Explant Type Research Articles

Implementation of biotechnological techniques for the propagation and introduction of Trichosanthes kirilowii Maxim

Trichosanthes kirilowii Maxim. is classified among the 50 most important medicinal plants used in traditional Asian medicine. This species is endemic and reproduction from seeds in the conditions of Central Europe is inefficient. The present study focused on the implementation of effective propagation of T. kirilowii, including standardisation of seed germination conditions, reproduction of the plant based on micropropagation, and introduction to field conditions. Four types of explants were tested on six types of media in various variants of culture conditions. Additionally, anatomical and histological analyses of specimens obtained through the in vitro cultivation were carried out, with particular emphasis on the site of initiation of aboveground and underground shoots. Microscopic analyses showed that the T. kirilowii shoots and roots obtained in the in vitro culture formed in the process of organogenesis via direct regeneration. Even though the callus did not participate in the regeneration of the entire plant, the micropropagation protocol proposed in the study was efficient: 6–8 shoots were obtained from 1 explant. The entire micropropagation process from the explant to an adult individual growing in the ground lasted 122 days. The protocol for T. kirilowii micropropagation in vitro cultures provides a fast, efficient, and robust approach to propagation of this species. The biotechnological applications in the reproduction of T. kirilowii will help not only to increase the number of daughter plants but also to multiply individuals of the appropriate sex, which will increase the probability of production of fruits in this dioecious species.

The Effect of Explant Type and Growth Regulators on Callus Induction in Passiflora quadrangularis

The Effect of Explant Type and Growth Regulators on Callus Induction in Passiflora quadrangularis

In Vitro Plant Regeneration of Sulla coronaria from Floral Explants as a Biotechnological Tool for Plant Breeding

Sulla coronaria L. Medik., a biennial forage legume typical of the Mediterranean basin, plays a significant role in foraging systems due to its high nutritional value, ability to increase ruminant live weight, and potential to reduce methane emissions. However, its allogamous nature complicates genetic improvement and the development of new varieties with desired traits. Given these challenges, this study aims to develop, for the first time, a successful protocol for the in vitro meristematic shoot regeneration of S. coronaria. The experiment utilizes four different flower explants (anther with filament, ovary, petals, and whole immature flower) collected from twenty distinct S. coronaria biotypes with three plant growth regulator (PGR) combinations and under both light and dark conditions. In terms of the regeneration response, the key factors appear to be the combinations of PGRs and the type of explant used. The interactions between all the factors do not seem to be significant.

OBTAINING ASEPTIC CULTURE IN VITRO OF RHODIOLA SEMENOWII L. FROM VARIOUS TYPES OF EXPLANTS

In vitro cultivation of callus cells allows for the stable production of secondary metabolites, being an alternative to the use of medicinal plant biomass from natural populations. Standardization of cultivation protocols and technological processes for the synthesis of secondary metabolites in vitro conditions determines significant interest in the use of tissue culture methods (PTC – Plant Tissue Culture). The aim of the study was to obtain an aseptic culture of Rhodiola semenowii in vitro from various types of explants, as well as the effect of the hormonal composition of the nutrient medium on the induction of callusogenesis in the culture of isolated organs of R. semenowii. As a result of the research, the optimal mode of obtaining R. semenovii aseptic cultures from various explants was determined. It has been shown that the use of 70% EtOH in combination with 0.01% HgCl2 or 20% NaClO makes it possible to obtain aseptic cultures from any type of R. semenovii explants. It has been established that the type of explant is R. semenovii influenced both the frequency of callus formation and the beginning of its formation. The least optimal types of R. semenovii explants are stem and rhizome segments, the frequency of callusogenesis varied from 5.8% to 75.6%. The induction of callusogenesis also depended on the hormonal composition of the nutrient medium: the highest percentage of callus formation in all types of explants was on media containing 0.5mg L−1 thidiazuron (TDZ) + 1.0 mg L−1 1-naphthaleneacetic acid (NAA) and 0.5mg L−12,4D + 2.0 mg L−1 6-Benzylaminopurine (BAP).

Establishment of Agrobacterium-Mediated Transient Transformation System in Desert Legume Eremosparton songoricum (Litv.) Vass.

