Artificial/synthetic Seeds

Synthetic seed technology is an excellent method for large-scale production of unique hybrids, valuable and unstable genotypes, and genetically modified plants that unable to produce plants that require symbiosis of mycorrhiza fungi for seed germination. This technology is now considered as an efficient way to propagate and store several important commercial orchids. The evaluation of physical properties of synthetic seeds has been recognized as one of the critical
aspects for seed quality enhancement, which resulted in higher germination and viability at storage duration. The main aim of this study was to optimize the encapsulation conditions to reach the physical properties of Phalaenopsis orchid synthetic seeds using the response surface methodology. A device was designed to control the dripping of alginate for a given temperature in order to wrap the protocorm. The central composite design has been used to investigate the effect of encapsulation variables on the physical properties of orchid synthetic seed such as volume, sphericity Index (SI) and Concentricity Index (CI). Four independent variables were considered to optimize the physical properties of orchid synthetic seed including two sodium alginate solution temperature (4 and 25 ◦C), three calcium chloride solution temperature (25, 30, and 35 ◦C), three alginate droplet height (10, 13, and 16 mm), and three stirring rate of calcium chloride solution (zero (no stirring), 1, and 2 rpm). To determine the synthetic seeds, digital images were taken, and response variables were extracted using the image J processing platform. The ANOVA results showed significant effects of linear and interaction terms of models on all studied factors (P <0.001). The results indicate that stirring rate has most significant effect on the synthetic seed sphericity Index (p < 0.0001). The results showed that the optimum values of volume, sphericity Index and concentricity Index CI were 1285.27 mm3, 0.927 %, and of 2.436 mm, respectively, with a desirability of 0.756. Optimum conditions for orchid protocorm  encapsulation were obtained with stirring rate of 0.83 rpm, droplet height of 10 mm, high calcium chloride solution temperature of 35 ◦C, and sodium alginate solution temperature of 25 ◦C. In general, results showed that the use of image J and the response surface methodology is a useful tool to obtain the optimal physical properties of synthetic seeds. The modeling and optimizing of physical characteristics of synthetic seed can be developed for encapsulation of any explants of plant species.

The present study demonstrates the potential of nutrient-alginate encapsulation of shoot tips of sunflower, Helianthus annuus (hyb. Azargol) for synthetic seed technology, which could be useful in germplasm distribution and exchange. Shoot tips from in vitro shoot cultures derived from mature seed explants were encapsulated in 3% sodium alginate and 100 mM CaCl2. 2H2O are supplemented with three different matrices (include distilled water, liquid MS medium and plant growth regulators) and they are stored for several periods (15, 30, 45 and 60 days) at 4°C. After each storage period for regeneration and regrowth evaluation, encapsulated and non-encapsulated shoot tips were cultured on hormone-free MS medium. The regrowth ability of encapsulated shoot tips affected by the storage duration and the presence or absence of MS nutrients in calcium alginate beads. Percentage response for the conversion of encapsulated and non-encapsulated shoot tips decreased gradually after storage at 4°C ...

Synthetic seed technology is an excellent method for large-scale production of unique hybrids, valuable and unstable genotypes, and genetically modified plants that unable to produce plants that require symbiosis of mycorrhiza fungi for seed germination. This technology is now considered as an efficient way to propagate and store several important commercial orchids. The evaluation of physical properties of synthetic seeds has been recognized as one of the critical aspects for seed quality enhancement, which resulted in higher germination and viability at storage duration. The main aim of this study was to optimize the encapsulation conditions to reach the physical properties of Phalaenopsis orchid synthetic seeds using the response surface methodology. A device was designed to control the dripping of alginate for a given temperature in order to wrap the protocorm. The central composite design has been used to investigate the effect of encapsulation variables on the physical properties of orchid synthetic seed such as volume, sphericity Index (SI) and Concentricity Index (CI). Four independent variables were considered to optimize the physical properties of orchid synthetic seed including two sodium alginate solution temperature (4 and 25 °C), three calcium chloride solution temperature (25, 30, and 35 °C), three alginate droplet height (10, 13, and 16 mm), and three stirring rate of calcium chloride solution (zero (no stirring), 1, and 2 rpm). To determine the synthetic seeds, digital images were taken, and response variables were extracted using the image J processing platform. The ANOVA results showed significant effects of linear and interaction terms of models on all studied factors (P < 0.001). The results indicate that stirring rate has most significant effect on the synthetic seed sphericity Index (p < 0.0001). The results showed that the optimum values of volume, sphericity Index and concentricity Index CI were 1285.27 mm3, 0.927 %, and of 2.436 mm, respectively, with a desirability of 0.756. Optimum conditions for orchid protocorm encapsulation were obtained with stirring rate of 0.83 rpm, droplet height of 10 mm, high calcium chloride solution temperature of 35 °C, and sodium alginate solution temperature of 25 °C. In general, results showed that the use of image J and the response surface methodology is a useful tool to obtain the optimal physical properties of synthetic seeds. The modeling and optimizing of physical characteristics of synthetic seed can be developed for encapsulation of any explants of plant species.

This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognize that its copyright rests with its author and that quotation from the thesis and no information derived from it may be published without the author's prior consent XIII 6.1.8. The effect of acclimation on the accumulation of dehydrin proteins in cauliflower microshoots .

Artificial seed production is an alternative technique for asexual propagation and conservation of plants with difficult natural seed propagation system, such as orchids. This technique is rapidly growing in germplasm conservation and plant gene pool exchanges between countries and in vitro floriculture trades. Artificial seed is formed by encapsulating somatic embryo with coating materials. Quality, durability and survival of artificial seed are very important and depend on the kinds of explant, concentration of encapsulation materials and other conditions. In this study, the effects of different sodium alginate (3, 4 and 5%) and calcium chloride (75, 100 and 150 mM) concentrations for capsule gel matrix were investigated on conversion capabilities and germination of Phalaenopsis artificial seeds. The ideal beads were obtained through a combination of 4% sodium alginate and 150 mM calcium chloride. Encapsulated protocorms showed the best germination percentage (100%) on MS medium after 3 weeks. Storage of synthetic seeds after desiccation significantly reduced the viability percentage of seeds and protocorms. Treatment with autoclaved distilled water after desiccation and storage resulted in high viability percentage in protocorms, whereas dehydrated protocorms were found to be less tolerant to the storage.

