In vitro study on the effect of cytokines and auxins addition to growth medium on the micropropagation and rooting of Paulownia species (Paulownia hybrid and Paulownia tomentosa)

This study represents an efficient preliminary protocol for in vitro mass production of two Paulownia species (Paulownia hybrid and Paulownia tomentosa) seedlings by using seed explant. Different concentrations of benzyladenine (BA) or Kinetin (Kin) (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L) were tested during multiplication stage. The number of shoots/explants was significantly increased with increasing either BA or Kin concentration; however, the shoot length significantly decreased. Data show that media fortified by BA (10 mg/L) combined with indole butyric acid (IBA) at 1.0 or 1.5 mg/L recorded the highest number of shoots/explant (9.13 and 9.25, respectively). After six weeks during the multiplication stage, data cleared that media fortified by benzyladenine (10 mg/L) combined with IBA at 0.5 mg/L recorded the highest shoot length (3.23 cm). The inclusion of indole butyric acid (IBA) or naphthalene acetic acid (NAA) at 1.0–1.5 mg/L to the medium significantly increased the number of roots/plantlets and the highest root length. The results indicated that IBA supplementation was more effective than NAA for in vitro rooting of both Paulownia species. The best treatment for multiplication was 10 mg/L and 8.0–10 mg/L BA for P. hybrid and P. tomentosa, respectively. Peat moss and sand (1:1, v/v) or peat moss and sand (1:2, v/v) were investigated as soil mixture during the adaptation stage. The results referred that Paulownia species plantlets were successfully survived (100 %) in soil mixture contained peat moss: sand (1:2, v/v). This mixture recorded the highest values of plantlet height and number of leaves/plantlets.     

In vitro propagation of ginger (Zingiber officinale Rosco)

An efficient and promising protocol for in vitro propagation of Zingiber officinale Rosco using sprouting buds was established. Sprouting buds were sterilized and cultured onto MS medium supplemented with different growth regulators. Augmentation of MS-medium with 4.5 mg/l BAP recorded the highest percentage of shootlets multiplication. Shootlets were highly rooted on half strength of B₅ medium supplemented with 1.0 mg/l NAA. The maximum percentage of acclimatization, hardening and rhizomes production of in vitro derived plants in greenhouse was 80–100%.     

Development of an efficient callus proliferation system for Rheum coreanum Nakai,a rare medicinal plant growing in Democratic People’s Republic of Korea

A clonal mass propagation to obtain mountainous sources of Rheum coreanum Nakai, a rare medicinal plant in Democratic People’s Republic of Korea was established by rhizome tissue culture. Whole plants were selected and collected as a vigorous individual free from blights and harmful insects among wild plants of R. coreanum grown on the top of Mt. Langrim (1.540 m above the sea) situated at the northern extremity of Democratic People’s Republic of Korea. Induction of the callus was determined using four organs separated from the whole plant and different plant growth regulators. The callus was successfully induced from rhizome explant on MS medium containing 2.4-D (0.2–0.3 mg/l). In the MS medium supplemented with a combination of BAP (2 mg/l) and NAA (0.2 mg/l), single NAA (0.5 mg/l), or IBA (0.5 mg/l), a higher number of shoot, root and plantlets was achieved. The survival rate on the mountainous region of the plantlets successfully acclimatized (100%) in greenhouse reached 95%, and yields of crude drug and contents of active principles were higher than those obtained by sexual and vegetative propagation. This first report of R. coreanum tissue culture provides an opportunity to control extinction threats and an efficient callus proliferation system for growing resources rapidly on a large scale.     

Rapid plant regeneration,validation of genetic integrity by ISSR markers and conservation of Reseda pentagyna an endemic plant growing in Saudi Arabia

Reseda pentagyna is the only endemic species among the seven species of the genera Reseda found in Saudi Arabia. Probably no information is available on regeneration by conventional method of regeneration through seeds or cuttings. Therefore, alternative method of tissue culture was attempted to regenerate and multiply the plant. High shoot regeneration (14.44 shoots/explant) was obtained after four weeks, when shoot cuttings cultured on MS containing BA at 1.0 µM. Other cytokinins e.g., Kn, 2iP and TDZ found to be less effective in bud induction and shoot multiplication. Individual shoots were rooted on MS medium supplemented with various auxins at 0.5–5.0 µM concentrations. The IBA (1.5 µM) supplemented MS media induced maximum (83.3%) rooting. The plantlets were acclimatized and hardened under greenhouse conditions in plastic pots containing soil and farm yard manure with 95.0% success. The protocol developed would help to multiply the plant as well as conserve them in natural habitat. This can also be utilized to obtain active constituents for pharmaceutics and genetic manipulations.     

A simple and efficient method for clonal propagation of Casuarina sumatrana (de Vriese) L. Johnson

A new and efficient method for clonal propagation of Casuarina sumatrana by rooting stem cuttings is described. High percentage (about 60–70%) of rooting was achieved with mature softwood stem cuttings. A quick-dip of 5 s in NAA (1–10mM) solution followed by sand culture under high humidity were required for a high rate of survival and rooting of stem cuttings. A simple, closed chamber propagation system, using fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film), was successfully developed for the rooting of stem cuttings without mist. Rooted cuttings inoculated with Frankia were easily transplanted and established in field conditions with very low (about 3%) mortality. The significance of these findings for mass clonal propagation of C. sumatrana is discussed.Abbreviations IBA indole-3-butyric acid - NAA -naphthaleneacetic acid     

Production of rhoifolin and tiliroside from callus cultures of Chorisia chodatii and Chorisia speciosa

The current work aims to stimulate the production of rhoifolin and tiliroside as two valuable phytochemicals from Chorisia chodatii Hassl. and Chorisia speciosa A. St.-Hil. callus cultures. A comparison between three explants from the in vitro germinated seedlings of both species for callus induction and accumulation of both flavonoids was carried out. Highly efficient calluses were induced from the leaves, stems and roots of C. chodatii seedlings on Gamborg’s B5 (B5) and Murashige and Skoog (MS) media containing 2.0 mg/l β-naphthalene acetic acid (NAA) and 0.5 mg/l 6-benzyladenin (BA) or kinetin (Kn), while those of C. speciosa seedlings efficiently produced calluses on both media supplemented with 0.5 or 1.0 mg/l NAA and 0.5 mg/l BA. Besides, the highest contents of rhoifolin (1.927 mg/g DW) and tiliroside (1.776 mg/g DW) from C. speciosa cultures were obtained from the calluses of seedlings’ roots and stems maintained on B5 medium containing 1.0 mg/l NAA and 0.5 mg/l BA, respectively. On the other hand, the maximum rhoifolin content (0.555 mg/g DW) from C. chodatii cultures was obtained from the calluses of seedlings’ stems grown on B5 medium supplemented with 2.0 mg/l NAA and 0.5 mg/l BA, whereas the highest tiliroside content (0.547 mg/g DW) was provided by the root explants on B5 medium containing 2.0 mg/l NAA and 0.5 mg/l Kn. Both flavonoids were bioaccumulated in greater amounts than the wild and cultivated intact plants, which provides a promising tool for their future commercial production under a controlled environment, independent of climate and soil conditions.     

Auxin-cytokinin synergism in vitro for producing genetically stable plants of Ruta graveolens using shoot tip meristems

An efficient micropropagation protocol was developed for Ruta graveolens Linn. using shoot tip meristems derived from a 4-month-old field grown plant. In vitro shoot regeneration and proliferation was accomplished on Murashige and Skoogs (MS) semi-solid medium in addition to different doses of cytokinins viz.6- benzyl adenine (BA), Kinetin (Kn) or 2-isopetynyl adenine (2iP), singly or in combination with auxins viz. indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Highest regeneration frequency (27.6%) was obtained on (MS) medium composed of BA (10 µM) with maximum number (9.4) of shoots and 4.3 cm shoot length after 4 weeks of incubation. Among various combinations tried best regeneration frequency (71%) of multiple shoot formation with highest number (12.6) of shoots per shoot tip explants were achieved in MS medium augmented with a combination BA (10.0 µM) and NAA (2.5 µM) after 4 weeks of incubation. The optimum frequency (97%) of rhizogenesis was achieved on half-strength MS medium having 0.5 µM IBA after 4 weeks of incubation. Tissue culture raised plantlets with 5–7 fully opened leaves with healthy root system were successfully acclimatized off in Soilrite? with 80% survival rate followed by transportation to normal soil under natural light. Genetic stability among in vitro raised progeny was evaluated by ISSR and RAPD markers. The entire banding pattern revealed from in vitro regenerated plants was monomorphic to the donor. The present protocol provides an alternative option for commercial propagation and fruitful setting up of genetically uniform progeny for sustainable utilization and germplasm preservation.     

Metabolic changes during rooting in stem cuttings of five mangrove species

Vegetative propagation through rooting in stem cuttings in five tree mangroves namely Bruguiera parviflora, Cynometra iripa, Excoecaria agallocha, Heritiera fomes, and Thespesia populnea using IAA, IBA and NAA was reported. Spectacular increase in the root number was noted in the cuttings of H. fomes and C. iripa treated together with IBA (5000 ppm) and NAA (2500 ppm). The highest number of roots was obtained with IBA (2500 ppm) and NAA (500 ppm) in E. agallocha. B. parviflora and T. populnea responded better to IAA and IBA treatment. The species specific variation in the rooting response to exogenous application of auxins was reflected in the metabolic changes during initiation and development of roots in cuttings. Biochemical analysis showed increase of reducing sugar in the above-girdled tissues at initiation as well as subsequent development of roots which was further enhanced by the use of auxins. Decreases in the total sugar, total carbohydrate and polyphenols and increase in total nitrogen were recorded in the girdled tissues and the high C/N ratio at the initial stage helped in initiation of roots in all the species. Interaction of IBA and NAA promoted starch hydrolysis better than IAA and IBA during root development and subsequently reduced the C/N ratio and increased the protein-nitrogen activity during root development which suggest the auxin influenced mobilization of nitrogen to the rooting zone.Abbreviations IAA Indole-3-acetic acid - IBA Indole-butyric acid - NAA A-naphthalene acetic acid     

Pharmacological properties and in vitro shoot production of Barleria argillicola – A critically endangered South African species

Barleria argillicola Oberm. is a critically endangered species, endemic to a small area in the KwaZulu-Natal Province, South Africa. Animals are known to forage on this plant species, suggesting its therapeutic or nutraceutical potential. This study investigated the antibacterial, acetylcholinesterase inhibition, antioxidant and phytochemical properties of this species with a view to exploring its medicinal potential. The possibility of in vitro propagation as a conservation strategy was also examined. Dichloromethane extract showed a good antibacterial activity (with minimum inhibitory concentration less than 1 mg/ml) against all the tested micro-organisms. Methanol extract exhibited a stronger antibacterial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa than the Gram-positive bacterium Staphylococcus aureus. Extracts obtained from the aerial parts and roots demonstrated a dose-dependent acetylcholinesterase inhibition and antioxidant activities. Higher iridoid, flavonoid and condensed tannin contents were recorded in the aerial parts compared to the roots although the total phenolic content was higher in the roots. The highest in vitro shoot proliferation of 4.60 ± 0.51 and 4.0 ± 0.47 shoots per explant was achieved using shoot-tip and single nodal explants respectively, after four weeks of culture in Murashige and Skoog medium supplemented with 5 μM benzyladenine riboside (BAR). Further supplementation of the medium with naphthalene acetic acid (NAA) or indole butyric acid (IBA) concentrations did not significantly increase shoot proliferation.     

Rapid in vitro micropropagation of Agapanthus praecox

In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influence of different growth regulators on shoot multiplication from shoot-tip explants of A. praecox was investigated. Prolific shoot multiplication (47.3 ± 1.96 shoots per explant) was achieved on Murashige and Skoog (MS) medium supplemented with 22.2 μM benzyladenine (BA), 2.9 μM indole-3-acetic acid (IAA), and 4.5 μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with 5.7 μM IAA and 2.5 μM 2-isopentenyladenine (2iP) with 11.3 ± 0.78 roots per shoot. The in vitro-raised plants were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with 100% survival. The elongated shoots (more than 5 cm in length) were treated for rooting and acclimatization in a moistened (5.7 μM IAA and 2.5 μM 2iP) vermiculite:sand (1:1 v/v) mixture, first in the misthouse and then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce phytomedicines.     

IBA浓度、扦插时间对江西杜鹃和百合花杜鹃扦插生根的影响

为探明有鳞大花亚组杜鹃扦插生根的最佳IBA浓度和扦插时间,该研究以江西杜鹃、百合花杜鹃为材料,分别采用腐叶土+河沙(1:1)、泥炭+珍珠岩+蛭石(3:1:1)基质,开展了4个IBA浓度和4个扦插时间的生根试验.结果表明:IBA浓度对除老叶留存数外的所有指标有显著影响,其中100 mg·L-1 IBA处理生根率、新梢长最大,腐烂率最低,其它指标也表现良好,为最佳生根浓度;50 mg·L-1 IBA处理根幅、新梢率最大,但不定根数最少,效果其次;200 mg·L-1 IBA处理促进根系生长,但生根率较低、特别是显著抑制新梢发育;对照处理生根效果最差.扦插时间对所有生根指标均有显著影响,早春(04-18)木质化硬枝扦插除老叶留存数较差外,其它指标均表现极佳,为最适扦插时间;秋季(10-19)半木质-木质化过渡枝扦插效果其次;夏季(06-21)嫩枝及(08-16)半木质化枝生根效果极差,不宜进行扦插育苗.物种、基质对生根指标也有显著影响,百合花杜鹃扦插生根能力强于江西杜鹃,泥炭+珍珠岩+蛭石(3:1:1)基质生根效果优于腐叶土+河沙(1:1).该研究结果首次发现早春新梢萌发前采用木质化硬枝扦插可以显著提高两种杜鹃的生根效果,为该亚组杜鹃的扦插育苗提供了科学依据.     

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

Phytochemical and antioxidant properties of unconventional leafy vegetables consumed in southern Africa

Indigenous leafy vegetables possess high horticultural potential based on their long utilisation history by local communities across Africa. Phytochemical and antioxidant properties of 50% aqueous methanol and water extracts of three indigenous as well as two commercial leafy vegetables commonly consumed in southern Africa were evaluated. The total extractable phenolic content was highest for Amarathus dubius (5.16 ± 0.12 mg GAE/g DW) followed by Cleome gynandra (3.94 ± 0.09 mg GAE/g DW). Total flavonoid concentration was highest for A. dubius (3.89 ± 0.28 mg CE/g DW) followed by C. gynandra (2.19 ± 0.11 mg CE/g DW) and Cucurbita maxima (1.55 ± 0.04 mg CE/g DW). No proanthocyanidins were detected in C. maxima and Brassica napus cv Covo whereas low concentrations were recorded in other vegetables. Total saponins were variable across the evaluated extracts, with the highest concentrations recorded for B. napus cv Covo (83.2 ± 16.58 mg DE/g DW). Total iridoid content was highest for C. gynandra (9.14 ± 0.20 mg HE/g DW). More potent DPPH radical scavenging activities were exhibited by 50% aqueous methanol extracts compared to water extracts. A similar trend was observed in the ferric-reducing antioxidant power assay. The antioxidant activity based on the rate of β-carotene bleaching was higher for water extracts compared to 50% aqueous methanol extracts. The indigenous vegetables evaluated in this study had higher levels of phytochemicals and also exhibited more potent antioxidant activity compared to the commercial varieties. These findings not only suggest the importance of the indigenous vegetables in a healthy diet, but also provide a motivation for exploring their horticultural potential.     

Production of schisantherin A and gomisin G in in vitro cultures of Schisandra chinensis

Methanolic extracts from the biomass of shoot-differentiating and undifferentiating callus cultures of Schisandra chinensis growing respectively on six and two different variants of the Murashige and Skoog (MS) medium, with different concentrations of plant growth regulators, BA (N⁶-benzyladenine) and NAA (α-naphthaleneacetic acid) were analyzed for the accumulation of two lignans–schisantherin A and gomisin G, using the HPLC method. The amounts of the two compounds in the biomass extracts from shoot-differentiating callus cultures were dependent on the concentration of plant growth regulators in the MS medium. The highest amounts of both lignans were obtained on the MS medium supplemented with 3 mg l⁻¹ BA and 1 mg l⁻¹ NAA. The maximum amount of schisantherin A (33.45 mg 100 g⁻¹ DW) was about 1.3 times higher than in the extracts from the leaves and fruits of parent plants. This is the most important result potentially promising from a practical point of view.     

Enhancing seedling growth of Jatropha curcas—A potential oil seed crop for biodiesel

The effects of aerosol smoke (AS), smoke-water (SW), potassium nitrate (KNO₃), naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) on germination and seedling growth of Jatropha curcas were investigated. Seed coat removal accelerated water imbibition and germination occurred within 48 h. Seeds subjected to AS failed to germinate over a 90 day period. There were no significant differences in germination percentage between the treatments and untreated control (intact- and shelled-seed). However, shelled-seeds had the shortest mean germination time (MGT). Seedlings developed from treated seeds were planted in trays under shade house conditions and growth traits measured after 3 months. Soaking intact-seeds in SW, KNO₃ and NAA (24 h) produced significantly heavier and longer seedlings, which resulted in higher vigour indices (VI) compared to the control treatments. These results provide empirical evidence of the stimulatory effect of SW, KNO₃ and NAA on J. curcas seedling growth and vigour and the continuation of the effect over time. The approach of treating intact-seeds of J. curcas with plant growth substances prior to planting will help in producing healthy seedlings and possibly improve crop productivity.     

Evaluation of edible mushroom Oudemansiella canarii cultivation on different lignocellulosic substrates

In this study, the mycelial growth rate, mycelial colonization time, yield, and biological efficiency of the edible mushroom Oudemansiella canarii were determined, and the effects of different substrate combinations on productivity, chemical contents and amino acids were evaluated. Lignocellulosic wastes, such as cottonseed hull, sawdust, corncob, and their combinations supplemented with 18% wheat bran and 2% lime, were used for the cultivation of O. canarii. The biological efficiency (BE) and essential amino acid content of treatment T1, which consisted of 80% cottonseed hull, were the highest among all the tested treatments. Mixtures that included sawdust, such as treatments T2 (80% sawdust), T4 (40% sawdust + 40% cottonseed hull), and T6 (40% sawdust + 40% corncob), exhibited lower yield and BE. Corncob was good for O. canarii production in terms of yield and BE, whereas the mycelial growth rate and colonization time were lower compared to those on other substrates. Comparing the BE, essential amino acids, and other traits of the six treatments, treatment T1 (80% cottonseed hull) was the best formula for O. canarii cultivation and should be extended in the future.     

Ex vivo implications of phytohormones on various in vitro responses in Leptadenia reticulata (Retz.) Wight. & Arn.—An endangered plant

Leptadenia reticulata (Retz.) Wight. & Arn. is an important medicinal plant, belongs to the family Asclepiadaceae. This plant is known for its medicinal uses since 4500 BC. Presently this is an endangered species (Arya et al., 2003). Six shoots (2–4 cm long) per node differentiated on MS medium + 5.0 mg/l of BAP + additives. Incorporation of additives in the culture medium promoted growth of cultures. The shoots differentiated per explant were repeatedly transferred on to fresh MS + 1.0 mg/l of BAP + 0.1 mg/l of NAA and additives. The regenerated shoots were subcultured for further multiplication on MS + 1.0 mg/l BAP + 0.5 mg/l Kin + 2-iP (0.5 mg/l) and 0.1 mg/l of NAA + additives regularly after an interval of 3 weeks. Addition of ammonium sulphate in the medium resulted in increase in shoot number and promoted elongation also growth of cultures was sustained even if subculturing was delayed (26 ± 2 days). Success was also achieved in defining protocol for in vitro regeneration of shoots from petiole derived callus. Shoots regenerated in vitro by both processes were rooted in vitro on 1/4 strength of MS medium + 3.0 mg/l of IBA after 15–20 days. Cent percent of the shoots rooted ex vitro, if the in vitro regenerated shoots were treated with 200 mg/l of IBA. The in vitro–ex vitro rooted plantlets were hardened under different regimes of temperature and humidity in a greenhouse. The hardened plantlets were transferred to soil in polybags. More than 95% plants survived in field conditions. Total dry biomass harvested per year was 2800 kg/acre.     

Genetic variability studies for morphological and qualitative attributes among Jatropha curcas L. accessions grown under subtropical conditions of North India

Evaluation of Jatropha curcas germplasm comprising seven accessions indicated a wide range of variability in vegetative growth and other qualitative attributes. These characteristics could be harnessed in future improvement programme of Jatropha curcas. Seed yield/plant had a positive and significant correlation with number of branches/plant, oil yield, plant spread (r = 0.806, 0.802, 0.782), plant spread had a highest correlation with plant height (r = 0.840). The seeds analyzed for proximate composition, fatty acid and physiochemical characteristics revealed that fiber and ash content in seed flour were high (16.5% and 4.35%). Oil content varied from 24.5% to 37.9%. The lower value of the viscosity suggests it as diesel oil. Accession JC006 could be an alternative source of linoleic acid (51%) while the accession JC001 could be a source for oleic acid (48%) and linoleic acid 42.4%. Stearic acid was highest in accession JC003 (42.9%).This evaluation has helped to identify cultivar with specific yield and vegetative growth features. Among all the seven accession evaluated accession JC007 is found to be promising which could be taken as productive genotype for commercial exploitation.