μM Indole-3-butyric Acid Research Articles

Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth

We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.

English

Javanese ginseng (Talinum paniculatum) is an herb and one of the traditional medicinal plants in Indonesia which accumulate saponin in the root organ. However, slow root growth hampers the accumulation of the compound in this plant. Therefore, in vitro culture of adventitious roots offers an alternative way to increase the production of saponin. Furthermore, this study was performed to investigate the effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the growth and saponin content in adventitious root of Javanese ginseng. Adventitious root culture was induced on solid Murashige and Skoog (MS) medium supplemented by 10 µM IBA (indole-3-butyric acid). Subsequently, adventitious roots were subcultured into a half strength MS liquid medium with the addition of different concentrations of MeJA or SA and incubated for 5, 10 and 15 days. Despite inhibition on the root growth, saponin production increased by 1.5 and 1.3 fold upon elicitation with 0.2 mM MeJA and SA for 15 days, respectively. Thus, MeJA and SA elicitations regulate saponin biosynthesis in adventitious root culture of Javanese ginseng as a time and dose-dependent manner. Key words: Adventitious root, elicitation, Javanese ginseng, saponin, Talinum paniculatum.

Successful stem cutting propagation of Patrinia rupestris for horticulture

This study was conducted to develop a protocol for the vegetative propagation of Patrinia rupestris (Pall.) Juss., to facilitate its use for ornamental and medical purposes. Cuttings of P. rupestris were treated with distilled water (control), commercial rooting powder (Rooton, 0.4% 1-naphthylacetamide), α-naphthalene acetic acid (NAA), and indole-3-butyric acid (IBA). The stem cuttings pretreated with 2685, 5370 µM NAA or 4920 µM IBA displayed the best rooting traits; the rooting percentage, number of adventitious roots, length of roots, and fresh weight of roots. It would be advisable to use the lower concentration, for economy in using the hormone. We recommend treating cuttings with 2685 µM NAA for the propagation of P. rupestris to produce large amounts of planting materials.

Influence of auxins on somatic embryogenesis in Haworthia retusa Duval

An efficient in vitro plant regeneration method through somatic embryogenesis has been established in Haworthia retusa. Somatic embryos were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and α-naphthaleneacetic acid (NAA) either alone or in combination with 4 μM thidiazuron (TDZ). Of the four auxins studied, IBA was found to be the most promising in terms of somatic embryo induction, followed in decreasing frequency by 2,4-D, IAA, and NAA. The highest somatic embryo induction (60.7%), with a mean of 20.7 embryos per leaf explant, was observed on MS medium amended with 20 μM IBA. The inclusion of 4 μM TDZ to the auxin-containing medium significantly (p < 0.0001) increased the somatic embryo induction frequency as well as the number of somatic embryos. The best combination for somatic embryogenesis was IBA + TDZ. The highest incidence of somatic embryo induction (100%), with a mean of 55.8 somatic embryos, was obtained on a culture medium containing 16 μM IBA + 4 μM TDZ. Somatic embryos germinated best on MS medium supplemented with 2 μM gibberellic acid. Morphological variations were observed among the regenerated plantlets. Well-developed plantlets obtained from germination media were acclimatized in the greenhouse.

Regeneration of Blueberry Cultivars through Indirect Shoot Organogenesis

Leaf explants derived from in vitro–grown shoots of blueberry cultivars Bluejay, Pink Lemonade, Sunshine Blue, and Top Hat were cultured on woody plant medium (WPM) supplemented with 9.12 μm 6-(4-hydroxy-3-methylbut-2-enylamino) purine or zeatin (ZT) in combination with 1.23, 2.46, or 4.92 μm indole-3-butyric acid (IBA). Calluses were induced from the explants and adventitious shoots were regenerated. ‘Sunshine Blue’ and ‘Top Hat’ produced more than four shoots per explant but shoot numbers were less than one for each ‘Pink Lemonade’ explant and about 0.2 per ‘Bluejay’ explant. The results indicate that there is significant difference among cultivars in indirect shoot organogenesis. The differences may be related to their diverse genetic background as they are polyploid hybrids. Microcuttings derived from adventitious shoots of ‘Sunshine Blue’ rooted in vitro in WPM medium supplemented with 9.84 μm IBA and also rooted ex vitro in a peat-based substrate after cuttings were dipped or not dipped in IBA solutions. Direct rooting of microcuttings in the peat-based substrate was effective, suggesting that in vitro rooting may not be necessarily needed. Survival rate of ex vitro–rooted plants in a shaded greenhouse was high, more than 90%. The established shoot regeneration protocols could be used for rapid propagation of ‘Sunshine Blue’ and ‘Top Hat’ and for cultivar improvement through genetic transformation.