Eremosparton songoricum (Litv.) Vass. is a desert legume exhibiting extreme drought tolerance and the ability to withstand various harsh environments, making it a good candidate for investigating stress tolerance mechanisms and exploring valuable stress-resistant genes. However, the absence of a genetic transformation system for E. songoricum poses significant limitations for functionally validating these stress-resistant genes in situ. In this study, we developed an Agrobacterium-mediated transient transformation system for E. songoricum utilizing the β-glucuronidase (GUS) gene as a reporter. We investigated three types of explants (seedlings, assimilated branches and callus) and the effects of different Agrobacterium strains, seedling ages, OD600 values, acetosyringone (AS) concentrations, sucrose concentrations and infection times on the transformation efficiency. The results reveal that the optimal transformation system was infecting one-month-old regenerating assimilated branches with the Agrobacterium strain C58C1. The infection solution comprised 1/2 MS medium with 3% sucrose and 200 μM AS at an OD600 of 0.8, infection for 3 h and then followed by 2 days of dark cultivation, which achieving a maximum transformation rate of 97%. The maximum transformation rates of the seedlings and calluses were 57.17% and 39.51%, respectively. Moreover, we successfully utilized the assimilated branch transient transformation system to confirm the role of the previously reported transcription factor EsDREB2B in E. songoricum. The overexpression of EsDREB2B enhanced drought tolerance by increasing the plant's reactive oxygen species (ROS) scavenging capacity in situ. This study established the first transient transformation system for a desert legume woody plant, E. songoricum. This efficient system can be readily applied to investigate gene functions in E. songoricum. It will expedite the exploration of genetic resources and stress tolerance mechanisms in this species, offering valuable insights and serving as a reference for the transformation of other desert plants and woody legumes.

Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters-such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride-were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

EXPRESIÓN EMBRIOGÉNICA DIRECTA EN Agave cupreata

<p><strong>Background:</strong> <em>Agave cupreata</em> is a specie that can only reproduce by seeds and could take up to 15 years until flowering, placing it at risk of extinction due to its agroindustrial exploitation to produce mezcal. <strong>Objective:</strong> To evaluate the type of explant, the vitamin complex and 2,4-D concentrations on the direct embryogenic response. <strong>Methodology:</strong> Explants of leaf, root and stem sections were evaluated. Also, the vitamins MS (Murashige and Skoog, 1962) and L2 (Phillips and Collins, 1979), five concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), (0.0, 0.1, 0.3, 0.6 and 0.9 mg L<sup>-1</sup>) and six concentrations of indole-3-acetic acid (IAA), (0.0, 0.1, 0.5, 1.0, 2.0 and 3.0 mg L<sup>-1</sup>) were assayed for the expression of somatic embryos directly. <strong>Results: </strong>The leaf explant, the vitamin L2 complex, and the concentration of 0.9 mg L<sup>-1</sup> of 2,4-D and 0.5 mg L<sup>-1</sup> of IAA turned out to be the most efficient for the formation of pro-embryogenic masses and the expression of direct somatic embryos in the globular state. <strong>Implications:</strong> These results lay the groundwork for the future regeneration of <em>A. cupreata</em> plants via direct somatic embryogenesis, potentially facilitating their conservation and mass propagation. This contributes to the sustainability of mezcal; boosts the local economy, preserves biodiversity, and ensures the continued production of this endangered species. <strong>Conclusions:</strong> For the first time, the successful expression of somatic embryos directly from <em>A. cupreata</em> explants was evidenced.</p>

12209 Validation Of A Novel Placenta Explant Protocol For Studying Circadian Rhythms In The Mouse Placenta In Early, Mid And Late Pregnancy

