Hypocotyl Segments Research Articles

Optimization of Agrobacterium-mediated transformation conditions for efficient Bacillus thuringiensis (Bt) cotton transformation

Cotton is one of the world’s largest fiber-producing crops. It is also known as the important cash crop of Pakistan. Cotton crop products contribute to economy as cottonseed oil, animal feed and the fiber support the textile industries, thus, playing a significant role in uplifting the economy of a country. But over time, bollworm attacks had escalated, severely reducing the yield of cotton and damaging cotton production. Bt (Bacillus thuringiensis) cotton is being widely used nowadays due to its enhanced resistance against cotton bollworms which are responsible for reducing crop production leading to high economic loss. It greatly affects the economy by increasing the cost of production and protection. Using resistant strains of Bt cotton not only makes it easier to combat bollworm attacks but also reduces the substantial expense issues that farmers face. Bt has many toxic proteins among which the two crystal proteins, Cry1Ac and Cry2Ab, are used to develop resistance against bollworms in cotton. This study aimed to optimize the developing transgenic callus containing Cry1Ac and Cry2Ab genes in cotton. The dual gene construct (Cry1Ac + Cry2Ab) provides more resistance to the cotton crop as compared to the single gene construct against whom the most bollworms have developed resistance. The vector was inoculated into the hypocotyl segments and the transformation process was preceded by shifting the hypocotyls at different mediums Agrobacterium tumefaciens strain LBA4404 was used for cotton transformation. The construct-containing vector was introduced into a standard cotton line. A molecular confirmation test was carried out utilizing PCR and gene-specific primers after the formation of the transgenic callus. As a result of the research an indigenous cotton line that expresses double Bt genes for insect resistance was developed.

The reaction of fiber flax (<i>Linum usitatissimum</i> L.) to the content of aluminum ions <i>in vitro</i> culture

Relevance. The influence of unfavorable environmental factors is one of the reasons for the low realization of the biological capabilities of modern flax varieties in production conditions. Cultivation of flax on soils with strongly acidic (pHKCl 4.5 and below) and neutral (pHKCl over 6.0) reactions leads to a significant decrease in the productivity of fiber and seeds. Biotechnological methods are effective in creating new forms with altered features and properties.Methods. The studies were conducted in vitro. The source material is seeds and hypocotyl segments of flax varieties-dolguntsa Impulse, Phoenix, Leader, S-108, Soyuz. The selective agent in the studies was aluminum salts in the form of AlCl3 at concentrations of 0 (control — H2O dist. / nutrient medium (without selective agent), 44 mg/l, 64 mg/l, 84 mg/l.Results. The toxic effect of AlCl3 on the germination of fiber flax seeds, the formation of primary roots and seedlings — hypocotyls of the varieties used was revealed. The indicator of the value indicating the length of primary roots in germinated fiber flax seeds decreased on the seventh day with an increase in the concentration of the AlCl3 solution. The highest sensitivity to the content of aluminum ions was shown by varieties S-108, Leader and Soyuz. This was expressed in a decrease in the length of the hypocotyl with an increase in the concentration of aluminum ions in the solution. In genotypes in all variants of studies on a selective medium containing AlCl3, a morphogenic callus was formed. The frequency of formation of morphogenic calli was different depending on the concentration of AlCl3 in the cultivation medium. A trend towards a decrease in the number of morphogenic cells with an increase in the concentration of aluminum ions was observed in all varieties. In less sensitive varieties (Impulse and Phoenix), the amount of morphogenic callus formed was higher than in more sensitive ones (C-108, Soyuz, Leader). Under selective conditions, shoots of flax of the studied varieties resistant to AlCl3 were obtained.

Direct organogenesis from cortical cells of hypocotyl segments in soybean

Direct organogenesis from cortical cells of hypocotyl segments in soybean

COMPARATIVE ANALYSIS OF THE EFFICIENCY OF THE METHODS
 OF REGENERATION FROM RAPESEED EXPLANTS OF MAGNAT AND
 LEADER VARIETIES

A comparative analysis of the effectiveness of organogenesis from the callus cells of hypocotyl segment explants in rapeseed varieties Leader and Magnat regionalized in Belarus was carried out. It was established that the Leader, compared to the Magnat variety, is more suitable for organogenesis from plant explants in vitro. It was shown that for the Leader variety the regeneration efficiency is up to 10 plants adapted ex vitro per explant, when using a method, according to which hypocotyl explants are first exposed to auxin (2,4-dichlorophenoxyacetic acid, 1 mg/l), and then to cytokinin (6-benzylaminopurine, 5–8 mg/l).

A 1-aminocyclopropane-1-carboxylic-acid (ACC) dipeptide elicits ethylene responses through ACC-oxidase mediated substrate promiscuity.

Plants produce the volatile hormone ethylene to regulate many developmental processes and to deal with (a)biotic stressors. In seed plants, ethylene is synthesized from 1-aminocyclopropane-1-carboxylic acid (ACC) by the dedicated enzyme ACC oxidase (ACO). Ethylene biosynthesis is tightly regulated at the level of ACC through ACC synthesis, conjugation and transport. ACC is a non-proteinogenic amino acid, which also has signaling roles independent from ethylene. In this work, we investigated the biological function of an uncharacterized ACC dipeptide. The custom-synthesized di-ACC molecule can be taken up by Arabidopsis in a similar way as ACC, in part via Lysine Histidine Transporters (e.g., LHT1). Using Nano-Particle Assisted Laser Desoprtion/Ionization (Nano-PALDI) mass-spectrometry imaging, we revealed that externally fed di-ACC predominantly localizes to the vasculature tissue, despite it not being detectable in control hypocotyl segments. Once taken up, the ACC dimer can evoke a triple response phenotype in dark-grown seedlings, reminiscent of ethylene responses induced by ACC itself, albeit less efficiently compared to ACC. Di-ACC does not act via ACC-signaling, but operates via the known ethylene signaling pathway. In vitro ACO activity and molecular docking showed that di-ACC can be used as an alternative substrate by ACO to form ethylene. The promiscuous nature of ACO for the ACC dimer also explains the higher ethylene production rates observed in planta, although this reaction occurred less efficiently compared to ACC. Overall, the ACC dipeptide seems to be transported and converted into ethylene in a similar way as ACC, and is able to augment ethylene production levels and induce subsequent ethylene responses in Arabidopsis.

Methodological aspects of increasing the intensity of callusogenesis and organogenesis of Linum usitatissimum L. in vitro

Aim. Improving methods for increasing the efficiency of obtaining callus cultures and somaclones of flax (Linum usitatissimum L.) in vitro. Methods. Hypocotyl segments were cultured on Murashige and Skoog nutrient medium supplemented with sucrose (30 g/l) and phytohormones at various concentrations. Other conditions: photoperiod 16 hours, illuminance 2500 lx, relative humidity 60–80%, air temperature 22–24°C. Results. The ability to form callus and somatic embryogenesis of flax depends on the phytohormonal composition of the nutrient medium, the size of the explants and the distance between them. Conclusions. For intensive callus formation and somatic embryogenesis in vitro, the optimal concentrations of BAP (mg/l) can be expressed as 1.0 ≤ BAP ≤ 1.75; the optimal concentrations of BAP for the medium supplemented with NAA (0.05 mg/l) 0.5 ≤ BAP ≤ 2.0; the optimal concentration of NAA for the medium supplemented with BAP (1.0 mg/l) 0.025 ≤ NAA ≤ 0.150; and the optimal concentrations of IAA for the medium supplemented with BAP (1.0 mg/l) 0.05 ≤ IAA ≤ 0.50. Addition of 0.5 mg/l GA3 to the medium with NAA and BAP is effective. It is optimal to use hypocotyl explants 3–6 mm long and place them at a distance of 1.5–2.5 cm from each other. Organogenicity of callus is significantly reduced in the process of subculturing.

