Somatic embryogenesis to overcome low seed viability and conserve wild banana (Ensete superbum (Roxb.) Cheesman)

The seed viability, ex vitro germination, and percentage of in vitro zygotic embryo germination were found to be very low in Ensete superbum (Roxb.) Cheesman. Only 33.33% of seeds were viable, and the ex vitro germination percentage was only 5%, while the percentage of in vitro zygotic embryo germination was 33%. Somatic embryogenesis experiments produced competent callus on Murashige and Skoog (MS) medium supplemented with 2.5 mg L⁻¹ 2,4-D and 3 mg L⁻¹ BAP from inflorescence explants. The embryogenic callus produced the maximum number of somatic embryos on MS basal medium kept in a dark chamber for 15 wk. Half-strength MS medium supplemented with 500 mg L⁻¹ glutamine was optimal for somatic embryo germination and development of plantlets. Regenerated plants had 80 to 90% survival rate. Therefore, somatic embryogenesis can be considered as an efficient method to overcome a drastic reduction in population and to achieve germplasm conservation.

     

An efficient callus-based in vitro regeneration protocol for Warburgia Ugandensis Sprague,an important medicinal plant in Africa

Warburgia ugandensis Sprague is a woody species in the family Canellaceae and an important source of medicines in Africa. Natural propagation of W. ugandensis is problematic due to its recalcitrant seeds and lack of an efficient in vitro regeneration system for this species. This study describes an efficient regeneration protocol. Petiole bases and shoot tips were used as explants. Callus tissue developed when the explants were cultured on Murashige and Skoog medium containing 30 g L⁻¹ sucrose and 7 g L⁻¹ agar (MS30 medium), supplemented with 1.0 mg L⁻¹ indole-3-butyric acid (IBA), 1.6 mg L⁻¹ 6-benzylaminopurine (BA), and 0.1 mg L⁻¹ thidiazuron (TDZ). Adventitious buds were efficiently induced from the callus when the MS30 medium was supplemented with 0.8 mg L⁻¹ BA and 0.2 mg L⁻¹ IBA. Root induction occurred within 7–10 d on half-strength MS30 medium supplemented with 0.8–1.0 mg L⁻¹ 1-napthalene acetic acid (NAA), 0.2 mg L⁻¹ IBA, and 0.03% (w/v) activated charcoal (AC). Roots were followed by root elongation on the same medium but lacking NAA and IBA. Approximately 50% of the plantlets cultured produced roots, while more than 80% of the plantlets survived and successfully grew to maturity.

     

Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl₂ for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L⁻¹ resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L⁻¹), along with IAA (0.5 to 2 mg L⁻¹), or Kn (0.5 to 5 mg L⁻¹), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

     

Use of bioreactors for large-scale multiplication of sugarcane (Saccharum spp.), energy cane (Saccharum spp.), and related species

The objective of this study was to set up a plant micropropagation facility to mass propagate sugarcane, energy cane, and related clonally propagated species. An efficient methodology for micropropagation of energy cane and perennial grasses using temporary immersion bioreactors was developed. Several different methods of tissue culture initiation, multiplication, and rooting were evaluated for several varieties of sugarcane (Saccharum officinarum L.) and sugarcane-related species such as Erianthus spp., Miscanthus spp., and Sorghum spp. × sugarcane hybrids, all from a germplasm collection. Apical meristem cultures were initiated for all genotypes that were micropropagated, when liquid or semisolid Murashige and Skoog (MS) medium was used, which was supplemented with 0.1–0.2 mg L⁻¹ BAP, 0.1 mg L⁻¹ kinetin, 0–0.1 mg L⁻¹ NAA, and 0–0.2 μg L⁻¹ giberellic acid. These cultures produced shoots between 4 and 8 wk after initiation. Shoot regeneration from leaf rolls or immature inflorescences was observed as early as 4 wk after initiation. Shoot multiplication was successful for all genotypes cultured in MS medium with 0.2 mg L⁻¹ BAP and 0.1 mg L⁻¹ kinetin. Energy cane had a significantly higher combined multiplication rate when grown under four or five LED lamps than when grown under three LED lamps, or under fluorescent lights in a growth chamber. The addition of 2 mg L⁻¹ NAA produced faster and better rooting in all of the genotypes tested. Shoots produced well-developed roots after one cycle of 15–21 d in the bioreactors. The maximum number of plantlets produced per bioreactor was 1080. Plantlets developed a vigorous root system and were ready to be transplanted into the field after 2 mo. A protocol was standardized for different energy cane clones that were recommended for their biomass production and cell wall composition. Different tissues were used to speed up or facilitate tissue culture initiation. Visual assessment of micropropagated plants in the field did not show any off-types, based on gross morphological changes of plant morphology or disease reaction, compared to plants of the same genotype derived from a traditional propagation method (stem cuttings). This is the first report of energy cane and Miscanthus spp. micropropagation using the SETIS bioreactor.

     

Organogenesis and embryogenesis inHelianthus tuberosus and in the interspecific hybridHelianthus annuus ×Helianthus tuberosus

A method of plant regeneration from cotyledons ofHelianthus tuberosus, Helianthus annuus ×Helianthus tuberosus and for the backcross of the interspecific hybrids onH. annuus was developed. Induction of somatic embryogenesis and plantlet regeneration from anther culture of the interspecific hybridsH. annuus ×H. tuberosus is reported.Cotyledons were cultured on Murashige and Skoog basal medium (MS) supplemented with indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin) or N⁶-benzylaminopurine (BAP). Shoot regeneration occurred on most of the media tested, but the best results were obtained on media with a high concentration of cytokinins (BAP or kinetin: 4 mg l^–1) and lower concentration of auxin (IAA: 0.5–1 mg l^–1).Embryogenic callus and adventitious buds were initiated from only two anthers of the hybridH. annuus ×H. tuberosus cultured on the MS medium containing BAP (0.2 mg l^–1) and 1-naphtalenacetic acid (NAA: 0.1 mg l^–1). Prolonged culture of these embryogenic calli and buds on the original medium with successive subculture on MS basal medium without growth regulators resulted in embryo formation and shoot differentiation. The plantlets, after rooting, were established in soil.     

Plant regeneration and production of embelin from organogenic and embryogenic callus cultures of <Emphasis Type="Italic">Embelia ribes</Emphasis> Burm. f.—a vulnerable medicinal plant

Embelia ribes, an important vulnerable medicinal liana, was regenerated through organogenesis and embryogenesis using leaf explants. Leaf explants produced organogenic calluses on MS medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzylaminopurine. Shoot regeneration was obtained from organogenic calluses on MS medium containing different concentrations of thidiazuron (TDZ) and indole-3-acetic acid (IAA). The frequency of shoot bud organogenesis was highest (23.9 shoots/explant) in MS medium containing 0.5 mg l⁻¹ TDZ and 0.1 mg l⁻¹ IAA. The best result for induction of embryogenic callus was noticed in the combination of 2.0 mg l⁻¹ TDZ and 0.5 mg l⁻¹ 2,4-D. This callus, maintained in the same medium, showed the highest differentiation of embryos (56.5%) after 6 wk of culture. Embryos were transferred to MS medium supplemented with different concentrations of TDZ, and this facilitated conversion of embryos into plants. After 6 wk of subculture, MS medium with 0.05 mg l⁻¹ TDZ favored the highest percentage (52.2%) embryo conversion. As per the present protocol, 52.2% of the embryos underwent conversion, and a mean number of 29.5 shoots per culture was obtained. Shoots developed from both types of calluses were rooted on half-strength MS basal medium supplemented with 1.0 mg l⁻¹ indole-3-butyric acid. HPLC-UV assay demonstrated the highest embelin content (5.33% w/w) in the embryogenic callus cultures. Embelin was isolated from embryogenic callus and was identified using IR and ¹H NMR studies.     

Highly efficient regeneration and medicinal component determination of Phellodendron chinense Schneid

Phellodendron chinense Schneid is an important Chinese herb with berberine and phellodendrine in stems and leaves, but with little information available on in vitro culture of this species. Disinfection of explants in 75% alcohol for 45 s, sterilization in 0.1% HgCl₂ for 20 min, and submersion in 1.0 mol L⁻¹ gibberellin3 (GA₃) solution for 24 h was the optimal condition for seed germination. Murashige and Skoog’s (MS) medium supplemented with 2.0 mg L⁻¹ 6-benzylaminopurine (6-BA) in combination with 1.5 mg L⁻¹ 1-naphthylacetic acid (NAA) was optimal for callus induction. MS medium supplemented with 2.0 mg L⁻¹ 6-BA was the appropriate medium for induction of adventitious shoots, and 1/2MS medium supplemented with 2.0 mg L⁻¹ indole-3-butytric acid (IBA) and 0.5% active carbon was the optimal medium for root induction. The 15-d survival rate of regenerated plantlets after transplanting to basins containing perlite and peat moss (1:4) was greater than 80%, and the berberine and phellodendrine accumulation was lower in callus compared with regenerated plantlets. The establishment of highly efficient regeneration system provides technical support for genetic breeding of Phellodendron chinense Schneid.

     

A rapid system for micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch-Ham. ex Wall.: an endangered medicinal herb via direct somatic embryogenesis

An improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness to induce somatic embryos. Leaf explants with abaxial side in the medium produced maximum number of somatic embryos. This system omits the callus stage and thus reduces the process of SE in S. chirata by 35–45 days. Embryos at different stages of development were observed. Maturation of heart stage embryos were observed on Murashige and Skoog (MS) medium containing 1 mg L⁻¹ 2,4-D. Upon transfer to the germination medium, they were converted to cotyledonary stage and then plantlets of 33% and 68% of them were converted to cotyledonary stage and then plantlets on MS medium supplemented with 0.05 and 0.1 mg L^-1 GA₃ respectively. The 2,4-D alone at 1.0 or 1.5 mg L⁻¹ was found to be better for embryogenic tissue initiation than 2,4-D in combination with indole-3-acetic acid or α-naphthalene acetic acid. For further embryo development, 2,4-D was combined with cytokinins such as 6-benzylaminopurine (BAP) and kinetin or plant growth regulator free medium or medium with 50% reduced concentration of the same hormone while subculturing. Mean germination and percentage of survival were maximum in the medium containing 1.0 mg L⁻¹ 2,4-D in combination with 0.1 mg L⁻¹ BAP. Regenerated plantlets were morphologically and genetically identical. This method offers a vast scope for the clonal propagation of endangered plants.     

Establishment and in vitro morphogenesis of sapucaia explants (Lecythidaceae)

Lecythis pisonis Cambess, popularly known as sapucaia, has great economic and socio-environmental potential. The objective of this study was to evaluate the establishment and in vitro morphogenesis of L. pisonis under the effect of disinfecting agents, plant growth regulators, and thermal stress. The study was divided into three experiments: (i) development of the disinfection protocol by testing different concentrations and times of exposure to sodium hypochlorite (NaOCl) and different concentrations and methods of amoxicillin application, (ii) in vitro budding induction by testing different concentrations of 6-benzylaminopurine (BAP) or kinetin (KIN) supplemented to Woody Plant Medium (WPM) and Murashige and Skoog (MS) culture media, and (iii) in vitro formation from plantlets by analyzing different concentrations of indole-3-butyric acid (IBA) with different exposure times to a thermal stress of 40°C. The disinfection of stem segments was effective using 3% NaOCl and 3.0 g L⁻¹ amoxicillin solution. MS culture medium supplemented with 0.25 mg L⁻¹ BAP induced more shoots in vitro. One milligram per liter IBA promoted greater rooting in vitro, and it is not necessary for thermal stress tolerance.

     

Pulvinus: an ideal explant for plant regeneration in <Emphasis Type="Italic">Caesalpinia bonduc</Emphasis> (L.) Roxb., an important ethnomedicinal woody climber

This study demonstrates the morphogenic potential of pulvinus, an important organ situated at the base of the petiole or rachis of leguminous plants. Plant regeneration via pulvinus-derived calli of Caesalpinia bonduc has been achieved. Organogenic calli have been derived from the explant 45 days after culture on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzylaminopurine (BA). Optimum callus induction (100%) occurred when the pulvini were cultured on MS medium fortified with 6 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BA. The highest shoot induction was obtained when the calli were transferred to MS medium supplemented with 5 mg l⁻¹ BA and 1 mg l⁻¹ indole-3-acetic acid (IAA). On this medium, 87% cultures responded with an average number of 4.2 shoots per culture. The maximum root induction from the regenerated shoots was observed on half strength MS medium containing 6 mg l⁻¹ indole-3-butyric acid (IBA). Here 100% shoots rooted with a mean number of 6.3 roots per shoot. The regenerated plantlets were acclimatized and subsequently showed normal growth. This efficient protocol will be helpful for propagating elite clones on a mass scale and could be utilized for genetic transformation study.     

Effects of cytokinin on callus proliferation associated with physiological and biochemical changes in <Emphasis Type="Italic">Vitis vinifera</Emphasis> L.

The essential role of 6-benzylaminopurine (BA) in plant tissue culture has been widely known; however, physiological and biochemical mechanisms behind BA requirement have not been fully understood yet. BA may have an important role on callus growth by regulating antioxidant enzyme activities and acting as an effective free radical scavenger. To test this hypothesis, the impact of exogenous BA concentrations on antioxidative system in Vitis vinifera L. cv. ‘Bogazkere’ callus was investigated under in vitro conditions. Relative fresh weight growth (RFWG) of calli, total phenolics (TP) content, endogenous hydrogen peroxide (H₂O₂), malondialdehyde (MDA), proline concentrations, percentage of electrolyte leakage (EL), and some of the antioxidant enzyme activities; such as superoxide dismutase (SOD), and guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were measured. Inhibitory effect of high concentrations of BA on antioxidant enzyme activities and RFWG was found. In the presence of BA at 0.1 mg L⁻¹, SOD, POD, and APX activities decreased, while CAT activity increased in comparison with the controls. Exogenous BA treatments higher than 0.1 mg L⁻¹ resulted in an increase in cellular TP, H₂O₂, MDA, proline contents, and percentage of EL, while RFWG of calli decreased. Based on the findings, it may be concluded that only 0.1 mg L⁻¹ BA concentration combined with NAA could play a direct role in reducing the level of free radicals and phenolic production associated with proliferation capacity of grape cells under in vitro conditions. Furthermore, cytokinin was effective in the antioxidative enzyme system, lipid peroxidation, and electrolyte leakage.     

In vitro organogenesis and plant regeneration of Thymus serpyllum L.: an important aromatic medicinal plant

An efficient in vitro propagation system has been developed for the rapid micropropagation of Thymus serpyllum L. (Banajwain), an aromatic medicinal herb from nodal explant on MS medium. Phenolic leaching and high rate of contamination was the most significant problem in establishing in vitro culture of Thymus serpyllum which was overcome by preparing explants in an antioxidant ascorbic acid (1000 ppm) at 6°C for 45 min and addition of the same antioxidant (50 mgl⁻¹) to the MS medium. The frequency of shoot production was influenced by different cytokinins (Kn, BAP, and Kn + BAP) and 95.56% shoot induction was observed when MS medium was supplemented with 1.0 + 2.0 mgl⁻¹ (Kn + BAP). The maximum average number of shoots 16.93 ± 2.15 and average length (3.98 ± 0.55) was recorded when MS medium have 0.5 + 2.0 mgl⁻¹ (Kn + BAP). The in vitro regenerated microshoots were rooted on MS and half strength MS medium and there was significant difference in root induction on both media under the influence of auxins (IAA, IBA, and NAA). The maximum average number (11.67 ± 3.03) and average root length (3.88 ± 0.71) was reported in half MS medium having 1.0 mgl⁻¹ IBA. The complete regenerated plantlets were acclimatized under growth chamber before transferring to the earthen pots and showed 90% survival.

Graphical abstract

     

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.     

A simple cryopreservation protocol of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. embryogenic calli by encapsulation-vitrification

Embryogenic calli of Dioscorea bulbifera L. were successfully cryopreserved using an encapsulation-vitrification method. Embryogenic calli were cooled at 6°C for 5 days on solid MS medium (Murashige and Skoog 1962) containing 2 mg L⁻¹ Kinetin (Kn), 0.5 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.5 mg L⁻¹ 2,4-dichlorophenoxy-acetic acid (2,4-D). These were prior precultured on liquid basal MS medium enriched with 0.75 M sucrose at 25 ± 1°C for 7 days. Embryogenic calli were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dropped in a 0.1 M CaCl₂ solution containing 0.4 M sucrose at 25 ± 1°C. After 15 min of polymerization, Ca-alginate beads (about 4 mm in diameter) were dehydrated for 150 min at 0°C in a PVS₂ solution [30% glycerol, 15% ethylene glycol, and 15% dimethyl sulfoxide (w/v)] containing 0.5 M sucrose. The encapsulated embryogenic calli were then plunged directly into LN (liquid nitrogen) for 1 h. After rapid thawing in a water bath (37°C; 2 min), the beads were washed 3 times at 10-min intervals in liquid basal MS medium containing 1.2 M sucrose. Following thawing, the embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, 0.09 M sucrose and 0.75% (w/v) agar (embryoid induction medium) and cultured under light conditions of 12-h photoperiod with a light intensity of 36 μmol m⁻² s⁻¹ provided by white cool fluorescent tubes after a 2-day dark period at 25 ± 1°C. After 30 days, the embryoids developed from embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L⁻¹, NAA 0.5 mg L⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium). After 60 days, the embryogenic calli developed normal shoots and roots. No morphological abnormalities were observed after plating on the regeneration medium. The survival rate of encapsulated vitrified embryogenic callus reached over 70%. This encapsulation-vitrification method appears promising as a routine and simple method for the cryopreservation of Dioscorea bulbifera embryogenic callus.     

Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67∼6.67 mg L⁻¹ h⁻¹), higher nitrite accumulation (47.3∼99.8 mg L⁻¹), and higher N₂O emission ratios (5∼283 mg L⁻¹/mg L⁻¹). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0∼0.05 mg L⁻¹) was found, and the absence of Cu resulted in massive N₂O emission due to poor synthesis of N₂O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5∼2.5 mg L⁻¹) > Cu(II) (0.5∼5 mg L⁻¹) > Ni(II) (2∼10 mg L⁻¹) > Zn(II) (25∼50 mg L⁻¹). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ > nirS ≈ cnorB > napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

     

Genetic homogeneity and high shoot proliferation in banana (Musa acuminata Colla) by altering medium thiamine level and sugar type

To enhance the multiplication rate in Musa acuminata Colla (banana; ‘Grand Nain’) organogenesis, higher amounts of thiamine along with different sugar types and concentrations were evaluated at the proliferation phase. Thiamine at 1, 10, 50, 100, and 200 mg L⁻¹ was compared with 0.1 mg L⁻¹ thiamine found in conventional Murashige and Skoog (MS) medium. Maximum proliferation of banana was induced with 100 mg L⁻¹ thiamine. Additionally, 15, 30, and 45 g L⁻¹ sucrose, glucose, fructose, and sorbitol combined with regular and optimal levels of thiamine were tested. Glucose at 30 g L⁻¹ most improved shoot proliferation alone and enhanced shoot proliferation further, when combined with 100 mg L⁻¹ thiamine, followed by sucrose and fructose, whereas sorbitol completely inhibited growth and caused tissue browning. All evaluated vegetative traits were significantly affected by sugar type and concentration, and thiamine levels, unlike the photosynthetic pigments. Moreover, genetic stability of the plants recovered from the enhanced protocol was confirmed by inter-simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. A total of 230 bands generated by both marker types were monomorphic for the randomly selected regenerated plants, compared with their mother plant. Thus, the proliferation medium supplemented with 30 g L⁻¹ glucose and 100 mg L⁻¹ thiamine could be recommended for banana organogenesis. Results herein are of great importance and helpful in enhancing the commercial in vitro propagation protocols of banana, without the need of increasing the number of subcultures, which can cause somaclonal variation.

     

Callus induction and plant regeneration in <Emphasis Type="Italic">Dorem ammoniacum</Emphasis> D., an endangered medicinal plant

Dorema ammoniacum D. Don. (Apiaceae), a native medicinal plant in Iran, is classified as a vulnerable species. Root, hypocotyl, and cotyledon segments were cultured on Murashige and Skoog (MS) (1962) medium supplemented with either 2,4-dichlorophenyoxyacetic acid (2,4-D) or naphathalene acetic acid (NAA), at 0–2 mg l⁻¹, alone or in combination with either benzyladenine (BA) or kinetin (KN), at 0–2 mg l⁻¹ for callus induction. The best response (100%) was observed from root segments on MS medium containing 1 mg l⁻¹ NAA and 2 mg l⁻¹ BA. The calli derived from various explants were subcultured on MS medium supplemented with BA (1–4 mg l⁻¹) alone or in combination with NAA or indole-3-butyric acid (IBA), at 0.2 or 0.5 mg l⁻¹ for shoot induction. Calli derived from hypocotyl segments showed significantly higher frequency of plantlet regeneration and number of plantlets than the calli derived from root and cotyledon segments. Therefore, MS medium supplemented with 2 mg l⁻¹ BA and 0.2 mg l⁻¹ IBA produced the highest frequency of shoot regeneration (87.3%) in hypocotyl-derived callus. The optimal medium for rooting contained 2.5 mg l⁻¹ IBA on which 87.03% of the regenerated shoots developed roots with an average number of 5.2 roots per shoots within 30 days. These plantlets were hardened and transferred to the soil. The described method can be successfully employed for the large-scale multiplication and conservation of germplasm this plant.     

Regeneration via organogenesis in callus cultures of Argyrolobium roseum

A reproducible protocol has been developed for high frequency plant regeneration from immature embryos of Argyrolobium roseum Jaub & Spach, an important medicinal legume. Green nodular calli were initiated from immature embryos excised from 10-d-old pods in 70 % of cultures within 3 weeks when grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm⁻³ benzylaminopurine (BAP) + 0.25 mg dm⁻³ indole-3-acetic acid (IAA). Subsequent transfer of 5 mm² callus pieces to MS medium supplemented with BAP (0.5 mg dm⁻³) alone or in combination with IAA (0.25 mg dm⁻³) facilitated regeneration of multiple shoots. Organogenic calli bearing multiple shoots when transferred to MS medium supplemented with BAP (0.5 mg dm⁻³) + IAA (0.25 mg dm⁻³) supported rapid shoot elongation. Shoot propagules subcultured to Gamborg's medium (B5) with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) rooted with 80 % frequency and developed into phenotypically normal plants. Plantlets were successfully acclimatized in a sterile mixture of sand and garden soil (1:1) under greenhouse and thereafter transferred to field beds.     

An <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation system using callus of <Emphasis Type="Italic">Zoysia tenuifolia</Emphasis> Willd. ex Trin

Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l⁻¹ 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l⁻¹ BA under darkness. For genetic transformation, calli were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, about 12% of the callus explants produced hygromycin resistant calli on MS medium supplemented with 2 mg l⁻¹ 2,4-D, 1 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, 500 mg l⁻¹ cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 0.2 mg l⁻¹ BA, 50 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted on 1/2 MS media supplemented with 50 mg l⁻¹ hygromycin, 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. About 5% of the total inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function.