Synthetic seed technology is a popular method in plant biotechnology and agricultural science because of its wide application in plant multiplication, short-term conservation, and germplasm exchange. The orchid is a precious ornamental plant that was studied in this project for encapsulation due to its medicinal and commercial value. The main objectives of this study were determining the physical properties of orchid synthetic seeds by image J platform and comparing experimentally two magnetic and air-bubble stirring methods for the production of artificial seed. Physical properties of encapsulated Phalaenopsis ' 'Beijing' orchid protocorms produced under three Na-alginate concentrations (3 %, 4 %, and 5 %), three CaCl 2-2H 2 O concentrations (100, 125, and 150 mM), and two stirring methods (magnetic and air bubbling) were investigated. Image J processing platform was applied to determine the physical properties (length, width, sphericity index (SI), and concentric index (CI)) of orchid artificial seeds extracted from digital images. Analysis of variance (ANOVA) demonstrated that interaction terms (Na-alginate * stirring method) and (Na-alginate * CaCl 2-2H 2 O) significantly affected all the dependent variables including weight, volume, SI, and CI (P < 0.05). Response Surface Method (RSM) was utilized for the prediction and optimization of the response variables. The optimum conditions were obtained with the air bubble stirring method having the desirability of 0.879, a weight of 0.15 g, volume of 0.14 cm3, sphericity index of 0.87, and concentric index of 0.13. This is while, the optimum conditions of the encapsulation process using the magnetic method with the desirability of 0.786 were recorded with weight, volume, SI, and CI values of 0.137 g, 0.169 cm3, 0.72, and 0.13, respectively. A comparison of the results showed that the air bubble method demonstrates better encapsulation performance in terms of sphericity index and concentricity index, while capsules produced using the magnetic method had higher weight and volume. Results of this study show the potential of the image J processing as a precision measurement tool which has been successfully applied here to determine the physical properties of orchid synthetic seeds.

Artificial seed production is an alternative technique for asexual propagation and conservation of plants with hard natural seed propagation such as orchids. This technique is rapidly growing in germplasm conservation and plant gene pool exchanges between countries and in vitro floriculture trades. Artificial seed is the beads formed by encapsulating somatic embryo with coating materials. Quality, durability and survival of artificial seed are very important and depend on the kinds of explant, concentration of encapsulation materials and other conditions. In this study the effects of different sodium alginate (3, 4 and 5%) and calcium chloride (75, 100 and 150 mM) concentrations for capsule gel matrix were investigated on conversion capabilities and germination of Phalaenopsis artificial seeds. The ideal beads were obtained through a combination of 4% sodium alginate and 150 mM calcium chloride. Encapsulated protocorms showed the best germination percentage (100%) on MS medium after 3 weeks. Storage following capsules desiccation significantly reduced the viability percentage of seeds and protocorms. Treatment with autoclaved distilled water after desiccation and storing resulted in high viability percentage in protocorms, whereas dehydrated protocorms were found to be less tolerant to the storing.

Artificial seed production is a valuable technology for commercial plant production and provides a viable option for preserving elite plant germplasm. The objective of this study was to determine the optimal concentrations of sodium alginates (3%, 4%, and 5%) and calcium chloride (75 mM, 100 mM, and 150 mM) for encapsulating Phalaenopsis orchid protocorms. The second objective was to determine the optimal temperature (4, 20, and 25 °C), light spectra (blue, red, green, far-red, white, and darkness), and storage duration (30, 60, 90, and 180 days) for preserving synthetic seeds. The optimal alginate concentration for producing uniform, circular, and sufficiently strong artificial seeds with the highest germination percentage was found to be 4% in a 100 mM calcium chloride solution. The synthetic seeds stored at 25 °C for 180 days had the shortest germination initiation time (2.33 days), the highest germination percentage (100%), and the highest number of leaves, roots, and protocorm-like bodies. The study on synthetic seeds found that storing them in darkness for 90 days is the most effective treatment, considering the interaction between light spectra and storage period. It is recommended to use green light during storage only when quick germination is necessary. Key Message Artificial seeds of Phalaenopsis made of 4% alginate with 100 mM CaCl 2 had the highest germination rate. The best storage condition was 25 °C for 180 days under LED lights, particularly green light.

Somatic embryos of Camellia japonica were hydrogel encapsulated using 3% sodium alginate and 0.1 M calcium chloride to produce synthetic seeds. Both germinability and repetitive embryogenesis capacity of the encapsulated embryos were investigated. The frequency of in vitro germination into plants of artificial seeds was affected by various nutrient additives included in the encapsulating matrix. The addition of Ca-free Murashige

Developing genetic variability of orchids via mutation is promising for orchid breeding. The objective of this research was to improve genetic variation of Spathoglottis plicata orchids through X-rayirradiation of the orchid seeds. The method involved X-ray irradiation of one month old mature orchid seeds at doses of 0, 6, 12, 18, and 24 rad. The X-ray irradiated seeds were sown on a half strength of MS medium and grown into protocorms (developing orchid embryo). Eight weeks-old protocorms were subcultured onto NP-SIM medium. Five months-old seedlings were then subcultured again into a new flask and morphological observations were recorded. Genetic variability detection was conducted using PCR RAPD based on nine primers, i.e. OPA1, OPA2, OPA11, OPA12, OPA14, OPB1, OPB4, OPD12, OPD14. The data was analyzed using GenAlex 6.1 software to obtain the genetic distance. Moreover, NTSys ver.2 was used to analyze the data for clustering and constructing a dendrogram based on Neighbour Joining model. The result showed that X-ray irradiation with doses of 12-18 rad was able to stimulate morphological variation of seedlings, especially characters of leaf, root and shoot. Interestingly, we found 2 in vitro early flowering plants among one years old X-ray irradiated plants, indicated that somehow X-ray mutation affect flowering time in orchid. Based on the dendrogram of genetic distance that the group of mutants was farther than the WT group and the percentage of polymorphism was bigger than that of wild type group, it is concluded that 12-18 rad doses of X-ray irradiation can be used for induction of genetic variability in Spathoglottis orchid.

Barbados aloe (Aloe vera (L.) Burm. F.) has traditionally been used for healing in natural medicine. However, aloe is now attracting great interest in the global market due to its bioactive chemicals which are extracted from the leaves and used in industrial preparations for pharmaceutical, cosmetic, and food products. Aloe originated from tropical and subtropical Africa, but it is also now cultivated in warm climatic areas of Asia, Europe, and America. In this review, the most important factors affecting aloe production are described. We focus on propagation techniques, sustainable agronomic practices and efficient post harvesting and processing systems.

