Shoot organogenesis from leaf explants of Dayaoshania cotinifolia W. T. Wang

Dayaoshania cotinifolia W. T. Wang is a rare and endangered member of the Gesneriaceae family which is endemic to China. To conserve this species, an efficient in vitro propagation and regeneration system via shoot organogenesis was established from young leaf explants. Adventitious shoot induction was possible within 50–60 d on basal Murashige and Skoog medium supplemented with 1–3 μM 6-benzyladenine, although 5 μM 6-benzyladenine induced hyperhydricity. Basal medium containing 1–5 μM thidiazuron induced fewer shoots, while 1–5 μM α-naphthaleneacetic acid induced numerous adventitious roots and a few adventitious shoots. However, when thidiazuron and α-naphthaleneacetic acid were combined, both the induction percentage and number of shoots increased. Leaf explants cultured on induction medium supplemented with 1–5 μM 2,4-dichlorophenoxyacetic acid become necrotic and died. Induction medium supplemented with 1 μM α-naphthaleneacetic acid and 1–3 μM 6-benzyladenine was optimal for inducing adventitious shoots as was the combination of 1–3 μM thidiazuron and 1 μM α-naphthaleneacetic acid. Induction medium containing 2.0 μM 6-benzyladenine and 0.5 μM indole-3-acetic acid was optimal for the multiplication of adventitious shoots. Rooting was achieved on half-strength MS medium supplemented with 3.0 μM indole-3-acetic acid or α-naphthaleneacetic acid and 0.1% activated charcoal. Plantlets were transplanted to a mixture of sand, vermiculite, and humus (1:1:1); 92% survived. This protocol is a unique and effective means to micropropagate this rare and important plant and could serve as a solution for in vitro and ex vitro conservation.     

TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

Regeneration of plants through somatic embryogenesis in Thymus hyemalis Lange,a potential medicinal and aromatic plant

A protocol for somatic embryogenesis was developed for Thymus hyemalis, a wild species in the Mediterranean region. First, the effects of explant type, plant growth regulators [kinetin (KIN) and 2,4-dichlorophenoxyacetic acid (2,4-D)], and genotype on callus induction were tested. For callus induction, the node was the best explant; Murashige and Skoog (MS) medium supplemented with 1.8 μM 2,4-D and 0.5 μM KIN was the best medium, and the genotype had a highly significant effect. To induce production of somatic embryos, the effects of KIN, 6-benzylaminopurine (BAP), and naphthalene acetic acid (NAA) were evaluated. After 5 wk of culture in the dark, MS medium supplemented with 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN gave the highest percentage (85%) of embryogenic callus and the highest number of somatic embryos (27.00) per 45 mg of callus. For germination and plant recovery, somatic embryos were transferred to MS medium without plant growth regulators and plantlet conversion from developed somatic embryos was 90%. In vitro plants with adequate growth and sufficient root systems were subsequently transplanted into a mixture of peat and vermiculite (2:1?v/v) under greenhouse conditions. The survival rate of the plantlets under ex vitro conditions was 80%.     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration and enhanced synthesis of plumbagin in root callus of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> L.—an important medicinal herb

Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47?±?0.63 mg g^?1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS?+?5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08?±?1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3?±?1.36 roots with an average length of 15.31?±?2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L^?1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.     

Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb.

Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus marsupium. Ninety percent of hypocotyl explants (excised from 12-day-old in vitro germinated axenic seedlings) produced callus on Murashige and Skoog medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid and 1 μM a 6-benzyladenine (BA). Induction of SE occurred after transfer of callus clumps (200 ± 20 mg fresh mass) to MS medium supplemented with BA at 2.0 μM, where a maximum of 23.0 ± 0.88 globular stage embryos per callus clump were observed after 4 weeks of culture. Subculturing of these embryos on MS medium supplemented with 0.5 μM BA, 0.1 μM α-naphthalene acetic acid and 10 μM abscisic acid significantly enhanced the maturation of somatic embryos to early cotyledonary stage, where 21.4 ± 0.32 embryos per callus clump were recorded after 4 weeks of culture. Of 30-well developed somatic embryos, 16.6 ± 0.33 germinated and subsequently converted into plantlets on half-strength MS medium supplemented with 1.0 μM BA. The morphologically normal plantlets with well-developed roots were first transferred to 1/4-liquid MS medium for 48 h and then to pots containing autoclaved soilrite and acclimatized in a culture room. Thereafter, they were transferred to a greenhouse, where 60% of them survived.     

