Plantlet Survival Research Articles

Arbuscular Mycorrhizal Symbiosis Improves Ex Vitro Acclimatization of Sugarcane Plantlets (Saccharum spp.) under Drought Stress Conditions.

The symbiotic associations between arbuscular mycorrhizal fungi (AMF) and plants can induce drought stress tolerance. In this study, we evaluated the effect of Glomus intraradices, a mycorrhizal fungus, on the ex vitro development and survival of sugarcane plantlets subjected to drought stress during the acclimatization stage of micropropagation. In vitro obtained sugarcane plantlets (Saccharum spp. cv Mex 69-290) were inoculated with different doses of G. intraradices (0, 100, and 200 spores per plantlet) during greenhouse acclimatization. Sixty days after inoculation, plantlets were temporarily subjected to drought stress. We evaluated the survival rate, total chlorophyll, total protein, carotenoids, proline, betaine glycine, soluble phenolic content, and antioxidant capacity every 3 days for 12 days. Symbiotic interaction was characterized by microscopy. Our results showed that the survival rate of inoculated plants was higher in 45% than the treatment without mycorrhizae. Total chlorophyll, protein, proline, betaine glycine content, and antioxidant capacity were increased in AMF inoculated plants. The soluble phenolic content was higher in non-inoculated plants than the treatment with mycorrhizae during the drought stress period. Microscopy showed the symbiotic relationship between plant and AMF. The early inoculation of 100 spores of G. intraradices per sugarcane plantlet during the acclimatization stage could represent a preconditioning advantage before transplanting into the field and establishing basic seedbeds.

Optimizing in vitro nutrient and ex vitro soil mediums-driven responses for multiplication, rooting, and acclimatization of pineapple

A comprehensive study was carried out on in vitro multiplication and rooting using the medium enriched with different plant growth regulators and acclimatization of pineapple cv. ‘Smooth Cayenne’ using different soil growing substrates. The significantly highest shoot buds (Avg. 16.7) were obtained on the medium comprising 2.0 mg L−1 BA (6-Benzylaminopurine). Results showed that 1.0 mg L−1 IBA (Indole-3-butyric acid) increased the thickness and length of white adventitious roots and resulted in a significantly highest number of roots (Avg. 8) and root length (6.15 cm). Plantlets with healthy, multiple roots were transplanted in several soil combinations of river silt, bolhari (yellow sand), and peat moss. However, the significantly highest survival (100%) of plantlets in the greenhouse was obtained on the soil medium containing only peat moss. Furthermore, soil mixtures of bolhari and peat moss (1:1) and river silt alone exhibited 98.9% and 95.1% survivability of plantlets, which was also considered equally significant (at 5% probability level). The in vitro and ex vitro protocols optimized in the current study can be applied commercially for pineapple crop worldwide.

In vitro Propagation of Curcuma caesia Roxb. via Bud Culture Technique and ISSR Profiling of the Plantlets for Genetic Homogeneity

An in vitro propagation protocol has been developed for Curcuma caesia Roxb., an endangered medicinal plant by Foundation for Revitalisation of Local Health Traditions and Central Forest Department of India. The plant bears poorly germinated seeds and produces two-storage organs: rhizomes and multiple root tubers. Only rhizomes have medicinal-economic values. They serve as propagules too, which results in a shortage of planting material. Therefore, a complete one-year production cycle of C. caesia has been standardized through in vitro propagation including explants establishment (one month), subculture cycles (seven months), rooting (one month) followed by primary hardening (one month) and secondary hardening (two months). Dormant shoot buds on rhizome served as explants for culture initiation on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA) and kinetin (KIN) in combination with citric acid (CA), adenine sulfate (AdS) and indole- 3-acetic acid (IAA). Maximum bud break (70%) was obtained on MS with 8 mg L-1 BA, 8 mg L-1 KIN, 100 mg L-1 CA, 200 mg L-1 AdS and 2 mg L-1 IAA (standard medium). Shoot production potential continued on this medium during the subsequent seven-month-long subculture cycle. The in vitro raised shoots rooted best on ½-strength MS containing 1 mg L-1 indole-3-butyric acid. Plantlet survival rate was >95% after acclimatization. The genetic homogeneity of plantlets with the mother plant was analyzed using Inter Simple Sequence Repeats which generated a monomorphic banding pattern to confirm the uniformity of in vitro raised plantlets of C. caesia.

