Highly efficient in vitro regeneration of the industrial oilseed crop Crambe abyssinica

A highly efficient regeneration protocol for oilseed crop Crambe abyssinica has been developed using hypocotyls as explants in this study. Crambe is a potential engineering oilseed crop for industrial purposes as it contains 55-60% erucic acid in its oil and, more importantly, it does not outcross with any food oil seed crops. However, the low regeneration frequency with the currently available protocols is still a limiting factor for genetic modification of Crambe. In this study, we investigated the effects of N-source, C-source, AgNO₃, cultural conditions as well as the concentration and combination of plant growth regulators (PGR) on the regeneration frequency of C. abyssinica. The results showed that all these factors, especially the N-source and PGR concentrations and combinations, played an important role in shoot regeneration. Among all the factors tested, the combination of using hypocotyls from C. abyssinica cv. galactica, the Lepiovre basal medium supplemented with 16 g l⁻¹ glucose, 0.5 g l⁻¹ AgNO₃, 2.2 mg l⁻¹ thidiazuron (TDZ), 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 2.5 g l⁻¹ Gelrite, seeds germinated in dark for 3 days and explants cultured in light, gave the best regeneration frequency (over 95%). The results also suggest that reducing the content of NH₄⁺ or keeping a suitable NO₃⁻/NH₄⁺ ratio in the regeneration medium would be crucial to Crambe shoot regeneration.     

Regeneration in Jatropha curcas: Factors affecting the efficiency of in vitro regeneration

Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl explants of Jatropha curcas were studied in the present investigation. Regeneration in J. curcas was found to be genotype dependent and out of four toxic and one non-toxic genotype studied, non-toxic was least responsive. The best results irrespective of genotype were obtained on the medium containing 0.5 mg L⁻¹ TDZ (Thidiazuron) and in vitro hypocotyl explants were observed to have higher regeneration efficiency as compared to ex vitro explant in both toxic and non-toxic genotypes. Adventitious shoot buds could be induced from the distal end of explants in all the genotypes. The number of shoot buds formed and not the number of explants responding to TDZ treatment were significantly affected by the position of the explant on the seedling axis. Explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas the regeneration response declined with increase in age of seedlings after 30 days. Transient reduction of Ca²⁺ concentrations to 0.22 g L⁻¹ in the germination medium increased the number of responding explants.Induced shoot buds, upon transfer to MS medium containing 2 mg L⁻¹ Kn (Kinetin) and 1 mg L⁻¹ BAP (6-benzylamino purine) elongated. These elongated shoots were further proliferated on MS medium supplemented with 1.5 mg L⁻¹ IAA (indole-3-acetic acid) and 0.5 mg L⁻¹ BAP and 3.01-3.91 cm elongation was achieved after 6 weeks. No genotype specific variance in shoot elongation was observed among the toxic genotypes except the CSMCRI-JC2, which showed reduced response. And for proliferation among the toxic genotypes, CSMCRI-JC4 showed highest number of shoots formed. Among the rest, no significant differences were observed. The elongated shoot could be rooted by pulse treatment on half-strength MS medium supplemented with 2% sucrose, 3 mg L⁻¹ IBA (indole-3-butyric acid), 1 mg L⁻¹ IAA, 1 mg L⁻¹ NAA (α-naphthalene acetic acid) and subsequent transfer on 0.25 mg L⁻¹ activated charcoal medium. The rooted plants could be established in soil with more than 90% success. No significant differences were observed in rooting of shoots in the different toxic genotypes. However, rooting response was reduced in non-toxic genotype as compared to toxic genotypes.     

Assessment of dermal wound healing and in vitro antioxidant properties of Avena sativa L.

Avena sativa L. (Poaceae) has been reported to have traditional utilization against skin diseases and inflammation. Therefore, in this study, the n-hexane, ethyl acetate, ethanol, and water extracts of A. sativa were investigated for their wound healing and antioxidant activities. Total phenol and flavonoid contents of the extracts were established spectrophotometrically. For the wound healing activity, linear incision and circular excision models on rats and mice were evaluated with a standard ointment Madecassol^®. Antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferrous ion-chelating, and ferric-reducing antioxidant power (FRAP) assays. Significant wound healing activity was observed with the ointment formulation of the ethanol extract at 1% concentration. The histopathological examination results also supported the outcome of both linear incision and circular excision wound models. All of the extracts exerted low antioxidant activity in the applied assays. The present study provides a scientific evidence for the traditional usage of A. sativa in the management of wound healing.     

NaCl plays a key role for in vitro micropropagation of Salicornia brachiata, an extreme halophyte

A simple and rapid method for micropropagation of succulent, salt accumulator and extreme halophyte Salicornia brachiata has been established for the first time using shoot tips and nodes. Individually, BA showed significant response compared to Kn and in combinations, improved shoot proliferation was observed with BA + NAA than BA + 2,4-D, however no significant response was observed with BA + IAA. Percentage of shoot response significantly increased with NaCl treatment in the combination of BA + NAA while BA + 2,4-D + NaCl combination showed reduced shoot proliferation followed by demises of most of cultures. Efficient shoot proliferation was observed with combinations BA (8.9 μM) + NAA (5.37 μM) + NaCl (500 mM) and BA (13.3 μM) + NAA (5.37 μM) + NaCl (250 mM) indicating that NaCl is required for the micropropagation. The developed method will facilitate functional analysis of novel salt responsive gene(s) isolated from S. brachiata and propagation of industrially important elite accessions.     

Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr.

The study revealed, for the first time, accumulation of spilanthol, an antiseptic alkylamide, in in vitro cultures of Spilanthes acmella Murr., a medicinal plant of immense commercial value. To achieve this, in vitro shoots were regenerated via direct organogenesis from leaf-disc explants of Spilanthes. Shoots were induced in the presence of N⁶-benzylaminopurine (BAP) alone or in combination with either α-naphthalene acetic acid (NAA) or Indole-3-acetic acid (IAA) in Murashige and Skoog medium. The best treatment for shoot regeneration was MS + BAP (5.0 μM) + IAA (5.0 μM), which promoted adventitious shoot proliferation in >82% cultures with an average of 5.3 shoots per explant. Regenerated shoots rooted spontaneously with a frequency of 100% on half strength MS medium (major salts reduced to half strength) containing 50 g l⁻¹ sucrose. The plantlets were acclimatized successfully with 90% survival rate. Additionally, ploidy stability of the regenerated plants was assessed by flow cytometry which showed that all investigated plants had the similar ploidy as that of the mother plant. For spilanthol identification, peaks eluted from HPLC were analyzed by mass spectrometry with its characteristic fragmentation pattern. For quantification studies, calibration curve was generated, which revealed a higher amount of spilanthol content (3294.36 ± 12.4 μg/g DW) in the leaves of in vitro plants compare to those of in vivo plants (2703.66 ± 9.6 μg/g DW of spilanthol). An efficient multiplication frequency, ploidy stability and enhanced spilanthol accumulation ensure the efficacy of the protocol developed for this industrially important medicinal plant.     

Azoxystrobin resistance of French Mycosphaerella graminicola strains assessed by four in vitro bioassays and by screening of G143A substitution

We tested here five distinct in vitro screening methods assessing Mycosphaerella graminicola resistance to strobilurin fungicides: (i) spotting on agar medium, (ii) spore germination on agar medium, (iii) growth in microplates without and (iv) with the addition of Alamar blue and (v) screening of the cytochrome b substitution conferring resistance G143A. The assays were performed by assessing resistance to azoxystrobin of 32 French M. graminicola strains and the two reference strains IPO323 and IPO94269 from the Netherlands. The microplate Alamar blue assay displayed high standard deviations from all growth averages, hence reporting the strong lack of reliability of this method. As expected, the two reference strains were found sensitive with all other methods. In agreement with the disruptive resistance of M. graminicola to single-site fungicides, the half maximal inhibitory concentration (IC₅₀) values of sensitive and resistant strains displayed a bimodal distribution pattern with each type of assay. All strains carrying G143 or A143 alleles were found sensitive or resistant respectively with each bioassay. Although the absolute IC₅₀ value for each strain varied among assays, the ranking of strains according to their IC₅₀ values was overall conserved with all assays. Finally, the frequency of strobilurin resistant haplotypes within France was investigated by screening G143A in 82 strains isolated in 2005 year from 12 localities. Results showed a marked gradual decrease in resistance distribution from 70% in northern to 30% and 0% in central and southern France, respectively. The accuracy of each method as well as the widespread and incidence of strobilurin resistance within the French population of M. graminicola have been discussed.     

Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb

Stevia rebaudiana is a valuable medicinal plant species and it is being used for the treatment of diabetes. Currently, there is a high demand for raw material of this medicinal herb due to ever increasing diabetes disorder among the population. In order to meet the increased demand an efficient in vitro propagation of S. rebaudiana was established. Nodal explants collected from the field were cultured on MS basal medium fortified with different concentrations of BAP (0.5-3.0 mg/l) and KIN (0.5-3.0 mg/l) individually for shoot bud induction. In vitro derived nodal buds were cultured on MS medium supplemented with different concentrations (0.5-3.0 mg/l) of BAP and KIN for multiple shoot bud regeneration. In the second experiment, in vitro derived buds were placed on MS medium supplemented with different concentrations of BAP (0.5-3.0 mg/l) in combination with 0.5 mg/l IAA or IBA or NAA for shoot bud multiplication. The highest frequency (94.50%) of multiple shoot regeneration with maximum number of shoots (15.69 shoots/explant) was noticed on MS medium supplemented with 1.0 mg/l BAP. For large scale plant production, in vitro derived nodal bud explants were cultured on MS medium fortified with 1.0 mg/l BAP, in which about 123 shoots/explant were obtained after three subcultures on the same media composition. Elongated shoots (>2 cm) dissected out from the in vitro proliferated shoot clumps were cultured on half-strength MS medium containing different concentrations of NAA (0.1-0.5 mg/l) and/or MS medium fortified with various concentrations (0.5-2.0 mg/l) of auxins (NAA, IAA and IBA) for root induction. Highest frequency of rooting (96%) was noticed on half-strength MS medium augmented with 0.4 mg/l NAA. The rooted plantlets were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and subsequently established in the greenhouse. The present in vitro propagation protocol would facilitate an alternative method for rapid and large-scale production of this important antidiabetic medicinal plant.     

In vitro starch digestibility and pasting properties of germinated brown rice after hydrothermal treatments

Germinated brown rice (GBR) recently has received renewed attention due to its enhanced nutritional value. Pasting properties and in vitro starch digestibility of GBR were examined before and after hydrothermal treatments. Steeping in water (30 °C, 24 h) raised the moisture content and germination percentage of brown rice. Pasting viscosity was substantially decreased but gelatinization temperatures and enthalpy were decreased only marginally by germination (30 °C, 48 h). However, annealing (50 °C, 24 h) and heat-moisture treatment (100 °C, 1 h at 30% moisture) after germination resulted in increased pasting viscosity and gelatinization temperatures. The hydrothermal treatments, however, induced browning reactions to darken the flour of GBR. The digestibility of starch in brown rice was increased by germination. The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) in the cooked brown rice were 47.3%, 40.8%, and 11.9%, respectively, but changed to 57.7%, 39.1%, and 3.2%, respectively upon germination. The hydrothermal treatments, however, decreased the digestibility of starch in GBR. The heat-moisture treatment decreased the RDS content in GBR near to that of native brown rice. The digestibility and physical properties of brown rice can be controlled by germination and hydrothermal treatments.     

Distribution of apple stem grooving virus and apple chlorotic leaf spot virus in infected in vitro pear shoots

The distribution patterns of Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) in in vitro-cultured pear plants were investigated using in situ tissue-printing hybridization (TPH) and tissue blotting immunoassay (TBIA) to detect viral RNAs and coating proteins. Both ASGV and ACLSV showed high concentrations in the tip of the pear shoots and lower concentrations in the middle stem. The highest viral RNA titers were found in the phloem parenchyma of vascular bundles. Monitoring of viral RNA concentrations was conducted on infected in vitro-cultured pear plants during thermotherapy using TPH combined with X-ray film exposure in serial cross sections. No viral RNA of ACLSV or ASGV was detected in less than 2 mm and 0.5 mm long tips, respectively. The heat treatment was less effective to reduce virus titers in the bottom shoot. The obtained results would assist in the selection of tips with proper sizes from pear shoots pre- and post-thermotherapy for the production of virus-free pear plants by meristem culture.     

In vitro pullulanase activity of wheat (Triticum aestivum L.) limit-dextrinase type starch debranching enzyme is modulated by redox conditions

Expression of a limit-dextrinase (LD) type starch debranching enzyme (EC 3.2.1.41) was observed in developing wheat (Triticum aestivum L.) endosperm and germinating grains, indicating a role for the enzyme in both biosynthesis and degradation of starch. A full-length cDNA, TaLD1, encoding LD in wheat developing kernels was isolated and predicted to encode a 98.6 kDa mature protein active in amyloplasts. Isolated cDNA was expressed in Escherichia coli to produce a recombinant His-tagged LD, which mainly accumulated in inclusion bodies as an inactive enzyme. Extraction of His-tagged LD from the inclusion bodies followed by dialysis under thiol/disulphide redox conditions allowed partial refolding of the protein and detection of pullulanase specific activities by zymogram analysis and enzyme assays. Several active conformations were demonstrated by the recombinant TaLD1 and pullulanase activity could be modulated by redox conditions in vitro. The results suggest that cellular redox conditions may regulate the level of LD activity in wheat tissues.     

Genetic stability of long-term micropropagated Opuntia ficus-indica (L.) Mill. plantlets as assessed by molecular tools: Perspectives for in vitro conservation

Randomly amplified polymorphic DNA markers (RAPD) were employed to assess the level of genetic stability of long term micropropagated prickly pear (Opuntia ficus-indica) plantlets.Thirteen micropropagated plantlets were chosen from a clonal collection of shoots that originated from a single mother shoot. This clonal collection had been maintained under in vitro culture conditions for at least 5 years, as achieved for the time by axillary branch multiplication in Opuntia ficus-indica.Twenty arbitrary primers were used to compare RAPD patterns between in vitro raised material and the mother plant. Only 11 primers were found to yield distinct and reproducible amplification products resulting in a total of 87 amplified products, out of which 82 bands were monomorphic across all the plantlets and 5 showed polymorphisms.Cluster analysis performed on the basis of similarity indices indicated that all micropropagated plantlets and their mother plant grouped together in one major cluster with a 91% level of similarity.Low level of genetic variation has been detected, as polymorphic bands accounted for just 2.79% of the total genetic variation. This very low level of genetic variation, despite more than 5 years of in vitro culture, demonstrates the genetic stability of Opuntia ficus-indica and indicates that the axillary branch multiplication method is highly reliable for the multiplication of genetically true-to-type plant material.The high degree of clonal fidelity detected here, recommend the use of axillary-branching micropropagation technique for the safe in vitro conservation of prickly pear interesting genetic resources.     

Conversion of starch from dry common beans (Phaseolus vulgaris L.) to ethanol

Dry common beans (Phaseolus vulgaris L.) were evaluated for potential conversion of starch to ethanol. Eight varieties of beans with average starch content of 46% (db) were assayed in a laboratory-scaled process based upon the commercial corn dry grind fermentation process. Ethanol yield was 0.43-0.51 g ethanol/g glucose (0.19-0.23 g ethanol/g beans). The average ethanol yield for the eight bean types was 92% of maximum theoretical yield, demonstrating that starch from beans could be efficiently converted to ethanol. Ethanol concentration obtained from 20% (w/w) solids loading was 3.5-4.4% (w/v). The residual fermentation solids contained, on a dry basis, 37.1-43.6% crude protein, 10.8-15.1% acid detergent fiber and 19.1-31.3% neutral detergent fiber.     

Effect of emulsifiers on complexation and functional properties of oxidized white sorghum (Sorghum bicolor) starch

The effect of addition of three commonly used emulsifiers namely GMS (glycerol monostearate), SSL (sodium stearoyl lactylate) and DATEM (diacetyl tartaric acid esters of monoglycerides) on complexation, thermal, pasting and textural properties of OWSS (oxidized white sorghum starch) was studied. The study is of interest as both oxidized starches and emulsifiers are present as co-ingredients in different food products and thus their complexation could affect the textural characteristics of foods. The complexation index (CI) reduced on oxidation of sorghum starch. The CI for native white sorghum starch (NWSS) was in the order GMS > SSL > DATEM whereas for OWSS, CI was in the order GMS > SSL > DATEM. Presence of emulsifiers significantly reduced gelatinization enthalpies of starches. Types I and II amylose–lipid complexes were observed in NWSS and OWSS on addition of GMS. Pasting temperature of NWSS increased while peak viscosity reduced on addition of SSL and GMS. Cold paste and setback viscosities of OWSS increased significantly (p ≤ 0.05) on addition of emulsifiers. Emulsifiers reduced firmness and rupture strength of NWSS and OWSS gels. Increase in firmness of gels on storage increased in NWSS and decreased in OWSS on addition of emulsifiers. Elasticity of OWSS on cold storage was higher in the presence of SSL and GMS.     

Study on melting and crystallization of short-linear chains from debranched waxy starches by in situ synchrotron wide-angle X-ray diffraction

In situ melting and crystallization of short-linear α-1,4-glucan (short-chain amylose, or SCA) from debranched waxy starches were investigated by synchrotron wide-angle X-ray diffraction. Amorphous SCA was prepared by dissolving completely debranched waxy starches in alkaline solution and neutralized by hydrochloric acid. When hydrated with 50% water at 25 °C, all amorphous SCA crystallized immediately and gave a B-type structure. The SCA from debranched waxy potato starch had a longer average chain length and a higher melting temperature but relatively lower crystallinity upon hydration; it was not completely melted at 100 °C and retained its original B-type structure during rapid cooling. In contrast, the SCA from debranched waxy wheat and waxy maize starches had a large portion of low molecular weight fractions, a higher crystallinity upon hydration, and a lower melting temperature. These differences suggest that amylopectin short chains crystallized more readily but their crystals were weaker than those of long chains. After the B-type crystals of hydrated SCA from waxy wheat and waxy maize starches melted, they reformed into the A-type polymorph upon rapid cooling. The thermal properties showed that the A-type polymorph of debranched waxy wheat and waxy maize starches had a higher melting temperature than their B-type structure.     

Effect of wheat bran addition on in vitro starch digestibility,physico-mechanical and sensory properties of biscuits

Biscuits contain high amount of fat and sugar thus having high calorie but low nutrient density. Wheat bran is a good source of dietary fibre (DF) and protein and is thus a good candidate for nutritional enrichment of cereal foods. The aim of this study was to understand the effect of bran incorporation and particle size reduction on biscuit microstructure, texture and in vitro starch digestibility. Five different biscuits containing 5–15% DF were produced. Two different particle sized wheat brans were used: coarse (450 μm) and fine (68 μm). Bran particle size reduction increased the elastic modulus and hardness of biscuits. Biscuits containing fine bran had visually more compact structure without any surface or internal defects than those with coarse bran. Fine bran containing sample had the highest hardness value. Sensory evaluation showed that roughness and breakdown of biscuits in the mouth was significant for the coarse bran with highest level of bran addition. The instrumental elastic modulus, stress and hardness were closely related to sensory hardness and strength to break. Increasing DF content from 5 to 15% increased hydrolysis index by 16%, from 32 to 37.     

Analysis of starch swelling power in Australian breeding lines of hexaploid wheat (Triticum aestivum L.)

Starch is the major component of wheat (Triticum aestivum L.) grain and is composed of two large glucan molecules, amylose and amylopectin. The ratio between the two polymers types influences the water absorbing properties of starch upon heating, and thus affects the end-use of grain and purified starch. In this study, we evaluated the starch swelling power (SSP) values in seven wheat populations developed from crosses involving low-SSP lines. Analysis of starch produced by the F₂ generation plants showed that the largest SSP variation (11.4–16.2) and lowest SSP mean (13.9) was obtained for a population derived from doubled haploid lines SM1028 (SSP = 14.5) and VK306 (SSP = 13.6). The population of 360 lines was advanced by single seed descent to the following generations for further studies. Starch analysis of grain produced by F₄ generation lines in two field locations during 2006 and in a greenhouse environment during 2005 showed that SSP values were relatively stably inherited. The average broad-sense heritability was 73% and significant (P < 0.001) genotype × genotype and genotype × environment interactions were seen. Starches with the highest and lowest SSP values were inversely related to amylose concentration determined by high pressure liquid chromatography (HPLC)–size exclusion chromatography (SEC) of debranched starch. Developed lines with the lowest SSP values surpassed 40% in apparent amylose concentration. The study illustrates that screening for SSP in early generations can be used to develop wheat lines with desired starch swelling characteristics.     

Differential response of pea (Pisum sativum) to Orobanche crenata, Orobanche foetida and Phelipanche aegyptiaca

Several broomrape species including Orobanche crenata, Orobanche foetida and Phelipanche aegyptiaca are reported to infect various grain and forage legumes in the Mediterranean and West Asia. Pea (Pisum sativum) is severely damaged by O. crenata, but there are no reports on O. foetida or P. aegyptiaca infection. We report here that pea can induce high germination of seeds of O. crenata, O. foetida and P. aegyptiaca but only O. crenata success infecting pea roots and developing further. Some differences in levels of infection by O. crenata were observed among pea accessions what can be exploited in pea resistance breeding programmes. On the contrary, all pea accessions studied were highly resistant to infection by both O. foetida and P. aegyptiaca, preventing any tubercle attachment and development. This makes pea a promising candidate as trap crop for O. foetida and P. aegyptiaca seed bank demise in infested soils.     

Digestibility of protein and starch from sorghum (Sorghum bicolor) is linked to biochemical and structural features of grain endosperm

Although a principal source of energy and protein for millions of the world's poorest people, the nutritional value of sorghum (Sorghum bicolor L. Moench) is diminished because of low digestibility of grain protein and starch. To address this problem, we analyzed the properties of two sorghum lines that have a common pedigree but differ in digestibility. Consistent with results based on a ruminal fluid assay, the protein and starch of one line (KS48) was more thoroughly digested than that of the other (KS51) using in vitro assays based on pepsin and α-amylase. The indigestibility of KS51 relative to KS48 was shown to be due to (i) a greater abundance of disulfide-bonded proteins; (ii) presence in KS51 of non-waxy starch and the accompanying granule-bound starch synthase; and (iii) the differing nature of the protein matrix and its interaction with starch. The current findings suggest that each of these factors should be considered in efforts to enhance the nutritional value of sorghum grain.