Abstract Disclosure: J.S. Lee: None. A.M. Yaw: None. H.M. Hoffmann: None. Introduction: The placenta serves as an interface facilitating the exchange of essential nutrients and oxygen between the mother and fetus. Preeclampsia, a complication of pregnancy driven by placental dysfunction, is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality. While deregulation of the genes driving circadian rhythm has been associated with preeclampsia, the role of circadian rhythms in the placenta largely remains unknown. To address this gap of knowledge, our goal was to establish and validate an organotypic ex vivo preparation to study placenta circadian rhythms at gestation day (GD) 10, GD14, and GD18 in the mouse. Methods: To establish an organotypic placenta culture GD10, GD14, and GD18 placental explants, we sectioned the placenta into 350μm sections using a vibratome. The explants were prepared from the validated circadian Per2::Luciferase reporter mouse, allowing automated circadian rhythms recording of the explants in a LumiCycle. We compared explant success rate and rhythm quality in 2x2mm section vs whole slice explants at GD14. Using hematoxylin and eosin (H&E) staining we validated the explants to be decidua which is strictly of maternal origin, labyrinth which is strictly of fetal origin, and the junctional zone which consists of binucleated cells fused of maternal and fetal cells. Results: Through H&E staining, we confirmed the decidua is enriched with neutrophils, the junctional zone contains spongiotrophoblasts and glycogen trophoblasts, and the labyrinth has red blood cells. Both explant types were enriched with >80% of cells from the targeted region. We next established a protocol to record Per2::Luciferase rhythms for <6 days. The fragility of the GD10 placentas required the whole explant protocol, whereas the GD18 2x2mm sectioned explants produced strong rhythms with a high success rate. At GD14, the whole explants resulted in stronger rhythms with higher amplitude and higher goodness of fit compared to the 2x2mm explants. Conclusion: We found that depending on GD recording success differs. Specifically, the whole placental explant provides a more robust and reliable model for studying circadian rhythms in GD10 and GD14, whereas 2x2mm explants are successful at GD18. This novel placental preparation protocol will be valuable in studying disease- and drug-induced changes to circadian rhythms in a placenta layer-specific manner in the mouse. Presentation: 6/1/2024

Importance of Media Composition and Explant Type in Cannabis sativa Tissue Culture.

Producing uniform Cannabis sativa (Cannabis) for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from "mother plants" in the cannabinoid industry, which also faces significant limitations. Cannabis tissue culture (TC) methods have been developed to overcome these challenges, but the long-term health and maintenance of Cannabis explants in TC have been largely overlooked in previous studies. The current study focused on the development of an efficient and optimized micropropagation protocol covering the entire process, with a specific focus on the health and performance in the multiplication stage. Multiplication media were formulated hormone-free to avoid longer-term vitrification issues, resulting in single-main-shoot cultures rather than multiple-shoot cultures. This instigated the use of stage II explant types different from the standard shoot tips previously used for multiple shoot cultures. Multiplication media were further improved from the basal salt composition via nitrogen and calcium additives. The optimized protocol was used on eight diverse Cannabis cultivars to test its applicability across various genetic backgrounds. Results indicated that the protocol was effective for conservation purposes across all cultivars and achieved good long-term multiplication rates for some but not all. The outcomes of this study mark a significant stride towards an efficient Cannabis TC methodology ready for more comprehensive industrial applications.

An Overview of Pharmaceutical Applications and In vitro Micropropagation Techniques for Rare and Endangered Plant Species

Many rare and endangered plant species possess valuable secondary metabolites with pharmacological applications. These bioactive compounds are often integral to traditional medicine systems, highlighting the cultural significance of these plants. The health benefits of many medicinal species are not fully validated by contemporary scientific research, and some may be facing extinction due to habitat loss, overharvesting or climate change. This situation highlights the urgent need for effective conservation strategies of the species and sustainable cultivation methods. Micropropagation is a valuable technique for producing large numbers of plants from a single explant, significantly aiding in the conservation and commercial cultivation of rare species. Among the various types of explants, shoot tips and nodal segments have been identified as the most effective explants for micropropagation. These explants can be induced to generate multiple shoots in Murashige and Skoog (MS) medium containing Benzyl aminopurine (BAP). Thidiazuron (TDZ), Kinetin (KIN), or 2,4-Dichlorophenoxyacetic acid (2,4-D) are commonly used in MS medium to promote shoot and root development in both direct and indirect organogenesis processes. Rooting of the plantlets was typically achieved using MS medium either supplemented with Indole-3-butyric acid (IBA) or devoid of auxins, depending on the species and the specific requirements for rooting.