Cleisostoma racemiferum, an epiphytic orchid of primary forest under threat in their natural habitat, was studied. Immature seeds of different developmental stages [(8-20 week after pollination (WAP)] were cultured on Knudson 'C', Mitra et al and MS media supplemented with sucrose (0-3%, w/v), coconut water (CW, 0-20%, v/v) and NAA (0-30 μM) + BA (0-8 μM) singly or in combination. After 7 week of culture, first sign of germination was recorded as nodular swelling of seeds. Amongst the three different basal media tested, better germination was supported by MS medium, followed by Mitra et al and Knudson 'C' media containing sucrose (3%) and NAA (10.0 μM) + BA (8.0 μM). Of various developmental stages of the seed, better germination was obtained from green pods of 16 week after pollination. Younger seeds did not show any sign of germination, while mature seeds exhibited delayed and deformed germination. Though incorporation of CW in the medium did not show much influence on seed germination, but 15% (v/v) CW in the initiation medium enhanced the early differentiation of protocorm-like bodies (PLBs) into plantlets. Within 14-16 week of culture on germination medium, the PLBs started releasing the first set of leaflets. The advanced stage PLBs were converted into rooted plantlets on MS medium containing IAA (10.0 μM) + Kn (9.0 μM). Although the medium containing NAA (10.0 μM) + BA (8.0 μM) resulted in multiple shoot buds, but the leaves were thin and etiolated. Further, the medium enriched with NAA (10.0 μM) + Kn (9.0 μM) resulted in stunted growth of plantlets, while presence of IAA (10.0 μM) + BA (8.0 μM) resulted in plantlets with poor roots.

Non-embryogenic, synthetic seeds were formed by encapsulating the nodal segments (NS) of Hemidesmus indicus R. Br. in calcium alginate hydrogel comprising of MS basal medium for short-term conservation. A 3% sodium aliginate (SA) with 100 mM CaCl 2 was found most suitable for the preparation of isodiametrical beads. Highest shoot regrowth (84.50 ± 0.35%) was recorded when the gelling matrix, i.e. 3% SA in Murashige and Skoog (MS) basal medium was supplemented with 5.0 µM 6-benzyladenine (BA) + 0.5 µM indole-3-butyric acid (IBA) and inoculated onto the nutrient medium comprised of MS + 5.0 µM BA gave 5.53 ± 0.096 shoots/encapsulated NS. Rooting in microshoots was obtained on the half strength liquid MS + 0.1 µM IBA. Plantlets achieved from preserved synthetic seeds were acclimatized and relocated in the natural condition successfully with the immortality rate of 89.3%. During the days of acclimatization, the obtained plants were subjected for the assessment of their pigment content and biochemical assay. The results obtained clearly exhibited the enhancement in the pigment content as the acclimatization period increases. A significant rise and fall in Superoxide dismutase (SOD), Catalase (CAT), Glutathione reductase (GR) and Ascorbate peroxidase (APX) were evident, suggesting their preventive role against the several environmental stresses during the acclimatization of the plant. Further gas chromatography-mass spectrometry (GC-MS) analysis of both mother and in vitro raised plants showed the occurrence of more than 60 diverse phyto-chemicals in the methanolic root extract. HPLC analysis of the root extract revealed the presence of 2-hydroxy-4-methoxybenzaldehyde (2H4MB), a potential phytoactive compound and flavouring agent. 2H4MB content quantified by high-performance liquid chromatography (HPLC) in methanol extract was 17.94 μg/ml. The genetic stability of the regenerated plantlets with mother plant was confirmed by the presence of monomorphic bands generated through inter simple sequence repeat (ISSR) markers. Key message A protocol with improved results has been developed for synthetic seed production and regeneration in H. indicus using nodal explants.

Synthetic seed is one of the most exciting methods in plant biotechnology, with the potential to be modified for horticultural and agricultural improvement in the present and future. Since all of the propagules used in synthetic seed preparation were created by in vitro clonal propagation, they did not experience two basic events of sexual reproduction: meiotic recombination (during crossing over) and gametic fusion of two separate parental genomes (cross-pollination), all of which can result in new forms of heterozygosity in zygotic plants. As a result, offspring resulting from synthetic seeds are still true to type when compared to their parent plant. Propagation by somatic embryogenesis and artificial seeds is the only way to save certain species, such as ornamental plants.&lt;br&gt;

Soma clonal variation presents an enormous opportunity in the horticultural industry for crop improvement in regards to using stable, heritable variations. The goal of micro-propagation of maintaining genetic infidelity and integrity resulting in clonal regenerants. In contrast to the latter, most often, in vitro conditions and, procedures do significantly account for epigenetic and genetic variability which leads to a mutation called soma clonal variation (SV). This ultimately may avail novel genotypes change that is invaluable if the variabilities are stable and heritable. Additionally, variability inducer (DNA methylation, transposons, karyotype change, mitotic crossing over, gene amplification polyploidy, aneuploidy) can lead to change(s) in gene expression or the genetic DNA sequence. In vitro variations can be isolated either by using selection or selection pressure against abiotic and biotic traits resistance. Some variations could increase undeniably the genetic diversity for plant species with narrow genetic bases, enhance useful secondary metabolites production in plants, evolutionary base phenotypic plasticity of plants response to environmental stresses through increased resistance. Consequently, most of these variations are unpredictable, less stable, non-heritable and not useful (like sterility) to their progeny. The epigenetic variations are most common and it requires intensive field trials to ascertain their suitability, stability and heritability. Embracing and utilizing the occurrence of useful soma clonal variation will contribute to knowledge bridge and crop improvement in line with resistance to pests, diseases and drought in the horticultural sector. This can significantly ameliorate food insecurity in many agriculturaldependent communities accredited to crop improvement to adapt to the current environmental condition.

Kalecik Karası, (Vitis Vinifera L.) Türkiye'de ıslah programlarında yaygın olarak kullanılan üzüm çeşitlerinden birisidir. Ancak, tohumlardaki düşük çimlenme oranı, ıslah çalışmalarında önemli bir sorundur. Kalecik Karası tohumlarında çimlenme oranını artırmak için tohumlar; benzilaminopürin (BAP), Gibberellik asit (GA 3), BAP+GA 3 ve Hidrojen peroksit (H 2 O 2) gibi büyümeyi düzenleyici maddeler ile ön muamele yapıldıktan sonra +4°C de 60 ve 90 gün süreyle katlamaya tabi tutulmuştur. En yüksek çimlenme oranı (%66.67) 1 g/l BAP + 3 g/l GA 3 uygulaması ve 60 gün katlama süresinde elde edilmiştir. Bunun yanı sıra, 90 gün katlama süresinde ise en yüksek çimlenme oranı %64 ile 0.5 g/l BAP+2 g/l GA 3 uygulamasında elde edilmiştir. Bu sonuçlara göre, Kalecik Karası tohumlarında en yüksek çimlenme oranı 0.5-1 g/l BAP + 2-3 g/l GA 3 uygulamalarında gerçekleşmiştir.

The apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) are among the major viral species that infect apple trees and are responsible for substantial damage and loss. The aim of this study was to assess the effectiveness of dropletvitrification cryotherapy technique in eradication of ASGVand ASPV in Malus prunifolia "Marubakaido" apple rootstock plants. The axillary shoot tips were excised from ASGV-and ASPV-infected plants and incubated for one day on preculture medium. Subsequently, they were exposed to plant vitrification solution 2 (PVS2) for 0, 20, 40, 50, 60 or 80 min at 22°C and immersed in liquid nitrogen for 1 h. Cryopreserved shoot tips had the highest survival (75 and 65%) and regrowth (53 and 58%) percentages with PVS2 exposure duration of 20 and 40 min, respectively. After six months of growth in a greenhouse, RT-PCR analyses revealed that all the tested plants were free of ASPV and 70% were free of ASGV. The high eradication percentage of ASGV and ASPV indicated that the droplet-vitrification cryotherapy technique is an effective tool to produce ASGV-and ASPV-free propagating material of "Marubakaido" apple rootstock. Keywords Cryopreservation. Malus prunifolia. Virus-free material. Propagation Viruses have a direct impact on apple plant productivity and fruit quality and can enhance susceptibility to other pathogens (Guerra et al. 2012). Viruses belonging to the species Apple stem pitting virus (Foveavirus), Apple stem grooving virus (Capillivirus), and Apple chlorotic leaf spot virus (Trichovirus) are most frequently responsible for causing substantial damage to apple production (Nickel and Fajardo 2014).

Polyploidy has been used in horticulture as a breeding tool to enhance characteristics such as flower size, flower number, plant size, increased width-to-length ratio of leaves and leaf thickness. Nitrous oxide gas (N2O) was applied to seedlings and zygotes of many plants as a polyploidizing agent instead of colchicine treatments. N2O application to obtain polyploid grapevines has not been tested before and has been used as a new approach in viticulture. In this study, N2O was applied in order to induce mitotic ploidy of 41B grapevine rootstock and 'Trakya İlkeren', 'Gök Üzüm' and 'Ekşi Kara' grape cultivars (Vitis vinifera L.) single node cuttings. The application of N2O was performed for 24 h at 12000 kPa and 25°C, after the cuttings sprouted and hand 2-3 leaf developed at different stages of mitosis. The effects were evaluated by stomata traits (density, length, width, and area) and by flow cytometry (FC) analysis using a high-performance multi-laser system. The results showed that stomatas were morphological changed by N2O applications at a significant level. The stomata density was significantly decreased (-28%) in 41B rootstock; grapevine cultivars were also significantly affected. On the other hand, the FC analysis did not detect any polyploid induced plants. The present study reveals that polyploid frequency obtained using N2O was low in the tested rootstock and grapevine varieties; this was likely is due either to the sensitivity of the diploid rootstock and grape cultivars genotypes or to the N2Otreatment method.

The aim of this study was to compare three cryotechniques, namely, droplet vitrification (DV), vitrificationcryo-plate (VCP) and dehydration-cryo-plate (DCP) on proliferating meristems of two Musa (AAA, Cavendish Subgroup) genotypes. The experiment was set up as factorial completely randomized design for the two Musa accessions (IC 250462 cv 'Borjahaji' and EC 493718 cv 'Williams') in five different dehydration duration (30-150 min). Regrowth after cryopreservation in all treatments was predominantly of two types-shoot regeneration and callusing. In both the cultivars, shoot regrowth of non-frozen and LN treated meristems were not impacted significantly by duration of dehydration exposure, however, callus growth was significantly different with increasing time of exposure. The mean shoot regrowth for all dehydration treatments in Borjahaji was not significantly different. However, the VCP method in Williams, with mean shoot regrowth of 75.5 ± 2.8% was found to be significantly (P<0.05) better than that obtained from DV (63.1 ± 2.5%) and DCP (64.8 ± 1.9%). Quantitatively, highest shoot regrowth was obtained after 120 min PVS2 exposure in both cultivars, but using VCP method in Williams (86.7 ± 7.7%) and DV method in Borjahaji (73.1 ± 2.7%). Taking into consideration practical aspects for each technique, any of the three cryopreservation approaches can be used for cryobanking of proliferation meristems of Musa, as DV offers cost-effectiveness, while VCP and DCP have ease of explant handling, and DCP avoids chemical toxicity of PVS2.

A well-organized procedure was established for the conservation and distribution of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] for the first time, using alginate-encapsulated nodal segments (NSs) as synthetic seeds. The ideal beads were obtained through a combination of 2.5% sodium alginate and 75 mM calcium chloride with 84.40 ± 2.20% rate of shoot emergence. The maximum regeneration (88.84 ± 2.24%) from synthetic seeds was achieved on liquid 1/2Murashige and Skoog (MS) medium in comparison to its other formulations. Furthermore, superior frequency (91.09 ± 2.24%) of complete plantlet (having both shoots and roots) formation was achieved when synthetic seeds were cultured on liquid 1/2MS (1.5% sucrose) fortified with 1.0 mg L −1 N 6-benzyladenine plus 0.25 mg L −1 α-naphthalene acetic acid. Synthetic seeds could be effectively stored at low temperature (8 °C) up to 90 days with a survival rate of 52.38 ± 3.06%, whereas higher temperature (25 °C) did not support regeneration after 75 days of storage. The plantlets were successfully acclimatized to natural conditions with ~ 89% survival frequency. To bypass the time-consuming in vitro culture step after encapsulation, synthetic seeds were directly regrown into complete plantlets ex vitro on sand, soil, and vermicompost (1:1:1; w/w). Regeneration rate of 42.22 ± 2.22% was attained when NSs were pretreated on 1/2MS medium containing 4.0 mg L −1 indole-3-acetic acid for 24 h in dark, prior to encapsulation. The random amplified polymorphic DNA and intersimple sequence repeat fingerprinting profiles demonstrated genetic uniformity amongst the regenerated plantlets, in vitro mother plant, as well as in vivo wild plant.

Application of traditional knowledge for the utilization of natural products, particularly of plant origin, has gained importance in the past several decades. For the tribal people of different parts of India, there is limited alternative to herbal medicines, which they have used for time immemorial. Along with other medicinal plants, orchids are considered to be an important source of herbal medicine. Orchids are among the most diverse of the flowering plant families, with over 181 genera and 1229 species specific to India. Orchids, which are well known for their floriculture value, are also used for curing several diseases. Due to over-exploitation for medicinal use and for the cutflower trade, many orchids have become either rare or endangered. This review attempts to summarize the use of micropropagation to conserve Indian orchids of medicinal significance.

Populations of threatened plants are declining rapidly in natural habitats due to various anthropogenic activities. Reinforcement of the dwindling populations through reintroduction is a promising aspect for conservation of threatened plants. However, due to lack of standardized propagation methods of such plants, mass production of planting materials has become a challenge, thereby constraining the replenishment process. Identification of factors constraining the seed germination of threatened plants and addressing it effectively, are among the most cost-effective strategies for large-scale multiplication and subsequent conservation of the threatened species. Similarly, conventional low-cost vegetative propagation techniques such as grafting, air layering, and regenerating plantlets from root-suckers, apical meristems, and stem cuttings often prove more successful for multiplication than relatively costly micropropagation techniques. In this article, we present a few case studies on low-cost mass propagation techniques of threatened plant species of India through seed, stem/apical shoot cutting and airlayering, that helped in the restoration of the species.

The apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) are among the major viral species that infect apple trees and are responsible for substantial damage and loss. The aim of this study was to assess the effectiveness of dropletvitrification cryotherapy technique in eradication of ASGVand ASPV in Malus prunifolia "Marubakaido" apple rootstock plants. The axillary shoot tips were excised from ASGV-and ASPV-infected plants and incubated for one day on preculture medium. Subsequently, they were exposed to plant vitrification solution 2 (PVS2) for 0, 20, 40, 50, 60 or 80 min at 22°C and immersed in liquid nitrogen for 1 h. Cryopreserved shoot tips had the highest survival (75 and 65%) and regrowth (53 and 58%) percentages with PVS2 exposure duration of 20 and 40 min, respectively. After six months of growth in a greenhouse, RT-PCR analyses revealed that all the tested plants were free of ASPV and 70% were free of ASGV. The high eradication percentage of ASGV and ASPV indicated that the droplet-vitrification cryotherapy technique is an effective tool to produce ASGV-and ASPV-free propagating material of "Marubakaido" apple rootstock. Keywords Cryopreservation. Malus prunifolia. Virus-free material. Propagation Viruses have a direct impact on apple plant productivity and fruit quality and can enhance susceptibility to other pathogens (Guerra et al. 2012). Viruses belonging to the species Apple stem pitting virus (Foveavirus), Apple stem grooving virus (Capillivirus), and Apple chlorotic leaf spot virus (Trichovirus) are most frequently responsible for causing substantial damage to apple production (Nickel and Fajardo 2014).

Vanilla planifolia (Orchidaceae) is the natural source of vanillin, which is the most widely appreciated flavour compound in the world. Although vanilla is cultivated throughout the tropics, the species' areas of natural distribution are being severely reduced due to anthropogenic effects. As a result, ex situ preservation actions are necessary to safeguard the threatened diversity of this species. In that sense, cryopreservation of vanilla shoot-tips has been previously reported using the dropletvitrification technique. However, survival and plant regeneration following this protocol were low and not very reproducible. In this study, we evaluated a new alternative for the cryopreservation of vanilla germplasm by using root-tips as explants and following the vitrification and droplet-vitrification protocols. Survival (~60%) and further regeneration (~40%) were obtained using the dropletvitrification technique by preconditioning root-tips from in vitro propagated plants on semi-solid MS with 0.3 M sucrose (7 days), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (20 min) followed by glycerol-sucrose plant vitrification solution PVS3 (45 min on ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on aluminium foils strips. Tissues were rewarmed by plunging the aluminium foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and bud induction were efficiently achieved by culturing cryostored root-tips on MS added with 1 mg L-1 KIN or BAP. Plant regeneration was reached by transferring the induced buds to MS media. This protocol has great potential for long-term conservation of V. planifolia germplasm and of other vanilla relatives.

Vanilla planifolia (Orchidaceae) is the natural source of vanillin, which is the most widely appreciated flavour compound in the world. Although vanilla is cultivated throughout the tropics, the species' areas of natural distribution are being severely reduced due to anthropogenic effects. As a result, ex situ preservation actions are necessary to safeguard the threatened diversity of this species. In that sense, cryopreservation of vanilla shoot-tips has been previously reported using the dropletvitrification technique. However, survival and plant regeneration following this protocol were low and not very reproducible. In this study, we evaluated a new alternative for the cryopreservation of vanilla germplasm by using root-tips as explants and following the vitrification and droplet-vitrification protocols. Survival (~60%) and further regeneration (~40%) were obtained using the dropletvitrification technique by preconditioning root-tips from in vitro propagated plants on semi-solid MS with 0.3 M sucrose (7 days), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (20 min) followed by glycerol-sucrose plant vitrification solution PVS3 (45 min on ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on aluminium foils strips. Tissues were rewarmed by plunging the aluminium foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and bud induction were efficiently achieved by culturing cryostored root-tips on MS added with 1 mg L-1 KIN or BAP. Plant regeneration was reached by transferring the induced buds to MS media. This protocol has great potential for long-term conservation of V. planifolia germplasm and of other vanilla relatives.

Polyploidy has been used in horticulture as a breeding tool to enhance characteristics such as flower size, flower number, plant size, increased width-to-length ratio of leaves and leaf thickness. Nitrous oxide gas (N2O) was applied to seedlings and zygotes of many plants as a polyploidizing agent instead of colchicine treatments. N2O application to obtain polyploid grapevines has not been tested before and has been used as a new approach in viticulture. In this study, N2O was applied in order to induce mitotic ploidy of 41B grapevine rootstock and 'Trakya İlkeren', 'Gök Üzüm' and 'Ekşi Kara' grape cultivars (Vitis vinifera L.) single node cuttings. The application of N2O was performed for 24 h at 12000 kPa and 25°C, after the cuttings sprouted and hand 2-3 leaf developed at different stages of mitosis. The effects were evaluated by stomata traits (density, length, width, and area) and by flow cytometry (FC) analysis using a high-performance multi-laser system. The results showed that stomatas were morphological changed by N2O applications at a significant level. The stomata density was significantly decreased (-28%) in 41B rootstock; grapevine cultivars were also significantly affected. On the other hand, the FC analysis did not detect any polyploid induced plants. The present study reveals that polyploid frequency obtained using N2O was low in the tested rootstock and grapevine varieties; this was likely is due either to the sensitivity of the diploid rootstock and grape cultivars genotypes or to the N2Otreatment method.

The apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV) are among the major viral species that infect apple trees and are responsible for substantial damage and loss. The aim of this study was to assess the effectiveness of dropletvitrification cryotherapy technique in eradication of ASGVand ASPV in Malus prunifolia "Marubakaido" apple rootstock plants. The axillary shoot tips were excised from ASGV-and ASPV-infected plants and incubated for one day on preculture medium. Subsequently, they were exposed to plant vitrification solution 2 (PVS2) for 0, 20, 40, 50, 60 or 80 min at 22°C and immersed in liquid nitrogen for 1 h. Cryopreserved shoot tips had the highest survival (75 and 65%) and regrowth (53 and 58%) percentages with PVS2 exposure duration of 20 and 40 min, respectively. After six months of growth in a greenhouse, RT-PCR analyses revealed that all the tested plants were free of ASPV and 70% were free of ASGV. The high eradication percentage of ASGV and ASPV indicated that the droplet-vitrification cryotherapy technique is an effective tool to produce ASGV-and ASPV-free propagating material of "Marubakaido" apple rootstock. Keywords Cryopreservation. Malus prunifolia. Virus-free material. Propagation Viruses have a direct impact on apple plant productivity and fruit quality and can enhance susceptibility to other pathogens (Guerra et al. 2012). Viruses belonging to the species Apple stem pitting virus (Foveavirus), Apple stem grooving virus (Capillivirus), and Apple chlorotic leaf spot virus (Trichovirus) are most frequently responsible for causing substantial damage to apple production (Nickel and Fajardo 2014).

The water content of dormant Malus buds is comparable throughout the stages of the cryopreservation protocol, regardless of whether they come from cultivars that survive cryopreservation well or poorly. However, thermal analysis of dehydrated and frozen/thawed buds suggests that the distribution of water within them is different, with those from cultivars that recover poorly from cryopreservation retaining a greater bulk volume of freezable water (estimated by simple thermal analysis [STA]) than tolerant cultivars. Implications for the design of successful cryopreservation protocols for dormant buds are discussed.

Dendrobium chryseum is an endangered epiphytic and lithophytic orchid species having medicinal and ornamental value. The objective of the present study is to develop micropropagation protocol for D. chryseum. The immature seeds of D. chryseum were used as an explant for the micropropagation. Protocorms were developed within 90 days in Murashige and Skoog (MS) medium without plant growth regulators. Protocorms were differentiated into micro shoots after 30 days of transfer to MS medium supplemented with 2 mg/L Benzyl amino purine (BAP), 1 mg/L kinetin and 10% coconut water. Among eleven different combinations of plant growth regulators (BAP, á-Naphthalene acetic acid (NAA), Adenine sulphate, Kinetin and coconut water) treated for shoot multiplication, maximum number of shoots were obtained in 0.5 mg/L BAP + 0.5 mg/L NAA (5.8 ± 0.53 SE shoots per explant). Longest shoot length was recorded in MS medium with Adenine sulphate (1 mg/L) (2.54 ± 0.03 cm SE). Root induction was carried out ...

Woodfordia fruticosa (L.) Kurz is a woody medicinal shrub (Lythraceae) commonly known as the "fire flame bush." W. fruticosa plant parts either alone or whole plant have a long history of recommended use in the Indian medicine systems of Ayurveda, Unani, and Siddha (AUS). This plant is prominently known for its pharmacological properties, viz., antimicrobial, anti-inflammatory, anti-peptic ulcer, hepatoprotective, immunomodulatory, antitumor, cardioprotective, analgesic, and wound healing activities. Its important phyto-constituents, woodfordin C, woodfordin I, oenothein B, and isoschimacoalin-A, exhibit in vitro or in vivo physiological activities beneficial to human health. As the plant is a rich storehouse of phyto-constituents, it is indiscriminately used in its wild habitats. Moreover, due to very poor seed viability and difficult-to-root qualities, it is placed under IUCN list of endangered plant species. For W. fruticosa, biomass production or to its conservation by in vitro regeneration is the best feasible alternative. Till date, only few important in vitro regeneration methods are reported in W. fruticosa. ISSR molecular markers based clonal fidelity and Agrobacterium-mediated transformation has been demonstrated, indicating that W. fruticosa is amenable to genetic manipulation and genome editing studies. This review presents concise summary of updated reports on W. fruticosa phyto-constituents and their biological activities, while a critical appraisal of biotechnological interventions, shortcomings, and factors influencing such potential areas success was presented. The unexplored gaps addressed here are relevant for W. fruticosa scientific innovations yet to come. In this paper, for the first time, we have presented a simple and reproducible protocol for synthetic seed production in W. fruticosa. Key points • Critical and updated records on W. fruticosa phytochemistry and its activities • In vitro propagation and elicitation of secondary metabolites in W. fruticosa • Key bottlenecks, in vitro flowering, value addition, and outlook in W. fruticosa Keywords Fire flame bush • Woodfordin • Pharmacological studies • Biotechnological cutting-edge tools • Synthetic seeds • Metabolic engineering Mahender Aileni and Mallesham Bulle contributed equally to this work.

A liquid overlay culture system in micropropagation improved shoot proliferation in many species. In the present investigation, we have developed an enhanced shoot proliferation protocol using the double phase culture system (DPS) for Vanilla andamanica, an endemic species of the Andaman and Nicobar Islands recognized under the vulnerable category by the IUCN. In vitro generated nodal explants were used for shoot proliferation experiments and were tested in Murashige and Skoog medium augmented with various cytokinins (BAP, KIN, 2iP) and auxins (IBA, NAA, IAA) to produce a maximum of 3.24 ± 0.08 shoots per explant in 5 µM BAP and 5.23±0.67 roots per shoot in basal MS medium. A remarkable increase in shoot regeneration was observed when the nodal explant was cultured on DPS system with optimum cytokinin (5 µM BAP). On evaluation of the influence of DPS and conventional single-phase systems (SPS), (solid medium) exhibits an improved multiplication rate on a DPS with optimal BAP (5µM) w...

