In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

Plant regeneration from leaf explants of Rhodiola fastigiata

Summary An efficient plant regeneration protocol for rapidly propagating Rhodiola fastigiata (Hk. f. et Thoms.) S.H.FU, a traditional Chinese medicinal plant, was developed. Shoot organogenesis occurred from the leaf explants inoculated on medium with appropriate supplements of plant growth regulators. Up to 5.3 shoots formed per leaf explant cultured on a medium containing 13.32 μM 6-benzylaminopurine (BA) and 0.54 μM 1-naphthaleneacetic acid (NAA). Regenerated shoots formed complete plantlets on a medium containing 1.48 μM indole-3-butyric acid (IBA), and mature plants were established, acclimatized, and thrived in greenhouse conditions. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite Chinese medicinal plant.     

Micropropagation of Aerides maculosum lindl. (Orchidaceae)

Summary Young leaf segments from plants growing both in vivo and in vitro were cultured on Murashige and Skoog (MS) medium supplemented with auxins [naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D)], cytokinins [kinetin (KN) and N⁶-benzyladenine (BA)] and coconut liquid endosperm (CW). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves growing in vitro developed protocorm-like bodies (PLBs) at their cut surfaces within 4–8 wk depending on the growth medium. An optimum of 18 PLBs developed from leaf explants on medium supplemented with 2.0 mg l⁻¹ (8.87 μM) BA. Upon subculture in basal MS medium, the PLBs differentiated into plantlets within 6–8 wk. The resulting plantlets were successfully transferred to vermiculite initially and subsequently to potting mixture; 84% of the plantlets survived after 3 mo. of transplantation.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue culture independent transformation for Corchorus olitorius

An efficient microprogation protocol has been developed for Dendrobium densiflorum Lindl. ex Wall., a traditional medicinal plant, through protocorm-like bodies (PLBs) from nodal stem segments using 6-benzylamino-purine (BAP) and the lanthanoid neodymium. The highest percentage of explants producing PLBs (72%), with an average of 15 PLBs per explant, was induced by culturing stem segments on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ BAP. The newly formed PLBs proliferated well on the basal MS medium and completely converted into shoots on MS medium containing 2.0 mg l⁻¹ BAP. Shoots produced an average of 22 roots per plantlet when cultured on MS medium supplemented with 2.0 mg l⁻¹ neodymium nitrate. Healthy plantlets with well-developed roots were successfully acclimatized. The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of an endangered medicinal plant Saussurea involucrata Kar. et Kir.

An efficient micropropagation system for Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant, has been developed. Shoot organogenesis occurred from S. involucrata leaf explants inoculated on medium with appropriate supplements of plant growth regulators. 66.0% of shoot regeneration frequency and 5.2 shoots per leaf explant were achieved when cultured on a medium containing 10 μM 6-benzylaminopurine (BAP) and 2.5 μM 1-naphthaleneacetic acid (NAA). Shoot organogenesis was improved further when the leaf explants were pre-incubated at low temperature, and 80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant at 4°C by 5-day pretreatment period. Up to 87.0% of the regenerated shoots formed complete plantlets on a medium containing 2.5 μM indole-3-acetic acid (IAA) within 28 days, and 85.2% of the regenerated plantlets survived and grew vigorously in greenhouse condition. The phytochemical profile of the micropropagated plants was similar to that of wild plants. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite medicinal plant.     

Somatic embryogenesis and plant regeneration in zygotic embryo explant cultures of rugosa rose

Rugosa rose (Rosa rugosa) is cultivated as a garden flower and an important genetic resource for the breeding of roses (R. hybrida). This study describes culture conditions for high frequency plant regeneration from zygotic embryo explants via somatic embryogenesis in rugosa rose. Mature zygotic embryo, cotyledon, and radicle explants formed embryogenic calluses at frequencies of 38, 6.7, and 8.8% when cultured on half-strength Murashige and Skoog medium (½MS) supplemented with 2.26, 9.05, and 9.05 μM 2,4-dichlorophenoxyacetic acid, respectively. Embryogenic calluses produced numerous somatic embryos, which then developed into plantlets on ½MS without growth regulators. Regenerated plantlets were grown to whole plants in a growth chamber.     

Propagation of Chelidonium majus L. by Somatic Embryogenesis

Direct somatic embryogenesis in celandine (Chelidonium majus L.) was achieved in epicotyl explants of seedlings after prolonged cultivation on Murashige and Skoog medium with or without plant growth regulators. Somatic embryos developed into plantlets which entered additional cycles of somatic embryogenesis. Cultures consisting of plantlets with prolonged embryogenic potential were maintained for five years on plant growth regulator free medium. Embryos which developed into rooted plantlets could be acclimated in a glasshouse enabling thus a continuous propagation scheme to be established.     

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l^-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l^-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 μM indole-3-acetic acid (IAA) and 2.2 μM N⁶-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and 8.8 μM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 μM IBA and 4.4 μM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 μM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.     

Regeneration of plantlets from leaf and petiole explants of Pelargonium x hortorum

Summary The in vitro plant regeneration potential of vegetatively propagated geraniums (Pelargonium x hortorum) has been investigated. Using various combinations of growth regulators and a choice of different explants, a regeneration protocol has been developed to raise in vitro plantlets from young petiole and leaf explants from three different cultivars of geraniums. In all three cultivars, very young petiole explants exhibited a higher regeneration potential as compared with leaf explants. Regeneration efficiencies were found to be highly dependent on the cultivar, with cv. Samba showing the highest regeneration potential, followed by cvs. Yours Truly and then Sincerity. Samba also showed the highest number of shoots from both the petiole [57 shoot buds per petiole explant in the presence of 3 μM zeatin and 1 μM indole-3-acetic acid (IAA) and leaf explants (43 shoots per leaf explant with 10 μM zeatin and 2 μM IAA). Shoot buds transferred to Murashige and Skoog (MS) medium supplemented with 0.44 μM N⁶-benzyladenine and 0.11 μM IAA grew vigorously and attained 1–2 cm in length in 3–4 wk. These shoots rooted with 100% efficiency on MS basal medium, and plants developed that showed normal growth and flowering under greenhouse conditions.     

High Frequency Plant Regeneration from Astragalus melilotoides hypocotyl and stem explants via somatic embryogenesis and organogenesis

An efficient and reproducible procedure is established for the plant regeneration from hypocotyl explants and hypocotyl-or stem-derived calli in Astragalus melilotoides. High frequency somatic embryo formation (98.3%) occurred direct on hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.69 µM NAA and 4.44 µM BA within 5 weeks. Three types of calli were induced from the hypocotyl and stem segments on MS medium containing 9.05 µM 2,4-D and 2.22–4.44 µM BA. Both somatic embryos and adventitious buds were initiated from hypocotyl-derived calli while only adventitious buds were formed from stem-derived calli in MS medium supplemented with 2.69 µM NAA and 4.44–8.89 µM BA. Somatic embryos or adventitious buds developed into plantlets following being cultured for 3 weeks on MS medium without any growth regulators or with 14.78 µM IBA, respectively. All the regenerated plants were normal with respect to morphology and growth characters, and produced fertile seeds after planting in soil.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

Characterisation of <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> Dun. root primordia culture with plant recovery

A liquid meristematic root primordia culture (RPC) of Solanum lycopersicoides Dun. based on persistent rhizogenesis in a modified Murashige and Skoog (1962) medium supplemented with NAA (15 mg·l⁻¹) or 2,4-D (1 mg·l⁻¹) was described. The meristematic clumps (2–3 mm in diameter) originating from NAA supplemented medium were capable of regenerating plants through the callus stage (up to 70 %). Efficient direct plant regeneration (up to 21 %) was possible from numerous single globular-shaped root primordia (RP) structures liberated from the parental aggregates in 2,4-D supplemented proliferation medium without NH₄NO₃ and with a 2.5 fold increase in KNO₃. The RP converted into plantlets (artificial seedlings) on solid or liquid media without growth growth regulators through the unipolar followed by the mace-shaped bipolar structure stages. The use of apical shoot bud, root apices or root segments as a primary explants brought about RPC induction and plant regeneration. The plants derived from 2 years old culture were phenotypically identical to their parental S. lycopersicoides plants and possessed the same ploidy.     

Efficient in vitro regeneration of fireweed,a medicinal plant

Epilobium angustifolium L. (fireweed) is a medicinal plant that has been used to treat diarrhea, mucous colitis, irritable-bowel syndrome, skin problems, prostate problems, menstrual disorders, asthma, whooping cough, and hiccups. A highly efficient and rapid regeneration system via multiple shoot formation was developed for fireweed. Explants (leaf, petiole, root, and stem segments) excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. Explant browning, a major problem for regeneration, was overcome by adding 100 mg/l ascorbic acid to all prepared media containing growth regulator combinations. Root explants formed more shoots than other explants. Best shoot proliferation was obtained from root explants cultured on media with 0.1 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were transferred to rooting media containing different concentrations of IAA, IBA, NAA or 2,4-D. Most shoots developed roots on medium with 0.5 mg/l IAA. Rooted explants were transferred to vermiculate in Magenta containers for acclimatization and after 3 weeks they were planted in to plastic pots containing potting soil and maintained in the plant growth room.