Micro-propagated Plants Research Articles

Micropropagation of Zuccagnia punctata, an argentine medicinal plant, by means of “in vitro” technology to promove its conservation and the sustainable production of bioactive compounds

Zuccagnia punctata Cav. (“jarilla poposa”) is a monotypic argentine native medicinal plant used by indigenous communities for the treatment of several pathologies. The pharmacological properties have been scientifically validated and its potential use in phytocosmetic and pharmaceutic industry as antiaged, hypoglycemic, anti-inflammatory, antitumoral, antioxidant, antibacterial and antimicotic has been previously demonstrated. However, the species has a low propagation rate in natural conditions and has been included in the preliminary red list of endangered plants in category 3. Therefore, it is necessary to develop methodologies leading to its conservation and propagation to achieve its sustainable use. The objective of this work is to perform a micropropagation protocol for Z. punctata species for the sustainable use and conservation of this species. Furthermore, the study evaluated also the impact of “in vitro” propagation on the content of bioactive compounds of ethanolic extracts obtained from Z. punctata plants obtained “in vitro” with collected wild plants. Seeds have been used as mother material. The seed germination to obtain explants were performed on Murashige and Skoog medium (MS) without plant growth regulators (PGRs) containing sucrose, agar, and ascorbic acid. The same medium was employed for the multiplication of shoots. “In vitro” rooting was achieved in liquid and semisolid medium, and a higher yield was obtained in the latter. The plants were transferred into commercial substrate and acclimatized in a greenhouse. The micropropagated plants (MP) had a high content of total phenolic compounds similar to those obtained from wild adult plants (WAP) collected in the Monte Desert. The flavonoid content of MP was higher than that of WAP. The main chalcones identified in WAP, 2′,4′ dihydroxychalcone (DHC) and 2′,4′-dihydroxy-3′-methoxychalcone (DHMC) were also present in the MP with a rate 1/1, and 1/3 in WAP. The extracts obtained from the MP showed antioxidant, antiinflammatory and antimicrobial activity, similar to WAP. The “in vitro” propagation and acclimatization of Z. punctata constitutes a fundamental tool to achieve large-scale multiplication of this species in the future and the sustainable production of bioactive compounds with commercial applications. Reimplantation in the natural habitat could prevent the loss of this medicinal genetic resource.

Harnessing the Beneficial Fungus Piriformospora indica for Climate Resilient Crop Production: A Review

Piriformospora indica, classified as an Agaricomycetes fungus, has garnered significant interest due to its remarkable capacity to enhance plant growth, fortification, and resilience to stress factors. Its ability to inhabit various plant species stems from its direct influence on plant hormone signalling pathways. This colonisation stimulates increased root proliferation by generating indole-3-acetic acid, facilitating better nutrient absorption and ultimately leading to heightened crop yields. Furthermore, P. indica initiates resistance against fungal and viral threats while reinforcing the plant antioxidant defences, fortifying its resilience to stressors. It aids in successfully acclimating micro-propagated plants upon transplantation. It can establish complex symbiotic relationships, including tripartite symbiosis, which enhances the population dynamics of plant growth-promoting rhizobacteria. Overall, P. indica is a multifaceted facilitator of plant growth, a source of biofertilisation, a barrier against pests and diseases, a regulator of biological functions, and a promoter of overall plant development. Understanding the physiological and molecular mechanisms underlying the mutualistic interaction between P.indica and crop plants can provide valuable insights for enhancing crop protection and productivity, thereby contributing to the sustainability of agricultural practices.

