In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Callus induction and plant regeneration from hypocotyl explants of the forage legume Astragalus adsurgens

An efficient procedure was developed for inducing callus and plant regeneration using hypocotyl segments of Astragalus adsurgens. The combinations and concentrations of different growth regulators were shown to be critical factors for both the frequency and the type of callus formation as well as for the potential of callus differentiation. Of the four morphologically distinct types of calli that were induced, a friable, yellow callus, i.e. type I, induced on MS medium supplemented with 9.0 μM 2,4-dichlorophenoxyacetic acid and 2.2 μM N⁶-benzylaminopurine (BA), and then transferred to MS medium containing 0.5 μM α-naphthaleneacetic acid and 8.9 μM BA, exhibited the maximum frequency of shoot regeneration (75%). After regenerated shoots were transferred onto half-strength MS medium without growth regulators, they rooted and complete plants were obtained. Plantlet regeneration from callus cultures required 7–8 weeks. Received: 26 February 1997 / Revision received: 28 August 1997 / Accepted: 13 September 1997     

Plant Regeneration from Immature Embryo Cultures of Vigna unguiculata

Micropropagated plants of two annual haloxerophytic Asiatic Salsola species (S. pestifer and S. lanata) were obtained from zygotic embryos cultured on Murashige and Skoog (MS) agar medium supplemented with 0.5 μM benzylamino-purine (BAP) and 0.3 μM indole-3-acetic acid (IAA) or with 0.5 μM 6 γ, γ-dimethylallylaminopurine and 0.3 μM IAA. The callus induction from shoot and leaf explants derived from plants propagated in vitro were obtained on MS agar medium with various concentration of auxins and cytokinins. The best medium for growth and proliferation of calluses of both studied species was MS medium containing 9.0 μM 2,4-dichlorophenoxyacetic acid. It was also determined that beginning of plant regeneration from callus of S. lanata was induced by 8.8 μM BAP. This revised version was published online in July 2006 with corrections to the Cover Date.     

Callus proliferation from leaf protoplasts using phenylurea type cytokinin, 4-PU,inBetula platyphylla VAR.Japonica

Summary Protoplasts were isolated from leaves ofBetula platyphylla var.japonica using a 0.6M mannitol solution containing 1% Cellulase Onozuka R-10 and 1% Driselase. The cell division and colony formation were largely enhanced using Murashige and Skoog (1962) liquid medium at half strength (1/2 MS), containing 0.6M mannitol, 0.09M sucrose, and factorial combinations of 0.1–30 μM N-(2-chloro-4-pyridyl)-N′-phenylurea (4-pu) and 0.1–10 μM 1-naphthaleneacetic acid (NAA) or 0.1–30 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimal protoplast density was 5–7 × 10⁴/ml. Continuous callus proliferation from protoplasts was achieved by transferring colonies to fresh 1/2 MS agar medium containing 1 μM NAA and 1 μM 4-pu with no mannitol. It appeared that supplementation of the medium with phenylurea type cytokinin, 4-pu gave the successful callus proliferation from the protoplasts ofB. platyphylla.     

In vitro establishment of nitrogen-fixing strawberry (Fragaria × ananassa) via artificial symbiosis with Azomonas insignis

Summary Artificial symbiosis was established between diazotrophic Azomonas insignis and strawberry (Fragaria × ananassa). The partnership was created by in vitro techniques through callus induction and organogenesis. Suitable micropropagation [M3=Murashige and Skoog (1962) (MS) basal medium supplemented with 2.5 μM N⁶-benzyladenine (BA), 0.3 μM gibberellic acid (GA₃), 2.2 μM indole-3-butyric acid (IBA), and 3% sucrose] and plant regeneration [R3=MS mineral salts+555 μM myo-inositol, 1.2 μM thiamine HCl, 4.4 μM BA, 0.5 μM IBA, 0.3 μM α-naphthaleneacetic acid (NAA), 0.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D)] media were developed for the test cultivar Fert?di F5. New shoots containing bacteria were rooted, acclimatized, and planted outdoors. The basis of the partnership during the in vitro phase is the bacterial dependence on the plant metabolic activity, using maltose in the medium as carbon and energy source that can be utilized by the plant cells only. The presence of bacteria in the intercellular spaces of the callus tissues and regenerated plants was proved by re-isolation and microscopic techniques. Nitrogenase activity was also detected in the plant tissues.     

Micropropagation of Heracleum candicans wall: A rare medicinal herb

Summary A protocol was developed for micropropagation of Heracleum candicans Wall. by axillary shoot proliferation. Maximum shoot proliferation was obtained on Murashige and Skoog medium supplemented with 0.5 mg (2.2 μM) 6-benzyladenine per 1 and 0.1 mg (0.4 μM) 1-naphthaleneacetic acid per 1. Regenerated shoots were rooted on MS medium fortified with 1 mg (4.9 μM) indole-3-butyric acid per 1. Complete plants were transferred to soil and all of these plants were morphologically and cytologically identical to the mother plant.     

Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill]

Summary Regeneration of several varieties of soybean [Glycine max (L.) Merrill] by somatic embryogenesis from cultured epicotyls and primary leaves has been demonstrated. Somatic embryogenesis was induced from epicotyls and primary leaves when cotyledon halves with the intact zygotic embryo axes were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg 1⁻¹ (45.2 μM) 2,4-D. Stable, continuously proliferating globular embryo cultures (GEC) were established from small groups of somatic embryos on MS medium supplemented with 20 mg 1⁻¹ (90.5 μM) 2,4-dichlorophenoxyacetic acid (2,4-D). Rapid multiplication of shoot tips from germinating somatic embryos was achieved on Cheng’s basal medium (CBO) containing 2.5 mg 1⁻¹ (11.3 μM) 6-benzyladenine. Fertile plants were obtained from individual somatic embryos and in vitro propagated adventitious shoot bud cultures.     

Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.)

We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 10⁶ cells/g fresh weight cells and then cultured at a concentration of 2.5×10⁵ cells/ml in NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 μM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 μM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 μM kinetin or 1 μM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants. Received: 7 March 1997 / Revision received: 16 June 1997 / Accepted: 5 July 1997     

Direct shoot formation and plant regeneration from cotyledon explants of rapid-cycling Brassica rapa

Summary An in vitro culture system for direct shoot regeneration from cotyledon explants of rapid-cycling Brassica rapa was developed. Cotyledons from 3-d-old seedlings, when cultured on Murashige and Skoog (MS) medium supplemented with 20 μM N⁶-benzyladenine (BA) and 2 μM α-naphthaleneacetic acid (NAA), regenerated shoots directly at a frequency of 20%. The addition of 2 μM aminoethoxyvinylglycine (AVG) to this medium increased shoot regeneration to 33%, but silver nitrate drastically inhibited shoot regeneration. Shoot regeneration occurred directly, at the petiolar cut ends of cotyledonary explants, between 10 to 17 d in culture. The highest percentage of regeneration (33%) was obtained from 3-d-old seedlings. NAA was the most effective auxin for root induction and development, with 49% of shoots producing roots after 2 wk on medium containing 1.0 μM NAA. Regenerated plantlets were grown to maturity in pots containing peat moss and vermiculite (1:1). These plants were morphologically normal and fertile. With this protocol, over 100 independently derived, flowering R₀ plants were obtained from 40 regenerating cotyledonary explants within 40 d after culture initiation.     

Micropropagation ofAcacia mearnsii

Summary A micropropagation system was developed forAcacia mearnsii De Wild., which is the principal source of the world’s tanbark and an excellent firewood. Shoot tips 5-mm long from 3-wk-old seedlings germinated in vitro served as explants. The seeds were germinated on hormone-free MS medium and the shoot tips were cultured on three-fourth-strength MS medium supplemented with combinations of auxins [indole-3-butyric acid (IBA) andα-naphthaleneacetic acid (NAA)] and cytokinins [kinetin and benzylaminopurine (BAP)]. Cultures were maintained at 25° ± 5° C and exposed to 12-h photoperiods of cool-white fluorescent light (70 μEm⁻²·s⁻¹). Multiple shoot formation was promoted by BAP at 2 mg · liter⁻¹ (8.87μM) and higher combined with or without 0.01 mg · liter⁻¹ (0.049μM) IBA. Cytokinins at concentrations of less than 1 mg · liter⁻¹ combined with 0.01 to 0.1 mg · liter⁻¹ auxin inhibited multiple shoot formation and promoted rooting of the shoot tip explants. Shoot multiplication cultures were maintained by transferring segments of multiple-shoot clusters onto a medium containing 2 mg · liter⁻¹ BAP and 0.01 mg · liter⁻¹ IBA. Although higher levels of BAP promoted more multiple shoot formation, this BAP level allowed shoot elongation as well as multiplication. In-vitro-produced shoots were induced to root on a range of NAA concentrations (0.0 to 0.8 mg · liter⁻¹[4.3μM]) supplemented to half- or full-strength MS medium. The highest frequency of root proliferation was on half-strength MS medium supplemented with 0.6 mg · liter⁻¹ (3.22μM) NAA. Plantlets survived in potting soil and exhibited normal growth under greenhouse conditions.     

Optimization of in vitro conditions for stigma-like-structure production from half-ovary explants of Crocus sativus L

Summary Studies were conducted to optimize the in vitro production of stigma-like-structures (SLS) that yielded the important biochemical constituents responsible for the color, taste, and aroma naturally found in the stigmas of autumn crocus. Immature half-ovary explants were evaluated for the frequency of proliferation of SLS by culture on five basal media supplemented with different combinations of plant growth regulators and vitamins. The optimum proliferation of SLS was observed on B5 basal medium containing α-naphthalene acetic acid (NAA) (5.4 μM), N⁶-benzyladenine (BA) (44.4 μM), MS organics, casein hydrolysate (0.05%), and l-alanine (11.2 μM) 50–60 d after inoculation. Some explants formed other structures (roots, corms, petals, leaves), the growth and development of which substantially reduced the development of SLS. Removal of brown tissues and other tissues during subculture (3-wk intervals) allowed continuous culture of halfovary explants for 9–10 mo. SLS that had deep red pigment and reached more than 0.5 cm length were removed from the explants and with concomitant reculturing of the ovaries, enabled SLS to be harvested three times. Activated charcoal (1%) added to B5 basal containing NAA (5.4 μM), BA (44.4 μM), and sucrose (3%) was found to be a helpful addendum to prevent browning of explants and accelerate the initiation, growth, and development of SLS. The amounts of crocin, crocetin glucosyl esters, picrocrocin, and safranal in SLS, as determined by high performance liquid chromatography analysis, were found similar to those in natural saffron.     

Rapid in vitro propagation of Saussurea lappa, an endangered medicinal plant, through multiple shoot cultures

Summary Rapid micropropagation of Saussurea lappa C. B. Clarke, an endangered medicinal plant endemic in the valley of Kashmir and western Himalayas of northern India, was achieved by culturing shoot tips (0.5–1 cm) of 2-wk-old seedlings on Murashige and Skoog’s medium (MS) supplemented with thidiazuron (TDZ, 0.45 μM). Although callus-free multiple shoots were obtained both on N⁶-benzyladenine- (BA) and TDZ-containing media, TDZ was most effective (90%) in inducing multiple shoots. Shoot tips containing proliferative buds were divided into equal halves and subcultured on MS liquid medium containing 0.225 μM TDZ for further multiplication and elongation. Multiplication of induced shoot buds was more effective when cultured in liquid medium than on agar-solidified medium. Shoots (8–10 cm) developed were rooted in MS medium containing naphthaleneacetic acid (NAA, 1.07 μM). Micropropagated plantlets were successfully transferred to soil after hardening.     

Morphological and molecular analysis of genetic stability in micropropagated Fragaria×Ananassa cv. pocahontas

Summary Micropropagated strawberry plants (Fragaria×ananassa L.) grown on 5 μM and 15 μM BA medium or cold-stored were grown in the field to examine morphological variation. Except for plant height, morphological characteristics did not differ for field-grown plants micropropagated on 5 μM and 15 μM BA medium. Cold-stored plants were less vigorous, both vegetatively and reproductively, than BA-treated plants. Random amplified polymorphic DNA (RAPD) markers were used to determine if cold storage or supraoptimal levels of N⁶-benzyladenine (BA) in the culture medium caused genetic changes leading to somaclonal variation. No mutations were observed in 246 loci amplified by the 29 primers tested. Possible changes in methylation patterns of ribosomal DNA genes of strawberries were also examined. Changes in methylation patterns were observed in only one DNA sample from plants grown on 15 μM BA medium and in one of the cold-stored plants. Length polymorphism was observed in two samples from plantlets derived from one explant. The low levels of RAPD variation and methylation observed, and the apparently epigenetic changes in morphological characteristics in plants used in this study, indicated that mutations had not occurred. Part of a thesis submitted by M. B. K. in partial fulfillment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

In vitro shoot tip multiplication of cowpea Vigna unguiculata (L.) walp

Summary Shoot multiplication was induced in cowpea, cv. Georgia-21, from shoot tip explants. Shoot tips, 5 mm long, were isolated from in vitro-grown seedlings and cultured on MS medium containing N⁶-benzyladenine (BA) at 1, 2.5, or 5 mg/liter (4.4, 11.1, or 22.2 μM) or 6-furfurylaminopurine (kinetin) at 1, 2.5, or 5 mg/liter (4.6, 11.6, or 23.2 μM) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.3 μM) or naphthaleneacetic acid (NAA) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.7 μM). Cultures were maintained at a 12-h photoperiod (40 μmol·m⁻²·s⁻¹) and 23 ± 2° C. Treatments with BA induced greater shoot proliferation than those with kinetin. The highest number of shoots was produced on 5 mg (22.2 μM) BA per liter in combination with NAA or 2,4-D at 0.01 mg/liter (0.05 μM). Callus proliferated from the basal ends of shoot pieces in all treatments. The cultures also formed roots in the presence of kinetin, but not on BA-containing medium. To produce whole plants, the shoots were separated and rooted on 0.1 mg (0.5 μM) NAA per liter. Resulting plants grew normally under greenhouse conditions. Shoot tips provide an excellent explant source for cowpea micropropagation and can be used for callus induction.     

Temporal effect of 2,4-D on induction of embryogenic nuclellar cultures and somatic embryo development of ‘Carabao’ mango

Summary Embryogenic nucellar cultures were established on B5 major salts, MS minor salts and organics, 400 mg/l⁻¹ glutamine, 60 g/l⁻¹ sucrose, 2 g/l⁻¹ gellan gum, and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). There was no clear relationship between developmental age of the nucellar explants and induction of embryogenic cultures. The temporal requirements for culture initiation and for induction of embryogenic competence from nucellar explants were determined by pulsing the cultures for 0, 7, 14, 21, 28, 35, 42, 49, 56, and 63 d. Culture initiation required a minimum 7–14 d pulse with 2,4-D, and was maximum after a 56-d pulse; however, embryogenic competence was optimum after a minimum of 28 d exposure to 2,4-D. Somatic embryogenesis occurred directly from the nucellar explants at low frequencies. Somatic embryo maturation only occurred following plating of suspensions onto semisolid medium, and was stimulated by 2.4–4.8 μM kinetin and 4.4 μM 6-benzyladenine.     

Direct somatic embryogenesis and plantlet regeneration from immature leaflets in chickpea

Summary Direct somatic embryo formation and plantlet regeneration was achieved from immature leaflets of chickpea (Cicer arietinum L.). Optimal somatic embryogenesis was obtained when immature leaflets were exposed to media supplemented with 15 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 7 d, to 2000 μM 2,4-D for 3 d, and to 50 μM 2,4-D for 10 d, followed by transfer onto Murashige and Skoog (MS) basal medium. Exposure of explants to high 2,4-D levels (200–2000 μM) for 3 d produced bottle-shaped embryos, while exposure to low 2,4-D levels (<50 μM) and 50–2000 μM for 10 d produced spherical-shaped embryos. Two percent of embryos converted into plants upon culture on MS medium containing 15 μM gibberellic acid and 1 μM 3-indolebutyric acid. All regenerated plants were phenotypically normal.     

Chromium-induced glucose uptake, superoxide anion production, and phagocytosis in cultured pulmonary alveolar macrophages of weanling pigs

The dose-dependent effects of chromium chloride (CrCl₃) and chromium picolinate (CrPic) were evaluated for their glucose uptake, superoxide anion (O ₂ ⁻ ) production, activity of glucose-6-phosphate dehydrogenase, and phagocytosis of incubated pulmonary alveolar macrophages in medium containing no or 5 × 10⁻⁸ M insulin. Glucose uptake was found to increase in cells treated with 20 μg/L CrCl₃. Incubation with 20 μg/L of CrPic enhanced glucose uptake and O ₂ ⁻ production in an insulin-dependent manner. However, the inclusion of CrPic to 100 μg/L in the medium absent of insulin also increased O ₂ ⁻ production. The activity of glucose-6-phosphate dehydrogenase was not affected by either the addition of Cr or insulin. The phagocytosis of Escherichia coli by macrophages was enhanced significantly (p<0.05) in medium containing 10–100 μg/L CrCl₃ or 20–100 μg/L CrPic in the presence of insulin. These results suggest that the addition of 10–20 μg/L CrCl₃ enhances directly the cellular activity of macrophages, whereas the effect of CrPic requires the cooperative action of insulin in enhancing their glucose uptake and phagocytosis.     

Proliferation and differentiation from endosperms of Carthamus tinctorius

The endosperms of Carthamus tinctorius cv. HUS-305, excised at globular to heart-shaped stages of zygotic embryo development, were cultured on Murashige and Skoog’s medium (MS) supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ), 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene-acetic acid (NAA). The highest incidence of callusing was on 2,4-D supplemented media. However, embryos differentiated only from the calli developed on media supplemented with BAP, kinetin or TDZ with the last eliciting maximum embryogenic response. The addition of a reduced nitrogen source, casein hydrolysate to MS medium supplemented with BAP and/or NAA, did not stimulate the response. However, adenine sulphate (100 mg dm⁻³) promoted the induction of somatic embryos. Upon transfer to MS basal medium or the same supplemented with 0.61 μM gibberellic acid (GA₃), plumular poles of few embryos elongated resulting in the development of shoots.     

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.     

Somatic embryogenic tissue establishment from mature Pinus nigra Arn. ssp. salzmannii embryos

Summary Somatic embryogenesis from mature zygotic embryos of salgare?o pine (Pinus nigra Arn. ssp. salzmannii) was induced after 2 wk of culture in L₁ medium [Murashige and Skoog mineral solution with 10.7 μM naphthaleneacetic acid (NAA) and 8.8 μM 6-benzyladenine (BA)] or L₂ medium [Gupta and Durzan mineral solution (DCR)] supplemented with the phytohormone combination as above. Four different combinations of growth regulators, NAA (2.6–10.7 μM) and BA (6.6–8.8 μM), were tried for subsequent passage. The best percentage of embryo induction and manifestation was obtained on DCR medium with 2.6 μM NAA and 6.6–8.8 μM BA. Transfer of isolated somatic embryos from the basal media to L₃ medium (Quoirin and Le Poivre modified mineral solution without hormones and supplemented with 0.3% activated charcoal), facilitated random embryo maturation and some development into plantlets.