVRA) in . About 90% micropropagated shoots rooted on pulse treatment of 500 mg L Indole-3-butyric acid (IBA; for 5 min) and produced 8.5 ± 0.97 roots per shoot with an average length of 9.40 ± 1.06 cm, after 5 wk. Over 80% of CEVRA plantlets were successfully transplanted to the soil in field. The defined protocol can be employed for conservation and restoration/rehabilitation/reintroduction of .

Plant genome editing, particularly CRISPR-Cas biotechnologies, has rapidly evolved and drawn enormous attention all around the world in the last decade. The cutting-edge technologies have had substantial impact on precise genome editing for manipulating gene expression, stacking gene mutations, and improving crop agronomic traits. Following the global trends, investigations on CRISPR-Cas have been thriving in Australia, especially in agriculture sciences. Importantly, CRISPR-edited plants, classified as SDN-1 organisms (SDN: site-directed nuclease), have been given a green light in Australia, with regulatory bodies indicating they will not be classified as a genetically modified organism (GMO) if no foreign DNA is present in an edited plant. As a result, genome-edited products would not attract the onerous regulation required for the introduction of a GMO, which could mean more rapid deployment of new varieties and products that could be traded freely in Australia, and potentially to export markets. In the present review, we discuss the current status and prospects of plant genome editing in Australia by highlighting several species of interest. Using these species as case studies, we discuss the priorities and potential of plant genome editing, as well as the remaining challenges.

Human population is expected to reach to about 10 billion by 2050. Climate change affects crop production, thus posing food security challenges. Conventional breeding alone will not bridge the gap between current level of crop production and expected levels in the decades to come in the food production systems. Rate of genetic gain with time has remained narrow considerably. Biotechnology-enabled crops developed through genome editing will have a part to play in improving crop productivity, meeting food, nutrition security besides catering to regional preferences and fetching valuable foreign exchange. Political, social, economical proposition, scientific will, retailer and consumer acceptance are a must for genome editing (GE) to succeed and add value in the food value chain. This will also help to make agriculture a lucrative profession and attract youth. Therefore, the present review looks into existing regulations governing crops developed using biotechnology in India, institutes involved in genome editing, prospects of new tools developed in this sphere such as DNA-free editing systems, nanotechnology, their applicability in crop improvement efforts, social and future prospects taking cue from recent global developments. This will make GE more appealing to stakeholders and defray any safety concerns.

Various genome-editing technologies have been embraced by plant breeders across the world as promising tools for the improvement of different crops to deliver consumer benefits, improve agronomic performance, and increase sustainability. The uptake of genome-editing technologies in plant breeding greatly depends on how governments regulate its use. Some major agricultural production countries have already developed regulatory approaches that enable the application of genome editing for crop improvement, while other governments are in the early stages of formulating policy. Central to the discussion is the principle of "like products should be treated in like ways" and the subsequent utilization of exclusions and exemptions from the scope of GMO regulations for these products. In some countries, the outcomes of genome editing that could also have been achieved through conventional breeding have been defined as not needing GMO regulatory oversight. In this paper, we provide a short overview of plant breeding and the history of plant biotechnology policy development, the different classes of current regulatory systems and their use of exemptions and exclusions for genome-edited plants, and the potential benefits of such approaches as it relates to achieving societal goals.

Solid-stemmed spring wheat cultivars ( L.) are resistant to the stem sawfly ( Nort.) and lodging. Anthers of 24 spring wheat cultivars with varying content of pith in the stem were used in the experiment. All were classified into three groups: solid, medium-solid and hollow stems. There was considerable influence of the cultivar on callus formation and green plant regeneration. The highest efficiency of green plant regeneration (24%) was observed for the solid-stemmed AC Abbey cultivar. There was no regeneration from the explants of four cultivars: CLTR 7027, Alentejano, Marquis and Bombona. Principal component analysis showed no differences between the cases under observation (callus induction and green plant regeneration) in their response to pre-treatment temperatures (4 and 8°C). The examination of the effects of various auxin types in the induction medium on callus formation and green plant regeneration revealed that the strongest stimulation of these processes was observed in the C17 medium with 2,4-D and dicamba. The efficiency of callus formation and green plant regeneration was greater in solid-stemmed cultivars than in hollow-stemmed cultivars.

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5-0.9 mm, 1.0-1.4 mm, and ≥1.5 mm), and transferred into 190-2 regeneration medium with different growth regulators: 0.5 mg L kinetin (KIN) and 0.5 mg L 1-naphthaleneacetic acid (NAA); 1 mg L zeatin (ZEA) and 0.5 mg L NAA; or 1 mg L dicamba (DIC), 1 mg L picloram (PIC), and 0.5 mg L kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0-1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L NAA and 0.5 mg L KIN, while the fewest germinated on medium with 1 mg L DIC, 1 mg L PIC, and 0.5 mg L KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.

The aims of the study were to evaluate the effect of media composition on the growth potential and morphology of the cultured biomass of three cultivars of , and on the production of flavonoids. Agitated shoot cultures were maintained in parallel on Linsmaier and Skoog (LS) and Murashige and Skoog (MS) media supplemented with 0.1-3.0 mg L of α-naphthaleneacetic acid and 6-benzylaminopurine. Methanolic extracts from the biomass collected after 3-wk growth cycles were analyzed quantitatively, for 21 flavonoids using high performance liquid chromatography. Three aglycones (kaempferol, luteolin, and quercetin) and three glycosides of quercetin (hyperoside, quercitrin, and rutoside) were detected in all of the extracts. The total amounts of the estimated compounds increased from 1.18- to 21.66-fold on LS media variants and from 1.52- to 17.34-fold on MS media variants. The main metabolite was quercetin (max. 210.55 mg 100 g dry weight [DW]). The maximum total amounts of all compounds in the biomass of 'Elixir,' 'Helos,' and 'Topas' were 328.53, 255.70, and 166.58 mg 100 g DW, respectively. The shoots of all cultivars cultivated on the LS and MS media containing low levels of plant growth regulators (0.1 mg L) accumulated high amounts of flavonoids. The highest amounts were accumulated in shoots of cultivar 'Elixir' grown on MS medium. This is the first comparison of flavonoid production in three cultivars of ('Elixir,' 'Helos,' and 'Topas') cultured , and the first report of flavonoid production in cultivars 'Elixir' and 'Helos.'

L. (white dogwood) is an important ornamental shrub having a wide range of applications such as reforestation programs and soil retention systems. The vegetative propagation of dogwood by cuttings may be slow, difficult, and cultivar dependent; therefore, an improved micropropagation method was developed. Nodal stem segments of cultivars 'Aurea' and 'Elegantissima' were cultured on media enriched with six different sources of macronutrients. Media were supplemented with either -benzyladenine (BA) or thidiazuron (TDZ) in combination with 1-naphthaleneacetic acid (NAA). Regardless of the cultivar, the best shoot proliferation was observed on Lloyd and McCown medium (woody plant medium (WPM)) at pH 6.2, containing 1.0 mg L BA, 0.1 mg L NAA, and 20-30 g L sucrose. Rooting of regenerated shoots was achieved by an method when different concentrations of NAA or indole-3-butyric acid (IBA) were tested. Microcuttings were rooted for 8 wk on medium enriched with 0.25 mg L NAA and potted into P9 containers in the greenhouse. The final survival rate of the plants after 20 wk was 80% for 'Aurea' and 90% for 'Elegantissima'. Genetic stability of the micropropagated plants was confirmed by using two DNA-based molecular marker techniques. A total of 30 random amplified polymorphic DNA (RAPD) and 20 inter-simple sequence repeat (ISSR) primers resulted in 197-199 and 184-187 distinct and reproducible band classes, respectively, in 'Aurea' and 'Elegantissima' plantlets. All of the RAPD and ISSR profiles were monomorphic and comparable with the mother plant.