The importance of tissue culture for clonal propagation in agriculture continues to increase each year. In general, commercial use of tissue culture propagation has been limited to crops that have a high per-unit value, such as ornamentals and fruit and nut trees. A lowcost, high-volume propagation system is not available, but could be of significant value to medium per-unit value crops such as lettuce, celery, and many others (Table 1). For these crops, highvolume propagation potential of somatic embryogenesis combined with formation of synthetic seeds for low-cost delivery would open a new field for clonal propagation. Candidate crops for synthetic seed production can be classified into two categories: 1) those that have a strong technological basis, such that high quality somatic embryos can currently be produced, and 2) those with a strong commercial basis. The latter category of crops are those in which seed costs are high because of fertility problems, gamete instability, labo...

We propose to develop transgenic grapevine rootstocks resistant to Pierce's disease (PD) using new transformation protocols for the commercially relevant grapevine rootstocks '101-14' and '1103-P.' Our genetic approach involves developing transgenic rootstocks that deliver therapeutic proteins like NE-CB that can protect an untransformed scion from PD. Our approach is to improve grapevine transformation, exploring the possibility of in planta transformation of shoot apical meristems (SAM). We have successfully isolated SAM tissues from grapevine rootstocks '101-14' and '1103-P' and from 'Thompson Seedless' as a control. These SAM tissues are being tested for regeneration potential using different media and hormone concentrations that have worked in other grapevine cultivars. The outcome of this research would be the successful development of a more efficient and rapid transformation system for commercially relevant grapevine rootstock and scion varieties using SAM. LAYPERSON SUMMARY Current strategies for Pierce's disease (PD) control emphasize deploying transgenic rootstocks that deliver PD control to the untransformed scion. Present technology must be improved to effectively generate transgenic, PD-resistant versions of preferred commercial rootstocks like '101-14' and '1103-P.' We propose to refine different components of the current transformation protocol for greater efficiency, allowing more rapid production and testing of plants. We will transform rootstock '110-14' with an existing vector that incorporates a chimeric protein (NE-CB) to provide enhanced resistance to PD in the scion. We have already initiated development of a more efficient and rapid transformation system for grape rootstocks.

Various concentrations of sodium alginate and calcium chloride were tested in order to optimize the shape, and texture of synthetic seeds developed through the encapsulation of Protocorm Like Bodies (PLBs) of Asparagus racemosus. Sodium alginate (2.5%) and CaCl2.2H2O (100 mM) solutions were found to be the optimal concentration for production of firm, transparent and uniform synthetic seeds. The beads showed 94.9% germination on MS basal medium within three weeks. The germinated seedlings formed complete plantlet having well developed root-shoot system. The developed protocol for synthetic seed production through PLBs can be used for conservation of A. racemosus.

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 transgenicmediated 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 present investigation highlighted the role of sodium alginate (Na-alginate) and calcium chloride (CaCl2) treatments on encapsulation of sugarcane (Saccaharum officinarum var. NSG-59) somatic embryos to produce synthetic seeds. Progress in the protocol for direct recovery of plants from man-made seeds under sterile conditions is of major importance. Although it is possible to produce a huge number of plants through tissue culture via embryogenesis or multiple shoot cultures, but their delivery is quite inconvenient. In vitro study pertaining to evaluate the efficient synthetic seeds formation from sugarcane somatic embryos was done in Plant Tissue Culture Lab, Department of Botany, University of Sargodha. Encapsulation of somatic embryos was achieved by using Na-alginate (as gelling agent) and CaCl2 (as complexing agent) respectively through dropping method under sterile conditions (inside Laminar air flow cabinet). Different concentrations of Na-alginate (0.5%, 1%, 2%, 2.5%, 3%, 3.5%, and 4%) and CaCl2 (50mM, 70mM, 90mM, 100mM, 150mM and 200mM) were applied in current process.The gelling matrix of 3% Na-alginate and complexing agent of 100mM CaCl2 yielded firm, clear, isodiametric and compact beads formation. It was observed that duration of Na-alginate treatment on bead formation has no effect however, CaCl2 treatment with 100mM Na-alginate for 15 minutes was more appropriate for isodiametric and compact beads formation. The synthetic seeds provide feasible handling, transport and large scale production of viable plants of sugarcane by direct sowing of synthetic seeds in the soil.

One of the obstacles for the production of Orchidaceae plants is the seed propagation. Thus, in in vitro cultivation, luminosity and the type of sealing of the bottles are factors that influence germination and initial development of orchids. Therefore, the objective was to evaluate the light condition, the type of sealing of the bottles and the evaluation time in germination and initial in vitro development of Dendrobium nobile Lindl. After sowing, cultures were subjected to two irradiance conditions according to the sealing of the flasks: 7.0 µmol m-2 s-1 (threaded cap) and 19.0 µmol m-2 s-1 (plastic film) and two light conditions: white fluorescent lamp and red fluorescent lamp. At 30, 60 and 90 days, the percentage of germination, survival, of protocorms in stage 1, stage 2, stage 3 and stage were evaluated 4. DIC was used, in a sub-divided plot scheme for 30 and 60 days after sowing and in a 2 x 2 factorial scheme at 90 days. The highest germination percentage was observed when...

The current study aimed to establish an efficient in vitro propagation and conservation protocol for Brunfelsia pauciflora plant. The effect of plant growth regulators types (BAP, Kin, 2ip and NAA) and concentrations at 0.0, 1.0, and 2.0 mg/l were studied during establishment phase in comparing with hormonal-free medium. In vitro proliferated shootlets were encapsulated using various levels of sodium alginate at 1, 2 and 3% (W/V) and calcium chloride at 2.5,5 and 10 %. Regrowth ability of encapsulated explants was evaluated after conservation for 3, 5, and 7 weeks and compared with non-encapsulated buds. The findings of the current study proved that during establishment phase, applying BAP at 2.0 mg/l gave the best results during the three successive subcultures. The best treatment 2ip (2.0mg/l) + BAP (2.0mg/l) + NAA (2.0mg/l) that was considered ideal propagation protocol for shooting and rooting of Brunfelsia. Successful plant recovery from encapsulated explants using low concentr...

The present investigation highlighted the role of sodium alginate (Na-alginate) and calcium chloride (CaCl2) treatments on encapsulation of sugarcane (Saccaharum officinarum var. NSG-59) somatic embryos to produce synthetic seeds. Progress in the protocol for direct recovery of plants from man-made seeds under sterile conditions is of major importance. Although it is possible to produce a huge number of plants through tissue culture via embryogenesis or multiple shoot cultures, but their delivery is quite inconvenient. In vitro study pertaining to evaluate the efficient synthetic seeds formation from sugarcane somatic embryos was done in Plant Tissue Culture Lab, Department of Botany, University of Sargodha. Encapsulation of somatic embryos was achieved by using Na-alginate (as gelling agent) and CaCl2 (as complexing agent) respectively through dropping method under sterile conditions (inside Laminar air flow cabinet). Different concentrations of Na-alginate (0.5%, 1%, 2%, 2.5%, 3%, 3.5%, and 4%) and CaCl2 (50mM, 70mM, 90mM, 100mM, 150mM and 200mM) were applied in current process.The gelling matrix of 3% Na-alginate and complexing agent of 100mM CaCl2 yielded firm, clear, isodiametric and compact beads formation. It was observed that duration of Na-alginate treatment on bead formation has no effect however, CaCl2 treatment with 100mM Na-alginate for 15 minutes was more appropriate for isodiametric and compact beads formation. The synthetic seeds provide feasible handling, transport and large scale production of viable plants of sugarcane by direct sowing of synthetic seeds in the soil.

From the medicinal orchid Dendrobium chryseum Rolfe, which is used in traditional and folk Chinese medicine, the protocorms were raised in Murashige and Skoog (MS) media in three strengths, full strength (FMS), half strength (1/2 MS), and quarter strength (1/4 MS), with or without the phytohormones 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) and coconut water (CW). The comparative cytotoxic activities of the wild and in vitro-raised protocorms were evaluated in human cervical carcinoma (HeLa) and human glioblastoma (U251) cell lines by MTT assay. In in vivo and in vitro, the methanol extracts of D. chryseum showed significant cytotoxic activities. Significant growth inhibition (%) and potent IC50 values were demonstrated in HeLa cell lines (49.79% (210.5 μg/mL) for in vitro-raised Dendrobium chryseum (DCT) versus 46.97% (226.5 μg/mL) for wild Dendrobium chryseum (DCW)). Similarly, activities against U251 cell lines exhibited also significant inhibition (28.76% (612....

In the unique, highly diverse ecosystem of Nepal, Orchidaceae is the largest family of flowering plants. This family comprises an estimated 454 species, of which 90 are reported to have beneficial medicinal values and 18 are endemic. Orchids are essential in the maintenance of Nepal's biodiversity, due to their epiphytic association with host species, mycorrhizal associations, and pollinators. Medicinal orchids are also important for their commercial value, and many species contain important chemical compounds, including alkaloids, bibenzyl derivatives, flavonoids, phenanthrenes, and terpenoids. Climate change, deforestation, over-exploitation, and illegal trade all pose serious threats to the orchid flora of Nepal, making their conservation a serious concern. Their germplasm is used in Nepal, to conserve both the threatened and commercially important medicinal orchids, ex situ. Various techniques of in vitro culture are used to propagate more than 30 species of medicinal orchids for large scale production. Community awareness and orchid species restoration programs have been conducted with the local people in central Nepal. Further, local people are encouraged to cultivate artificially propagated species as a source of long-term economic benefit.

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 transgenicmediated 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.

Introduction Physalis peruviana L. is a species from the family Solanaceae and genus Physalis, commonly known as Cape gooseberry (also known as physalis, golden berry, etc.). It is an herbaceous, semishrub, perennial plant, native to tropical Peru and other warm temperate and subtropical regions throughout the world (Ramirez et al., 2013). Cape gooseberry resembles tomato in flavor and appearance, though the sour and sweet taste is richer in this tropical fruit. This plant has increasing popularity due to its nutritional and medicinal values. Cape gooseberry is a source of health-related compounds found in the fruits and other parts of the plant, including leaves and stems. It has been widely used for treating diabetes, hepatitis, ulcers, and several other diseases (Mayorga et al., 2002; Arun and Asha, 2007). It also contains high levels of vitamins A, C, and B-complex, as well as compounds with antiinflammatory and antioxidant properties (Strik, 2007). Wu et al. (2009) reported that supercritical carbon dioxide extracts of P. peruviana leaves induced cell cycle arrest and apoptosis in human lung cancer cells. Similarly, Çakir et al. (2014) reported that phenolic content of ethanolic leaf extracts (100 µg mL-1) possessed high cytotoxic effects on HeLa cells (an immortal cell line). The use of tissue culture methods for the selection of the best cultivars for agricultural practices and clonal propagation might be conducive for micropropagation, especially when taking into account high consumption due to value-added natural compounds, limited plant production, and requirements of manpower, transport, and storage (Rodrigues et al., 2013a). Though P. peruviana was reported as recalcitrant for shoot organogenesis through adventitious regeneration from leaf explants (Torres, 1991), there are a limited number of in vitro regeneration studies concerning the micropropagation of P. peruviana (Rodrigues et al., 2013b), not containing information about the synergistic effects of plant growth regulator (PGR) combinations on growth parameters. Several studies on in vitro propagation of other Physalis species, including P. minima, P. ixocarpa, and P. pruinosa,

The aims of this study were to determine the seed size and the effects of temperature and light on germination of Plantago algarbiensis and P. almogravensis seeds, two endemic species from Portugal that are at risk of extinction. The temperatures tested were a constant 15 or 25°C and an alternating temperature regime of 25/15°C, combined with 16 / 8 h light / dark photoperiod (light) or constant darkness. The results showed that P. algarbiensis and P. almogravensis have non-dormant seeds. Although P. algarbiensis seeds are larger and heavier, seeds of both species have similar germination requirements. The best germination results were obtained at 15°C under either light or darkness, along with the shortest mean germination time. The results are crucial for developing conservation strategies for these Plantago species.

The aims of this study were to determine the seed size and the effects of temperature and light on germination of Plantago algarbiensis and P. almogravensis seeds, two endemic species from Portugal that are at risk of extinction. The temperatures tested were a constant 15 or 25°C and an alternating temperature regime of 25/15°C, combined with 16 / 8 h light / dark photoperiod (light) or constant darkness. The results showed that P. algarbiensis and P. almogravensis have non-dormant seeds. Although P. algarbiensis seeds are larger and heavier, seeds of both species have similar germination requirements. The best germination results were obtained at 15°C under either light or darkness, along with the shortest mean germination time. The results are crucial for developing conservation strategies for these Plantago species.