Tea (Camellia sinensis L. O. Kuntze) callus development based on morphological and phytochemical traits applied by variations in plant growth regulators (PGRs)

A variation of the clonal propagation system for tea has been established through in vitro methods, utilizing shoots and leaves derived from primary explants of mature field-grown plants. The research aimed to assess the diversity of tea calluses based on morphological and phytochemical traits derived from leaf and shoot explants on MS media induced by the application of plant growth regulators, including auxin and cytokinin. The research was conducted at the Seed Technology Unit Tissue Culture Laboratory, Faculty of Agriculture, Jatinangor, from April 2022 to February 2023. The experiment employed MS-modified multiplication media and PGR, with BAP at 9 mg/L, TDZ at 1.0 mg/L, Zeatin at 0.1 mg/L, NAA at 0.01 mg/L, and 1 mg/L. Results indicated that applying PGR, auxin, and cytokinin with MS media influenced morphological and phytochemical traits of tea's callus derived from leaves and shoots. Specifically, the treatment of 1 mg/L NAA + 1 mg/L BAP showed the best performance on callus weight and total phenolic and flavonoid contents compared to other treatments., i.e. 9.038% % and 0.05% %, respectively. The success of clonal propagation in tea through in vitro methods is expected to increase by carefully selecting the appropriate source and type of explant, planting media, PGR, and addressing potential contaminants.

Conquering Limitations: Exploring the Factors that Drive Successful Agrobacterium-Mediated Genetic Transformation of Recalcitrant Plant Species.

Agrobacterium-mediated transformation is a preferred method for genetic engineering and genome editing of plants due to its numerous advantages, although not all species exhibit transformability. Genetic engineering and plant genome editing methods are technically challenging in recalcitrant crop plants. Factors affecting the poor rate of transformation in such species include host genotype, Agrobacterium genotype, type of explant, physiological condition of the explant, vector, selectable marker, inoculation method, chemical additives, antioxidative compounds, transformation-enhancing compounds, medium formulation, optimization of culture conditions, and pre-treatments. This review provides novel insights into the key factors involved in gene transfer facilitated by Agrobacterium and proposes potential solutions to overcome existing barriers to transformation in recalcitrant species, thereby contributing to improvement programs for these species. This review introduces the key factors that impact the effectiveness of a molecular breeding program using Agrobacterium-mediated transformation, specifically focusing on recalcitrant plant species.

Agrobacterium-mediated transformation of B. juncea reveals that BjuLKP2 functions in plant yellowing.

A stable Agrobacterium-mediated transformation system was constructed for B. juncea, and BjuLKP2 was overexpressed, leading to plant yellowing. A stable and efficient transformation system is necessary to verify gene functions in plants. To establish an Agrobacterium-mediated transformation system for B. juncea, various factors, including the explant types, hormone combination and concentration, infection time and concentration, were optimized. Eventually, a reliable system was established, and two BjuLKP2 overexpression (OE) lines, which displayed yellowing of cotyledons, shoot tips, leaves and flower buds, as well as a decrease in total chlorophyll content, were generated. qRT-PCR assays revealed significant upregulation of five chlorophyll synthesis genes and downregulation of one gene in the BjuLKP2 OE line. Furthermore, antioxidant capacity assays revealed reduced activities of APX, CAT and SOD, while POD activity increased in the BjuLKP2 OE26. Additionally, the kinetic determination of chlorophyll fluorescence induction suggested a decrease in the photosynthetic ability of BjuLKP2 OE26. GUS assays revealed the expression of BjuLKP2 in various tissues, including the roots, hypocotyls, cotyledons, leaf vasculature, trichomes, sepals, petals, filaments, styles and stigma bases, but not in seeds. Scanning electron revealed alterations in chloroplast ultrastructure in both the sponge and palisade tissue. Collectively, these findings indicate that BjuLKP2 plays a role in plant yellowing through a reduction in chlorophyll content and changes in chloroplasts structure.

Influences of Thidiazuron Concentrations and Exposure Duration on Axillary Shoot Proliferation of Faba Bean [Vicia faba (L.)] Genotypes

Background: Faba bean is one of the most important feed grain legume crops in the world. However, this crop is a recalcitrant species for in vitro regeneration. Methods: In this study, in vitro shoot proliferation of ten faba bean genotypes, that are classified according to their seed size into small, medium and large, were investigated in response to different light regimes and thidiazuron (TDZ) treatments. Result: Incubation under different light regimes, explant type as well as genotypes influenced seedling growth and axillary shoot multiplication of faba bean. The explants with single cotyledon and incubated under continuous darkness for 6 days resulted higher number of shoot than nodal explants. Medium size seeded genotype (ILB4347) produced the highest shoot and root length and shoot proliferation as compared with large (Luz) or small (Triple White) seeded genotypes. The effects of TDZ concentrations (2-10 mM/L) and exposure duration (10 and 20 days) on axillary shoot multiplication were investigated for the ten faba bean using cotyledonary explants. The overall mean of TDZ time exposure showed that treatment of 20 days exposure had higher number of shoot compare to 10 days exposure treatment with 3.77 shoot per explant. On the basis of genotype used, the three most responsive genotype in tissue culture were medium sized seeds genotypes (ILB4347, Hassawi 2 and Misr 3 with 4.76, 4.40 and 4.15 shoot per explant, respectively). ILB4347 were the most responsive genotype in tissue culture. Treatment of 8 mM TDZ after 20 days followed by 2 weeks on MS medium without PGRs generated the highest proliferation with 6.63 shoot per explant. These findings contribute towards effective in vitro propagation of faba bean.

An Efficient and Rapid Protocol for Somatic Shoot Organogenesis from Juvenile Hypocotyl-Derived Callus of Castor Bean cv. Zanzibar Green.

Aseptic seedlings of different ages derived from surface-sterilized mature seeds were applied as an explant source. Various explants such as 7- and 21-day-old hypocotyl fragments, 42-day-old nodal stem segments, and transverse nodal segments of stem, as well as leaf petioles, were cultured on the agar-solidified Murashige and Skoog (MS) basal medium supplemented with 0.1 mg/L IAA, 5 mg/L AgNO3 and different types and concentrations of cytokinin (1 mg/L zeatin, 0.25 mg/L thidiazuron (TDZ), and 5 mg/L 6-benzylaminopurine (6-BAP)). Consequently, it was found that 7- and 21-day-old hypocotyl fragments, as well as nodal stem segments obtained from adult aseptic seedlings, are characterized by a high explant viability and callus formation capacity with a frequency of 79.7-100%. However, the success of in vitro somatic shoot organogenesis was significantly determined not only by the culture medium composition and explant type but also depending on its age, as well as on the size and explant preparation in cases of hypocotyl and age-matched nodal stem fragments, respectively. Multiple somatic shoot organogenesis (5.7 regenerants per explant) with a frequency of 67.5% was achieved during 3 subcultures of juvenile hypocotyl-derived callus tissue on MS culture medium containing 0.25 mg/L TDZ as cytokinin source. Castor bean regenerants were excised from the callus and successfully rooted on ½ MS basal medium without exogenous auxin (81%). In vitro plantlets with well-developed roots were adapted to ex vitro conditions with a frequency of 90%.

Plantlet regeneration of Cucumis melo L. Glamour cv. using different types of cytokinin and explants

Plantlets or clones’ regeneration in Cucurbitaceae species can be performed in vitro by applying the tissue culture techniques. Hypocotyls, cotyledons, leaves, cotyledonary nodes and petioles can be used as the explants or starting materials to initiate the regeneration under laboratory condition. BAP (Benzyl aminopurine) and TDZ (Thidiazuron) were among the well-known cytokinin used in tissue culture as plant hormones to regulate the plant growth in vitro. The effectiveness of BAP and TDZ was investigated in this study to determine the effect of different types of cytokinin hormone on C. melo explants since both hormones might show differences in their activity towards C. melo explants. This study found that BAP was superior at all used concentrations compared to TDZ during most of the growing stage of the explants (cotyledons and hypocotyls). Based on this finding, the experiment was repeated by using different types of explants which were nodal explants, petioles and young shoots along with cotyledons and hypocotyls, by using BAP that showed excellent plantlet regeneration. Cotyledon and nodal explants successfully regenerated plantlets with excellent height and significant number of shoots compared to the rest of the explants. The plantlets from cotyledons and nodal explants were regenerated via direct regeneration without any formation of callus. The direct shoot regeneration was the preferred method for mass production via tissue culture because the chances of somaclonal variation is low. Contrarily, the hypocotyls, petioles and shoot tips mostly developed callus and only several treatments formed very small plantlets.

Scanning electron microscopic and morphological studies on in-vitro derived callus of low-chill peach (Prunus persica L. Batsch)

In this investigation the effect of collection seasons of explants (winter, spring and summer), type of explants (leaf disc and intermodal segments) and length of explants (0.5, 1.0 and 1.5 cm) for callusing in low-chill peach were standardized. The maximum callus induction (97.78%) in the low-chill peach was obtained from the intermodal segments of 0.5 cm in length used as an explant collected during spring season. The structural changes on the surface of the callus (5–7 weeks old yellowish green compact callus) during the progress of somatic embryogenesis of low-chill peach from the both intermodal segment as well as leaf disc derived callus were also examined with the use of scanning electron microscope (SEM). The SEM studies indicated that callus derived from internodal segment explant had the highest frequency of somatic embryos than callus from leaf discs. The SEM investigation, also demonstrated the sequential events/steps leading to low-chill peach somatic embryogenesis which was originating from somatic embryo mother cells through one unicellular pathway. Two types of calli were morphologically distinguished in both leaf disc and intermodal segment generated callus and these were the compact, well organized yellowish green embryogenic callus, containing large number of small, rich cytoplasmic, starch containing meristematic cells and soft and unorganized non-embryogenic callus containing sparsely cytoplasmic, vacuolated, and large cells devoid of metabolic reserves. The present SEM studies clearly demonstrated that somatic cells from peach explants generated callus could develop into fully differentiated somatic embryos through the characteristic embryological patterns of differentiation.

Application of various growth regulators in clonal micropropagation of black currant (Ribes Nigrum L.)

Currently, the optimization and development of new techniques for microclonal reproduction of black currant (Ribes nigrum L.) are one of the traditional objects of research of domestic and foreign scientists. It is noted that the success of reproduction depends on a number of factors: the timing of introduction into culture in vitro, the type of explants, the sterilizing agent, the composition of the nutrient medium. At the same time, the survival and growth of explants at each stage of reproduction depends not only on the salt composition of the nutrient medium, but also on growth regulators, mainly cytokines and auxins. This article discusses the theoretical aspects of the use of various growth regulators at different stages of clonal micropropagation of black currant, provides methods of microclonal reproduction developed by scientists of leading research organizations.

Regenerative potential and its variability in different topophysical zones of Kalanchoe daigremontiana leaves in in vitro culture conditions

Plants of the genus Kalanchoe are the most frequently purchased potted plants because of their decorative qualities and easy cultivation. Modern commercial plants have unique shapes that are usually not genetically determined; hence, in vitro cultures are used to propagate such specimens. The efficiency of regeneration is significantly affected by the type of explant used when that explant was previously located on one of several parts of a complex plant organ. The study aimed to investigate the influence of the topophysical location of collected explants on the regeneration efficiency of adventitious shoots. Various regeneration pathways were observed in the in vitro micropropagation of Kalanchoe daigremontiana leaf fragments, i.e., direct and indirect organogenesis from the callus stage. The results show a dependent relationship between the location of the collected explant and its regenerative potential. The most remarkable regenerative capacity of K. daigremontiana was found in leaf blade fragments taken from the middle and proximal zones. In addition, the explants collected from the marginal zone of the leaf blade were notably more valuable than those from the part located near the midrib. The research results indicate that the growth and development of new seedlings propagated vegetatively with the in vitro method depends on their location on the mother plant.

Optimization of Rhizobium rhizogenes-mediated transformation, regeneration and characterization of Malus domesticaBorkh. Ri lines

This study explored the establishment and optimization of Ri (root inducing) technology for apple breeding, using the bacterium Rhizobium rhizogenes to obtain Ri lines with compact shoots and stronger root systems. The transformation and shoot regeneration for Malus domestica cultivars was studied in detail. Various R. rhizogenes strains, scion and rootstock genotypes, explant types, wounding methods and explant orientations were tested for hairy root induction. Most of the 16 tested strains, especially those with plasmid type III, induced hairy roots in the rootstock genotype ‘M26’. Although apple genotypes differed in response, in most of them roots were successfully induced using strains ATCC 15834, LMG 63 and LMG 150, with leaf blades outperforming petioles as explants. Wounding by scratching or sonication further improved transformation efficiency, as did placing leaf blades with their abaxial side upward on root induction medium. The majority (94%) of roots formed in one transformation experiment were tested PCR-positive for at least one T-DNA gene. Shoot regeneration experiments investigated salt concentrations, gelling agents, cytokinin types, concentrations, and a resting period on hormone-free medium. Shoot regeneration was highly genotype-dependent varying between 0 and 83%, whereas only minor, non-significant effects were observed for the treatments tested. Copy numbers of T-DNA genes were estimated using digital PCR for the first time in apple Ri lines. In the greenhouse, two Ri lines showed compact shoots and shorter leaves, but no enhanced root system. The improved protocol provides a valuable tool for breeders and scientists to obtain and further use Ri lines.