Study of influence of organochlorine pesticides and their destruction products on tomatoes in in vitro

Aim. Given the prospects of the method of detoxification of organochlorine contaminants in environment using microorganisms, the aim of the work was the investigation of the effect of microbial destruction products of pesticide hexachlorocyclohexane (HCCH) on morphogenetic and morphophysiological reactions of tomato in vitro. Methods. To test the effects of HCCH and its microbial degradation products obtained by strains of Bacillus megaterium, Pseudomonas putide 3, Stenotrophomonas maltophilia 6, these compounds were added to nutrient medium for tomato cultivation. As explants the segments of hypocotyls of L. esculentum seedlings were used. For plant regeneration, MS medium supplemented with 1 mg/l of zeatin and 1 mg/l IAA was used. Results. It was found that the products of HCCH destruction stimulate the shoot regeneration, but inhibit their rooting, whereas HCCH inhibits the shoot formation and provokes further morphophysiological changes in plants. Conclusions. A pronounced negative effect of pesticide HCCH on cells and tissues of tomato in vitro was revealed. It was found that the products of HCCH destruction do not affect the morphogenetic processes of isolated tomato cells and tissues and do not cause morphophysiological changes in obtained in vitro tomato plants.

Agrobacterium-mediated transformation of spring camelina with yeast genes of trehalose biosynthesis

Aim. The aim of the study was the obtaining of Camelina sativa (L.) Crantz lines with yeast genes of trehalose synthesis TPS1 and TPS2 to increase their resistance to drought. Methods. Seeds of C. sativa genotype FEORZhYaF-1 were used for in vitro culture establishment. For this hypocotyl segments and shoot meristems of 5-days-old camelina seedlings were cultivated on three different nutrient media for regeneration supplemented with various hormone combinations. Vector constructions pGWB2-TPS1 and pGWB2-TPS2 with TPS1 and TPS2 genes have been used for genetic transformation. Results. The highest efficiency of plant regeneration from hypocotyl explants was found on medium supplemented with 1 mg/l BAP and 0.1 mg/l NAA, and from meristem explants – on medium with 1.5 mg/l BAP and 0.5 mg/l NAA. Agrobacterium-mediated transformation was conducted out, and camelina lines were picked up on corresponding medium with selective concentration of hygromycin. Transgenic nature of obtained plants was confirmed by PCR-analysis. Conclusions. The efficiency of in vitro plant regeneration of C. sativa genotype FEORZhYaF-1 has been investigated. Two types of explants and two vector constructions pGWB2-TPS1 and pGWB2-TPS2 with TPS1 and TPS2 yeast trehalose synthesis genes have been used for obtaining of transgenic camelina lines.

INFLUENCE OF MINERAL COMPOSITION OF SELECTIVE MEDIUM ON FLAX MORPHOGENESIS IN IN VITRO CULTURE

The studies were carried out on the basis of the laboratory of breeding technologies of Federal State Budgetary Scientific Institution "Federal Scientific Center of Bast Crops" (Tver region) in 2016, 2018, 2021. The purpose of the research is to determine appropriate mineral composition of the selective medium for effective in vitro selection for resistance to anthracnose. Various mineral composition media were studied. It was found that it is possible to use nutrient media Gamborg, MS, SH-2, which do not contain vitamins, chelate complex and phytohormones, in order to obtain cultural filtrates of fungus strains - the causative agent of flax anthracnose. The culture filtrates obtained on the basis of these media were highly toxic and it was possible to use them for in vitro selection. It was noted that the callus formed on the basis of hypocotyl segments had a higher ability for morphogenesis compared to the callus formed on the basis of immature embryos. As for selective medium consisting of mineral salts of Gamborg medium and cultural filtrate, the proportion of formed morphogenic callus was the smallest in all genotypes taken in the study and amounted to 0.1–6.8%. Mineral composition of the selective medium and the morphogenetic potential of the genotype influenced the formation of morphogenic callus based on primary callus. Depending on the genotype, the proportion of morphogenic calli formed on Gamborg medium + CF was 0.1 - 2.2%, on MS medium + CF - 3.3 - 8.0%, on Sh-2 medium + CF - 3.4 – 8.1%, respectively. Mineral composition of the selective medium had no significant effect on formation of morphogenic cells based on transplanted callus. The proportion of morphogenic callus was 3.0–5.2% for Lenok variety; 3.0 - 5.4% - Rosinka; 0.1 - 4.3% - Zaryanka; 3.1 - 5.5% - for L 2053-5-11 line; 6.1 - 7.4% - L 957-8-7. The breeding lines L 2053-5-11 and L 957-8-7 had a high morphogenetic potential.

In vitro regeneration and Agrobacterium-mediated genetic transformation of Dragon\u2019s Head plant (Lallemantia iberica)

Dragon’s head plant (Lallemantia iberica), is a flowering species belongs to the mint family (Lamiaceae). The species contains valuable essential oils, mucilage and oil which are used in pharmaceutical and food industries. Tissue culture is a feasible strategy to attain large‐scale production of plantlets with a huge potential to produce plants with superior quality. The objective of this study was to develop a simple and efficient method for regeneration and transformation of L. iberica. To reach this goal, the regeneration ability of various explants including leaf, cotyledonary node, hypocotyl and cotyledon segments was investigated in MS medium supplemented with diverse concentrations of NAA (Naphthalene acetic acid) and BAP (6-Benzyl Amino Purine). According to the results, cotyledonary nodes showed the best regeneration response. The maximum rate of regeneration (and number of induced shoots was achieved in 1 mg l−1 BAP in combination with 0.05 mg l−1 NAA from the cotyledonary nodes. Additionally, through the optimized regeneration technique Agrobacterium-mediated transformation of L. iberica was successfully accomplished. Gene transfer was assessed on leaf samples from regenerated plantlets under a fluorescent microscope to detect the GFP signals. Moreover, transgene integration and its expression were confirmed by PCR and RT-PCR analysis, respectively. The establishment of these efficient regeneration and genetic transformation methods paved the way for further application such as plant improvement, functional analysis and gene editing.

In vitro regeneration of hybrid squash (Cucurbita pepo L.) cv. First Runner from cotyledonary node explants

Squash (Cucurbita pepo L.) is highly polymorphic in nature which could contribute its different regeneration potentiality for different genotypes. The present investigation was carried to know the effects of plant growth regulators, explant types, explants age and AgNO3 on in vitro regeneration potentiality of hybrid squash genotype and to optimize regeneration protocol. In this protocol, cotyledon and hypocotyl segments were grown on MS medium supplemented with 1.5 mg/l 6-Benzylaminopurine (BA) and 0.2 mg/l α-Naphthalene acetic acid (NAA), successfully induced callus and subsequently shoots for C. pepo L. cv. First Runner. The height percentage of shoot production frequency was 66.7% from 6 days old cotyledon explants in MS + 1.5 mg/l BA + 0.2 mg/l NAA combination. The shoot regeneration frequency was increased up to 86.67% when 2.0 mg/l AgNO3 was added with MS + 1.5 mg/l BA + 0.2 mg/l NAA media combination by using 5 days old cotyledon explants. Shoot regeneration potentiality of four other C. pepo genotypes were also investigated with this system and showed a substantial amount of regeneration potentiality for different genotypes. Regenerated shoots induced the height 93.33% rooting frequency which promoted long, thick roots on 10 to 12 days in MS medium supplemented with 0.1 mg/l NAA. The regenerated plantlets with long and thick roots were acclimatized in pot soil and eventually grown in natural environment with culture facility. The present study describes a simple and efficient protocol for in vitro plant regeneration of hybrid C. pepo genotypes and may be utilized for further transgenic development.

Effect of 1-naphthylacetic and indol-3-acetics acid on the intensity of callusogenesis and organogenesis of Linum usitatissimum L. in vitro

Aim. Investigate the effect of auxins of exogenous origin in nutrient medium in vitro on the germination and organogenesis intensity in Linum usitatissimum L. convar. elongatum (‘Hlinum’ variety) at the constant concentration of 6-benzylaminopurine (BAP). Methods. Hypocotyl segments were cultured on Murashige and Skoog nutrient medium supplemented with sucrose (30 g/l) and phytohormones at various concentrations. Other conditions: photoperiod 16 hours, relative humidity 60–80%, air temperature 22–24°C. Results. Common flax has a great capacity to form callus and shoots under the effect of the following factors: 1) only auxins, 2) only cytokines, 3) combinations of auxins and cytokines. Somatic embryogenesis is also possible on a nonhormonal nutrient medium. Conclusions. For somatic embryogenesis in vitro, the optimal concentrations of BAP can be expressed as 1.0 ≤ BAP ≤ 1.75, the optimal concentrations of BAP for the medium supplemented with 1-naphthylacetic (NAA, 0.05 mg/l) 0.5 ≤ BAP ≤ 2.0, the optimal concentration of NAA for the medium supplemented with BAP (1.0 mg/l) 0.025 ≤ NAA ≤ 0.150, and the optimal concentrations of indol-3-acetics acid (IAA) for the medium supplemented with BAP (1.0 mg/l) 0.05 ≤ IAA ≤ 0.50. Keywords: Linum usitatissimum L., in vitro, phytohormones, callus, organogenesis.

Genotype-Independent Transformation and Genome Editing of Brassica napus Using a Novel Explant Material.

Agrobacterium-mediated transformation of canola (Brassica napus) via hypocotyl segments has been a commonly used method for the past 30 years. While the hypocotyl-based method is well-established, it is not readily adapted to elite germplasm and the prolonged process is not ideal for a production transformation setting. We developed an Agrobacterium-mediated transformation method based on epicotyl and higher stem (internodal) segments that is efficient, rapid and amenable for high-throughput transformation and genome editing. The method has been successfully implemented in multiple canola genotypes. The method appears to be genotype-independent, with varying transformation efficiencies. Internodal segment transformation was used to generate transgenic events as well as CRISPR-Cas9-mediated frameshift gene knockouts.

The Influence of Effectors of the Ca2+ Signaling System and Oligosaccharin OSRG on IAA-Induced Formation of Adventitious Roots on Explants of Buckwheat Hypocotyls

The interplay between different signaling pathways that occurs during the development of a root system is investigated by considering, by way of example, the role of effectors of the Ca2+ signaling system and a rhizogenesis-stimulating oligosaccharin (OSRG) on the indoleacetic acid (IAA)-induced formation of adventitious roots. By identifying the sensitivity of different stages of rhizogenesis in cultivated buckwheat hypocotyls (Fagopyrum esculentum Moench) to IAA, Ca2+, and the oligosaccharin, it is shown that both Ca2+ and OSRG affect the root formation processes at its initial stages, preceding the effect of the hormone. Auxin can be added to the medium with some delay but no later than 1 h after the explant cultivation was started; while Ca2+ must be present in the medium from the very beginning of cultivation process. The number of IAA-induced adventitious roots on segments of buckwheat hypocotyls decreases in the presence of Ca2+ channel blockers (verapamil and diltiazem), compounds affecting Ca2+-release from intracellular storages (ruthenium red, neomycin), and calmodulin-antagonists (chlorpromazine and fluphenazine). Using calcium channel blocker (diltiazem), it is shown that the effect caused by the oligosaccharin does not depend on the functioning of potential-dependent channels of plasma membrane. The analysis of transcriptome profiling data for the cultivated explants identifies the expression of 149 genes (TGR > 5) relevant to Ca2+ signaling in investigated specimens. The expression profile of these genes changes markedly during IAA-induced root formation, while the addition of OSRG affecting the expression of only few of those.

Eucalyptus nitens plant regeneration from seedling explants through direct adventitious shoot bud formation

A protocol for true-to-type plantlets production by direct adventitious bud proliferation from Eucalyptus nitens hypocotyl segments was developed. Hypocotyls, cotyledons, and expanded leaves of Eucalyptus nitens were cultured in Murashige and Skoog (MS) medium containing naphthalene acetic acid or indole-3-acetic acid (IAA) and 6-benzyl adenine (BA). The most effective treatment for shoot bud formation, 0.5 μM IAA and 2.5 μM BA, was used in additional experiments. After 30 days of culture, the highest rate of regeneration (40 ± 5.8%) and the maximum number of buds differentiated per hypocotyl explant (10.3 ± 4) were obtained when explants were subjected to darkness for the first 10 days and then transferred to light-emitting diode lights mimicking daylight for 20 days of incubation. Histological examination confirmed direct shoot regeneration of E. nitens. For shoot growth, half-strength MS plus 0.09 M sucrose under forced ventilation yielded elongated shoots after 1 month. After 12 weeks from the onset of an experiment, 40 ± 11.5% of elongated shoots produced 5.3 ± 0.8 roots after pretreatment with indole-3-butyric acid aqueous solution and culture on basal medium without plant growth regulators under a temporary immersion system. Inter simple sequence repeat (ISSR) marker analysis revealed the genetic uniformity among the in vitro raised plants, demonstrating the reliability of the procedure.

Callus formation, organogenesis and microclonal reproduction in different species of the genus Linum L. in vitro

Purpose. To reveal the frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction of various species of the genus Linum L. (Linaceae) in vitro. Methods. For in vitro induction of callus formation and organogenesis, hypocotyl segments of species Linum usitatissimum L. convar. elongatum and convar. usitatissimum, L. tenue Desf., L. bienne Mill., L. corymbulosum Pchb., L. nervosum Waldst. & Kit., L. flavum L., L. campanulatum L., L. perenne L., L. austriacum L., L. grandiflorum Desf., L. strictum L. were cultivated on Murashige and Skoog medium supplementedwith 0.05 mg/l 1-naphthylacetic acid and 1.0 mg/l 6-benzyl aminopurine at 22–24 °C, relative humidity of 60–80%,with 16 hours photoperiod (2500 flux). For microclonal reproduction Murashige and Skoog, White, Gamborg and Eveleigh media and their modifications were used. The measurement results were interpreted by the arithmetic mean, standard error for the sample mean, the leastsignificantdifference and ranked. Results. Different species of the genus Linum to a large extend are capable of forming callus and regenerating shoots under the specified cultivation conditions. The frequency of callus formation for the studied samples on the 35th day of cultivation varied within 81.25–100%, the mass of callus from one explant – 0.21–1.64 g, the frequency of organogenesis – 12.50–100%, the number of shoots – 1.8–7.6 pcs. and the height of the shoots was 0.82–2.12 cm. The following species: L. usitatissimum convar. elongatum, L. tenue, L. bienne and L. strictum were distinguished by a high intensity of callus formation. Intensive organogenesis was pecular to L. tenue, L. bienne, L. flavum, L. austriacum and L. grandiflorum. The efficiency of somaclone obtaining was quite low in L. nervosum and L. campanulatum. In total, for the microclonal reproduction of species of the genus Linum Murashige and Skoog, Gamborg and Eveleighmedia supplementedwith 12.5 g/l glucose were optimal. At the final stages of microclonal propagation, before transferring microclones in vivo, it is advisable to use White medium, which contributes to a high frequency of rhizogenesis. Varieties of L. usitatissimum convar. elongatum and convar. usitatissimum had diffe­rent responses to in vitro culture. Conclusions. The frequency and intensity of callus formation and organogenesis, the effectiveness of microclonal reproduction depended on the genotype of a particular species; therefore it is advisable to select the composition of the nutrient medium and growth regulators for each of them. Some species of the genus Linum have not yet been studied in vitro, so the obtained results allow expanding the scope of their use in practice, in particular in breeding as a new source material with somaclonal variation, interspecific crosses, and ornamental floriculture.

Efficient and reproducible somatic embryogenesis and micropropagation in tomato via novel structures - Rhizoid Tubers.

A dual in vitro regeneration system consisting of indirect organogenesis and somatic embryogenesis (SE), applicable to several varieties of tomato—Solanum lycopersicum (cv. Riogrande, cv. Roma, hybrid 17905 and model cv. M82) has been established. This system is both improved and highly reproducible compared to current methods. Callus initiation, plant regeneration and SE was developed for one-week-old cotyledon explants. Indirect organogenesis via callus induction (CI) was developed for all four varieties of tomato used in this study. One-week-old tomato seedlings were used as a source of cotyledon and hypocotyl segments as explants. The explants were subsequently cultured on Murashige and Skoog (MS) medium supplemented with different combination and concentrations of plant growth regulators (PGRs). Substantial trends in regeneration and propagation response were observed among the varieties and treatments. For commercial varieties cvs. Riogrande and Roma, maximum CI was observed at 2 weeks in CIMT9 (0.5 mg/L NAA, 1 mg/L BAP) and CIMT12 (2 mg/L IAA, 2 mg/L NAA, 2 mg/L BAP, 4 mg/L KIN). However, cv. M82 responded after 4 weeks to a combination of treatments CIMT9 (0.5 mg/L NAA + 1 mg/L BAP) and CIMT13 (2 mg/L IAA + 2 mg/L NAA + 2 mg/L BAP + 4 mg/L ZEA) for the production of calli. Subsequent shoot and root organogenesis were optimized for all four varieties. Cv. Riogrande, exhibited fastidious in vitro regeneration potential and selected for induction of somatic embryos via SE involving novel structure: rhizoid tubers (RTBs). Numerous fine hair like rhizoids (~23/explants) were first developed from cotyledon and hypocotyl explants cultured on MS medium supplemented with 0.5 or 2 mg/L NAA at pH 4.0 in dark conditions. Further incubation of each rhizoid under light conditions on MS media supplemented with 5 mg/L TDZ or BAP at pH 4.0 led to the formation of a novel structure—rhizoid Tubers (RTBs). Thus, as evident from histology, SE in Riogrande tomato species requires a medium with pH of (4.0) and higher concentration of cytokinins (BAP/TDZ) to form on average 40–45 RTBs from both explants. Histological and morphological studies revealed that RTBs develop through different stages of embryogenesis to multiple plantlets, on MS medium with 5 mg/L TDZ/BAP at normal pH (5.8). The results obtained indicated that the induced somatic embryos of tomato with lower pH are a more efficient mode of propagation than the organogenesis with or without callus formation. The RTBs led to a complete plantlets regeneration in 45 days compared to indirect organogenesis at 60 days.

Efficient plant regeneration and histological evaluations of regenerants through organogenesis and somatic embryogenesis in Spermacoce hispida L.—An underutilized medicinally important plant

Shaggy button weed (Spermacoce hispida L.) is a therapeutically important herb widely used in Indian System of medicine particularly Ayurvedic, Siddha, and Unani. S. hispida is recalcitrant during germination in natural habitat. Plant tissue culture is an alternate approach for mass multiplication of such vulnerable plants. Plant regeneration by organogenesis and somatic embryogenesis in S. hispida was attempted using cotyledonary leaf, shoot tip, hypocotyl, and nodal explants. Maximum shoot regeneration frequency (72%) and number of shoots (10.5 ± 0.3) through direct organogenesis was achieved from nodal explants cultured on Murashige and Skoog (MS) medium amended with 2.0 mg/dm3 of 6-benzylaminopurine (BAP). Further, MS medium containing 0.5 mg/dm3 2, 4-dichlorophenoxyacetic acid (2, 4-D) produced higher frequency of callus induction (86.25%) from hypocotyl segments. Optimum shoot regeneration response (69.5%) through indirect organogenesis was observed from hypocotyl-derived callus on MS medium fortified with 1.0 mg/dm3 kinetin with 6.1 shoots per callus. The highest regeneration frequency (68%) through somatic embryogenesis was recorded from nodal explants on MS medium supplemented with 2.0 mg/dm3 BAP and 0.8 mg/dm3 kinetin. The regenerated plantlets were acclimatized successfully under ex vitro conditions with normal phenotypic appearance. Shoot tips and nodes did not induce organogenesis (indirect), whereas nodal segments showed better regeneration via organogenesis (direct) and somatic embryogenesis in MS medium with either BAP or BAP with kinetin. Histological examinations confirmed the distinctive developmental stages and bipolar structure of somatic embryos. The described regeneration protocols can be effectively utilized for the large-scale multiplication, germplasm conservation, pharmacological and genetic manipulation studies.

Agrobacterium-mediated genetic transformation of ajowan (Trachyspermum ammi (L.) Sprague): an important industrial medicinal plant

Biotechnology-based breeding methods can improve useful industrial medicinal plants in a faster way. Tissue culture-based and in planta Agrobacterium-mediated gene transformation methods were applied to enhance drought and salinity tolerance in ajowan medicinal plant using betaine aldehyde dehydrogenase (BADH) gene. The pBI121 binary vector harboring kanamycin resistance gene (nptII) and BADH gene in its T-DNA region was applied to transfer BADH gene. Different concentrations of the selective antibiotic were tested on 15 day-old hypocotyl segments and 75 mg/L kanamycin was identified as the minimum inhibitory concentration to screen for transformed cells of ajowan. The different parameters of gene transformation including Agrobacterium optical density (OD), Agrobacterium strain, Agrobacterium killing antibiotic, acetosyringone concentration, and inoculation duration were tested during tissue culture-based Agrobacterium-mediated transformation. The OD600 = 0.6-0.8 of LB4404 strain of Agrobacterium × 160 mg/L timentin × 250 μmol/L acetosyringone × 30 min inoculation duration were identified as the best gene transformation parameters. Successful integration and expression of transferred gene were confirmed in T0 plants using polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) methods. Five transgenic lines with 2.94% transformation efficiency were achieved by regular tissue culture-based Agrobacterium-mediated gene transformation method, whereas the dip flora in planta transformation method has led to one transgenic line with 1.42% transformation efficiency. Bioassay analysis revealed enhanced drought and salinity tolerance in transformed plants in comparison with non-transformed plants. The morphological characteristics of plant height and seedling fresh weight of BADH-transformed plants were improved under salinity stress. Under drought stress, the proline content and relative water content of transgenic plants were more than non-transformants. The results of gas chromatography mass spectrometry (GC–MS) revealed significant increase in thymol content of BADH-transformed plants (55.07%) compared with non-transformed plants (39.20%) under drought stress condition. The obtained transgenic plants are cultivable in unfavorable environments to give an acceptable level of useful secondary metabolites. The optimized protocol is applicable for gene transformation of other medicinal plants of Apiacea family for different purposes.

Advancement in protocol for in vitro seed germination, plant regeneration and cryopreservation of Viola cornuta.

The aim of this study was to develop a fast, reliable and true-to-type protocol for in vitro plant regeneration and long-term storage of horned pansy (Viola cornuta L). Seed germination over 60% was recorded after 12weeks of growth at 10°C or 4°C. Calli formation and shoot induction were obtained in petiole and hypocotyl culture on half-strength MS mineral salts with full concentration of Na-FeEDTA and vitamins (½MS medium) with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.1mg/L) and 6-benzylaminopurine (BAP, 2.0mg/L) and leaf culture on ½MS medium with thidiazuron (TDZ,1.0mg/L). The highest frequency of adventitious shoot induction (50%) with six shoots/explant was achieved in hypocotyl culture from top hypocotyl segments, close to epicotyl which was grown 8 weeks at 16h light/8h dark photoperiod. Subsequent shoot multiplication was achieved on ½MS medium with α-naphthaleneacetic acid (NAA, 0.1 or 0.5mg/L) and BAP (1.0mg/L). Rooting of shoots was obtained on ½MS medium with low concentration (0.1mg/L) of auxins: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or NAA, or without growth regulators. In vitro-derived plantlets were acclimatized under greenhouse conditions. All plants developed normally, bloomed and set seeds. Shoot tips were cryopreserved succssefully using modified plant vitrification 3 (PVS3-based vitrification procedure). Cold acclimation for 2weeks significantly improved shoot regrowth (64%) after thawing in comparison to non-acclimated shoots (39%). Clonal fidelity of regenerated plantlets at ploidy level was confirmed by chromosome counting. The presented protocol can be useful for mass propagation, genetic transformation studies and long-term storage of valuable Viola spp.