High-efficiency somatic embryogenesis and plant regeneration in brinjal (Solanum melongena L.) var. Mattu Gulla

ABSTRACT The organogenesis protocol was established for a unique brinjal (Solanum melongena L.) variety, “Mattu Gulla”, which has a distinct size, color and flavor. The objective of this study was to develop a reliable and efficient protocol for the initiation and maturation of somatic embryos into plantlets. The explants were cultured on Murashige and Skoog medium augmented with 2,4-dichlorophenoxyacetic acid (2,4-D) (1.0 mg/L) and benzyl aminopurine (BAP) (1.5 mg/L) for callus initiation. The developing calli (45-days old) were transferred onto an MS medium augmented with plant growth regulators to determine the embryogenic potential of the explants. The proportion of embryogenic callus was higher in hypocotyl-derived calli (HC) with indole butyric acid (IBA) (1.5 mg/L) and BAP (1.0 mg/L with 2,4-D and thidiazuron (TDZ) (1.0 mg/L) than in cotyledon and leaf-derived calli. The embryo initiation was recorded on the 23rd day after subculture from HC with the 2,4-D and TDZ (1.0 mg/L) combination, and germination was recorded. The somatic embryos developed from cotyledon-derived calli showed the highest number of plantlets and a significant percentage of ex vitro survival. In contrast, the lowest number of plantlets was noted from the embryos of hypocotyl-derived calli and ex vitro survival of plantlets. Under greenhouse conditions, the acclimatized plantlets thrived and produced fruits with viable seeds. The established protocol in this study should serve as a platform for large-scale somatic embryogenesis and micropropagation of plantlets. The somatic embryo-based manipulation should be exploited as a biotechnological tool in crop breeding and improving desired agronomic traits.

Optimization of Bacillus subtilis-based fermentation of anaerobic digestate and biohazard-free application in endophyte-assisted hardening of micropropagated plantlets for increasing survivability

Anaerobic digestion of agricultural waste helps to decarbonize the energy sector while also increasing the use of stabilized residues in agriculture. Although digestate (Dg) contains amino acids, vitamins, humic acids, and micronutrients, using it directly poses an unknown risk to crops due to the presence of pathogens, heavy metals, and excessive ammonia, necessitating processing for agricultural application as fertilizer. The study aimed to minimize the undesired effects of Dg by optimizing fermentation with Bacillus subtilis CW-S and using it as a potential inoculant for endophytic-assisted hardening of micropropagated potato plantlets. The single factor test results revealed that Dg concentrations of 70%, 80%, and 90% had a greater positive impact on cell concentration than concentrations of 50%, 60%, and 100%. According to the Box-Behnken design, diluted (80%) Dg produced a higher cell concentration (4.36 × 108 CFU/mL) than undiluted (100%) (1.18 × 108 CFU/mL). In hardening experiments, Bacillus subtilis CW-S fermented digestate (FDg) had a significantly higher plantlet survival rate of 90.25% and a root length of 5.22 (cm) with greater Serendipita indica infection, whereas Dg (unprocessed) had a lower survival rate of 67.88% and a root length of 3.13 (cm) with less Serendipita indica infection. According to the findings, FDg could be employed as a non-threat bioinoculant for widespread agricultural application.

Establishment of an acclimatization protocol for pepper (Piper nigrum L.) plantlets post-in vitro stage

This study was conducted with the aim of determining the factors that affect the growth and development of in vitro-derived pepper plantlets variety Vinh Linh in greenhouse. The results indicated that gradual adaptation of in vitro plantlets to ex vitro environment by placing enclosed plantlets-containing culture vessels in greenhouse for 2 weeks improved the survival and growth of pepper plantlets upon transfer to acclimatization substrate. The plantlets were required to reach the weight of 0.7-0.9 g before transplanting into nursery bags containing substrate that was red soil:cow dung:coconut husk:rice husk (5:2:2:1). Alternative application of NPK 0.2% and foliar fertilizer Nupe 0.2% every 2 weeks positively affected plant growth and the plants were ready for transplanting into the field after about 5 months. The research results establish an acclimatization protocol for pepper plantlets post-in vitro stage that helps to complete the protocol for pepper production using micropropagation method.

