Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

Abstract

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

The Effect of Plant Growth Regulators and Sucrose on the Micropropagation and Microtuberization of <Emphasis Type="Italic">Dioscorea nipponica</Emphasis> Makino

The effects of sucrose, plant growth regulators, MS (Murashige and Skoog), and ½MS salt media formulations were investigated for the development of shoot cultures, microtuber induction, and plantlet regeneration in Dioscorea nipponica. The cytokinin N-benzyladenine (BA) in the range of 0.5–2.0 mg/l showed strong enhancing effects on microtuber induction only when used in conjunction with the auxin alpha-naphthalene acetic acid (NAA), with the effect that NAA increased from 0.5 to 2.0 mg/l. Murashige and Skoog salt media supplemented with sucrose at 3% (w/v) gave the highest frequencies of shoot induction (86%) when BA was present at 2.0 mg/l and NAA at 1.0 mg/l. Sucrose at 7% (w/v) was the single most significant medium constituent for microtuber growth. The heaviest microtubers were formed on media containing 1.0 mg/l BA and 2.0 mg/l (0.073 g), especially with 7% sucrose (3.46 g). With media containing ½MS, 2% sucrose, and 0.1% (w/v) activated charcoal, the percentage of rooting was maximal when supplemented with 1.0 mg/l BA and 0.5 mg/l NAA for the in vitro produced shoots (95%) and BA and NAA both at 0.5 mg/l for the microtubers (100%). When removed from culture flasks and transferred into sterilized soil in a greenhouse, most of the hardened plantlets survived (over 91% after 1 week), and they were suitable for field planting after 1 month.     

Somatic embryogenesis and organogenesis in Dendrocalamus hamiltonii

In this study, mature zygotic embryos, plant growth regulators, and various media were tested with the aim of developing an efficient regeneration system for plantlets of the bamboo species Dendrocalamus hamiltonii. Callus formation was induced in explants cultured in Murashige and Skoog (MS) medium supplemented with 1.0–3.0 mg/l 2,4-dichlorophenoxyacetic acid. Optimal shoot differentiation and subsequent shoot growth were also obtained in MS medium supplemented with 2 mg/l benzyladenine, 1 mg/l kinetin, and 1 mg/l naphthaleneacetic acid. Root induction was enhanced by the addition of 5 mg/l indole-3-butyric acid to the culture medium. Histological analysis revealed that both somatic embryogenesis and organogenesis were induced during callus initiation, shoot differentiation, and the development of plantlets from the mature zygotic embryos. Our data provide a useful basis for developing culture protocols for the regeneration of bamboo plants.     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.     

Mass propagation of <Emphasis Type="Italic">Plectranthus bourneae</Emphasis> Gamble through indirect organogenesis from leaf and internode explants

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.     

In vitro propagation of an endemic and endangered medicinal plant <Emphasis Type="Italic">Notopterygium incisum</Emphasis>

An efficient system in vitro propagation for Notopterygium incisum Ting ex H. T. Chang, an endemic and endangered medicinal plant, was established to address increased demand and germplasm conservation goals. Optimum response in callus induction (CI) was observed on Murashige and Skoog (MS) medium supplemented with 1.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/l 6-benzylaminopurine (BAP), which the induction rate and growth of callus were 84.44% and 0.67 g respectively. The highest shoot regeneration frequency (76.97%) and maximum number of shoots (3.6 shoots per callus) were achieved on MS medium with 1.5 mg/l BAP and 0.2 mg/l naphthalene acetic acid (NAA). Half-strength MS medium supplemented with 0.6 mg/l indole-3-butyric acid (IBA) was determined to be the best rooting medium, resulting in the maximum number of roots (18.6 roots per shoot) and the highest rooting frequency (92.28%). An approximate 83.8% survival rate among the regenerated plantlets was recorded after they were transplanted in the field at an altitude of 3200 m. An HPLC analysis showed that the content of two main chemical constituents, notopterol and isoimperatorin, in the rhizomes of 3-year-old regenerated plantlets was higher (3.84 mg/g and 4.05 mg/g, respectively) than that in commercially marketed crude drugs. This first report of complete regeneration in vitro could provide an alternative method for the rapid, large-scale production and conservation of this valuable, rare, and endangered medicinal plant.     

Hairy root induction and plant regeneration of medicinal plant Dracocephalum kotschyi

An efficient hairy root induction system for an important endangered medicinal plant, Dracocephalum kotschyi, was developed through Agrobacterium rhizogenes-mediated transformation by modifying the co-cultivation medium using five bacterial strains, A4, ATCC15834, LBA9402, MSU440, and A13 (MAFF-02-10266). A drastic increase in transformation frequency was observed when a Murashige and Skoog medium lacking NH₄NO₃ KH₂PO_4, KNO₃ and CaCl₂ was used, resulting in hairy root induction frequencies of 52.3 %, 69.6 %, 48.6 %, 89.0 %, and 80.0 % by A4, A13, LBA9402, MSU440, and ATCC15834 strains, respectively. For shoot induction, hairy roots and unorganized tumors induced by strain ATCC15834 were placed on an MS media supplemented with 0.1, 0.25, 0.5, and 1 mg/l BA plus 0.1 mg/l NAA. The high frequency of shoot regeneration and number of shoot were obtained in the medium containing 0.25 mg/l BA and 0.1 mg/l NAA. Root induction occurred from the base of regenerated shoots on the MS medium supplemented with 0.5 mg/l IBA after 10 days.     

Asymbiotic seed germination and in vitro seedling development of Cymbidium aloifolium (L.) Sw.: a multipurpose orchid

Cymbidium aloifolium is a multipurpose economically important epiphytic orchid grows on tree trunk in the primary forests. Its population in natural habitat is downsized due to different anthropogenic activities. A successful attempt was made for asymbiotic immature embryo culture and in vitro mass scale production of plantlets. For successful culture initiation seed pods of various developmental ages, various nutrient media, sucrose concentrations, different quality and quantity of plant growth regulators were surveyed. Immature embryos of 9 months after pollination was successfully germinated on MS medium containing sucrose (2%) (w/v) and α-naphthalene acetic acid (NAA) and benzyl adenine (BA) (3 and 6 μM respectively in combination) within 45 days of culture where 90% germination was recorded. The germinated seeds formed PLBs on the optimum germination medium within two passages. The protocorm like bodies (PLBs) differentiated into rooted plantlets within 3 weeks on regeneration medium containing sucrose (3%), casein-hydrolysate (0.1 gl^?1) and BA 3 μM. Amongst the three media studied, optimum regeneration was registered on MS medium where as many as 12 shoot buds developed per explants per subculture of 4 weeks duration. The well rooted plantlets of 6–7 cm long with 3–4 roots were hardened in vitro 3–4 weeks before they were transferred to potting mix. The potted plants were exposed to full sunlight periodically and watered at regular interval. About 70–80% transplants survived after 2 months of potting.     

Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones.)

A reproducible and efficient callus-mediated shoot regeneration system was developed for the large-scale production of Valeriana jatamansi Jones., a highly medicinal plant species of global pharmaceutical importance. Effect of Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) on callus induction and production of valepotriates accumulation was studied by using different explants. In V. jatamansi, the degree of callus induction varied significantly depending on explants type and the growth regulators used. Among different explants used, rhizomes have the highest callus induction potential followed by leaf. The callus induction frequency was found to be optimum in rhizome explants on media supplemented with 0.5 mg/l 2,4-D. The regenerative ability of proliferated compact calli was studied by the application of cytokinins alone and in combination with auxin. MS medium fortified with 0.75 mg/l thidiazuron in combination with 0.5 mg/l NAA showed the highest regeneration frequency (88.6 %) and produced the maximum number of shoot buds (15.20 ± 0.20) capable of growing into single plants. Vigorous callus obtained from MS medium supplemented with different concentrations of 2,4-D, NAA and IBA were used for industrially important valepotriates [acevaltrate (ACE), valtrate (VAL) and didrovaltrate (DID)] analysis. High performance liquid chromatography analysis of callus revealed that medium with 2,4-D (1 mg/l) was found responsible for increasing ACE and DID yield, whereas VAL production was higher in case of medium supplemented with NAA (1 mg/l). However, the accumulation of valepotriates in callus decreased in logarithmic phase after 8 weeks. IBA was not beneficial for the valepotriate synthesis, as it helped to accumulate significantly lower concentration of ACE, VAL and DID. Micropropagated plantlets with well-developed root system were successfully acclimatized in greenhouse condition, in root trainers containing garden soil with a survival frequency of 100 %. As Indian valerian is a highly traded medicinal plant due to extensive use of its industrially important secondary metabolites, the present system can be utilized to obtain mass multiplication of the species as well as for the stable biomass and continuous valepotriate production for the pharmaceutical industries.     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

Haploid plants from anther cultures of poplar (Populus × beijingensis)

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial woody plants using conventional breeding techniques is currently a challenge due to a long juvenile period, high heterozygosity, and substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In the present study, we report the regeneration of haploid lines of poplar (Populus × beijingensis) via anther culture. Anthers at the uninucleate stage were induced to produce callus using three basic media. Two auxins (naphthalene acetic acid [NAA] and 2,4-dichloro-phenoxyacetic acid [2,4-D]), and two cytokinin (kinetin [KT] and 6-benzyladenine [BA]) were tested to explore the influence of plant growth regulators on callus response. H medium (Bourgin and Nitsch 1967) supplemented with 1.0 mg/L NAA and 1.0 mg/L KT induced the highest rate of callus formation. When callus obtained from anthers were subcultured in MS medium containing 1.0 mg/L BA and 0.2 mg/L NAA, followed by transfer to half-strength MS medium supplemented with indole-3-butyric acid (0.2–0.5 mg/L), the formation of regenerated plantlets increased dramatically. Inclusion of gibberellic acid (0.02–0.2 mg/L) in addition to a combination of BA (0.6 mg/L)-NAA (0.2 mg/L) in the culture medium resulted in enhanced frequency of shoot development, as well as greater internode elongation. Ploidy analysis of 580 regenerated plants, using both flow cytometry and chromosome counting, revealed 10.3 % haploid and 1.0 % triploid plantlets. The remaining plantlets were all diploid.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (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 supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

High frequency multiple shoot induction from nodal segments and rhinacanthin production in the medicinal shrub Rhinacanthus nasutus (L.) Kurz

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.     

Rapid in vitro multiplication and ex vitro establishment of Caribbean copper plant (Euphorbia cotinifolia L.): an important medicinal shrub

An efficient micropropagation protocol was developed for E. cotinifolia by utilizing mature nodal segments for axillary shoot proliferation. The nodal explants from a 2-year-old plant were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5 and 10.0 μM) of 6-benzyladenine (BA), Kinetin and 2-isopentenyl adenine singly as well as in combination with α-naphthalene acetic acid (NAA) or Indole-3-butyric acid (IBA) (0.1, 0.5 and 1.0 μM). The highest regeneration frequency (92 %) with multiple shoots (13.0 ± 1.15) and shoot length (4.23 ± 0.14 cm) was achieved on MS medium supplemented with 5.0 μM BA and 0.1 μM NAA after 8 weeks of culture. Further experiments were performed to test the effects of medium type, medium strength, pH and subculture passages on shoot induction and proliferation. An enhancement in average number of shoots (16.6 ± 0.45) per explant was obtained after four subculture passages. Micro shoots exhibited in vitro rooting on half strength MS medium containing 2.5 μM Indole-3-butyric acid (IBA) after 4 weeks of culture. The in vitro raised healthy plantlets with well-developed roots and shoots were successfully acclimatized in plastic cups containing sterile soilrite for 8 weeks under culture room conditions (150 PPFD) prior to field transfer. Through the acclimatization period (0–56 days), photosynthetic pigments (Chlorophyll a, b and Carotenoid content) decreased during the initial 2 weeks followed by significant increase during the successive period (21–56 days) of acclimatization. At the same time, all the tested antioxidant enzymes (SOD, CAT, APX and GR) exhibited an increasing trend throughout the acclimatization period. The culture room acclimatized plantlets were successfully established in earthen pots containing garden soil in greenhouse with 70 % survival rate.     

High-frequency direct shoot regeneration from <Emphasis Type="Italic">Drymaria cordata</Emphasis> Willd. leaves

An efficient and reproducible procedure is described for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine. The regeneration frequency varied with the plant growth regulator concentrations, orientation of the explants, and the carbon source and basal salts present in the regeneration medium. The highest mean number of shoots per explant (10.65 ± 1.03) was recorded on MS plates containing 3% sucrose and 0.8% agar supplemented with 0.1 mg/l NAA and 1.0 mg/l BAP. Shoot buds were induced in the basal parts of the leaf explants. Concentrations of NAA exceeding 1 mg/l suppressed shoot regeneration. Explants bearing the entire lamina and petiole were much more responsive than those having only the lamina. The plantlets that regenerated from the leaf explants were rooted successively on MS medium alone or in combination with indole butyric acid (IBA). The highest mean number of root organogenesis, with 25.67 ± 3.68 roots per leaf segment, was obtained in the presence of 1 mg/l IBA. Histological investigations of the regenerating shoots showed that the shoot buds had emerged from epidermal cells without callus formation. More than 90% of the in vitro-propagated plants survived when transferred to a greenhouse for acclimatization. Thus, this optimized regeneration system may be used for rapid shoot proliferation and genetic transformation.     

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

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

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.