GENETIC VARIATIONS OF IN VITRO PROPAGATED MD2 PINEAPPLES USING DNA-BASED MARKERS

Genetic variation determinations among micro-propagated plants is often tested to prevent the occurrence of morphological abnormalities which are deemed unsuitable for commercial use. Although the MD2 pineapple has been widely propagated clonally, there is no genetic fidelity testing on the micro-propagated MD2 pineapple. This study aims to determine genetic variations among the micro-propagated MD2 pineapples using Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. The amplification of both markers across 22 pineapple genotypes generated 120 bands with polymorphism percentages of between 93% and 73%, respectively. The evaluation through Rp, PIC and MI showed RAPD more informative (Rp = 7.08, PIC = 0.34, MI = 1.72) compared to ISSR (Rp = 4.17, PIC = 0.32, MI = 1.00). The PCA resulted in indefinite clustering patterns while the dendrograms portray the inability of the markers to correlate the plant morphology with their genetic structure. An analysis of molecular variance (AMOVA) analysis found high genetic variation (>70%) within the pineapples under review. Meanwhile, the Phi statistic estimate showed a wide genetic variation among the studied genotypes. To conclude, both markers suggest that the plant morphology did not inevitably correlate to its genetic structure. This study may form the basis for the MD2 breeding programme and explore other molecular markers that can potentially correspond to phenotypic polymorphisms.

Results of Propagation of Three Cultivars of Japanese Quince (Chaenomeles Japonica)

Abstract The aim of the trial was to determine the optimal propagation methods for three Japanese quince cultivars. Rooting trials with cultivars ‘Darius’, ‘Rasa’, and ‘Rondo’ were conducted in 2017–2019 in a greenhouse, to investigate the rooting and shoot quality of softwood cuttings, using different treatments. At the beginning of fruit production, growth and amount of the first yield was evaluated depending on propagation method. Softwood cuttings of Japanese quince rooted well (average 82–93%) in a greenhouse with heated substrate and partly automated temperature and moisture control, in spite of different weather conditions during three vegetation periods. The best results for rooting were found for cultivar ‘Rondo’. On average, better results of rooting for all cultivars were obtained when cuttings were soaked in indolylbutiric acid solution and trichodermin was added to the substrate. Significant differences in length and diameter of the bush shoots found between years for both propagation methods and also between years for one type of propagation. On average, microproagated plants had the highest shoot length. After evaluating the first yield, no significant differences were found between cultivars during the first two trial years, but micro-propagated plants had significantly higher yield per bush.

Comparative cytological studies of naturally grown plants and in vitro grown plants of Gynura procumbens – an important medicinal plant species

Summary Introduction: Gynura procumbens is an important medicinal plant that contains different types of chemical constituents that show tremendous curative effects. Objective: The aim of this research work was comparative karyomorphological analysis between in vitro and in vivo grown selected medicinal plant species. Methods: The roots were collected properly from naturally grown plants and in vitro propagated plants and pretreated with 1,4-para-dichlorobenzene (PDB) for 2.00 – 3.00 hours at room temperature. After pretreating with PDB the roots were washed thoroughly and kept in 1:3 (v/v) aceto-alcohol for 24 hours and then, transferred to 70% alcohol (v/v) for preservation. After that completing staining process, prepared slides were observed under microscope for karyotype analysis & ideogram preparation. Results: This research work reveled that both mother plant and tissue culture raised plants had 2n=20 chromosomes in somatic cell. No telocentric or acrocentric chromosome observed in the basic set of chromosomes. Both plants had 3 submetacentric and 7 metacentric chromosomes in the basic set of chromosomes. The centromeric formula for both plants was 3sm + 7m. The length of individual chromosome of in vivo plants ranged from 6.20 to 11.24 µm, whereas chromosome length of in vitro grown plants ranged from 6.08 to 11.22 µm. The total form percent (TF%) of naturally grown plants and micro-propagated plants were same (43.93%). Mother plant had 2A type of karyotype, the formula being 2Lsm + 4Msm + 4Mm + 10Sm. In vitro grown plant had also 2A type of karyotype which could be expressed by the formula, 2Lsm + 4Msm + 2Mm + 12Sm. Conclusion: The results of cytological studies point out that chromosome numbers of naturally grown plants and micro-propagated plants were same. There were very little karyotypic variations observed in case of both micropropagated and mother plants.