Constitutive expression of the L. (maize) morphogenic transcription factors () and () in maize can not only greatly increase transformation efficiency but can also induce phenotypic abnormalities and sterility. In an effort to alleviate the pleiotropic effects of constitutive expression, a genome wide search was undertaken to find suitable maize promoters to drive tissue and timing-specific expression of the transformation enhancing genes and . A promoter from a maize phospholipid transferase protein gene (- ) was identified based on its expression in leaves, embryos, and callus while being downregulated in roots, meristems, and reproductive tissues. When driving was transformed into immature maize embryos along with a expression cassette driven by the nopaline synthase promoter ( ::) abundant somatic embryos rapidly formed on the scutella. These embryos were individual and uniformly transformed and could be directly germinated into plants without a callus phase. Transformed plants could be sent to the greenhouse in as little as 1 mo and regenerated plants matched the seed-derived phenotype for the inbred and were fertile. However, T1 seed from these plants had poor germination. Replacing with a maize auxin-inducible promoter ( ) in combination with ::, allowed healthy, fertile plants to be regenerated. Single-copy T1 seed germinated normally and had a predominantly wild-type inbred phenotype. For maize, this callus-free transformation process has worked in all inbred lines tested.

Since the isolation of the first cytokinin almost 60 yr ago, cytokinins have become critically important for ornamental and agricultural crops in plant tissue culture. Despite the extensive research on this class of compounds, little information is available on the chemical stability of cytokinins in solution or following an autoclave cycle with Murashige and Skoog (MS) basal medium. This work describes the stability in aqueous solutions of five widely used adenine-based cytokinins: trans-zeatin (tZ), 6-(γ,γ-dimethylallylamino) purine (2iP), kinetin, benzyladenine (BA), and -topolin. High pressure liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS) were used to quantify and identify their degradation. BA, kinetin, 2iP, and -topolin were stable at 1.0 mg mL in 0.05 N KOH, with no statistically significant concentration changes ( > 0.05) after 90 d of storage at temperatures of -20°C, 2-6°C, or 25°C. The cytokinin tZ was used as a model compound to evaluate stability under alkaline and acid conditions as well as after repeated freeze-thaw cycles. Trans-zeatin retained >90% of the initial concentration of 1.0 mg mL when dissolved in 0.01 N KOH and stored at -20°C and 2-6°C for 90 d, with only the 2-6°C temperature treatment showing a statistical significant concentration change ( = 0.03). The 1.0 mg mL tZ solution in 0.01 N KOH was stable through six repeated freeze-thaw cycles over 90 d without any significant change in concentration compared to the initial freeze-thaw. Yet, tZ showed highly significant concentration changes when dissolved at 50 mg mL and 0.5 N KOH. All of these adenine-based cytokinins showed exceptional stability following an autoclave cycle at 121°C, 110 kPa for 30 min when in solutions of 1.0 mg mL in 0.05 N KOH, with no significant degradation detected. Trans-zeatin was also found to be stable after one autoclave cycle with 1× MS-basal salts.

Wheat ( L.) is a temperate cereal with an optimum temperature range of 15-22°C during the grain filling stage. Heat stress is one of the major environmental constraints for wheat production worldwide. Temperatures above 25°C during the grain filling stage significantly reduced wheat yield and quality. This reduction was reported due to the inactivation of the soluble starch synthase, a key heat-labile enzyme in starch transformation of wheat endosperm. To improve wheat productivity under heat stress, the rice soluble starch synthase I, under the control of either a constitutive promoter or an endosperm-specific promoter, was expressed in wheat and the transgenic lines were monitored for expression and the effects on yield-related traits. The results showed that the transgenic wheat events expressed rice soluble starch synthase I at a high level after four generations, and transgenic plants produced grains of greater weight during heat stress. Under heat stress conditions, the thousand kernel weight increased 21-34% in T and T transgenic plants compared to the non-transgenic control plants. In addition, the photosynthetic duration of transgenic wheat was longer than in non-transgenic controls. This study demonstrated that the engineering of a heat tolerant soluble starch synthase gene can be a potential strategy to improve wheat yield under heat stress conditions.