In Vitro Techniques for Shipping of Micropropagated Plant Materials

Shipping of in vitro micro-cuttings in tubes or jars is a frequently used method as the plants are more likely to quickly reproduce and comply with quarantine regulations in plant germplasm distribution. However, these containers are fragile during transportation. To diminish the risk associated with the long-distance shipping of in vitro plants, a safe and widely applicable packing and conservation technique based on microplate and slow growth was developed in this study. Potato cultivar ZHB and ginger cultivar G-2 were used to optimize the system with microplates (96 wells), vacuum-sealed packaging, and slow-growth techniques. Under regular culture conditions, packing in vacuum-sealed microplates reduced the survival of ZHB and G-2 micro-cuttings to 85.8% and 20.0%, respectively, and regeneration to 61.8% and 0%, respectively. Reducing the temperature to 10 °C maintained the survival of ZHB and G-2 micro-cuttings in the range of 83.3–100% after 60 days. Exposure to darkness decreased the survival of G-2 and inhibited regrowth. Thus, conservation in darkness at 10 °C is suggested. The effects of iron concentration and plant growth retardants were further assessed. The addition of 1/4 MS medium combined with 100 mg/L chlormequat chloride (CCC) resulted in full survival and growth inhibition of plantlets, without malformation identified. Finally, incubation with 1/4 MS medium supplemented with 100 mg/L CCC in vacuum-sealed microplates at 10 °C in the dark resulted in high survival and suppressed germination. Sweet potato HXS was incubated as well to test the broad-spectrum applications of the technique; 100% survival and 6.7% germination was gained. Morphological indices of released cuttings recovered to control levels after two cycles of subculture in MS medium. A 0.1–0.2% genetic variation was detected by SSR and ISSR, suggesting genetic stability of the conserved samples. Finally, micro-cuttings were safely transported to cities located thousands of kilometers away without package and sample damage. Our results enable easy distribution of in vitro plant germplasms.

Prescriptions for the Control of a Clonal Invasive Species Using Demographic Models.

Until recently, little research has focused on determination of the population dynamics of invasive species and evaluating their genetic variation. Consequently, not much is known of what drives clonal invasive species and their demography. Here, we describe the population dynamics of Kalanchoe delagoensis (Crassulaceae), considered invasive to several countries. We quantified the demography of a population in central Mexico using integral projection models (IPM) in a population that reproduced asexually exclusively through plantlets. The effect of clonal recruitment on population growth rate () was evaluated by changing plantlet survival and simulating management scenarios that used previous data of watering and seven experimental herbicide treatments. The finite rate of population increase indicated that this Kalanchoe delagoensis population is growing (above one) and with water availability, growth rates will only accelerate. The IPM showed that plantlet survival and recruitment were the most critical steps in the cycle for the population, and simulations of different management scenarios showed that reducing plantlet survival significantly decreased only in two out of the seven herbicides used.

Development of an Improved Micropropagation Protocol for Red-Fleshed Pitaya ‘Da Hong’ with and without Activated Charcoal and Plant Growth Regulator Combinations

Micropropagation protocols for red-fleshed Hylocereus species (Cactaceae) have been developed; however, these methods prolong the sprout duration from areoles and produce irregular micro-propagules in ‘Da Hong’ pitaya. Thus, the present study aimed to establish an improved micropropagation protocol for this cultivar. Shoot regeneration and root induction of self-pollinating seedling segments were evaluated in response to combinations of activated charcoal (AC; 200 mg/L), α-naphthaleneacetic acid (NAA; 0.05, 0.10, and 0.20 mg/L), and 6-benzylaminopurine (BAP; 1.00, 2.00, and 4.00 mg/L). The correlations among plantlet growth characteristics and plantlet survival rate after transplantation under field conditions were calculated. Increasing the NAA concentration increased the number of roots but reduced root length. The addition of AC enhanced shoot length and prevented the regeneration of dried-out, clustered, and abnormal shoots. Plantlets treated with 200 mg/L AC and 0.10 mg/L NAA produced the highest number of shoots, i.e., 4.1 shoots, which however, were shorter and lighter than those cultured with AC alone. Plantlets grown on medium supplemented with BAP showed no advantage in shoot number, shoot weight, plantlet surface area, or plantlet volume. The weight and shoot surface area of plantlets were strongly correlated. All plantlets grew well at 4 weeks post-transplantation. Overall, these results support this improved micropropagation method to regenerate robust ex vitro plantlets.