Establishment of dedifferentiated callus of haploid origin from unfertilized ovaries of tea (Camellia sinensis (L.) O. Kuntze) as a potential source of total phenolics and antioxidant activity

This is the first report of induction of haploid callus with significant antioxidant activity from unpollinated ovary cultures of tea. Out of the five cultivars tested, TV18 gave the highest percentage of callus induction. Within 1 wk of induction, ovules swelled to almost double their original size, and white, friable callus emerged. A high cytokinin/auxin ratio, provided by 8.5 μM benzyl adenine and 4.5 μM 2,4-dichlorophenxyacetic acid, and high-temperature treatment (33°C) for 10 d in the dark promoted maximum callus induction. Callus was maintained on MS medium containing 22.2 μM benzyl adenine and 9.8 μM indolebutyric acid (callus line RM 1) in the light at 25°C. Well-developed tracheids were formed within 4 wk in callus subcultured on MS medium containing 1.8 μM thidiazuron and 5.0 μM 2,3,5-triiodobenzoic acid (line RM 2). Flow cytometric analysis revealed that most cells were haploid. Both RM 1 and RM 2 produced phenolic compounds with significant antioxidant capacity. Phenolic content showed a positive linear correlation with antioxidant activity. The total phenolic content of RM 1 was 3.47?±?0.21 gallic acid equivalents (GAE) mg/g dry weight and that of RM 2 was 2.39?±?0.12 GAE mg/g dry weight. Antioxidant activity was measured using IC₅₀, a measure of inhibitory concentration; a lower IC₅₀ value reflects greater antioxidant activity. The IC₅₀ value of RM 1 was 2,530 μg/ml and that of RM 2 was 3,170 μg/ml. The results suggested that the phenolic compounds contributed significantly to the antioxidant capacity of the in vitro cell lines.     

In vitro plantlet regeneration of Capsicum chinense Jacq. cv. ‘Bhut jalakia’: hottest chili of northeastern India

Chili (Capsicum chinense) cv. ‘Bhut jalakia’ is used in India for extraction of oleoresin and capsaicin as it is characterized by a very high capsaicin content. The conventional method of propagation of ‘Bhut jalakia’ is through seeds, but this is beset by short viability and low germination rates. Developing a suitable regeneration protocol for ‘Bhut jalakia’ was the focus of this study; as to date, in vitro regeneration for this cultivar has not been investigated. Cotyledon and shoot tip explants were cultured on Murashige and Skoog (MS) media supplemented with different concentrations of cytokinins and auxins. In the case of cotyledon explants, MS medium supplemented with 6-benzylaminopurine (BAP) at 35 μM and kinetin (KIN) at 15 μM were found to be optimal (4.00?±?0.57) for induction of multiple shoots per explant, whereas BAP at 14.8 μM and KIN at 60 μM were best (5.00?±?0.57) for growth of shoot tip explants. Shoots developed from cotyledon explants produced the maximum (8.67?±?0.32) number of roots on MS medium supplemented with low concentration (2.6 μM) of 2-naphthaleneacetic acid (NAA). Supplementation of indole-3-butyric acid (IBA) at 5 μM was found optimal for root formation (16.67?±?2.60) for shoots derived from of shoot tip explants. One month after transfer of in vitro regenerated plantlets to various potting mixes, the highest survival rate (40%) was observed in a mixture of sand, soil, and cow dung in a ratio of 1:1:1. Thus, both shoot tip and cotyledon explants may be cultured on MS medium modified with BAP, IBA, NAA, and KIN to regenerate ‘Bhut jalakia’ chili plants within 90 d.     

Application of in vitro pollination of opened ovaries to obtain Brassica oleracea L. × B. rapa L. hybrids

This study presents the results of experiments concerning: (1) interspecific hybridization of Brassica oleracea × Brassica rapa via application of in vitro placental pollination and (2) embryological analysis of the process of resynthesis of Brassica napus. In order to overcome certain stigma/style barriers, B. rapa pollen was placed in vitro on an opened B. oleracea ovary (with style removed). Pollinated ovaries were cultured on Murashige and Skoog (MS) medium. After 24-d culture, the developing embryos were isolated from immature seeds and transferred onto MS medium supplemented with 0.47 μM kinetin, 0.49 μM 1-naphthaleneacetic acid, and 10% (v/v) coconut water. When the embryos had turned green, they were immediately placed onto MS medium with 100 μM kinetin. After development of the seedling, plantlets were transferred to soil. Chromosome doubling was achieved after another week. Cytometric analysis of nuclear DNA confirmed the hybrid nature of the plants. Resynthesis of B. napus can be performed through interspecific hybridization of B. oleracea × B. rapa followed by embryo rescue and genome doubling.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

<Emphasis Type="Italic">In vitro</Emphasis> growth profile and comparative leaf anatomy of the C<Subscript>3</Subscript>–C<Subscript>4</Subscript> intermediate plant <Emphasis Type="Italic">Mollugo nudicaulis</Emphasis> Lam.