Organogenesis and efficient in vitro plantlet regeneration from nodal segments of Allamanda cathartica L. using TDZ and ultrasound assisted extraction of quercetin

The present investigation was carried out to evaluate the instigative effect of thidiazuron (TDZ) on multiple shoot induction from nodal segments of Allamanda cathartica and estimated the flavonoid yield among the regenerants. High rate of shoot bud induction was achieved on Murashige and Skoog (MS) medium augmented with 0.3 µM TDZ from nodal segments exposed for 30 days. However, for shoot proliferation and elongation, TDZ exposed cultures were further cultured on MS medium devoid of TDZ and/or supplemented with different concentration of 6-benzyladenine (BA) and Kinetin (Kn). BA at 2.5 µM gave the maximum mean number of shoots (44.00 ± 1.30) and shoot length (7.50 ± 0.21 cm) per explant after 12 weeks of incubation in the secondary medium. The response of explant was influenced by the collection time. The highest rooting in the microshoots (5 cm) was achieved on 1/2 MS liquid medium supplemented with 0.5 µM Indole-3 butyric acid (IBA) which produced 4.50 ± 0.16 mean roots/shoot with 4.05 ± 0.17 cm mean root length. The leaves of 30 day old acclimatized plantlets were used for phytochemical screening. Ultrasonication mediated extraction and quantification of bioactive flavonoid namely quercetin through colorimetry and mass spectrometry analysis from the leaves of regenerants. Extraction was processed in methanol using 2 g leaf sample through sonication. Total yield of flavonoids and quercetin content was found to be maximum in 2.5 µM BA treated plants with respect to control and other treated samples. The concentration of total flavonoids was estimated to be 172.90 mg QE/g which yielded 51.39 mg/g quercetin. The study ensures a rapid cultivation of plantlets, thus enhancing the biomass production which may be utilized in the isolation and quantification of other biological potential compound for the use in treatment of various ailments.

Direct Shoot Organogenesis from Seedling Derived Shoot Tip Explants of Endangered Medicinal Plant Saussurea costus (Falc.) Lipsch.

An efficient direct in vitro plant regeneration protocol for Saussurea costus (kuth) was developed using shoot tip explants derived from in vitro grown seedlings to generate genetically uniform plants. Murashige and Skoog (MS) medium supplemented with 1.14 µM thiadiazuron and 2.68 µM naphthalene acetic acid was found to be most optimal for highest for average shoot regeneration (73.33%) with maximum average number of shoots (11.4) and average shoot length (4.17 cm). For in vitro multiplication, shoots cultured on MS medium supplemented with 4.44 µM benzyl adenine, 2.32 µM kinetin and 1.44 µM gibberellic acid were found to grow best with 20.7 average shoot multiplication rate after 5–6 weeks. MS medium supplemented with 2.46 µM indole butyric acid was found to be the best medium for rooting of microshoots (77.78% rooting with 6.0 average number of roots and 3.07 cm average root length). The in vitro raised plantlets were successfully acclimatized under ex vitro conditions. The true to type nature of the in vitro raised plants was confirmed using DNA based ISSR markers for assessment of genetic stability of micropropagated plants. The standardized micropropagation protocol under present study may be tuned towards mass production of roots which are the most economical part of the plant. This would be useful as prerequisite for carrying out any genetic transformation studies while concentrating on any desirable trait in the same herb.

Exogenous methyl jasmonate acts as a signal transducer in the enhancement of camptothecin (CPT) production from in vitro cultures of Ophiorrhiza mungos L. var. angustifolia (Thw.) Hook. f.

Ophiorrhiza mungos var. angustifolia is a member of Rubiaceae family can be a gold standard for the in vitro production of high-value anticancer compound camptothecin to meet the increasing market demand for medicine. Multiple shoot cultures of Ophiorrhiza mungos var. angustifolia was induced in the nodal explant using half-strength Murashige and Skoog solid media supplemented with different concentrations of cytokinins (4.44–22.19 μM Benzyl adenine and 4.65–23.23 μM Kinetin either individually or in combination) and auxins (4.92–24.6 μM Indole butyric acid, 5.37–24.6 μM Indole acetic acid and 5.71–28.54 μM in combination with Benzyl adenine) for three weeks. Among the plant growth regulators treated, benzyl adenine (8.88 μM) was found to be the most effective in multiple shoot induction (22 ± 3.90 shoots/explant), thus further shoot multiplication is achieved in fresh media with the same composition. Increased camptothecin production is achieved by the elicitation of multiple shoots using methyl jasmonate (50, 100, 150 and 200 μM), salicylic acid (50, 100, 150 and 200 μM) and chitosan (10, 50,100 and 150 mg/l) separately under a different period of incubation. The results showed 7 fold increase in the production of camptothecin (0.74 ± 0.05 mg/g dry weight of shoots) in shoots elicited with 150 μM methyl jasmonate for 72 h compared to the control. (0.14 ± 0.01 mg/g dry weight of shoots). Additional shoot bud formations were not observed irrespective of elicitor used. Camptothecin was estimated using high-performance liquid chromatography. The results obtained in the present experiment clearly demonstrate and strongly endorse the suitability of using Methyl jasmonate as a promising and reliable elicitor for enhanced production of camptothecin in Ophiorrhiza mungos var. angustifolia.

Effect of concentration, method of auxin application and cultivation conditions on in vitro rooting of bog blueberry (Vaccinium uliginosum L.)

BACKGROUND: Bog blueberry, Vaccinium uliginosum is one of the newer fruit crops. Choosing the most appropriate methods for mass vegetative propagation of V. uliginosum cultivars, including in vitro propagation, is crucial. OBJECTIVE: We investigated the effect of growth regulators and light on in vitro rhizogenesis of V. uliginosum to determine the optimal method of propagation. METHODS: The following cultivation techniques were tested on microshoots of bog blueberry varieties ‘Golubaya rossyp’, ‘Nektarnaya’, ‘Shegarskaya’ and ‘Iksinskaya’: 1) ½ Anderson (A) medium supplemented with 3 and 10 μM indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA) and 2) 24-h aqueous treatments of microshoots with 148 μM IBA or 171 μM IAA and further cultivation in ½ A medium. In addition, we tested precultivation in darkness for 3 d versus a 16/8-h photoperiod. RESULTS: 100% rooting was observed in ‘Golubaya rossyp’ in all treatments of the experiments and with ‘Nektarnaya’ on the growth-regulator-free ½ A medium and after treating the microshoots with 171 μM IAA solution. Precultivation in darkness produced a positive effect on rooting in ‘Shegarskaya’ and ‘Iksinskaya.’ CONCLUSIONS: Response to in vitro rooting techniques varied between cultivars of V. uliginosum. Treatment of microshoots with auxin solutions improved in vitro rooting.

Rapid in vitro shoot multiplication of the recalcitrant species Juglans nigra L.

Black walnut (Juglans nigra L.) has long been prized for its timber, leading to commercial cultivation and significant breeding efforts for improving marketable traits. Vegetative and in vitro black walnut propagation techniques, however, are variable and highly genotype dependent. Optimizing plant growth regulator type and concentration are integral for developing a successful micropropagation protocol. The addition of meta-topolin (MT) combined with the novel use of a liquid medium has led to the development of a rapid shoot multiplication system. The objective of this research was to develop a reproducible and dependable micropropagation protocol for elite black walnut genotypes. In vitro shoot cultures were established from nodal explants cultured on semi-solid Driver and Kuniyuki walnut (DKW) medium supplemented with 8.9 μM benzyladenine, 0.005 μM indole-3-butyric acid (IBA), 200 mg L−1 casein hydrolysate, 50 mg L−1 adenine hemisulfate, 2 mL L−1 Plant Preservative Mixture™, and 4.1 μM MT. Long-term survival and proliferation of microshoots was achieved when nodal segments of in vitro grown shoots were cultured in liquid initiation medium in 3-L polycarbonate Fernbach-style flasks on a rotary shaker (100 rpm) under a 16-h photoperiod at 25°C. Elongated microshoots (5–7 cm in length) were rooted in a slurry-like medium composed of half-strength DKW medium with 0.11% (w/v) Phytagel™ and coarse vermiculite (2:1, v/v) supplemented with 50 μM IBA for 5 wk. Rooted shoots were acclimatized to ambient culture room conditions, but plantlets did not survive once transferred to the greenhouse.

Biochemical and histological changes during in vitro rooting of microcuttings of Bacopa monnieri (L.) Wettst.

The present study was conducted to investigate the biochemical changes vis-a-vis histological changes during adventitious rooting of microcuttings of Bacopa monnieri (L.) Wettst. The rooting in these microcuttings was induced on basal MS medium and medium supplemented with different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA). Presence of lower auxin concentration (1.0 µM) in the medium enhanced rooting and significantly improved number of roots per shoot but maximum root length was observed on basal MS medium. Histological studies were conducted to identify different phases of rooting in these microcuttings. The root meristemoids with distinct polarity become visible after 3 days and mark the beginning of in vitro root initiation phase. It was followed by primordia elongation, root emergence and visible rooting on the 5th day of culture on medium supplemented with auxins. Biochemical studies were also conducted from basal portions of microcuttings cultured on MS medium supplemented with 1.0 µM IBA and control (basal MS medium) from 0 to 7 days. Total carbohydrate content was lower during initial periods (up to day 1) and was found to increase during root initiation and primordia development, which reflects high energy demands for active cell divisions. A significantly higher level of phenols was recorded in microcuttings on medium supplemented with IBA. Polyphenol oxidase, peroxidase (POX), ascorbate peroxidase activities were also found to vary during different phases of rhizogenesis. Early phases were also marked with the lower activities of POX and IAAO. This study revealed significant role of enzymes, sugars and phenols during different phases of rooting.

In vitro shoot regeneration and enhanced synthesis of plumbagin in root callus of Plumbago zeylanica L.—an important medicinal herb

Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47 ± 0.63 mg g−1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS + 5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08 ± 1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3 ± 1.36 roots with an average length of 15.31 ± 2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L−1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.

Synergetic effect of TDZ and BA on minimizing the post-exposure effects on axillary shoot proliferation and assessment of genetic fidelity in Rauvolfia tetraphylla (L.)

The stimulatory effect of different concentrations of thidiazuron (TDZ) on in vitro bud induction and proliferation of a potent medicinal woody shrub R. tetraphylla were investigated. Multiple shoots were induced from nodal explants on Murashige and Skoog medium augmented with different concentrations (0.2–1.0 µM) of TDZ. Continuous exposure beyond 5 weeks of singly supplemented TDZ resulted in hyperhydricity, fasciation and distortion of cultures. To avoid these deleterious effects, benzyl adenine (BA) was supplemented to the media. It not only mitigated the negative effects of prolonged TDZ exposure, but also enhanced shoot multiplication rate. About 30 shoots per explant with an average shoot length of 5.8 cm were obtained after 20 weeks of culture on MS medium fortified with 0.4 µM TDZ and 2.0 µM BA, regeneration frequency was also enhanced by 24% on this media as compared to singly supplemented TDZ. Microshoots (~ 5 cm) were successfully rooted on filter paper bridge in MS liquid medium augmented with different concentrations of indole-3-butyric acid (IBA) after 4 weeks of incubation. Best rooting response (80%) was achieved on MS medium supplemented with 0.3 µM IBA with (9.3 ± 1.45) mean number of roots and (3.3 ± 0.33 cm) root length per shootlet. The in vitro raised healthy plantlets with well developed root and shoot were successfully hardened off and acclimatized in thermocol cups containing sterilized planting substrate, Soilrite® for 8 weeks under culture room conditions prior to field transfer and successfully established in earthen pots with 90% survival rate. The genetic fidelity of in vitro regenerated plantlets was evaluated using random amplified polymorphic DNA markers. No polymorphism was observed in banding pattern among micropropagated and the donor plant, thus, exhibiting their genetic uniformity and clonal fidelity.

Micropropagation of Epilobium canum garretti (Firechalice) by Axillary Shoot Culture

Epilobium canum subsp. garrettii (firechalice) is an herbaceous wildflower with landscape potential, but its seeds are difficult to germinate because of dormancy requirements. The objective of this study was to develop a complete micropropagation procedure for a selected accession of firechalice. Single-node stem explants from the plant were examined for their ability to establish on Murashige and Skoog (MS) medium or Woody Plant Medium (WPM). Shoot explants on MS medium supplemented with 4.4 μm benzyladenine (BA) produced more than double the number of axillary shoots compared to explants on WPM (12.6 vs. 4.9 shoots, P = 0.0001). Benzyladenine, kinetin (kin), 6-(γ,γ-dimethlyallylamino)purine (2iP), thidiazuron (TDZ), and meta-topolin (mT) at concentrations of 0, 1.1, 2.2, 4.4, or 8.8 μm were evaluated for shoot proliferation efficacy. Stem explants treated with 8.8 μm of BA or mT produced the most shoots, 11 or 15, respectively. Benzyladenine, 2iP, and kin failed to affect shoot height even at the highest concentrations used, but 4.4 or 8.8 μm TDZ reduced shoot height to less than half of the heights of control shoots (3.1 vs. 1.2 cm, P = 0.0001). Firechalice shoots formed three to four roots easily without auxin added to the medium, but four to six roots formed per shoot when using up to 9 μm of indole-3-butyric acid (IBA). In contrast, 9 μm naphthaleneacetic acid (NAA) prevented root formation. When using 0–9 μm IBA for rooting, 82.5% of the rooted shoots survived transplanting. Based on these results, optimum micropropagation of firechalice may be achieved with shoots established on MS medium plus 4.4 µm BA, a concentration of 4.4 or 8.8 μm BA or mT used for shoot proliferation, and use of up to 6 µm IBA during root induction should result in &gt;80% shoot survival after transplanting.

TDZ-induced plant regeneration in Brassica oleracea L. var. botrytis: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB5) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB5 medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB5 medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB5 medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB5 medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA3). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.

Micropropagation and Subsequent Enrichment of Carotenoids, Fatty Acids, and Tocopherol Contents in Sedum dasyphyllum L.

A promising micropropagation protocol has been systematically established and demonstrated for the enhanced production of carotenoids, tocopherol and fatty acids in shoot tissues of Sedum dasyphyllum. Shoot tip explants were grown on Murashige and Skoog (MS) medium. Different concentrations of N6-benzyladenine (BA) or thidiazuron (TDZ) alone or in combination with α-naphthaleneacetic acid (NAA) were tested in order to stimulate multiple shoot production. Ideal shoot induction (100%) and maximized shoot numbers (36.4) were obtained on explants cultured on media incorporated with 2 μM BA and 1 μM NAA combinations. The in vitro-developed shoots rooted best on half-strength MS media incorporated with 2 μM indole 3-butyric acid. Plantlets were effectively acclimatized in the greenhouse with 100% survival rate. The composition and contents of bioactive compounds such as carotenoids, tocopherol and fatty acids in shoot tissues of S. dasyphyllum were investigated using HPLC and GC-MS. The most abundant carotenoid in the shoot tissue was all-E-lutein (40.3–70.5 μg g−1 FW) followed by 9′-Z-neoxanthin (5.3–9.9 μg g−1 FW), all-E-violaxanthin (4.4–8.2 μg g−1 FW), and all-E-β-carotene (1.6–3.6 μg g−1 FW). The α-tocopherol contents of in vitro-raised shoots was 6.5-fold higher than shoots of greenhouse-grown plants. The primary fatty acids found in shoot tissues were α-linolenic acid (32.0–39.3%), linoleic acid (27.4–38.2%), palmitic acid (13.3–15.5%), and stearic acid (5.2–12.2%). In all, summarizing the findings, the micropropagated S. dasyphyllum showed significant enrichment of valuable bioactive carotenoids (92.3 μg g−1 FW), tocopherols (14.6 μg g−1 FW), and α-linolenic acid (39.3%) compared to their greenhouse counterparts. The protocol demonstrated here could be applied for the mass propagation and production of enhanced bioactive compounds from S. dasyphyllum with credibility.

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.

Micropropagation of blueberry ‘Bluejay’ and ‘Pink Lemonade’ through in vitro shoot culture

Abstract This study systematically evaluated effects of culture media, cytokinins, and auxins on axillary shoot induction as well as auxins and media or substrates on in vitro and ex vitro rooting of a highbush cultivar Bluejay (Vaccinium corymbosum L.) and a V. ashei rabbiteye hybrid derivative ‘Pink Lemonade’. Optimal protocols for in vitro shoot culture of the two cultivars were developed. Two-node stem explants derived from axillary shoots were cultured on Anderson medium supplemented with 13.68 μM 6-(4-hydroxy-3-methylbut-2-enylamino) purine (ZT) and 0.27 μM naphthaleneacetic acid (NAA) for inducing axillary shoots of ‘Bluejay’ and 9.12 μM ZT and 0.05 μM NAA for ‘Pink Lemonade’ shoot proliferation. Microcuttings derived from the axillary shoots were cultured on half-strength Anderson medium supplemented with 0.49 μM indole-3-butyric acid (IBA) for in vitro rooting. The rooting percentages for ‘Bluejay’ and ‘Pink Lemonade’ were 75% and 65% in two months, respectively. However, by dipping microcuttings into 4.92 μM IBA for 10 s and rooting ex vitro in a substrate composed of 90% peat and 10% perlite, the rooting percentages could be improved to 89% for ‘Bluejay’ and 97% for ‘Pink Lemonade’. After being acclimatized in a shaded greenhouse for three months, rooted plants were ready for planting in the field. The established methods could be used for rapid propagation of highbush and rabbiteye hybrid derivative cultivars for commercial blueberry production.

Effects of indole‐3‐butyric acid on growth, pigments and UV‐screening compounds in Nostoc linckia

SUMMARYIn the present research, the effect of indole‐3‐butyric acid (IBA) on the growth, and the production of some primary and secondary metabolites was studied in Nostoc linckia. In this respect, algae cultures were supplied with 0, 0.01, 0.1, 1, 10, and 100 μM IBA for 14 days. IBA at concentrations of 10 and 100 μM induced algal growth expressed as fresh weight in N. linckia. Treatment with IBA at all concentrations stimulated heterocyst formation. In addition, low concentrations of IBA (0.01, 0.1, and 1 μM) had a stimulatory effect on chlorophyll a and carotenoids accumulation. In contrast, higher concentrations of IBA induced the accumulation of phycocyanin, allophycocyanin, and phycoerythrin in the treated algae. In this case, IBA at the concentration of 10 μM was more effective. A significant decrease in protein content was observed in the algae treated by 0.01 μM IBA. All concentrations of IBA caused a decrease in sugar content, but lower concentrations were more effective. IBA application in all of the concentrations except 100 μM increased oligosaccharide‐linked mycosporine‐like amino acids (OS‐MAAs) content. Lower concentrations had a more significant effect on increasing OS‐MAAs content. However the concentrations of 10 and 100 μM IBA decreased scytonemin content. These results indicated the stimulatory impact of IBA on weight, heterocyst formation, and photosynthetic pigments in N. linckia.

Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra

Black ash (Fraxinus nigra) is an endangered hardwood tree species under threat of extirpation by the emerald ash borer (EAB), an aggressive exotic phloem-feeding beetle. We have developed an efficient regeneration system through adventitious shoot organogenesis in F. nigra using in vitro-derived leaf explants. Two types of leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators to induce callus and adventitious shoot bud formation. Significant effects of explant, and plant growth regulator interactions were found. The frequency of callus formation ranged from 77.8 to 94.4% and 88.9–100% from single leaflets and intact compound leaves, respectively, with no significant difference between treatments. For adventitious shoot bud induction, however, 22.2 µM 6-benzylaminopurine (BA) combined with 31.8 µM thidiazuron (TDZ) was the best treatment regardless of the initial leaf explant type, showing 21.1 and 28.8% shoot bud induction, with 1.5 and 1.9 adventitious shoots per explant, from single leaflets and intact compound leaves, respectively. The regenerated shoot buds were elongated on MS medium supplemented with Gamborg B5 vitamins plus 2 mg L−1 glycine (MSB5G), 13.3 µM BA, 1 µM indole-3-butyric acid (IBA), and 0.29 µM gibberellic acid. The elongated shoots were continuously micropropagated through nodal stem sectioning until used for rooting. An average of 85.2% of the microshoots were successfully rooted in woody plant medium containing 5.7 µM indole-3-acetic acid plus 4.9 µM IBA with a 10-day initial dark culture, followed by culture under a 16-h photoperiod. Rooted plantlets were acclimatized to the greenhouse and showed normal plant growth and development with 100% survival. This regeneration protocol would be useful for mass propagation for conservation of F. nigra and for use in genetic transformation for EAB resistance.