Biotization of tissue culture banana plantlets with Methylobacterium salsuginis to enhance the survival and growth under greenhouse and open environment condition

Aim: This study aimed to investigate the impact of M. salsuginis TNMB03 biotization on tissue culture banana cv. Grande Naine plantlets growth and survival under greenhouse and open environmental condition (exposed to direct sunlight). Methodology: Banana plantlets were transferred from culture flasks to protray and maintained under the greenhouse and open environmental condition for 30 days with or without M. salsuginis TNMB03 treatment. After 30 days, plant growth parameters like pseudostem height, girth, number of leaves, leaf area, fresh and dry biomass, root parameters, plantlet survival, chlorophyll a and b, total chlorophyll, carotenoids and soluble protein and Methylobacterium population in upper and lower surface of leaf, as well as endophytic population were assessed. Results: This study showed that the plantlets biotized with M. salsuginis TNMB03 had better acclimatization response under both the experimental condition than that of uninoculated plantlets. Positive influence on the survival and growth of M. salsuginis TNMB03 biotized plantlets was observed when transferred directly to greenhouse and open environmental condition. Inoculation of M. salsuginis TNMB03 increased the plant height, girth and number of leaves, root length, lateral root and biomass in comparison to the uninoculated plantlets in greenhouse and open environment. Uninoculated plantlets kept under open environment had lower chlorophyll content and sun scorching damages compared to M. salsuginis TNMB03 inoculated plants, which had dark green leaves and increased chlorophyll content. Interpretation: This study shows a new potential technique of using M. salsuginis TNMB03 in tissue culture plantlets, which can help in enhancing the growth of plantlets transferred from culture vessel to greenhouse or open environmental condition without undergoing the routine acclimatization procedure.

Variables Affecting Shoot Growth and Plantlet Recovery in Tissue Cultures of Drug-Type Cannabis sativa L.

Tissue culture approaches are widely used in crop plants for the purposes of micropropagation, regeneration of plants through organogenesis, obtaining pathogen-free plantlets from meristem culture, and developing genetically modified plants. In this research, we evaluated variables that can influence the success of shoot growth and plantlet production in tissue cultures of drug-type Cannabis sativa L. (marijuana). Various sterilization methods were tested to ensure shoot development from nodal explants by limiting the frequency of contaminating endophytes, which otherwise caused the death of explants. Seven commercially grown tetrahydrocannabinol (THC)-containing cannabis genotypes (strains) showed significant differences in response to shoot growth from meristems and nodal explants on Murashige and Skoog (MS) medium containing thidiazuron (1 μM) and naphthaleneacetic acid (0.5 μM) plus 1% activated charcoal. The effect of Driver and Kuniyuki Walnut (DKW) or MS basal salts in media on shoot length and leaf numbers from nodal explants was compared and showed genotype dependency with regard to the growth response. To obtain rooted plantlets, shoots from meristems and nodal explants of genotype Moby Dick were evaluated for rooting, following the addition of sodium metasilicate, silver nitrate, indole-3-butyric acid (IBA), kinetin, or 2,4-D. Sodium metasilicate improved the visual appearance of the foliage and improved the rate of rooting. Silver nitrate also promoted rooting. Following acclimatization, plantlet survival in hydroponic culture, peat plugs, and rockwool substrate was 57, 76, and 83%, respectively. The development of plantlets from meristems is described for the first time in C. sativa and has potential for obtaining pathogen-free plants. The callogenesis response of leaf explants of 11 genotypes on MS medium without activated charcoal was 35% to 100%, depending on the genotype; organogenesis was not observed. The success in recovery of plantlets from meristems and nodal explants is influenced by cannabis genotype, degree of endophytic contamination of the explants, and frequency of rooting. The procedures described here have potential applications for research and commercial utility to obtain plantlets in stage 1 tissue cultures of C. sativa.

Componența substratului pentru aclimatizarea vitroplantulelor de goji

The survival of wolfberry plantlets obtained by tissue culture, under ex vitro conditions, depends directly on the artificially created environmental conditions at stage I of acclimatization: protection from water stress, excessive sunlight, thermal extremes, air currents and establishing an optimal substrate for adequate growth.

Effect of Culture Media and Plant Growth Regulators on Shoot Proliferation and Rooting of Internode Explants from Moroccan Native Almond (Prunus dulcis Mill.) Genotypes

In this study, several methods have been used to facilitate shoot formation from nodal explants of local almond ecotypes known as “Beldi” grown in Eastern Morocco. Nodal segments of divers old local genotypes were cultured on various concentrations of auxin (indole-3-butyric acid (IBA)) and cytokinins (6-benzyl-aminopurine (BAP), thidiazuron (TDZ), and kinetin (KIN)) added to two different media (Murashige and Skoog (MS) and Heller medium). The results showed that TDZ was more effective than the other tested hormones for in vitro proliferation of the “Beldi” ecotype. TDZ at the concentration of 1 mg/L significantly improved the nodal shoot proliferation rate, with the highest percentage (63.6% ± 0.63) and number of regenerated shoots (13 ± 0.54) recorded for S1 genotype inoculated on MS medium, while the most significant rooting rate (60.41% ± 0.81) of proliferated shoots and number of roots per shoot (7.3 ± 1.36) were achieved for S2 genotype on 1 mg/L of IBA incorporated to a half-strength MS medium. With 80% of plantlets survival, the rooted shoots were successfully adapted to the in vivo conditions and were grown vigorously in the greenhouse without any morphological abnormalities.

Stem endophytes increase root development, photosynthesis, and survival of elm plantlets (Ulmus minor Mill.)

Long-lived trees benefit from fungal symbiotic interactions in the adaptation to constantly changing environments. Previous studies revealed a core fungal endobiome in Ulmus minor which has been suggested to play a critical role in plant functioning. Here, we hypothesized that these core endophytes are involved in abiotic stress tolerance. To test this hypothesis, two core endophytes (Cystobasidiales and Chaetothyriales) were inoculated into in vitro U. minor plantlets, which were further subjected to drought. Given that elm genotypes resistant to Dutch elm disease (DED) tend to show higher abiotic stress tolerance than susceptible ones, we tested the endophyte effect on two DED-resistant and two DED-susceptible genotypes. Drought stress was moderate; endophyte presence attenuated stomata closure in response to drought in one genotype but this stress did not affect plant survival. In comparison, long-term in-vitro culture proved stressful to mock-inoculated plants, especially in DED-susceptible genotypes. Interestingly, no endophyte-inoculated plant died during the experiment, as compared to high mortality in mock-inoculated plants. In surviving plants, endophyte presence stimulated root and shoot growth, photosynthetic rates, antioxidant activity and molecular changes involving auxin-signaling. These changes and the observed endophyte stability in elm tissues throughout the experiment suggest endophytes are potential tools to improve survival and stress tolerance of DED-resistant elms in elm restoration programs.

Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with A Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein.

We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

THE GENETIC VARIATION ASSESSMENT OF IN VITRO IRRADIATED TOMATO (Lycopersicon esculentum Mill) BY SCoT AND ISSR MARKERS

