High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

Plant regeneration <Emphasis Type="Italic">in vitro</Emphasis> directly from cotyledon and hypocotyl explants of <Emphasis Type="Italic">Perilla frutescens</Emphasis> and their morphological aspects

A rapid plantlet regeneration system for Perilla frutescens was established from cotyledon and hypocotyl explants. A maximum of 91.06 % cotyledon and 76.4 % hypocotyl explants could directly produce shoots (3.09 ± 0.18 shoots per explants) on Murashige and Skoog (MS) medium. The optimum hormone combinations were 4.44 μM 6-benzylaminopurine (BA) for cotyledon and 2.22 μM BA + 2.85 μM indole-3-acetic acid (IAA) for hypocotyls. Rooting was induced on half-strength hormone-free MS medium. After transplantation to soil, approximate 80 % of the regenerated plantlets could survive, flower and fruit. Moreover, some morphological abnormalities were found among the regenerated plants.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

Direct shoot organogenesis and assessment of genetic stability in regenerants of <Emphasis Type="Italic">Solanum aculeatissimum</Emphasis> Jacq.

A simple and efficient procedure was developed for in vitro propagation of Solanum aculeatissimum Jacq. using leaf and petiole explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). Effects of various plant growth regulators, explant types, carbohydrates, and basal salts on induction of adventitious shoots were also studied. Leaf explants appeared to have better regeneration capacity than petiole explants in the tested media. The highest regeneration frequency (79.33 ± 3.60%) and shoot number (11.33 ± 2.21 shoots per explant) were obtained in leaf explants in MS medium containing 3% sucrose and 0.8% agar, supplemented with 0.1 mg/l NAA and 2.0 mg/l BA, whereas petiole explants were more responsive to 0.1 mg/l NAA and 1.0 mg/l thiadiazuron. Developed shoots rooted best on MS medium with 1.0 mg/l indole acetic acid (IAA), producing 18.33 ± 2.51 roots per shoot. Histological investigation showed that the shoot buds originated mainly from epidermal cells of wounded tissues, without callus formation. The regenerated plantlets were successfully acclimatized in a greenhouse, where over 90% developed into morphologically normal and fertile plants. Results of flow cytometry analysis on S. aculeatissimum indicated no variation in the ploidy levels of plants regenerated via direct shoot formation and showed almost the same phenotype as that of mother plants. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies of S. aculeatissimum.     

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.     

Shoot regeneration from in vitro-derived leaf and root explants of <Emphasis Type="Italic">Centaurea ultreiae</Emphasis>

In vitro culture is currently used to produce plant material for ex situ conservation of endangered species. In this study, an efficient protocol for shoot regeneration from leaves and roots was developed for Centaurea ultreiae, a critically endangered species. Organogenesis from leaf and root explants was promoted by incubating these explants on half-strength Murashige and Skoog (MS) medium in the presence of one of four different cytokinins [6-benzyladenine (BA), zeatin, kinetin or N⁶-(2-isopentenyl) adenine (2iP)], each provided at five different levels. Shoot organogenesis was induced in both explants. The best response, 90% of leaf explants producing a mean of 2.48 shoots per explants and 94.3% of root explants producing a mean of 5.60 viable shoots per explants, was observed when explants were incubated on a medium containing 0.55 μM BA. Histological studies revealed connectivity between vascular tissues of regenerated shoots and cambial cells of leaf explants. Moreover, adventitious shoots were derived from pericycle cells of root explants and parenchymatic cells of callus tissues.     

Micropropagation of <Emphasis Type="Italic">Sapindus trifoliatus</Emphasis> L. and assessment of genetic fidelity of micropropagated plants using RAPD analysis

An efficient in vitro propagation system has been developed for rapid micropropagation of Soapnut (Sapindus trifoliatus Linn.), a medicinally and economically important tree from nodal (axillary bud) segments of seedlings. The frequency of shoot regeneration from seedling node explant was influenced by the age of the seedlings, growth regulators and successive transfer of the mother explant. Explants from 4-week-old seedlings yielded the maximum shoot regeneration frequency (97.22%) on full-strength MS medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP). After harvesting the newly formed shoots, the mother explants transferred to same medium subsequently produced a maximum of 5.16 shoots per explant after third passage. Further improvement in the morphogenic response occurred when the nodal explants excised from in vitro regenerated shoots were employed, and 6.89 shoots per explant were obtained on the same medium after the third subculture. Optimal rooting (91.67%) was obtained by placing the micro-shoots in liquid MS medium with 1.0 mg l⁻¹ IBA for 24 h and then transferring to the agar solidified MS medium devoid of IBA. The micropropagated shoots with well-developed roots were acclimatized and successfully transplanted to soil with 90% survival rate. Genetic stability of the regenerated plants was assessed using random amplified polymorphic DNA (RAPD). The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants. This is the first report of an efficient protocol for regeneration of S. trifoliatus through organogenesis, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

