In vitro micropropagation of Basella rubra L. through proliferation of axillary shoots

In vitro micropropagation protocol for Basella rubra regeneration was tried through proliferation of axillary shoots of the potted mature plant. The improved seed germination (70%) was recorded upon 2% urea treatment. The nodal shoot segments from matured potted plant were used to initiate the multiple shoot proliferation. The shoot segments exhibited 70% shoot initiation when cultured on Murashige and Skoog (MS) medium supplemented with Indole-3-acetic acid (IAA)?+?N₆ – Benzylaminopurine (BAP) (0.25?+?2.0 mg/L) and BAP?+?Kinetin (Kin) (2.0?+?0.5 mg/L) respectively. Multiple shoots (5–6) were obtained on MS medium supplemented with BAP?+?Kin and IAA?+?BAP respectively. When compared with silver nitrate (AgNO₃) (2–40 µM) and activated charcoal (AC) (0.1–1.0%), the MS medium devoid of any plant growth regulator showed good number of shoots (5.48?±?2.42), elongation (15.64?±?2.42 cm) and root length (14.52?±?2.78 cm). Upon transferring of regenerated microshoots to MS medium, simultaneous elongation of shoots with more shoot number, shoot length and rooting was achieved during four subcultures that carried out at 6 weeks’ interval. The regenerated in vitro shoots showed 100% rooting in MS medium and also in MS medium supplemented with 0.1–1.0% AC. Hundred percent survival of micropropagated shoots well rooted was established successfully under greenhouse condition and the plants were subsequently acclimatized and transferred to the field conditions wherein 90% success rate was noted.

     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Micropropagation,berberine content and antitumor activity of Jeffersonia dubia (Maxim.) Benth et Hook

An efficient micropropagation protocol was developed for Jeffersonia dubia using sucker explants. High frequency of multiple shoot formation was induced when the sucker explants were cultured on Chu’s (N6) medium with different concentrations of thidiazuron (TDZ) plus 0.54 µM α-naphthaleneacetic acid (NAA). The maximum frequency of shoot formation (96.2 %) was obtained on N6 medium with 2.27 µM TDZ plus 0.54 µM NAA. The highest mean number of shoots per explant (13.6) was obtained in temporary immersion system using an immersion frequency of 30 s every 30 min. The highest frequency of rooting (100 %), number of roots per shoot (5.8), and root length (6.3) was observed in half-strength N6 medium supplemented with 2.69 µM NAA. The regenerated plantlets (30 days old) were successfully acclimatized in the greenhouse with 98 % survival rate. The berberine content and cytotoxicity were higher in in vitro-developed calli and shoots than in leaves of field-grown plants. The greatest content of berberine was found in shoots (1381 μg g⁻¹) followed by calli (1092 μg g⁻¹) and leaves of field-grown plants (92 μg g⁻¹). At 1000 μg mL⁻¹ concentration, growth inhibition rate of berberine, callus, shoot, and leaf (in vivo) extracts were 68.4, 57.1, 54.2, and 17.7 %, respectively.

     

Recalcitrant effects associated with the development of basal callus-like tissue on caulogenesis and rhizogenesis in <Emphasis Type="Italic">Sclerocarya birrea</Emphasis>

In the micropropagation of woody plant species, adventitious root and shoot formation remain some of the major bottlenecks due to their recalcitrance to in vitro manipulation. Some plant growth regulators may ameliorate these recalcitrant effects and improve in vitro caulogenic and rhizogenic processes. Shoot induction on shoot meristems, hypocotyls and epicotyls was evaluated using equimolar concentrations of benzyladenine (BA), meta-topolin (mT), meta-topolin riboside (mTR), and meta-methoxytopolin riboside (MemTR). Three auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) were used in the induction of adventitious roots. Moderately high shoot formation (62.7%) was achieved at a concentration of 8.0 μM mT after 8 weeks in culture. The highest number of adventitious shoots per explant (2.4 ± 0.3) and the longest shoots (23.5 ± 3.16 mm) were recorded on 8.0 μM mT, though not significantly different from BA treatments. Most shoots progressively produced brown basal callus, which is a potential sink for cytokinin conjugates that are inhibitory to further proliferation of adventitious shoots. Good adventitious shoot formation occurred in 55% of hypocotyl explants on 8.0 μM mT. The highest rooting (91.6%) was achieved with IBA-treated shoots at a concentration of 4.0 μM. The use of mT and IBA provide an efficient micropropagation method for S. birrea, though further research is required especially in overcoming ex vitro plantlet survival challenges.     

