Shoot regeneration from cotyledonary leaf explants of <Emphasis Type="Italic">Jatropha curcas</Emphasis>: a biodiesel plant

A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l⁻¹ activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.     

Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of <Emphasis Type="Italic">Encyclia mariae</Emphasis> (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21–4.4 μM 6-benzylaminopurine (BA) in combination with 2.69–10.74 μM naphthalene acetic (NAA), 2.07–8.29 μM indole-3-butyric (IBA), or 2.85–11.42 μM indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 μM BA and 10.74 μM NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 μM IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 μM BA and 5.37 μM NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 μM IAA or 4.14 μM IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.     

ABA enhances plant regeneration of somatic embryos derived from cell suspension cultures of plantain cv. Spambia (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Creamy friable calli were induced from meristems (scalps) of proliferating shoots of plantain (Musa sp.) cv. Spambia (genome AAB) incubated on a semi-solid modified Murashige and Skoog (MS) medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 μM zeatin. About 25% of shoot-tip explants formed scalps, and about 98% of scalps developed embryogenic calli. Small dense aggregates of cells, were obtained when these calli were transferred to liquid MS medium supplemented with 4.5 μM 2,4-D and 1.0 μM zeatin. Upon transfer to semi-solid MS medium of the same composition as described above, aggregates of cells formed somatic embryos. In the presence of 2.5 μM abscisic acid (ABA), maturation of somatic embryos was 2.6-fold higher than that of control (lacking ABA), and regardless of the type of cytokinin used in the medium. Upon transfer to MS medium supplemented with 1.25 μM 6-benzyladenine (BA), 80% of germinated embryos developed into plantlets.     

Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.     

Thidiazuron Induced Multiple Shoot Induction and Plant Regeneration from Cotyledonary Explants of Mulberry

A rapid micropropagation protocol through induced multiple shoots from the cotyledonary explant of mulberry (Morus alba L) is described. The highest number of shoots (20.3) was obtained when explants from 14-d-old embryos were cultured on Murashige and Skoog (MS) medium supplemented with 7 μM thidiazuron for 45 d. Of the three cultivars used, cv. S-36 was the best followed by cv. K-2 and S-1. The shoots were transferred to MS medium supplemented with 5 μM 6-benzylaminopurine for elongation. The elongated shoots were rooted on half strength MS medium containing 1 – 7 μM indole 3-butyric acid or 1-naphthalene acetic acid. The rooted plants were transplanted to soil with 90 % success. The emerged shoot primordia probably initiated from the pre-existing meristems since the shoot bud show definite vascular connection to the major vascular tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regeneration of <Emphasis Type="Italic">Aeschynanthus radicans</Emphasis> via direct somatic embryogenesis and analysis of regenerants with flow cytometry

Plant regeneration through direct somatic embryogenesis in Aeschynanthus radicans ‘Mona Lisa’ was achieved in this study. Globular somatic embryos were formed directly from cut edges of leaf explants and cut ends or on the surface of stem explants 4 wk after culture on Murashige and Skoog (MS) medium supplemented with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA), TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D), or 6-benzylaminopurine (BA) or kintin (KN) with 2,4-D. MS medium containing 9.08 μM TDZ and 2.68 μM 2,4-D resulted in 71% of stem explants producing somatic embryos. In contrast, 40% of leaf explants produced somatic embryos when induced in medium containing 6.81 μM TDZ and 2.68 μM 2,4-D. Somatic embryos matured, and some germinated into small plants on the initial induction medium. Up to 64% of stem explants cultured on medium supplemented with 9.08 μM TDZ + 2.68 μM 2,4-D, 36% of leaf explants cultured on medium containing 6.81 μM TDZ and 2.68 μM 2,4-D had somatic embryo germination before or after transferring onto MS medium containing 8.88 μM BA and 1.07 μM NAA. Shoots elongated better and roots developed well on MS medium without growth regulators. Approximately 30–50 plantlets were regenerated from each stem or leaf explant. The regenerated plants grew vigorously after transplanting to a soil-less substrate in a shaded greenhouse with more than a 98% survival rate. Three months after their establishment in the shaded greenhouse, 500 plants regenerated from stem explants were morphologically evaluated, from which five types of variants that had large, orbicular, elliptic, small, and lanceolate leaves were identified. Flow cytometry analysis of the variants along with the parent showed that they all had one identical peak, indicating that the variant lines, like the parent, were diploid. The mean nuclear DNA contents of the variant lines and their parent ranged from 4.90 to 4.99 pg 2C⁻¹, which were not significantly different statistically. The results suggest that the regenerated plants have a stable ploidy level, and the regeneration method established in this study can be used for rapid propagation of ploidy-stable Aeschynanthus radicans.     

Regeneration of salvia (<Emphasis Type="Italic">Salvia officinalis</Emphasis> L.) via induction of meristematic callus

Nodular meristematic callus was induced on the basal cut surface of apical shoot explants of salvia cultured on Murashige and Skoog (MS) medium supplemented with 4.5, 13.5, or 22.5 μM thidiazuron (TDZ). Cultures were incubated in the dark for 1 wk and then transferred to light conditions for 4 wk. A higher percentage of explants developing callus was observed on medium containing either 4.5 or 13.5 μM TDZ, although explants on 4.5 μM developed larger calluses. The callus was maintained on medium containing 4.5 μM TDZ and 0.45 mM ascorbic acid. Shoot differentiation, after each of three successive maintenance passages, was induced from callus grown on medium containing either 4.4 or 8.8 μM benzyladenine (BA). A greater number of shoots were harvested from callus differentiated on BA (4.4 or 8.8 μM) medium with 0.45 mM ascorbic acid added. Shoots developed roots on MS medium supplemented with 4.9 μM of indole-3-butyric acid. The addition of ascorbic acid to the shoot differentiation medium enhanced rooting, number of roots per shoot, and survival rate. Approximately 75% in vitro plantlets were acclimatized to ex vitro conditions. Histological investigations confirmed both adventitious meristem initiation during the callus induction phase, and subsequent organogenic shoot development on the differentiation medium. The novel protocol for the meristematic callus induction and plant regeneration in this study may be useful for biotechnological applications for salvia improvement via genetic transformation or mutagenesis and in vitro propagation approaches.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of Indian Kino (<Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.) using Thidiazuron

An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2 ± 0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4 ± 0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4 ± 0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.     

High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledons of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L.

The effect of copper sulphate on differentiation and elongation of shoot buds from cotyledonary explants of Capsicum annuum L. cv X-235 was investigated. Shoot buds were induced on medium supplemented with 22.2 μM BAP and 14.7 μM PAA. Elongation of shoot buds was obtained on MS medium containing 13.3 μM BAP + 0.58 μM GA₃. Both shoot induction and elongation media were supplemented with different levels of CuSO₄ (0–5 μM). The levels of CuSO₄ in the induction as well as elongation medium highly influenced the shoot bud formation and their subsequent elongation. Highest number of shoot buds per explant was obtained when the concentration of CuSO₄ was increased 30 times to the normal MS level. Shoot buds formation frequency i.e., the number of shoots formed per explant was increased two fold as compared to those formed on control. Elongation both in terms of percentage and length of shoots was better than that on control. Healthy elongated shoots were rooted on MS medium supplemented with 5.7 μM IAA. Rooted plantlets were transferred to field conditions.     

Somatic embryogenesis and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated) was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible ‘annatto pigment,’ which mainly constitutes an apocarotenoid called bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The maximum of 28 somatic embryos were produced in 16–18 weeks when immature zygotic embryonic stalks were inoculated onto Murashige and Skoog (MS) medium containing B5 vitamins supplemented with 0.44 μM benzyladenine (BA), 0.054 μM α-naphthaleneacetic acid (NAA), 2.89 μM gibberellic acid (GA₃), 0.02 μM triiodobenzoic acid (TIBA), and 0.011 μM triacontanol (TRIA). Callus initiation from hypocotyl explants was obtained on MS medium supplemented with 1.07–2.14 μM NAA and 10.2 μM BA. In 3 months, somatic embryos were produced when callus was inoculated onto MS medium supplemented with 4.44 μM BA, 40 μM AgNO₃, and 0.011 μM TRIA. Somatic embryos were efficiently regenerated on MS basal solid and liquid media supplemented with 0.44–4.4 μM BA, 0.54–2.69 μM NAA, 4.92 μM 2iP, 2.1 μM calcium d-pantothenate, 0.21 μM biotin, 227.7 μM cysteine HCl monohydrate, and 108.6 μM adenine sulfate. Agrobacterium tumefaciens GV 3101 harboring pCAMBIA 1305.2 binary vector-mediated stable transformation of somatic embryos exhibited a transformation frequency of 2.56%. As somatic embryogenesis in any perennial system is useful in terms of both commercial and scientific nature, this somatic embryo-based transformation protocol for the commercially important dye-yielding tropical plant B. orellana is useful for its improvement through genetic engineering.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

Plant regeneration through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

Influence of explants, genotypes and culture vessels on sprouting and proliferation of pre-existing meristems of cotton (Gossypium hirsutum L. and Gossypium arboreum L.)

Summary A simple and efficient method for multiple shoot induction and proliferation was achieved in six Indian cotton cultivars from the pre-existing meristems of 21-d-old in vitro-grown seedlings. Combinations of naphthalene acetic acid (0.3–10.7 μM) and 6-benzylaminopurine (BA; 2.2 or 4.4 μM) were used for induction of shoots. The shoots proliferated and were maintained on MS (Murashige and Skoog) medium supplemented with 4.4 μM BA. Simultaneous elongation of shoots was obtained in the same medium. Optimum multiplication was observed in cv. LRK-516 (19.7±4.6), in cotyledonary nodes isolated from the adjoining node and cultured individually in 250 ml flasks. This indicates lateral inhibition of adjoining meristems. A positive influence of culture flasks as opposed to test tubes on the proliferation of multiple shoots was observed in all six cultivars tested. The morphogenic response varied with genotype and the nature of explants. Rooting of elongated shoots was achieved on MS medium devoid of growth regulators. The plantlets were transferred to the field after hardening in the greenhouse. All plants flowered and formed bolls on maturity.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and plant establishment of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. F.) Merrill: Petiole callus culture

Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.     

<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.     

In vitro organogenesis and plant formation in Acacia sinuata

In vitro morphogenesis via organogenesis was achieved from callus cultures derived from hypocotyl explants of Acacia sinuata on MS (Murashige and Skoog, 1962) medium. Calli were induced from hypocotyl explants excised from 7-day-old seedlings on MS medium containing 3% sucrose, 0.8% agar, 6.78 μM 2,4-dichlorophenoxyacetic acid and 2.22 μM 6-benzylaminopurine. Regeneration of adventitious buds from callus was achieved when they were cultured on MS medium supplemented with 10% coconut water, 13.2 μM 6-benzylaminopurine and 3.42 μM indoleacetic acid. Addition of gibberellic acid (1.73 μM) favored shoot elongation. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 7.36 μM indolebutyric acid. Rooted plantlets, thus developed were hardened and successfully established in the soil. This protocol yielded an average of 20 plants per hypocotyl explant over a period of 4 months. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (<Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.     

Micropropagation of <Emphasis Type="Italic">Pongamia pinnata</Emphasis> through enhanced axillary branching

A complete protocol is presented for the first time for the micropropagation of Pongamia pinnata, a biofuel tree, using cotyledonary nodes derived from axenic seedlings. Multiple shoots were induced in vitro from nodal segments through forced axillary branching. Murashige and Skoog (MS) medium supplemented with 7.5 μM benzylaminopurine (BAP) induced up to 6.8 shoots per node with an average shoot length of 0.67 cm in 12 d. Incorporation of 2.5 μM gibberellic acid (GA₃) in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Single use of GA₃ during the first subculture eliminated the need for prolonged culturing on BAP medium. Further use of GA₃ in the medium was not useful. Shoot culture was established for at least two subcultures without loss of vigor by repeatedly subculturing the original cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 16–18 shoots were obtained in 60 d. Shoots formed in vitro were rooted on full-strength MS medium supplemented with 1.0 μM indole butyric acid (IBA). Plantlets were successfully acclimated, established in soil, and transferred to the nursery.     

In vitro morphogenesis of organogenic nodules derived from <Emphasis Type="Italic">Sclerocarya birrea</Emphasis> subsp. <Emphasis Type="Italic">caffra</Emphasis> leaf explants

Sclerocarya birrea (marula) is an indigenous South African tree with highly valued medicinal and nutritional properties. Induction of nodular meristemoids from leaf explants was achieved on Murashige and Skoog (MS) and woody plant medium (WPM) supplemented with 6-benzyladenine (BA) in combination with naphthalene acetic acid (NAA), indole-3-butryric acid (IBA) and indole-3-acetic acid (IAA). Induction of nodular meristemoids from 86% of the leaf cultures was achieved on MS medium with 4.0 μM BA and 1.0 μM NAA. High levels (78–100%) of induction were also achieved on WPM with different concentrations of BA (1.0–4.0 μM) and IBA (1.0–4.0 μM). The highest conversion of meristemoids into shoots was only 22% for 4.0 μM BA and 1.0 μM NAA on MS initiation medium. This was improved to 62% when nodular clusters were cultured in a MS liquid medium. Histological studies revealed the globular stage of the nodular meristemoids. This protocol has potential for application in mass micropropagation and plant breeding of S. birrea.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.