Tomato (Lycopersicon esculentum Mill.) are considered major and important globally vegetable crops and in Egypt in particular. Tissue culture techniques have encouraged the utilization of mutation methods in crop improvement. The mutation induction in vegetative crops through tissue culture may be the optimal method to improve these crops. Induced genetic variation in tomato plantlets by using gamma radiation and identified these changes through SCoT and ISSR markers. Egyptian tomato cultivar Idkawy explant was cultured onto MS medium supplemented with 0.2 mg-l BAP. The resulted plantlets were irradiated with γ radiation doses (50, 100, 150, 200 or 250 Gy). The survival, growth rate, and mean of shoot length were decreased with increasing gamma radiation dose. The irradiated plantlets survival percentages were ranged from 78.75% to (50 Gy) and 18.75% to (250 Gy), whereas, the shoot length decreased by a rate of 2.71 cm for the dose (50 Gy) and 1.2 cm for dose (250 Gy). Genetic diversity was evaluated by SCoT and ISSR markers using ten primers for each. It was noticed that the polymorphism percentage mean of SCoT marker (60.53%) is higher than the ISSR marker (39.6). The PIC values average for both markers SCoT and ISSR were 0.429 and 0.347, as well, MI values were 0.345 and 0.156, respectively. On the other hand, the effective no. of alleles (Ne), Nei’s genetic diversity (H) and Shannon’s information index (I) parameters, it was found that the dose 100 Gy caused the highest genetic variation compared with other doses using SCoT marker, however, in ISSR marker was dose of 150 Gy the highest dose for induced genetic variation. The obtained results demonstrate that SCoT marker was more accurate and efficient than ISSR marker for distinguishing and genetic variation analysis of irradiated tomato plantlets. The relationships within treatments were assessed through cluster analysis (UPGMA) based on SCoT and ISSR analysis.

Requirements for efficient plantlet regeneration using cotyledonary nodal explants of purple coral tree (Erythrina fusca Lour.)

There are over hundred species in the genus Erythrina which are of practical importance in multiple ways. The requirements for Erythrina plantlet regeneration in vitro were rarely studied. Surface-sterilized seeds of purple coral tree (Erythrina fusca Lour.) exhibited a high germination rate (80%) in vitro. The cotyledonary nodes from the one-month-old purple coral tree seedlings grown in vitro were used as explants. About 70% of the cotyledonary nodal explants exhibited shoot regeneration when the explants were cultured vertically with the cut ends inserted into basal Murashige and Skoog (MS) medium compared to only 10% of those placed lying horizontally on the surface of the medium. Supplementation of the basal MS medium with 1 mg L−1 of BAP, 1 mg L−1 kinetin and 1 mg L−1 TDZ resulted in the greatest shoot elongation and the highest number of shoots developed from the cotyledonary nodal explants. The regenerated shoots were able to root (100%) on basal MS medium while supplementation with 2 mg L−1 of either NAA or IBA was inhibitory to root elongation. This is the first study on plantlet regeneration of purple coral tree which seems to have more complex requirements for shoot regeneration in vitro than those reported for the other three Erythrina species. Since only 30% of the rooted in vitro shoots survived during acclimatization following a previously published protocol for acclimatization of another plant, it is recommended that future studies are to investigate the requirements of higher plantlet survival for the rooted in vitro shoots of purple coral tree.

An efficient plant regeneration, detection and identification of secondary metabolites from propagate plants of Peperomia pellucida (L.) for mass cultivatione

Several species in the genus Peperomia Ruiz and Pav have giant chloroplasts in the palisade parenchyma of their leaves. Because of this unique feature, Peperomia pellucida L. has become a valuable model plant for studying plastid biology. However, the use of Peperomia for molecular studies has been limited by the lack of efficient regeneration and transformation protocols. This study describes an effective protocol for in vitro shoot induction, plant regeneration and rooting from internode of explant of Peperomia pellucida using Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The multiple shoots were induced from internodes cultured on MS medium containing Kinetin (KN) (0.5 mg L-1) alone which induced six shoots per internodal explant. In combination Benzyladenine (BA) (1.5 mg L-1) and KN (1.5 mg L-1) induced 10 shoots per explant, while, length of shoots were significantly increased in BA (1.0 mg L-1) and KN (3.0 mg L-1) (0.8 cm per explant); KN (0.5 mg L-1) (0.7 cm per explant) respectively. Adventitious root induction was achieved on MS media with different concentrations of Indole-3-Butricacide (IBA) and Indole-3-Acetic Acid (IAA). The successful root induction was observed in MS medium supplemented with IBA and shown 100% responses while plantlets survival was 90% at greenhouse garden soil. Rooted plantlets were successfully acclimatized to pot containing soil, sand and farm manure (1:1:1) and established in tissue culture room. In vitro methanol and ethanol extracts gave the highest amount of Apiol and Phytol content in GC-MS analysis. This protocol could be very useful for mass cultivation of Peperomia pellucida for industrial exploitation with selected and standardized plants productiont.