Mollugo nudicaulis Lam., commonly known as John’s folly or naked-stem carpetweed, is an ephemeral species of tropical regions. The plant is ideal to study the eco-physiological adaptations of C₃–C₄ intermediate plants. In the present report, in vitro growth profiling of the plant and comparative leaf anatomy under in vitro and ex vitro conditions were studied. In vitro propagation of the plant was carried out on Murashige and Skoog (MS) basal medium augmented with additives and solidified with 0.8% (w/v) agar-agar or 0.16% (w/v) Phytagel?. The concentration of plant growth regulators (PGRs) in the basal medium was optimized for callus induction, callus proliferation, shoot regeneration, and in vitro rooting. The optimum callus induction was obtained from M. nudicaulis seedling hypocotyls. The highest regeneration induction of about 88% or nearly 41 shoots with about 142 leaves per culture vessel was observed from friable callus on MS basal medium solidified with Phytagel? and containing 4.44 μM 6-benzylaminopurine, 4.65 μM kinetin, 2.69 μM naphthaleneacetic acid, and 0.91 μM thidiazuron. In leaf anatomy, differences related to photosynthetic tissue organization were observed in leaves of in vitro and ex vitro plants, which indicated that changes in the environment affected the anatomy of subsequent leaves in plants. This is the first report of an efficient micropropagation protocol for M. nudicaulis, using an indirect organogenesis method. Efforts were made to optimize the concentrations of various PGRs and organic compounds for in vitro growth of regenerated shoots.     

Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth

We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.

     

Ιn vitro plant regeneration from leaf explants of the cherry rootstocks CAB-6P,Gisela 6, and MxM 14 using sodium nitroprusside

Conventional multiplication of cherry (Prunus cerasus L.) rootstocks utilizes division, cuttings, and propagation through seed, which are relatively slow and labor intensive and result in genetic variability. Tissue culture, on the other hand, ensures rapid, large-scale, and low-cost production of genetically identical, physiologically uniform, and pathogen-free plants. In the cherry rootstocks CAB-6P, Gisela 6, and MxM 14, sodium nitroprusside (SNP) promoted callus induction, in vitro shoot proliferation, and rooting from leaf explants in a medium containing 17.6 μM benzyladenine and 2.68 μM α-naphthaleneacetic acid. CAB-6P explants treated with 10 μM SNP gave the maximum shoot number (5), whereas 30 μM SNP gave the longest shoots and the greatest shoot induction rate (26.67%). Best rooting was obtained with 50 μM SNP. In Gisela 6 rootstock, the shoot number (10) and shoot length (20.5 mm) were maximal in the control group without plant growth regulators. The shoot induction rate was enhanced (40%) with 40 μM SNP. SNP at 40 μM resulted in root formation, while 30 μM produced the largest callus size, and 10 μM SNP resulted in the maximum callus fresh weight. MxM 14 leaves treated with 30 μM SNP gave the maximum shoot number (3), root number (7.56), and shoot induction rate (40%), whereas 40 μM SNP gave the longest shoots (12 mm) and roots (20 mm). Best results for callus size, callus fresh weight, and callus induction rate (100%) in the CAB-6P and MxM 14 rootstocks were observed with 30 and 40 μM SNP, respectively. Rooted explants with shoots were gradually acclimatized to the external environment with a high survival percentage (85%). An efficient protocol of indirect organogenesis was established for the three cherry rootstocks using SNP.     

In vitro propagation of two Iranian commercial pomegranates (Punica granatum L.) cvs. ‘Malas Saveh’ and ‘Yusef Khani’

An efficient in vitro propagation is described for Punica granatum L. using shoot tip and nodal explants. The influence of two basal medium, WPM and MS, and different plant growth regulators was investigated on micropropagation of the Iranian pomegranate cultivars, ‘Malas Saveh’ and ‘Yousef Khani’. For proliferation stage, media supplemented with different concentrations (2.3, 4.7, 9.2 and 18.4 μM) of kinetin along with 0.54 μM NAA was used. WPM proved to be more efficient medium compared to MS. The best concentrations of kinetin were 4.7 μM for ‘Malas Saveh’ and 9.2 μM for ‘Yousef Khani’, resulting in the highest number of shoots per explants, shoot length and leaf number. For both cultivars, half-strength WPM medium supplemented with 5.4 μM NAA was most effective for rooting of shoots. Rooted plantlets were successfully acclimatized and transferred into soil. The micropropagated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants.     

Callus induction and plant regeneration from anthers of Dendrocalamus latiflorus Munro

Bamboo varieties are very difficult to improve by traditional breeding methods. Here, we established an efficient plant-regeneration system for Dendrocalamus latiflorus (tropical giant bamboo) by anther culture. Culture conditions, especially the plant growth regulators required for callus induction and shoot differentiation, were optimized by orthogonal design. M8 medium supplemented with 5.37 μΜ α-naphthaleneacetic acid (NAA), 1.33 μM?N ⁶ -benzyladenine (BA), 110.17 μM phenylacetic acid (PAA), and a pretreatment time of 3 d produced the highest rate (5.08?±?0.61%) of callus induction. The maximum shoot differentiation rate reached 28.3?±?4.29% in M8 medium supplemented with 2.32 μM kinetin (KT), 8.89 μM BA, 1.08 μM NAA, and 110.17 μM PAA. The results of the ploidy level test showed that most of the regenerated plants were dodecaploid (96/100), a few were hexaploid (3/100), and one was triploid (1/100). The average chlorophyll content of dodecaploid lines was significantly higher than that of hexaploid lines. The present study provides an innovative method for bamboo ploidy breeding and a useful method for genetic improvement.     

Cardenolide estimation in callus-mediated regenerants of Digitalis lamarckii Ivanina (dwarf foxglove)

Digitalis cardenolides can regulate heart rhythms and are effective agents in cancer chemotherapy, in particular, for treating prostate and breast cancer. In this study, an optimized and efficient plant tissue culture protocol was established using callus cultures of Digitalis lamarckii Ivanina, commonly known as dwarf foxglove. Lamina explants developed callus when cultured on Linsmaier and Skoog (LS) medium containing different concentrations of 6-benzyladenine (BA; 4.4, 13.3, or 22.2 μM) and α-naphthalene acetic acid (NAA; 2.7, 5.4, or 10.8 μM). The highest incidence of callus formation (100%) was achieved on LS medium containing 13.3 μM BA and 10.8 μM NAA. Indirect shoot regeneration was achieved when the callus explants were cultured on LS medium supplemented with varying concentrations of BA (0.4, 1.1, or 2.2 μM) and/or gibberellic acid (0.7 or 1.4 μM) for 8 wk. Following the rooting of shoots on LS medium supplemented with either indole-3-acetic acid (ranging from 1.4 to 5.7 μM) or NAA (1.3 to 5.2 μM), lamina and petiole tissues of the 4-mo-old regenerated plants were compared for their cardenolide contents. Lamina extracts showed nearly three times higher cardenolide accumulation than petiole extracts. Of the cardenolides analyzed by reverse-phase high-performance liquid chromatography, neo-odorobioside G and glucogitoroside were abundant in lamina extracts (170.3 and 143.9 mg/kg dry weight, respectively). The regeneration protocol described in this study can be used for the in vitro production of certain cardenolides from D. lamarckii.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated 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 2.25 μM BAP and 8.5 μM IAA. 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 four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

In vitro organogenesis and somatic embryogenesis from leaf explants of Leucosceptrum canum sm.

Plantlets were obtained from leaf explants of a Labiatae tree — Leucosceptrum canum Sm. using plant tissue culture techniques. Two types of calli proliferated from the leaf explants when grown on different media, one of which was amenable to somatic embryogenesis. Differentiation of the embryoids started from the fourth passage of culture and continued up to the seventh passage. The number of embryoids decreased with the age of the callus. The capacity of such embryoids to form entire plantlets was studied using different nutrient mileux. Embryoids formed plantlets on Murashige and Skoog's (MS) medium fortified with benzylaminopurine plus indolebutyric acid. Organogenesis was observed in shoot-buds derived from explants of in vitro regenerated plantlets on MS basal medium supplemented with benzylaminopurine. Culture regenerated plantlets were transferred to MS medium without sucrose and growth hormones; finally transferred to pots containing sterile vermiculite where they are growing.Abbreviations MS Murashige and Skoog's medium - 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - Kn kinetin - BAP benzylaminopurine - CW coconut water