Transformation of <Emphasis Type="Italic">Liquidambar formosana</Emphasis> L. via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using a mannose selection system and recovery of salt tolerant lines

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l⁻¹ induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l⁻¹ α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l⁻¹ TDZ, 0.5 mg l⁻¹ NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l⁻¹ BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l⁻¹ indole-3-butyric acid (IBA) and 1.5 mg l⁻¹ activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.     

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.     

Morphogenetic pathway in petiole derived callus of Amorphophallus albus in vitro

Two different morphogenetic pathways, adventitious bud and corm-like structure (CLS), were observed on organogenic calli derived from the petioles of Amorphophallus albus in vitro. The organogenic calli was established via culture of petiole segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.0 mg l⁻¹ 6-benzyladenine (BA) and subculture of the petiole-derived calli on MS medium with 0.5 mg l⁻¹ NAA and 0.5 mg l⁻¹ BA. These organogenic calli were used to induce morphogenesis via culture on MS medium with various concentrations of NAA and BA. BA alone favoured adventitious bud differentiation (57.0 ± 8.3% at maximum) from the organogenic calli but inhibited CLS formation. In the presence of NAA and BA, both adventitious bud and CLS were observed in a same culture system. The maximum CLS formation (71.2 ± 9.3%) were found on MS medium with 0.5 mg l⁻¹ NAA and 2.0 mg l⁻¹ BA, associated with 26.7 ± 8.6% adventitious bud differentiation. A small part of the adventitious buds developed into normal shoots which needed rooting culture phase to form complete plants. About 80% survival rate was obtained with these plants after transplantation to soil. More than 90% of the CLSs produced complete plants with shoots and root systems, regardless of the rooting media tested. Transplantation of the CLS-derived plants to soil gave 100% survival rate. Histological observations revealed both the two morphogenetic events originated from the meristematic cells located in superficial layers of callus tissue.     

Regeneration of <Emphasis Type="Italic">Pelargonium × hederaefolium</Emphasis> ‘Bonete’ from petiole explants

The adventitious shoot regeneration from petiole explants of Pelargonium × hederaefolium ‘Bonete’ was achieved via a mixed pathway i.e. organogenesis and somatic embryogenesis. The histological study of regenerated structures revealed the presence of both shoot primordia and embryo-like structures. The initial growth in petiole explants occurred on media with BAP + auxin or TDZ alone. However, the most effective regeneration (24 structures/explant) was noted in the presence of TDZ (2 mg l⁻¹) and IBA (0.1 or 0.2 mg l⁻¹). Moreover, the application of TDZ in the induction phase reduced the time needed for the formation of adventitious structures and positively influenced the further shoot development on the medium containing m-topolin and IBA.     

Adventitious shoot regeneration of scotch spearmint (menthaxgracilis Sole)

Summary The effect of different cytokinins on in vitro adventitious shoot regeneration from internodal explants of Menthaxgracilis Sole (scoth spearmint) was investigated. Murashige and Skoog (MS) medium containing 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% (w/v) sucrose, 10% (v/v) coconut water and supplemented with 4.5 μM thidiazuron (TDZ) was effective in inducing adventitious shoot formation from callus. The greatest percentage of explants with shoots (85%) with the highest mean number of shoots per explant (29) was obtained with explants from the 1st and the 2nd internodes from 2-wk-old stock plants growing on a medium containing MS basal salts, 2% sucrose, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, at TDZ 4.5 μM and 10% (v/v) coconut water and solidified with 0.2% (w/v) phytagel. The regenerated shoots rooted on a medium containing MS basal salts, 100 mg l⁻¹ myo-inositol, 0.4 mg l⁻¹ thiamine-HCl, 2.0% sucrose, and 0.054 μM naphthalene acetic acid (NAA). Micropropagated plantlets were transplanted into soil and acclimated to greenhouse conditions. This is the first report describing adventitious shoot regeneration of scotch spearmint.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Regeneration of Syngonium podophyllum ‘Variegatum’ through direct somatic embryogenesis

A simple and effective method of regenerating Syngonium podophyllum ‘Variegatum’ via direct somatic embryogenesis has been established. Leaf and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with either α-naphthalene acetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos directly formed at one or two sides of petiole explants on MS medium supplemented 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA or 2.0 mg l⁻¹ TDZ with 0.2 mg l⁻¹ NAA or with 0.2 and 0.5 mg l⁻¹ 2,4-D, respectively. The frequency of petiole explants with somatic embryos produced was as high as 86% when cultured on medium containing 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA. Up to 85% of somatic embryos were able to germinate after transferring onto medium containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ NAA. Approximately 50–150 plantlets were regenerated from a single petiole explant. However, there was no somatic embryo formation from leaf explants regardless of growth regulator combinations used. Regenerated plantlets from petiole explants were stable and grew vigorously after transplanting to a soilless container substrate in a shaded greenhouse.     

Thidiazuron stimulates adventitious shoot regeneration in different safflower explants

Adventitious shoot regeneration from root, hypocotyl, cotyledon and primary leaf explants of safflower (Carthamus tinctorius L.) was studied. Shoot regeneration was promoted by benzyladenine (BA) + naphthaleneacetic acid (NAA), BA + indole-3-butyric acid (IBA), kinetin + NAA and thidiazuron (TDZ) + NAA incorporated in Murashige and Skoog (MS) basal medium. High frequency of shoot regeneration and high number of shoots per regenerating explant were obtained on a wide range of TDZ + NAA combinations. Proliferated shoots were elongated in MS + 0.5 mg dm⁻³ kinetin and well-developed shoots were rooted in half strength MS + 0.5 mg dm⁻³ NAA. Rooted shoots were successfully acclimatized and established in soil.     

In vitro plant regeneration from cotyledon explants of Swainsona salsula Taubert

An effective protocol has been developed for plant regeneration from cotyledon explants of Swainsona salsula Taubert (Saline swainsona), a medicinal and agronomic shrub. Adventitious shoots were obtained from 83.2% of cotyledon explants from 3-day seedlings cultured on Murashige and Skoog (MS) medium containing 2.0 mg l⁻¹ thidiazuron (TDZ), with an average of 9.3 shoots per explant. Individual elongated shoots were rooted on half strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid (IBA), with 59.3% success. Regenerated plants with well developed shoots and roots were successfully transferred to soil, without detectable variants. Histological observation revealed that shoots developed from cotyledon explants via organogenesis, with little callus. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">In vitro</Emphasis> direct organogenesis and regeneration of <Emphasis Type="Italic">Medicago sativa</Emphasis>

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm⁻³ thidiazuron (TDZ) and 3 mg dm⁻³ AgNO₃. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm⁻³ α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.     

Direct organogenesis and plantlet regeneration from mature zygotic embryos of masson pine (Pinus massoniana L.)

Mature zygotic embryos of masson pine were cultured as initial explants to investigate the process of direct organogenesis. Adventitious buds were initiated on DCR medium (Douglas-fir cotyledon revised medium) supplemented with 0.5 mg l⁻¹ N⁶-benzyladenine (BA) and 0.05 mg l⁻¹ indolebutyric acid (IBA) or α-naphthaleneacetic acid (NAA). The highest induction frequency of adventitious buds was 99.3%. Subsequent transfer of buds to medium with lower concentrations of plant growth regulators in time was necessary for differentation of high quality adventitious buds. After culturing on elongating medium, in which the proportion of cytokinins to auxins was reduced, shoots higher than 2 cm were transferred for root induction to GD medium with half of the concentration of macro-salts (½ GD) and with 2 mg l⁻¹ IBA and 0.05 mg l⁻¹ BA. The average root frequency was over 70%. After adventitious roots had appeared, the shoots were transferred to ½ GD medium with a lower concentration of IBA (0.2 mg l⁻¹) for further root development.     

Pretreatment in thidiazuron improves the in vitro shoot induction from leaves in Curculigo orchioides Gaertn., an endangered medicinal plant

Leaf regeneration via direct induction of adventitious shoots obtained from an endangered medicinal plant, Curculigo orchioides Gaertn. by pretreating with thidiazuron. C. orchioides is an endangered medicinal herb belonging to the family Hypoxidaceae. Direct inoculation of leaf pieces on MS medium supplemented with various concentrations of BAP (2–8 μM) or TDZ (2–8 μM) alone or in combination with NAA (0.5 and 1.0 μM) produced low shoot induction both in terms of % response and number of shoots per explant. Hence, leaf explants were pretreated with 15, 25 or 50 μM thidiazuron (TDZ), for 6, 24 or 48 h with the aim of improving shoot regeneration from cultured explants. After pretreatment, explants were transferred to an agar solidified MS medium that was supplemented with BAP (4 μM), TDZ (6 μM), BAP (4 μM) + NAA (1.0 μM), TDZ (6 μM) + NAA (0.5 μM). Control explants were incubated directly on the medium without any pretreatment. The pretreatment of explants with 15 μM TDZ for 24 h significantly promoted the formation of adventitious shoots and the maximum response was observed on MS medium supplemented with 6 μM TDZ. In this medium, 96 % cultures responded with an average number of 16.2 adventitious shoots per explant. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when TDZ pretreated leaves were cultured onto MS medium supplemented with BAP or TDZ alone or in combination with NAA. The rooted plantlets were successfully transplanted to soil with 90% success. The present investigation indicated the stimulatory role of TDZ pretreatment in regulating shoot regeneration from leaf explants of C. orchioides.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis

The common techniques for the in vitro production of Alstroemeria plants are based on rhizomes as explants, which have low multiplication rates and a high risk of carrying viral diseases. To overcome these problems, we developed a protocol for the in vitro regeneration of Alstroemeria cv.‘Yellow King’, by testing for shoot induction several explant sources (leaf, stem apices, rhizomes and immature inflorescence apices), temperature and light/dark regimes, hormone and salt concentrations. For shoot multiplication and rooting, several hormone concentrations were tested. We found that only the young floral apices produced adventitious shoots by direct organogenesis. The highest shoot induction rate (10.4 shoots per explant) was obtained by incubation in the dark for 15 days at 8 °C followed by 15 days at 25 °C and a 16-h/8-h light/dark regime, on a Murashige and Skoog (1962) liquid medium at 50% of the salt concentration, supplemented with 2.5 mg l⁻¹ KIN, 1.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, using a piece filter paper to support the explant. The highest shoot multiplication rate (9 shoots per explant) was obtained on a liquid MS medium at full strength supplemented only with BA at 1.0 mg l⁻¹. In vitro rooting of shoots was induced also on a liquid MS medium, either with or without plant hormones.     

Micropropagation of Gymea Lily (<Emphasis Type="Italic">Doryanthes excelsa</Emphasis> Corrêa) from New South Wales,Australia

The physiological effects of three auxins [indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d)] and two cytokinins [thidiazuron (TDZ) and N⁶-benzylaminopurine (NAA)] on in vitro morphogenesis of Doryanthes excelsa were measured. Longitudinal bud sections derived from immature inflorescences were used as a source of explants. Callus regeneration was observed at the highest frequencies (46.2%) when grown on media containing 50 μmol L^-1 NAA and 0.5 μmol L⁻¹ TDZ. Adventitious shoot organogenesis was observed at the highest frequency (56.8%) when grown on media containing 0.5 μmol L⁻¹ NAA and 50 μmol L⁻¹ TDZ. Regenerated shoots were rooted ex vitro after 6 weeks when dipped in a solution of 50 μmol L⁻¹ NAA or no plant growth regulators were applied.     

Micropropagation ofCereus peruvianus mill. (cactaceae) by areole activation

Summary Currently,Cereus peruvianus plants can be rapidly clonedin vitro via adventitious organogenesis using callus cultures; however, somaclonal variation is a problem. A method is described herein using lateral bud explants to produce multiple shoots for clonal propagation. Apical and lateral explants were cultured on MS (Murashige and Skoog, 1962) media with factorial combinations of the auxins indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), and cytokinins 6-ben-zyladenine (BA) and N-(2-furanyl-methyl)-1-purine-6 amine (kinetin) at the concentrations 0.0, 0.01, 0.1, 1.0 mg“l⁻¹. Positive results were obtained from the lateral explants in all conditions tested, but apical explants did not respond toin vitro multiplication ofC. peruvianus cactus at all growth regulator combinations tested. Formation of axillary shoots inC. peruvianus seems most frequent in medium containing BA at 1.0 mg·l⁻¹ (4.44 μM) and IAA or NAA at 1.0 mg·l⁻¹ (5.71 μM or 5.37 μM respectively), but the frequency of shoot formation in the BA or kinetin and NAA or IAA combinations indicated that any of the combinations tested can be used for multiplication ofC. peruvianus plants regenerated from callus tissue culture. Root formation occurred in all (100%) of the cactus shoots after 9 wk in the same culture medium. All the cacti that developed at the different auxin and cytokin combinations continued growth after transfer to a potting mix of red earth (Paleudult) and ground river sand (1∶1).     

High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro (15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l⁻¹ sucrose, 2.2 g l⁻¹ Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO₃). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA₃). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an average of 40 plants per leaf explant with a culture period of 98 days.     

Indirect regeneration of the Cancer bush (<Emphasis Type="Italic">Sutherlandia frutescens</Emphasis> L.) and detection of <Emphasis Type="SmallCaps">l</Emphasis>-canavanine in <Emphasis Type="Italic">in vitro</Emphasis> plantlets using NMR

The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l⁻¹) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation (97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l⁻¹ TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l⁻¹ IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate.