Callus Origin Research Articles

Каллусные культуры in vitro в экспериментальной оценке засухоустойчивости хлебных злаков (обзор)

One of the directions in plant biotechnological research is the use of callus cultures in vitro. The callus is an integrated system that is formed both exogenously (as a result of the proliferation of surface cells of various tissues of the plant organism) and endogenously (in the depth of these tissues). Initially, callus consists of homogeneous cells gradually transformed into a system of groups of heterogeneous cells with specific morphogenetic potencies. These potencies are realized by various ways of morphogenesis in vitro, including those leading to the formation of full-fledged plants-regenerants. In this review, using cereals as an example, we analyze the literature and our data on the use of calli in vitro as experimental systems for studying the plant stress-resistance to abiotic factors, especially to the drought factor. The advantages and limitations of using callus cultures in vitro are considered. The prospects of studying the mechanisms of action of abiotic stressors and resistance to them at the cellular and tissue levels under model conditions of callus culture in vitro are shown. Attention is paid to the assessment of the drought resistance of cereals under selective conditions simulating drought in vitro by such a parameter as the growth activity of calli. The issue of studying anti-stress effects in callus cultures in vitro is analyzed on the example of brassinosteroids. The prospects of using the model system “immature embryo in vivo–embryonic morphogenic callus in vitro” for rapid assessment of the effect of anti-stress plant growth regulators are considered. It is emphasized that the basis for using calli as the model system is both the significant role of the cell in all morphogenetic events of the plant organism in vivo and in vitro and the similarity of the responses of plants in vivo, calli in vitro, regenerants of callus origin in vitro and ex vitro.

Получение и фитохимический скрининг каллусных и суспензионных культур клеток вздутоплодника сибирского Phlojodicarpus sibiricus (Steph. ex Spreng.) К.-Pol.

Callus and suspension cell cultures have been obtained from aseptic seedlings of Siberian bloat fruit (Phlojodicarpus sibiricus), a producer of coumarins. P. sibiricus callus cultures were characterized by white-yellow coloration, a combination of loose and dense cell aggregates, and satisfactory growth. The dry biomass growth index for cultures of leaf, hypocotyle or root origin was 7-9, 10-12 and 11-13, respectively. Suspension cell cultures were initiated from calluses of leaf and hypocotyle origin; these cultures were also white-yellow and consisted mainly of cell aggregates of the meristem-like and parenchyma-like types with different aggregation degrees depending on the origin. Cell viability during the growth cycle was at the level of 70-80%. In contrast to the original callus cultures, the suspension culture of leaf callus origin had the highest growth characteristics (growth index of about 10). Preliminary phytochemical screening by UPLC ESI MS showed the presence of khellactone-group coumarins in the biomass of P. sibiricus primary calluses (1st-3rd growth cycle) and suspension cultures derived from them. Sibirian bloat fruit, Phlojodicarpus sibiricus, callusogenesis, suspension cell culture, coumarins The works on obtaining and growing P. sibiricus cells, as well as studying their growth characteristics were financed by the Russian Scientific Foundation grant no. 74-00097. The study of phytochemical characteristics of P. sibiricus cell cultures was supported by the state assignment of the Ministry of Education and Science of Russia (FSRG-2020-0019).

Cytological Characteristics of Different Origin-Derived Calli and Adventitious Roots and Phenolic Compounds Profiling in Camellia japonica

The aim of this study was to compare the two in vitro culture systems callus and adventitious root by investigating the biomass and phenolic compounds in calli and adventitious roots induced from four different explants (leaf, root, petal, and ovary) in Camellia japonica. The biomass of calli and adventitious roots was examined after 4 and 8 weeks of cultivation, respectively, and 22 phenolic compounds were analyzed using high-performance liquid chromatography (HPLC). The biomass of the ovary-derived calli (2.0 g･mass-1) was 1.5-fold that of the leaf-derived calli. The dry weight (DW) was highest in ovary-derived calli; however, the highest dry matter content was obtained from leaf-derived calli. Differences in the investigated characteristics depended on the callus origin. In adventitious roots, the highest biomass was achieved in the leaf-derived adventitious root system; its fresh weight was 2.3-fold (89 mg･ea-1) higher, and its DW was 1.8-fold (16 mg･ea-1) higher than those of ovary-derived adventitious root system. Active cell division was detected in petal-derived lines in both the calli and adventitious roots. Results of the HPLC analysis revealed that the total content of 22 phenolic compounds was highest in ovary-derived calli and ovary-derived adventitious roots. Our experiments confirmed that the calli and adventitious roots of C japonica have different cytological characteristics and bioactive compounds depending on the explant origin. In addition, callus culture was a more suitable system than adventitious root for producing phenolic compounds when the duration of the culture period and biomass were considered.

Impact of the Leaf Cut Callus Development Stages of Populus on the Tetraploid Production Rate by Colchicine Treatment

This paper examined the impact of the leaf incision callus developmental status of Populus on the tetraploid production efficiency. Using diploid full-sib progeny [(Populus pseudo-simonii × P. nigra ‘zheyin3#’) × (P. × beijingensis)] as experimental material, leaf explants were gathered from seedlings of five genotypes selected randomly from full-sib hybrid progeny cultured in Murashige and Skoog basal medium with 0.4 mg/L 6-benzyladenine and 0.05 mg/L 1-naphthaleneacetic acid. The morphological and cytological characteristics of the incision callus (from the callus origin and adventitious bud development) were observed and divided into five stages based on the characteristics of the callus. The incision callus from each of the five stages was treated with 30 mg/L colchicine for 3 days. Then, the polyploidy level of the regenerated plants was confirmed by flow cytometry analysis and chromosome number counting. The results indicate that the rate of tetraploid production was significantly correlated with the callus development stage of Populus leaves; the most likely stage for chromosome doubling was Stage II, in which the calli initially formed around the cut end. To validate that callus developmental Stage II was the optimal callus developmental stage for chromosome doubling of diploid full-sib progeny, ten full-sib progeny genotypes were treated with 30 mg/L colchicine for 3 days at Stage II. All ten genotypes of the diploid progeny obtained tetraploid with no mixoploid production; the percentage of tetraploid induction was 7.9–13.2%. There were significant differences in the morphological characteristics of the leaves and roots of diploid and tetraploid plantlets.

Erratum to: Developing biotechnology tools for ‘beautiful’ vavilovia (Vavilovia formosa), a legume crop wild relative with taxonomic and agronomic potential

Beautiful vavilovia, the closest species to the common now extinct ancestor of the whole tribe Fabeae holds significant taxonomical interest and also for breeding within this group of species, which includes the most cultivated leguminous pulses in the world. In spite of this, vavilovia has attracted very scarce research to date and is in danger of complete extinction. Thus, as a part of the research carried out by an informal international group of researchers from various countries, we report here various experiments for the development and exploitation of a range of biotechnology tools for vavilovia, ranging from standard in vitro propagation, to plant regeneration from explant-derived callus, and also from protoplasts. Plants were successfully recovered following propagation from nodes, and by regeneration through organogenesis from callus derived from internodes (which provided the best responses) and leaves. Also, protoplasts were isolated from leaves and stems from in vitro shoots and from callus derived from these two explants, with the latter undergoing sustained division. Subsequently, protoplasts isolated from internode callus proliferated and also underwent organogenesis coupled with whole plant recovery at a low frequency, while protoplasts from leaf callus origin followed both organogenesis and embryogenesis simultaneously but failed to yield viable plants. Flow cytometry assessments permitted to ascertain the genetic fidelity of both propagated and regenerated plants irrespectively of the source tissue from which they were derived (i.e., either callus from explants or from protoplasts). Finally, flow cytometry also permitted us to provide the first record on the relative nuclear DNA content and genome size for Vavilovia formosa.

CALLUS AND PLANTLETS PRODUCED FROM IN VITRO CULTURE OF ANTHERS OF ACTINIDIA CHINENSIS

Homozygous genotypes are valuable for genomic and genetic studies in higher plants, but obtaining homozygous perennial plants using conventional breeding technologies is challenging, with the long times and large field areas required for several back-crossed generations. It is especially difficult with Actinidia because of dioecy, long generation times, high heterozygosity and variation in ploidy from diploid to octoploid. In vitro androgenesis through anther or microspore culture offers a route to develop the haploid and dihaploid plants often used in modern crop breeding, genetics and genomic studies. I report in vitro culture of anthers of six genotypes of diploid Actinidia chinensis resulting in production of callus and regeneration of plantlets. Preliminary results showed that genotype, plant growth regulators, the stage of microspore development and pre-treatments (cold, heat shock) all affected callus formation. Confirmation of their callus origin, homozygosity and determination of the ploidy levels of plantlets are underway.

КАЛЮСОГЕНЕЗ ТА РЕГЕНЕРАЦІЯ РОСЛИН Deschampsia antarctica Desv. (Poaceae) У КУЛЬТУРІ in vitro

Conditions for induction of callus formation from root and stem explants and long-term maintenance of Deschampsia antarctica Desv. tissue culture have been specified. Ability to callus formation depended on mineral composition of nutrient medium, combination of growth regulator concentrations, place of donor-plant vegetation and type of explant. The optimal for callus tissue generation was Gamborg, Eveleigh — B 5 nut rient medium, supplemented with 0.9–1.0 mg/l 2.4-dichlorophenyl acetic acid and 0.09–0.1 mg/l of cytokinine benzylaminopurine. Callus formation potency from the root explants considerably exceeded (1.5–2 times) that of from stem ones. The shoots were derived through spontaneous indirect organogenesis. Regeneration efficiency was found to be affected by nutrient medium composition and callus origin. Regenerated shoots were rooted and conditions for growth of regenerated plants in vitro were specified.

Improvement of Selected Induction Culture Media on Callus Induction in Anther Culture of Anthurium and a Histological Study on its Callus Formation

Improvement of selected induction culture media on callus induction in anther culture of anthurium and a histologicalstudy on its callus formation were studied at the tissue culture laboratory of the Indonesian Ornamental CropsResearch Institute from February to October 2008. The objectives of the study were to optimize selected media forcallus formation, reveal cell origin of callus derived from anther culture and shoot formation process. Selectedmedia improved in the study were 1) MMS-TBN containing 0,5 mg/l TDZ, 1,0 mg/l BAP and 0,01 mg/l NAA (Winartomedium, WM) and 2) MMS III supplemented with 1,5 mg/l TDZ, 0,75 mg/l BAP and 0,02 mg/l NAA (Winarto andRachmawati medium, WRM). Improvement treatments were carried out by omission and application of 2,4-D in 0.5mg/l and reduction of medium strength of full, half, quarter, one eighth, one sixteenth, and zero strength. Afactorial experiment was arranged using a randomized complete block design with four replications. Results ofthis study indicated that the highest callus induction was clearly established in WRM. The medium stimulatedpotential growth of anther (PGA) up to 81% with 49% of percentage of anther regeneration (PAR) and 2.7 number ofcallus formed per replication (NCF). Significant improvement in callus formation was also recorded by reduction ofmedium strength of WRM to one eighth compared to others. The reduction induced PGA up to 58% with 29% of PARand 1.8 NCF. From histological studies it was well recognized that regenerated callus on half anthers cultured wasoriginated from middle layer cells of anther wall. The morphogenic response of anther wall cells caused primarilyon no androgenesis effect in microspore cells.

Stress treatments and in vitro culture conditions influence microspore embryogenesis and growth of callus from anther walls of sweet pepper (Capsicum annuum L.)

Production of doubled haploids (DHs) is a convenient tool to obtain pure lines for breeding purposes. Until now, the easiest and most useful approach to obtain pepper DHs is via anther culture. However, this method has an associated possibility of producing calli from anther wall tissues that would be coexisting in the anther locule with embryos derived from microspores. Using two established protocols for anther culture, Dumas de Vaulx et al. (Agronomie 2:983–988, 1981) and Supena et al. (Sci Hort 107:226–232, 2006a; Plant Cell Rep 25:1–10, 2006b) callus and embryo development was assessed in four sweet pepper cultivars. For all genotypes tested, the protocol of Dumas de Vaulx et al. (Agronomie 2:983–988, 1981) promoted both embryo development and callus growth, whereas the protocol of Supena et al. (Sci Hort 107:226–232, 2006a; Plant Cell Rep 25:1–10, 2006b) produced no callus but only embryos. However, differences in embryo production were observed among these genotypes. In parallel, anthers were exposed to a 35 °C inductive heat shock for 4, 8, 12 and 16 days, prior to culture at 25 °C. The duration of the heat shock had significant effects in embryo production, but also in callus generation. Callus generation increased with prolonged exposures to 35 °C. Embryo and callus origin was analyzed by flow cytometry, light microscopy and molecular markers. Tests conducted demonstrated a gametophytic origin for all of the embryos tested, and a sporophytic origin for all of the calli. Together, our results reveal that culture conditions have a significant influence on the presence of calli derived from anther walls, which could be minimized by reducing heat shock exposure and/or using a shed-microspore approach.

Regeneration and Agrobacterium-mediated transformation of multiple lily cultivars

To pursue genetic improvement of lily, efficiency of both regeneration and transformation from callus cultures induced from different explants were evaluated in multiple cultivars. Thirty-five callus lines induced from filaments or styles and one control callus line derived from bulb scales of in total twenty lily cultivars representing Lilium longiflorum, Oriental × Trumpet and Longiflorum × Asiatic hybrids were maintained on a medium with 8.3 μM picloram (PIC). In this study, they were tested for their regeneration potential by transferring them onto a regeneration medium supplemented with 0.4 μM PIC and 0.044 μM 6-benzyladenine. Regeneration was obtained in all cultivars examined and the percentage varied from zero to 89 % in the 36 callus lines. Regeneration frequency was significantly influenced by the genotype (cultivar). Subculturing the calli every 4 weeks by refreshing the regeneration medium contributed positively to bulblet formation, when compared to an eight week subculture frequency. It was found that the regeneration ability generally decreased with an increasing age of the callus cultures for all cultivars. The origin of the callus (style or filament) did not lead to significant differences in regeneration frequency, but there was an interaction between callus origin and genotype. Calli of eight randomly chosen cultivars were co-cultivated with Agrobacterium tumefaciens strain AGL0 carrying binary vectors with the gus gene as reporter and putative transgenic plants were produced. GUS histochemical assays demonstrated transient and stable expression of the gus gene in both calli and regenerated lily plants. Transient expression frequencies ranged from 0.3 to 20.6 % while stable transformation was much lower, only 1.4 % as the maximum.

Embryonic dormancy persists in the calli derived from apple embryo

Growth rate and differentiation ability of calli obtained form apple embryos depended on the depth of embryo dormancy and on the organ of callus origin. We wanted to answer whether some dormancy – related metabolic activities persist also in calli and whether they are affected by the dormancy status. The activities of invertases and proteases were determined in the calli obtained from axes and cotyledons of embryos after different periods of cold stratification. Patterns of changes in activities of alkaline invertase, albumine hydrolysing protease and cystine-aminopeptydase in extracts of different calli corresponded precisely to changes of these activities in axes and cotyledons of intact embryos submitted to stratification. These findings indicate that embryonic dormancy persists in undifferentiated cells of calli. The involvement of an unknown factor responsible for maintenance of the dormancy, which is transmitted to daughter cells during callus growth is suggested.

벼에서 캘러스 유기원와 계대배양 기간에 따른 재분화 계통의 주요 농업형질 변이

Variations of agronomic traits were evaluated on the progenies of regenerated rice plants of different callus origin and subculture period. From eighty-eight percent to ninety percents of ovary culture-derived plants (OCP) and anther culture-derived plants (ACP) were not segregated within the lines in major agronomic characters. Compare to ACP and OCP lines, the seed culture derived lines (SCP) showed more segregation (17％) in major agronomic traits among the lines. The most frequent segregating traits were grain fertility (6.1％) in ACP, leaf color (4.2％) in OCP and grain fertility (4.9％) and leaf color (4.9％) in SCP lines. The SCP line showed more variation in culm length, panicle length and heading date than those of OCP and ACP lines. The variation of agronomic traits in SCP lines was tended to increase with prolonged subculture. Culm and panicle length were shorter than those of original cultivar in all three types of tissue culture-derived lines (OCP, ACP and SCP).

Production of petroselinic acid from cell suspension cultures of Coriandrum sativum

The fatty acid pattern and petroselinic acid content in calli, somatic embryos and cell suspension cultures of Coriander sativum are described. The petroselinic acid content was 0.15–0.23 mg g −1 fresh wt of calli, somatic embryo and cell suspension cultures and varied little with callus origin and culture conditions. The ratio of unsaturated: saturated fatty acid was ca 4: 1 in calli. However, the unsaturated fatty acid content was greater in somatic embryos and cell suspension cultures.

Histological Observation of Callus Formation in Mungbean(Vigna radiata(L.) Wilczek) Cotyledon Culture.

Histological observation was conducted to evaluate the origin, initiation site and development of callus in mungbean cotyledon culture. Results showed that callus formation in mungbean occurred in three sites: 1) in the peripheral area of the provascular strands where the initiation of endogenous callus was noted; 2) at the cut portion of the cotyledon where external primary callus developed; and 3) in the ruptured epidermal layer where the primary callus emerged. Callus formation differed in each site. Endogenous callus formed due to the rapid cell division of the cells in the peripheral zone of the provascular tissues. The primary callus from the cut portion of the cotyledon and reptured portion of the epidermal layer were mostly formed out of cell enlargement of the parenchymatous cells. Although the primary callus cells soon became highly vacuolated, this does not seem to contribute to the increase in callus cell mass. Owing to low dividing activity of the primary callus cells, the cytoplasm-rich and meristematic callus cells are apparently derived from the small callus cells which were located on the periphery of the original provascular tissues. And so, some cells in the provascular cells could be seen as the prominent origin of callus in the excised cotyledon system of mungbean.

Plant regeneration from cotyledon and cell suspension protoplasts of apple (Malus x domestica cv. Starkrimson)

Protoplasts were isolated from field and in vitro-grown leaves, cotyledons and cell suspension cultures (of ovule callus origin) of the scion apple cultivars Starkrimson, Rainier, Qiujin and Liaofu. Fast-growing calluses were obtained from leaf, cotyledon and cell suspension derived protoplasts of the four genotypes. The best proliferation responses were obtained from cell suspension protoplasts. For all genotypes tested, nodular calluses were obtained from protoplasts that had originally been cultured on K8P medium, but only those of cultivar Starkrimson underwent organogenesis. In this cultivar shoot buds were produced on callus derived from both cotyledon and cell suspension protoplasts and complete plants. This is the first example of whole plant regeneration from protoplasts isolated from an undifferentiated tissue in apple.

Factors affecting transient gene expression in protoplasts isolated from very slowly growing embryogenic callus cultures of wheat (Triticum aestivum L.)

Protoplasts isolated from embryogenic ('Mustang' and 'Chinese Spring') and non-embryogenic ('Mit') calli of wheat (Triticum aestivum L.) genotypes transiently expressed β-glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm(-1) and 250 μF for 2 s using 50 μg/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed.

Somatic embryogenesis in European black pine (Pinus nigra Arn.)

Embryogenic callus was initiated from immature zygotic embryos of black pine on medium DCR supplemented with 2 mg 1-1 2,4-D and 0.5 mg 1-1 BAP. The diploid number of chromosomes confirmed the origin of callus from zygotic embryos. The callus was white, glossy, mucilaginous and contained somatic embryos consisted of an embryonic region with densely cytoplasmic cells and suspensor region with long vacuolated cells. Although somatic embryos with green cotyledons were recognisable after ABA treatment and subsequent transfer to growth-regulator free media whole plants have not yet been obtained.

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH4NO3 and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.

Characterization of aLycopersicon esculentum xSolanum lycopersicoides somatic hybrid lacking a glutamate oxaloacetate transaminase isozyme

Morphological, cytological, isozyme and chloroplast DNA analyses were used to determine possible mechanism(s) for the loss of glutamate oxaloacetate transaminase-4 (GOT-4) isozyme activity in a somatic hybrid. Plant 204-1, derived by cell fusion between tomato (Lycopersicon esculentum) andSolanum lycopersicoides, was characterized for bothGot-4 and acid phosphatase-2 (Aps-2), two isozyme loci which are closely linked (recombination 2.5 cM). This hybrid was determined to be chimeric for bothGot-4 andAps-2. TheS. lycopersicoides plant used to provide cells for the fusion was determined to be heterozygous for bothGot-4 andAps-2. Only oneS. lycopersicoides allelic form ofAps-2 andGot-4 was found in plant 204-1. This observation indicated that either the alternative copy of theS. lycopersicoides chromosome region encodingGot-4 andAps-2 is deleted or the entire chromosome is absent. Plant 204-1 was cytologically determined to be aneuploid with approximately 62 chromosomes. Sixty-two somatic hybrids of separate callus origin were analysed for GOT-4 and a high proportion (27%) lacked theS. lycopersicoides form ofGot-4. The loss of this allele and the linkedAps allele most likely occurred in the suspension culture ofS. lycopersicoides used to provide cells for fusion.

Androgenic response from cultured anthers of a leguminous tree,Cassia siamea Lam.

Anthers ofCassia siamea, on culture on B5 medium supplemented with coconut milk (15%, v/v), 2,4-D (2 mg/l), and kinetin (0.5 mg/l), split open after one to two weeks of inoculation and eject a callus mass. Microscopic examination of the anthers cultured at the late uninucleate or early bi-celled stages, after 7–14 days of culture, revealed many multicellular structures at various stages of development, thus indicating the pollen origin of callus. Callus cells also showed the haploid chromosome number (n=14).