Maximizing Hybrid Seedlings Recovery and Early Identification of Highly Polyembryonic Acid Lime (Citrus aurantifolia Swing.) × Lemon (Citrus limon Burm.) Hybrids using SSR Markers

Abstract Nucellar embryony is the major obstacle in getting hybrid seedlings in interspecific crosses in citrus. Hence, the present study was conducted to standardize embryo age, culture media for maximizing germination, and subsequent identification of simple sequence repeat (SSR) markers to differentiate the hybrids. A factorial experiment was conducted with three embryo ages – 80–90, 110–120, and 130–140 days after pollination (DAP). The germination of the rescued immature embryos of acid lime × lemon crosses was initiated on three different growing media. The fruits harvested at 130–140 and 80–90 DAP had a higher number of functional seeds (90%). Older embryos (130–140 DAP) and middle-aged embryos (110–120 DAP) germinated in 100% on Murashige and Skoog (MS) medium supplemented with 500 mg·l−1 malt extract, 1.5 mg·l−1 gibberellic acid (GA3), and 0.02 mg·l−1 naphthalene acetic acid (NAA). MS medium supplemented with 500 mg·l−1 malt extract proved better for the germination of embryos taken at 80–90 DAP. Plantlet survival was the highest in younger embryos (80–90 DAP) cultured on MS basal medium (84.21%) and the lowest in older embryos cultured on MS medium supplemented with 500 mg·l−1 malt extract plus 1.5 mg·l−1 GA3 and 0.02 mg·l−1 NAA. The seedlings obtained from the culture 80–90 DAP had the highest root length (4.9 mm) and shoot length (5.3 mm) at 60 days after inoculation (DAI) on the above medium. SSR marker analysis revealed that CCSM-4 and CAC-33 markers expressed polymorphism between female and male parents, proving their ability to identify the hybrids of ‘Kagzi’ acid lime × ‘Konkan Seedless’ lemon.

Calcium-Dependent Changes in Cellular Redox Homeostasis and Heat Resistance of Wheat Plantlets under Influence of Hemin (Carbon Monoxide Donor)

Carbon monoxide (CO) is considered to be an important molecule-gasotransmitter involved in the regulation of the functional activity of plants, including in the processes of adaptation to stress factors. However, the associations of CO with other participants of signaling in the plant cells remain poorly studied. Using an inhibitory method, the role of different calcium pools in realization of the influence of hemin (carbon monoxide donor) on generation and neutralization of reactive oxygen species (ROS) in the root cells of wheat (Triticum aestivum L.) plantlets and their resistance to damaging heating (45°C, 10 min) was studied. The treatment of plantlets with 5 μM hemin caused a transient increase in the activity of extracellular peroxidase in roots and an increase in the generation of ROS with the maximum in 1.5–2 h after the beginning of treatment. The chelator of extracellular calcium EGTA and the inhibitor of inositol-1,4,5-phosphate formation neomycin, which reduces calcium influx into the cytosol from intracellular compartments, almost completely eliminated an increase in the activity of extracellular peroxidase caused by exogenous CO. At the same time, EGTA (completely) and neomycin (partially) leveled an increase in the content of hydrogen peroxide in the roots of plantlets occurring under the influence of CO donor. The treatment of plantlets with hemin also induced an increase in the activity of superoxide dismutase, catalase, and intracellular peroxidase in roots. Both calcium antagonists eliminated these effects. In the presence of EGTA and neomycin, there was also no positive effect of the treatment of hemin on the state of biomembranes and survival of plantlets after the damaging heating. It was concluded that both extracellular calcium and that deposited in intracellular compartments is involved in CO donor-induced increase in the formation of ROS, induction of the antioxidant system, and development of heat resistance of wheat plantlets.