Overexpression of <Emphasis Type="Italic">AtATM3</Emphasis> in <Emphasis Type="Italic">Brassica juncea</Emphasis> confers enhanced heavy metal tolerance and accumulation

The eucalypt Corymbia torelliana × C. citriodora is planted widely in India, Brazil and Australia although plantation establishment has been limited by inadequate seed supply and low amenability to propagation via cuttings. This study optimised node culture and organogenic culture methods for in vitro propagation of Corymbia hybrids by identifying explant position (topophysic) effects on rooting, shoot elongation and shoot proliferation. Strong, negative morphogenic gradients in shoot elongation and proliferation capacity were evident from the cotyledonary node to the fourth or fifth node of seedlings when their nodes were transferred to node culture (without benzyladenine). These topophysic effects were related to differences in rooting capacity of individual nodes. Root formation in node culture was associated with formation of long multi-nodal axillary shoots, and so higher rooting of shoots from the cotyledonary node or first true-leaf node was associated with higher shoot proliferation. However, all nodes were equally capable of shoot proliferation in organogenic culture (with 2.2 μM benzyladenine), where rooting and rapid stem elongation did not occur. Most shoots (61–100%) from both node culture and organogenic culture were converted to plantlets, with plantlet conversion and primary root number not differing significantly among explant node positions. The strong topophysic effect in node culture, combined with the lack of a topophysic effect in organogenic culture, provides for an optimised clonal propagation system based on segregation of nodes from the same seedling into separate node and organogenic culture pathways.     

In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

Ferulic acid impairs rhizogenesis and root growth,and alters associated biochemical changes in mung bean (Vigna radiata) hypocotyls

The present study investigated the effect of ferulic acid (FA; 0–1000 µM) on early growth, and rhizogenesis in mung bean (Vigna radiata) hypocotyls and associated biochemical changes. FA severely affected the radicle elongation and number of secondary roots after 72 h. The root and shoot length, number and length of secondary roots, and seedling dry weight of one-week-old seedlings of mung bean were decreased by 64%. The rooting potential (percent rooting, number and length of adventitious roots) of mung bean hypocotyls under in vitro conditions was significantly inhibited in response to 1–100 µM FA. At 1000 µM there was complete cessation of rooting. FA caused a reduction in the contents of water-soluble proteins and endogenous total phenolics, whereas the activities of proteases, peroxidases, and polyphenol peroxidases increased. The study concludes that FA inhibits root growth and development, and in vitro rooting process in mung bean by interfering with biochemical processes that are crucial for root formation.     

The role of cytokinins on in vitro shoot production in Salix tetrasperma Roxb.: a tree of ecological importance

A valuable tropical tree, Salix tetrasperma Roxb. commonly known as Indian willow has been investigated for its in vitro regeneration potential using nodal explants obtained from a 30-year-old elite tree. Agar-solidified Woody Plant Medium (WPM) containing different concentrations of Plant Growth Regulators (PGRs) was used in the study. Shoot induction response was best on WPM supplemented with 6-benzyladenine (5.0 μM) where 90% explants responded with an average shoot number (4.40 ± 0.50) and shoot length (0.92 ± 0.04) after 6 weeks of culture. However, multiplication and elongation was best recorded when BA (5.0 μM) treated shoot clusters were transferred to WPM containing BA (1.0 μM) + NAA (0.5 μM) where 18.40 ± 0.92 well-grown healthy shoots with an average shoot length of 5.30 ± 0.43 cm were obtained on completion of 12 weeks culture period. In vitro rooting of shoots was best achieved in half-strength WPM containing 0.5 μM IBA. Well-rooted plantlets were successfully hardened off and acclimatized in plastic cups containing sterile Soilrite. These plantlets were then transferred to pots containing normal garden soil followed by transfer to greenhouse and ultimately to field under full sun.     

In vitro acclimation to prolonged metallic stress is associated with modulation of antioxidant responses in a woody shrub Daphne jasminea

Comparative effect of meta-topolin and other cytokinins was assessed to develop an efficient and reliable regeneration protocol for Tecoma stans, using mature nodal explants. The morphogenic effect of benzyl adenine (BA), kinetin (Kin), meta- topolin (mT) and 2-iP (2-iso pentenyl adenine) at various concentrations (1.0–10 µM) was studied individually or in combination with auxins (IAA, IBA or NAA). Superior multiplication rates were achieved on MS medium supplemented with mT and NAA. Of the tested combinations, maximum shoot regeneration (95%), mean shoot number (19.6?±?0.60) and length (5.26?±?0.73 cm) was recorded on MS medium supplemented with 7.5 µM mT?+?0.5 µM NAA after 8 weeks of incubation. Among the different auxins employed for in vitro root induction, 92.5% microshoots rooted on MS medium enriched with 1.0 µM IBA with 10.8?±?0.20 mean root number and 5.62?±?0.17 cm length after 4 weeks of incubation. The acclimatized plants grew well in green house with 90% survival rate. The gas chromatography–mass spectrometry (GC–MS) analysis of ethanol leaf extract of in vitro-raised plants yielded a higher number of compounds than control plant. The assessment of genetic fidelity among regenerants, using ISSR markers did not reveal any somaclonal variation. Therefore, the protocol developed appears to be simple and reliable for mass production of clones with higher diversity of secondary metabolites.

     

Micropropagation of <Emphasis Type="BoldItalic">Embelia ribes</Emphasis> Burm f. through proliferation of adult plant axillary shoots

Micropropagation of Embelia ribes was achieved through proliferation of axillary shoots obtained from mature plants. Nodal shoot segments, collected March–May, exhibited high-frequency (75%) shoot initiation when cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) at 1.13 μM and indole-3-butyric acid (IBA) at 0.49 μM. Subculture of sprouted shoots from the original explants on medium containing TDZ (1.13 and 0.45 μM) during the first and second subcultures was found essential for further shoot proliferation, while inhibition of shoot elongation by TDZ could be overcome by transferring shoot cultures onto MS medium containing 6-benzylaminopurine (BAP; 11.10 μM) for the third subculture. Treating the explants with an antioxidant mixture of 568 μM ascorbic acid, 119 μM citric acid, and 307 μM glutathione prior to inoculation, coupled with subculture at 2-wk intervals onto fresh medium, both helped to reduce browning of the explants and facilitated production of five to six shoots/explant. MS medium supplemented with BAP (4.44 μM) and IBA (0.49 μM) induced shoot multiplication, producing five to six shoots/explant with a shoot length of 3 to 4 cm over a 4-wk culture period. Shoots of 3 to 4 cm in length exhibited 100% rooting within 4 wk after transfer to media containing half the nutrient salt concentration of MS medium with 3.69 μM IBA. Ex vitro rooting in the greenhouse from the in vitro shoots treated with 4.93 μM IBA for 30 min exhibited 95% rooting in soilrite™ medium in a 4-wk period. About 85% of micropropagated plants were established successfully in root trainers. Three-month-old, hardened plants could further be successfully established in the field. In 1 yr, by using the above protocol, 3,200 plants could be produced from a single shoot and 2,700 could be established in the field.     

Comprehensive in vitro regeneration study with SCoT marker assisted clonal stability assessment and flow cytometric genome size analysis of Carthamus tinctorius L.: an important medicinal plant

An efficacious and reproducible in vitro regeneration technique for safflower was established using varying concentrations and composition of plant growth regulators (PGRs) supplemented Murashige and Skoog (MS) medium. Successful in vitro seed germination in half strength MS (H-MS) with 1.4 µM GA₃ resulted in procurement of sterile explants (cotyledons, apical meristems) for in vitro study. Callogenesis (2.2 µM BAP?+?2.7 µM NAA), indirect organogenesis of shoot buds (0.54 µM NAA?+?9.08 µM TDZ), somatic embryogenesis (2.2 µM BAP?+?5.4 µM NAA) and somatic embryo germinated plantlets (H-MS?+?1.4 µM GA₃?+?2.2 µM BAP?+?5.4 µM NAA) were successfully obtained. Histological study and scanning electron micrographs of embryogenic callus revealed pre-globular, heart-shaped and torpedo stages of dicot embryogeny. H-MS?+?8 µM NAA showed maximum rhizogenic response with a mean root and shoot length of 17.5 mm and 48.50 mm respectively in 2.2 µM BAP?+?0.54 µM NAA bearing an average of 9 capitula per plantlet with 70% post transplantation survival rate. True to type nature of the regenerates was confirmed using Start Codon Targeted (SCoT) marker, exhibiting 100% and 97.3% monomorphic bands for direct and somatic embryo regenerated plants respectively. Flow cytometry method (FCM) was employed for 2C DNA content analysis. The histogram peaks of 2C nuclear DNA content of in vitro regenerated safflower (direct and embryo derived) were similar to the peak of field grown donor plant. 2C nuclear DNA content of field grown, direct and somatic embryo regenerated C. tinctorius was 2.65?±?0.04 pg, 2.62?±?0.06 pg and 2.68?±?0.04 pg respectively, further verifying genetic homogeneity. All things considered, the above protocol is insusceptible to genetic alteration and can be used for large scale production and sustainable utilization of desired genotype.

     

Adventitious plant regeneration on leaf explants from adult male kiwifruit and AFLP analysis of genetic variation

The present work reports on a study of plant regeneration carried out with callus from the leaf blades and petioles of field-grown male adult kiwifruit plants (Actinidia deliciosa (Chev.) Liang and Ferguson). The cultivars used were ‘Tomuri’ and clone A, a selected male plant grown in north western Spain. The best shoot induction conditions were obtained in ‘Tomuri’ leaf blades cultured in K(h) medium in the presence of 23 μM Zeatin and 0.1 μM NAA. Under these conditions, more than 80% of organogenic callus induction was observed, with an average of 14 new shoots in the second subculture. The initial length of the shoots affected shoot elongation, which was accomplished by culturing isolated shoots in K(h) medium with half-strength salts, supplemented with 0.4 μM Zeatin and 0.1 μM NAA. A possible detrimental long-term effect of cytokinins on shoot elongation can account for the results, since elongation was not observed until 1 month of culture in elongation medium. For rooting, shoots (1 cm in length) were basally immersed in a 5 mM IBA solution for 15 s, and transferred to half-strength K(h) basal medium. Regenerated plants were acclimated in a sterile peat:perlite substrate for 10 days, and then transferred to soil. AFLP analysis was accomplished with 15 primer combinations from which 13 showed reproducible and well-resolved bands, producing a total of 1321 fragments from which 1281 were polymorphic (97%). A dendrogram was constructed using both monomorphic and polymorphic bands, showing genetic variation among field-grown plants and tissue culture-derived regenerants.     

High frequency shoot organogenesis and somatic embryogenesis in juvenile and adult tissues of seabuckthorn (<Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.)

Seabuckthorn (Hippophae rhamnoides) is a multipurpose small tree with unique berries of high nutritional and pharmaceutical values. A clonally propagated plant originating from a 20-year-old tree of H. r. rhamnoides × mongolica hybrid cultivar Julia and seedling offspring of this cultivar were investigated regarding induction of shoot organogenesis in leaf explants and in roots of intact seedlings, and induction of direct somatic embryogenesis in explants from shoot tissue. The highest percentage of leaf explants showing shoot organogenesis was achieved (juvenile explants, 65%; adult explants, 75%) when incubated in Murashige and Skoog (MS) medium supplemented with either 4.5 μM of the phenylurea cytokinin thidiazuron (TDZ) or 2.25 μM TDZ plus 2.2 μM 6-benzyladenine (BA), for juvenile and adult explants, respectively, both supplemented with 0.53 μM α-naphthaleneacetic acid (NAA). Juvenile explants developed on average 18 shoots per explant in the MS medium supplemented with 4.5 μM TDZ, a four fold increase over those incubated on the medium supplemented with 2.25 μM TDZ and 2.2 μM BA. Adult leaf explants grown on medium containing 2.25 μM TDZ and 2.2 μM BA medium produced 12 shoots per explant, while those grown on medium containing 4.5 μM TDZ produced 5 shoots per explant. Shoot organogenesis was observed in roots of intact seedlings pre-cultured on plain medium lacking nutrients (PM) or woody plant medium (WPM) salts and then grown on WPM salts supplemented with 4.4 μM BA, 0.29 μM gibberrelic acid (GA3), and 57.0 μM indoleacetic acid (IAA). The number of shoots formed on each seedling root system was ten fold higher when the pre-culture was in WPM medium indicating a promoting effect of mineral nutrients in the pre-culture medium. Somatic embryogenesis was induced in both juvenile and adult leaf explants in 65 and 78% of the explants, respectively, in MS-based medium supplemented with 2.0 μM N-(2-Chloro-4-pyridyl)-N ¹-phenylurea (CPPU), 0.53 μM NAA and varying concentrations of BA. There was an interaction effect between MS salt strength and BA concentration. The most effective medium for inducing somatic embryogenesis in juvenile explants contained half strength MS salts and 2.2 μM BA and full strength MS salts and 13.2 μM BA for adult explants.     

Direct shoot organogenesis from <Emphasis Type="Italic">in vitro</Emphasis>-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl⁻¹ IAA and 2 mgl⁻¹ BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl⁻¹ BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl⁻¹ of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.     

Micropropagation of Semecarpus anacardium L. from mature tree-derived nodal explants

A protocol was established for micropropagation of Semecarpus anacardium L. from mature tree-derived twigs. Sixty percent of aseptic cultures were obtained by surface sterilization with Bavistin, liquid detergent, and cefotaxime. Elongated twigs collected before flowering were optimum for in vitro culture initiation. Meristematic activity was triggered at all concentrations of thidiazuron (TDZ) incorporated into Woody Plant Medium. TDZ suppressed elongation of axillary buds, resulting into swollen meristems and upon its elimination multiple shoot primordia formation and differentiation were noted. Differentiation and shoot elongation were slower in explants pre-cultured with higher concentrations of TDZ. Swollen axillary meristems pre-cultured on TDZ (9.08 and 13.62 μM) failed to differentiate, whereas TDZ at 2.27 μM was optimal for shoot differentiation and elongation. Multiple bud induction was favored by 4.45 μM of TDZ. Differentiation of multiple shoot primordia by repeated subculturing on growth regulator-free medium and rooting was 100% in filter-paper supported half-strength liquid medium containing 7.38 μM IBA. Rooting was 90% in shoots placed directly in half-strength liquid medium with 2.46 μM IBA. Rooted plantlets hardened in soil:sand mixture (1:1) were transferred to green house. Genetic uniformity of in vitro raised clones with mother plant was confirmed by Inter-Simple Sequence Repeat markers.     

Organogenesis of Phaseolus angularis L.: high efficiency of adventitious shoot regeneration from etiolated seedlings in the presence of N6-benzylaminopurine and thidiazuron

A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established. Pre-culture of 5-day old seedling explants with MS (Murashige and Skoog (1962) Physiol Plant 15:473–493) + B₅-vitamins (Gamborg et al. (1968) Exp Cell Res 50:151–158) liquid medium containing either 5.0 μM TDZ or 5.0 μM BAP under dark condition was essential for organogenesis. Bud growth and shoot multiplication were stimulated by reducing the BAP concentrations from 5.0 to 2.5 μM after 3 weeks. The maximum frequency of shoot induction was 65.2% (33.8 ± 2.54 shoots/explant) in cultivar KS-8 followed by KS-7 34.6% (23.4 ± 1.91 shoots/explant) and KS-6 30.6% (21.2 ± 2.28 shoots/explant). The multiplied buds elongated after transferring to solid MSB₅ medium supplemented with 4.0 μM GA₃, 12.5 μM AgNO₃ and 0.4 μM IBA. Up to 98% rooting efficiency of was obtained when the shoots were pulse-treated with liquid medium containing 4.5 μM IBA for 10 min. The rooted plantlets were transferred to pots in the greenhouse, where they grew, mature, flowered and bared pod normally. The efficient shoot bud induction capability was found to be cultivar dependent. All the three cultivars tested formed multiple shoots. This efficient and rapid regeneration system may also be helpful for Agrobacterium- or particle gun-mediated transformation for this important legume crop.     

Thidiazuron: a potent cytokinin for woody plant tissue culture

Thidizuron (TDZ) is among the most active cytokinin-like substances for woody plant tissue culture. It facilitates efficient micropropagation of many recalcitrant woody species. Low concentrations (<1 µM) can induce greater axillary proliferation than many other cytokinins; however, TDZ may inhibit shoot elongation. In some cases it is necessary to transfer shoots to an elongation medium containing a lower level of TDZ and/or a less active cytokinin. At concentrations higher than 1 µM, TDZ can stimulate the formation of callus, adventitious shoots or somatic embryos. Subsequent rooting of microshoots may be unaffected or slightly inhibited by prior exposure to TDZ. The main undesirable side effect of TDZ is that cultures of some species occasionally form fasciated shoots. The high cytokinin activity and positive response of woody species to TDZ have established it as among the most active cytokinins forin vitro manipulation of many woody species.     

Shoot regeneration and determination of iridoid levels in the medicinal plant <Emphasis Type="Italic">Castilleja tenuiflora</Emphasis> Benth.

Castilleja tenuiflora is a medicinal plant that grows in pine–oak woods primarily in southern and central Mexico. It is highly valued for its medicinal properties, which have been attributed to aucubin-like iridoids. In the present study, we developed an efficient protocol for in vitro shoot proliferation and ex vitro rooting of C. tenuiflora. Using a colorimetric method, we determined total iridoid contents of various different tissues of propagated plants. The shoots were induced from nodal explants cultured on Murashige and Skoog (MS) (1962) medium supplemented with indole-3-butyric acid (IBA) (0 and 0.5 μM) and different concentrations of thidiazuron (TDZ), 6-benzyladenine (BA), or kinetin (KIN) (0–20 μM). Of the cytokinins tested, KIN was more effective for shoot induction than TDZ or BA, and the highest shoot proliferation rate was achieved with 5 μM KIN (4 shoots per explant). Plantlets were rooted on MS medium, nutrient solution, or potting mix, alone or in combination with auxins. The best responses (100% rooting efficiency) were obtained by dipping shoots in half-strength MS medium containing 7.5 μM IBA before transfer to potting mix. On average, each shoot formed 9 roots of 39.3 ± 3.8 mm in length after 21 days. These roots appeared to be more functional than those that developed in nutrient solution, and were associated with a high survival rate (95%) during acclimatization and cultivation in a greenhouse, where flowering occurred after 4 months. Propagated plants accumulated iridoids, thus representing a potential source of pharmacologically useful compounds.