Comparative Karyomorphological Analysis of In vivo and In vitro Grown Plants of Plumbago zeylanica: An Important Medicinal Plant

The present report describes the comparative cytological analysis of in vivo and in vitro grown plant species of Plumbago zeylanica. Somatic chromosome number of in vivo and micro-propagated plants was confirmed to be 2n=28. Individual chromosome length of mother plant and micro-propagated plant was ranged from 2.26 to 5.54 µm and 2.26 to 5.46 µm, respectively. The total length of the haploid complement of in vivo and in vitro grown plants was 49.35 and 52.98 µm. The total form percent (TF%) of mother and in vitro grown plants was 45.11% and 45.58%, respectively and according to Stebbins classification (1971) both plants karyotype was fell into 2B symmetric type. The centromeric formula was for in vivo and in vitro grown plants were 3sm + 11m.

DNA-barcoding, SCoT and SRAP Based Somaclonal Variation in Micropropagated Withania somnifera Plantlets

Ashwagandha (Withania somnifera) is one of the recognized plant species that considered of most traditional natural supplements. Tissue culture is an efficient method as fast and affordable in plant propagation. Few studies have discussed the genetic impact of such method on ashwagandha plant. The aim of this research was to identify the genetic stability of micropropagated plantlets and to assess the impact of in vitro-propagation on somaclonal variability in ashwagandha using start codon-targeted (SCoT), sequence-related amplified polymorphism (SRAP) and DNA-barcoding assays. SCoT marker assay produced a total number of 132 bands with an average of 11 bands per primer, where scorable PCR fragments were generated from all primers. The phylogenetic tree constructed using SCoT binary data, revealed genetic variability among studied plant samples. SRAP primer combinations showed a total of 78 bands by an average of 11.1 bands / combination, in which all combinations produced scored PCR fragments. Over SRAP assay, one specific band was obtained that was present in different ashwagandha micropropagated plant samples compared to the control (mother plant). This PCR fragments were obtained using me1F/em1R primer combination (287 bp). The phylogenetic tree constructed using SRAP data was successful to differentiate between micro-propagated plants and the control. The DNA- barcoding analysis using chloroplast gene RNA polymerasel (rpoCl) gene was used to detect the soma- clonal variation between control and one micro-propagated plant of ashwagandha. The phylogenetic tree constructed using DNA-barcoding sequences was successful to differentiate between the two samples, where control and micropropagated plantlets were grouped in two different groups. This study suggests the valuableness of using SRAP and DNA-barcoding in detecting soma-clonal variation among micropropagated plantlest of ashwagandha.

Development of an efficient protocol for production of healthy sugarcane seed cane through Meristem culture

Healthy seeds are a prerequisite for improving the productivity of sugarcane crop. The best way to ensure the production of disease-free sugarcane seeds is via a technique called ‘Meristem Culture’ (Tissue Culture). An efficient protocol for meristem culture of sugarcane has been developed in this study. The potting mix for the survival of meristem cultured plantlets in a greenhouse was standardized, and agronomy for growing tissue culture plantlets in the field was developed. Multiple shoots were observed on Murashige and Skoog's (MS) medium supplemented with BAP 0.2 ​mg/l and Kinetin 0.1 ​mg/l. Best rooting was observed on MS media supplemented with IAA 1 ​mg/l and NAA 1 ​mg/l. Soil mixture containing [Soil ​+ ​Vermiculite ​+ ​Sand in 4: 1: 1 proportion (by volume)] ​+ ​Vermicompost @ 25% by volume of medium ​+ ​NPK is best suited for hardening tissue cultured plants in a greenhouse. Two feet distance between 2 plants and 3 feet distance between 2 rows was found as an appropriate method for planting tissue cultured plantlets in the field. Setts obtained from tissue cultured plants planted at 15 ​cm spacing gave higher yield than the other spacing. Setts obtained from micro-propagated plants (M) gave the highest yield as compared to setts from conventional sugarcane raising. The protocol developed in this study, shows potential for development of sugarcane meristem cultured plantlets and use of these plants for planting in field to obtain healthy sugarcane seed.

Field Performance and Genetic Stability of Micropropagated Gooseberry Plants (Ribes grossularia L.)

Gooseberry (Ribes grossularia L.) is a small fruit crop producing valuable fruits, which is constantly gaining importance. In vitro propagation of this species can significantly support the production of virus-free planting material and accelerate the introduction of new cultivars to the market. The aim of presented study was to assess field performance and genetic stability of micropropagated plants (MPs) of four gooseberry cultivars, “Captivator”, “Hinnonmaki Rot”, “Invicta”, and “Resika”. The growth vigor and yield of MPs and plants propagated by standard methods from softwood cuttings (ST) were evaluated in a field experiment. Microscopic observations of the number and length of the stomata of MP and ST plants were carried out. Two DNA-based techniques, amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR), were used to assess genetic stability of MP plants. For analysis of genetic stability of ST plants, the ISSR technique was applied. For three cultivars, Captivator, Hinnonmaki Rot, and Invicta, the plants’ growth vigor and fruit yield were greater in MP plants than in ST plants. In the case of Resika, most of these parameters were higher in ST plants. Microscopic observations of the stomata indicated a lack of differences in the length between MP and ST plants, while the stomata frequency on leaves of MP plants was higher than that of ST plants. The genetic variability of MP plants, assessed by AFLP, ranged from 0.35% for Hinnonmaki Rot to 2.12% for Resika. The results of ISSR analysis of MP plants showed variability from 0% in the case of Hinnonmaki Rot and Resika to 4% and 8.69% for Captivator and Invicta, respectively. No polymorphism was detected among ST plants of all analyzed gooseberry cultivars.

Performance evaluation of plants raised through different propagation methods in pomegranate (Punica granatum)

Disease free and elite planting material propagated through in vitro propagation may prevent the spread of diseases particularly bacterial blight through infected planting material. However, there are certain misapprehensions about fruit quality of harvest from micro-propagated plants as compared to air layered or hardwood cutting raised plants. Keeping these facts under consideration, an elaborate study on comparative qualitative and quantitative evaluation of harvest from different types of planting material had been carried out during 2015-17 at ICAR-NRC on Pomegranate, Solapur. The terminal bearing non-significantly ranged from 25 to 28.30% across the different types of planting material. Fruit weight and aril to fruit ratio were found at par in harvest from the three types of planting material. Rind thickness and PLW, which generally play critical role in governing fruit shelf life, were also found at par in fruits from all the three types of planting materials. The rind to fruit ratio of fruits from hardwood cutting raised plants (0.41) was found significantly higher than air layered plants. The maximum ‘L*’ and ‘b*’ values of fresh fruits were recorded for fruits from air layered plants (60.76 and 31.65, respectively) and ‘a*’ value for fruits from TC raised plants (39.70). The results proved parity among fruit quality of harvests from different types of planting material in pomegranate (Punica granatum L.) cv. Bhagwa.

A 1H NMR-based metabolomic approach to study the production of antimalarial compounds from Psiadia arguta leaves (pers.) voigt

Psiadia arguta (Asteraceae) is endemic to the island of Mauritius in the Indian Ocean. The species is traditionally used to treat various ailments, such as its use as an expectorant or for the treatment of bronchitis and asthma. Preliminary biological screenings have displayed the antimalarial (Plasmodium falciparum) and anticancer (HeLa human cell line) potential of P. arguta leaves. The phytochemical investigation of this plant has led to the isolation and characterization of sixteen compounds including five antiplasmodial molecules. The accumulation of the antiplasmodial compounds during the growth of the plant was studied by a 1H NMR-based metabolomic approach. In order to identify factors influencing the production of bioactive compounds, young plants of P. arguta were multiplied using in vitro culture techniques, and micro-propagated plants at different stages of development were acclimatized and followed for the experiments. The multivariate data analysis showed an accumulation of four bioactive compounds in the leaves of P. arguta when these plants were challenged with a biotic stress: labdan-13(E)-en-8α-ol-15-yl acetate, labdan-8α-ol-15-yl acetate, labdan-13(E)-ene-8α-ol-15-diol, and (8R,13S)-labdan-8,15-diol.

First attempts to obtain Actinidia deliciosa plants inoculated with endophytes antagonistic against Psa

Severe outbreaks of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) have been reported in Veneto and Friuli Venezia Giulia (north-east Italy) since 2010-2011. Since in some orchards with severe epidemics of Psa some Hayward plants were less affected by Psa, we carried out a study aiming at the selection of potential biocontrol agents (BCAs). From asymptomatic plants a bacterial strain 4A7 closely related to Pseudomonas asplenii was isolated and demonstrated in vitro antagonistic activity against Psa. In this study P. asplenii 4a7 (Pa4A7) was used for inoculation of micro-propagated plantlets 'Hayward' to obtain propagated material containing the antagonistic endophyte. To confirm the success of Pa4A7 inoculation in micro-propagated plants, we developed a specific and sensitive molecular method for its detection and quantification. The presence of Pa4A7 has been confirmed up to 100 days from the beginning of the test. This work represents the first step in trying to obtain plants with characteristics of tolerance to the pathogen based on inoculation of antagonistic endophytes.

Evaluation of Somaclonal and Ethyl Methane Sulfonate-Induced Genetic Variation of Mexican Oregano (Lippia graveolens H.B.K.)

Lippia graveolens, commonly known as Mexican oregano, is an aromatic plant of great industrial, nutritional, and medicinal value, principally for its essential oils. Regeneration via axillary buds was established in MS medium supplemented with 6-benzyladenine (BA) (0.5 mgL−1) as a growth regulator. Three genotypes and three stages of cultivation were considered in the study. On average, 3.5, 4.2, and 6.4 shoots induced per explant were obtained for genotypes B, C, and D, respectively. Several doses (0.1, 0.3, and 0.5%) of ethyl methane sulfonate (EMS) and different exposure times (1, 2, and 3 h) were applied to investigate the effect of the chemical mutagen on the formation of axillary buds. Genetic variation among the collected plants, the micro-propagated plants during three sub-cultivations, and the plants regenerated in the presence of the mutagen was evaluated by means of randomly amplified microsatellite polymorphism (RAMP) markers. A high genetic stability was observed in the micro-propagation of Mexican oregano for the three genotypes and three sub-cultivations, presenting 100% of monomorphic bands. The genetic variation observed in the different collections of wild populations (A, R, and V) and after treatment with EMS regarded 34 and 35% of polymorphic loci, respectively.

Photosynthetic Effects of Light-emitting Diode (LED) on in Vitro-derived Strawberry (Fragaria x Ananassa cv. Festival) Plants Under in Vitro Conditions

Purpose of this study was to investigate the effect of different photosynthetic photon flux density (PPFD) conditions using LED lamps on culture of shoot explants derived from in vitro shoots of Fragaria x ananassa cv. Festival. To examine the combined effect of 55% red LED, 15% far red LED, 10% blue LED, 15% green LED and 5% warm light LED light on in vitro growth of plantlets, fresh and dry plant biomass, plant height, leaf area, number of shoot, shoot length, number of root and percentage of rooting and various growth of micro-propagated plants were assessed under four light intensities (25, 50, 75 and 100 μmol m−2 s−1 PPFD; TRT2, TRT3, TRT4, TRT5, respectively). Un-rooted strawberry shoots were cultured in the “Culture Pack”-rockwool system with MS medium under CO2-enriched condition. The best response for regeneration of shoots and root induction was observed for shoot explants obtained on MS supplemented with BAP 1 mg/l and NAA 0.5 mg/l at TRT3 (50 μmol m−2 s−1 PPFD). Chlorophyll and net photosynthesis were optimal in plants grown under TRT4 (75 μmol m−2 s−1 PPFD). Stomatal resistance and Fv/Fm values were highest at low light irradiance (TRT2). The highest efficiency and high frequency of shoot formation occurred after 30 days. Elongation of shoot buds was obtained at TRT4 (75 μmol m−2 s−1 PPFD) and TRT5 (100 μmol m−2 s−1 PPFD) on the same MS medium. Regenerated shoots rooted best on the same medium of elongation. Irradiance at 75 μmol m−2 s−1 PPFD was suitable for the acclimatization of strawberry plants. We concluded that the present protocol can be efficiently used for mass propagation of the strawberry.

Effect of sucrose on physiological and biochemical changes of proliferated shoots of Bambusa vulgaris Schrad. Ex Wendl in temporary immersion

Tissue culture systems have contributed to the massive propagation of bamboos. However, micro-propagated plants show a low percentage of rooting and ex vitro survival due to anatomical and physiological anomalies induced by in vitro factors. This study aims to determine the effect of sucrose on physiological and biochemical changes in Bambusa vulgaris Schrad. Ex Wendl shoots cultured in a temporary immersion system and in plantlets growing under greenhouse conditions. For this purpose, two concentrations of sucrose (20 and 30 g L−1) and a control treatment (sucrose-free medium) were used in a rooting culture medium. Our data revealed that the absence of sucrose in the culture medium improved the morpho-physiological, biochemical and anatomical development of the shoots. However, a high sucrose concentration decreased the total chlorophyll content and leaf area in shoots. Additionally, the hydrogen peroxide content and malondialdehyde increased in shoots cultured with 30 g L−1 of sucrose. On the other hand, scanning electron microscopy revealed that leaves of shoots which grow on medium containing 30 g L−1 sucrose displayed anatomical changes in the stomata of plants, whereas those from sucrose-free medium exhibited normal structural development. Finally, shoots cultured without sucrose showed a high survival rate and allowed a better adaptation of plantlets in the greenhouse. The temporary immersion systems combined with culture medium without sucrose offer new prospects for improving the physiology and biochemistry of plantlets during in vitro to ex vitro transition.

Effect of soil P addition on growth, nutritional status and photosynthesis of potted grapevine plants grown under soil copper excess

The continuous use of copper (Cu)-based fungicides in viticulture to counteract fungal diseases has resulted in an accumulation of Cu in the topsoil, sometimes reaching toxic levels that can cause plant stress and reduce soil fertility. A possible strategy to mitigate the negative effects of Cu could include its incorporation into chemical compounds with phosphorous (P). The aim of this study was to evaluate the effectiveness of soil P addition on alleviating the toxic effect of Cu when applied in excess to potted grapevines plants. Micro-propagated plants of 1103 Paulsen rootstock were grown according to a factorial experimental design with two factors: Cu (4 levels: 0, 100, 200 and 300 mg kg-1 dw) and P (2 levels: 0 and 100 mg kg-1 dw) and three replicates. Sixty days after transplant, P was not effective in preventing the accumulation of Cu in vines. The total plant biomass was not influenced by soil Cu addition, while P increased plant growth by enhancing plant nutritional status. In addition, Cu reduced nutrient (P, K and Zn) content in plants. Net photosynthesis and efficiency of photosystem II decreased as a consequence of the increase of Cu concentration in soil. The effect of P in mitigating Cu toxicity was mainly related to the ability of plants to restore nutrient uptake.

Comparing nursery behavior, field plant yield and fruit quality of in vitro and in vivo propagated strawberry mother plants

In different countries, mostly in EU, rules for strawberry nursery propagation impose the use of micropropagation only to produce stock virus free plant followed by at least three nursery production cycles in the field before selling these plants to growers. This limit has been imposed by problems concerning true-to-type plants observed in micro-propagated plants of some cultivars, with higher genotypic and phenotypic instability, and the incorrect use of higher concentrations of cytokinin in the proliferation phase. This production system of certified plants has high economic and environmental costs without a final guarantee on the sanitary state of the plants. New knowledges on in vitro techniques and new cultivars with higher genetic stability can offer the opportunity to improve the use of micropropagation in strawberry nursery industry. With the aim to propose a new protocol for strawberry propagation, an experimental trial was set up to compare the behavior of frigo and micro-mother plants of cv. Alba, a well know commercial cultivar in EU, in two cycles of nursery runner production and the yield, to verify in two successive cycles the fruit quality of daughter plants grown in open field conditions. The results confirmed the equivalence of the two types of mother plants both in plant nursery production and subsequently in field fruit production. Therefore, the direct use of micro-mother plants for nursery productions of frigo-plants for growers offer an alternative option for large scale frigo-plant nursery production.

Soil-applied phosphorous is an effective tool to mitigate the toxicity of copper excess on grapevine grown in rhizobox

Abstract The aim of the present study was to evaluate the effectiveness of P soil addition to mitigate the toxic effect of excess of Cu on grapevine on sandy non-calcareous soil. Micro-propagated plants of 1103 Paulsen rootstock were grown according to a factorial experimental design with two factors: Cu addition (0, 100, 200 and 300 mg kg −1 dw) and P addition (0, 50 and 100 mg kg −1 soil dw). The experiment was conducted in the greenhouse and plants were grown on a sandy soil in rhizoboxes. At the end of the trial, plants were destructively harvested, the biomass and the nutrient concentration of organs were determined. Root growth was evaluated during the trial and the concentration of organic acid in root tissue and exudates was determined. Shoot length and plant biomass declined as Cu concentration in soil increased; the application of P enhanced plant growth and nutritional status. Root biomass decreased at Cu > 100 mg kg −1 and increased in P at 100 mg kg −1 . Phosphorous stimulated root length, diameter and the number of apexes. Root citric acid increased as a response of Cu at 300 mg kg −1 , root ascorbic acid decreased with the increase of Cu (200 and 300 mg kg −1 ) and P at 100 mg kg −1 . The concentration of citric acid from root exudations was higher in Cu at 300 mg kg −1 and Cu at 200 mg kg −1 (compared to control) and P at 100 mg kg −1 soils. The application of P fertilization seems to be a valuable strategy to overcome the toxicity of high concentration of Cu in soil.

Genetic Stability of Cassava Plants Regenerated Through Organogenesis Using Microsatellite Markers

Tissue culture technology of cassava (Manihot esculenta Crantz) is a viable alternative to currently adopted techniques for mass propagation, germplasm conservation and genetic improvement. However, somaclonal variation is a common phenomenon in tissue culture which makes it mandatory to monitor the genetic stability of plants. Therefore, the objective of this study was to evaluate the genetic stability of cassava plants regenerated from axillary bud explants through direct organogenesis using simple sequence repeat (SSR) markers. High shoot regeneration (81.2 – 90.0%) occurred in MS medium supplemented with 10 mg/L 6-bnzylaminopurine (BAP) and multiple shoots (2 – 4 shoots per from axillary bud explant) were formed for all the three cultivars (TME14, TMS60444 and Kibandameno) tested. High frequency of rooting (100%) was obtained after transferring the plantlets to cassava basic medium (CBM) and the rooted plants were successfully hardened and acclimatized in the glasshouse with 100% survival rate. Three-month old plants exhibited normal morphological characters comparing with the mother plant. A total of 10 SSR markers were used to validate the genetic homogeneity amongst five randomly selected plants along with the donor mother plants. DNA fingerprints of axillary bud regenerated plants displayed monomorphic bands similar to mother plant, indicating homogeneity among the regenerated plants with donor mother plant. The effect of subculture frequency on genetic stability of axillary bud-derived regenerants and micropropagated plants was also assessed using SSR markers. All the SSR profiles from axillary bud regenerants and micropropagated plants were monomorphic and comparable to mother plants from 1st to 5th subculture, confirming the genetic stability among clones and mother plants. At the 6th subculture, similarity indicators between the progenies and the mother plants ranged from 0.95 to 1.0 and such a similarity indicated a very low polymorphism. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis of the 6th subculture revealed 96% similarity amongst axillary bud regenerants and micropropagated plants with donor mother plant. This low polymorphism ratio between mother plants, axillary bud regenerants and micro-propagated plants indicates the little effect of somaclonal variations, the high genetic similarity between mother plants and progenies and demonstrates the reliability of this propagation system for cassava. These results suggest that direct organogenesis from the axillary buds is the safest method for regeneration of true-to-type plants and this system can be used for clonal mass propagation, germplasm conservation and genetic transformation of cassava.

Efficient in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood) and assessment of genetic fidelity of micropropagated plants.

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3%) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5µM) along with 7.5µM 2-isopentenyl adenine (2-iP). An optimal of 10.16±2.24 shoots were regenerated on medium supplemented with 2.5µM BAP+7.5µM 2-iP. Quarter strength MS medium supplemented with 10µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.