Immunoadjuvant saponin production in seedlings and micropropagated plants of <Emphasis Type="Italic">Quillaja brasiliensis</Emphasis>

The leaves of Quillaja brasiliensis, a native tree species of southern Brazil, accumulate saponins which have adjuvant activity in vaccines. An efficient micropropagation system for Q. brasiliensis was developed. Plants were obtained from aseptically germinated seeds. High rates of germination were observed on filter paper and aseptic medium substrate (sucrose and light exposure did not affect the germination response). Stem nodal segments were inoculated into Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine. Each explant produced multiple shoots, which elongated satisfactorily on the same medium. Elongated shoots were rooted in a modified MS medium with or without auxins. Best rooting responses were observed in medium containing 10 mg l⁻¹ indole-3-acetic acid under continuous exposure. The rooted explants were acclimatized and successfully transferred to soil, yielding approximately 95% survival after 10 mo. Leaf content of immunoadjuvant saponins in micropropagated plants was not affected by auxin type used for rooting and was comparable to that of field-grown trees. The results indicate that Q. brasiliensis represents an alternative and readily renewable source of biomass for the production of bioactive saponins.     

Rapid micropropagation of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> using shoot tip explants pre-cultured in thidiazuron supplemented liquid medium

An efficient protocol has been developed for rapid micropropagation of Ocimum basilicum. Multiple shoots were induced by culturing shoot tip explants excised from mature plants on a liquid Murashige and Skoog (MS) medium supplemented with 5–100 μM of thidiazuron (TDZ) for different treatment duration (4, 8, 12 and 16 d). The optimal level of TDZ supplementation to the culture medium was 50 μM for 8 d induction period followed by subculturing in MS medium devoid of TDZ as it produced maximum regeneration frequency (78 %), mean number of shoots (11.6 ± 1.16) and shoot length (4.8 ± 0.43 cm) per explant. A culture period longer than 8 d with TDZ resulted in the formation of fasciated or distorted shoots. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA). The micropropagated shoots with well developed roots were successfully established in pots containing garden soil and grown in greenhouse with 95 % survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Regeneration and micrografting of lentil shoots

Summary A protocol for regeneration and micrografting of shoots of lentil (Lens culinaris Medik) was developed. Multiple shoots (4–5) were regenerated from cotyledonary node explants on Murashige and Skoog (MS) medium containing 8.8 μM 6-benzylaminopurine. In vitro regenerated shoots were micrografted on rootstocks with 96% efficiency. The successful grafts were transplanted to pots in Redi-earth^TM, hardened off and were grown to maturity with 100% success. The success of the micrografting was independent of the nature and concentration of growth regulator used in shoot initiation medium and the time period for induction of shoots. The protocol was successful with several cultivars of lentil. The advantages of micrografting over in vitro rooting are discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the tennessee coneflower (<Emphasis Type="Italic">Echinacea tennesseensis</Emphasis>)

Summary Tennessee coneflower [Echinacea tennesseensis (Beadle) Small] was regenerated from flower stalks, leaf sections from flowering plants, and hypocotyls and cotyledons from seedlings. Murashige and Skoog medium (MS) supplemented with naphthaleneacetic acid (NAA) at 0.54 μM and thidiazuron (TDZ) at 22.7 μM yielded the most shoots per leaf explant. NAA and 6-benzylaminopurine concentrations for optimal shoot regeneration from leaf, flower stalk, cotyledon and hypocotyl explants in MS media were 0.54 and 24.6μM, respectively. All explant types generated shoots; however, those derived from leaves and flower stalks produced the highest number of shoots per explant and highest percentage of explants with shoots. Explants cultured on media containing high levels of NAA (5.4–27 μM) formed calluses but no adventitious shoot. Leaf explants responded to a wider range of NAA concentrations than the other explant types but shoots generated from flower stalks grew the fastest. While all cytokinins tested increased the number of shoots per explant, the number of shoots in media containing TDZ was increased by nearly threefold. Regenerated shoots from all explant types cultured on MS medium supplemented with 0.25 μM indole-3-butyric acid initiated roots within 4 wk; NAA was not effective for root induction. All vernalized plantlets developed into plants that were morphologically identical to the source material.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.) from leaf explants and accumulation of esculin

Summary An efficient protocol has been developed for the regeneration of plantlets from leaf explants of witloof chicory (Cichorium intybus L.). Regeneration via callus was obtained on modified Murashige and Skoog semisolid medium (MS) containing 2.0 μM indole-3-acetic acid +5.0 μM 6-furfurylaminopurine (kinetin), and 1000 mgl⁻¹ casein hydrolyzate. At least five or more shoots regenerated from each callus. The shoots were rooted on MS +0.2 μM indole-3-butyric acid. The plantlets thus obtained were successfully established in soil after bardening. Esculin accumulation was recorded in plant tissues at different stages of differentiation in in vitro cultures and compared with in vivo-grown, plants. The esculin accumulation was higher in in vitro plants.     

An efficient plant regeneration system with <Emphasis Type="Italic">in vitro</Emphasis> flavonoid accumulation for <Emphasis Type="Italic">Hylotelephium tatarinowii</Emphasis> (Maxim.) H. Ohba

An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid was as high as 2.0 mg l⁻¹ in combination with 0.5 mg l⁻¹ 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed to 1.0 mg l⁻¹ 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile of callus and regenerated plants was similar to that of wild plants.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea pallida</Emphasis> from leaf explants

Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important medicinal plant.     

Plant regeneration of <Emphasis Type="Italic">Erigeron breviscapus</Emphasis> (vant.) Hand. Mazz. and its chromatographic fingerprint analysis for quality control

An efficient micropropagation system for Erigeron breviscapus (vant.) Hand. Mazz., an important medicinal plant for heart disease, has been developed. Shoot organogenesis occurred from E. breviscapus leaf explants inoculated on a medium supplemented with a combination of plant growth regulators. On average, 17 shoots per leaf explant were produced after 30 days when they were cultured on MS basal salts and vitamin medium containing 5 μM 6-benzylaminopurine (BAP) and 5 μM 1-naphthaleneacetic acid (NAA). All the regenerated shoots formed complete plantlets on a medium containing 2.5–10 μM indole-3-butyric acid (IBA) within 30 days, and 80.2% of the regenerated plantlets survived and grew vigorously in field conditions. Based on the variation in common peaks and the produced amount of the most important bioactive component, scutellarin, a high performance liquid chromatography (HPLC) fingerprinting system was developed for quality control of these micropropagated plants. Chemical constituents in E. breviscapus micropropagated plants varied during plant development from regeneration to maturation, the latter of which showed the most similar phytochemical profile in comparison with mother plants. The regeneration protocol and HPLC fingerprint analysis developed here provided a new approach to quality control of micropropagated plants producing secondary metabolites with significant implications for germplasm conservation.     

RETRACTED ARTICLE: Influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types

A comparative performance of two explants types (CN and Nodal) for their efficiency to induce multiple shoot regeneration in Clitoria ternatea has been carried out. Thidiazuron (TDZ) in different concentrations (0.05–2.5 μM) was used as a supplement to the Murashige and Skoog’s (MS) basal media. Explant type apart, two factors viz. concentration and exposure duration to TDZ played an important role in affecting multiple shoot regeneration. Cotyledonary node explants produced the best results at 0.1 μM TDZ, while in nodal explants the highest rate of shoot formation was achieved on MS medium supplemented with 1.0 μM TDZ. In both the explants, shoot multiplication increased when the regenerated shoots were subcultured on hormone free MS medium after 4 weeks of exposure to TDZ. Among the two, cotyledonary node explants produced considerably higher number of shoots at a comparatively lower concentration of TDZ than nodal explants. The regenerated shoots rooted best on MS medium containing 1.0 μM indole-3-butyric acid (IBA) and were successfully established in pots containing garden soil with 88 % survival rate. All the regenerated plants showed normal morphology and growth characteristics.     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid