Fast versatile regeneration of <Emphasis Type="Italic">Trifolium alexandrinum</Emphasis> L.

Trifolium alexandrinum L. (Egyptian clover) is one of the most important forage crops in the world. Its regeneration in tissue culture has been described in a few reports but the efficiency, accurate time scales and applicability to various genotypes of the described procedures are uncertain. Therefore their suitability for genetic transformation is unclear. In this study, were report new fast procedures for regeneration of Egyptian clover that are applicable to the regeneration of various genotypes (Mescawi-ahaly, Sakha3 and Sakha4). Shoots were regenerated from intact and wounded cotyledons as well as hypocotyls of Mescawi-ahaly on naphthaleneacetic acid/benzyladenine (NAA/BA) and naphthaleneacetic acid/thidiazuron (NAA/TDZ) media. The highest shoot regeneration frequencies were obtained from intact cotyledons on NAA/BA (0.05 mg l⁻¹ NAA combined with 2.0 mg l⁻¹ BA) and NAA/TDZ (0.05 mg l⁻¹ NAA combined with 1.0 mg l⁻¹ TDZ) media (66.2 and 43.1% respectively) compared to 18.4 and 10.1% for wounded cotyledons on NAA/BA and NAA/TDZ respectively. 21.0% shoot regeneration frequency was observed for hypocotyls on NAA/BA (2.0 mg l⁻¹ NAA combined with 0.5 mg l⁻¹ BA) medium but no regeneration was obtained on NAA/TDZ medium. Rooting of the regenerated shoots was induced on indole butyric acid (IBA: 0.24 mg l⁻¹) or NAA (2.0 mg l⁻¹) media where IBA medium supported significantly higher frequencies of rooting as well as survival of the whole plantlets after transfer to soil. However, the rooting and survival frequencies also depended on the type of explant and the medium used for shoot regeneration. The two cultivars Sakha3 and Sakha4 were regenerated using the culture conditions optimized for Mescawi-ahaly with comparable efficiencies, indicating that the described procedure is not genotype dependent. The time scale of whole plantlet regeneration ranged from 7.5 weeks for intact and wounded cotyledons to 10 weeks for hypocotyl explants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> mediated genetic transformation of summer squash (<Emphasis Type="Italic">Cucurbita pepo</Emphasis> L. cv. Australian green) with <Emphasis Type="Italic">cbf-1</Emphasis> using a two vector system

Efficient plant regeneration via shoot tip provided a basis for the optimization of the genetic transformation protocol. Therefore, experiments were conducted to establish an efficient in vitro regeneration protocol in summer squash for genetic co-transformation. 6-benzylaminopurine at 0.05 mg l^−l was found to be optimum concentration of direct regeneration from shoot tip. Effective root system was induced in shootlets in indole-3-aceticacid 0.5 mg l^−l. Two vectors namely pCAMBIA 2200 harboring marker gene nptII and pCAMBIA 0390 harboring gene, encoding C-repeat binding factor (cbf1) were used for co-transformation taking shoot tips as explants from in vitro germinated seeds. Explants were selected after co-cultivation on kanamycin supplemented medium and shoots and roots were induced. The transgenic plants were confirmed by polymerase chain reaction (PCR) and further southern blot analysis confirmed the integration of nptII and cbf1 genes in genome of summer squash with co-transformation efficiency of 0.7 percent.     

Adventitious regeneration in blackberry (<Emphasis Type="Italic">Rubus fruticosus</Emphasis> L.) and assessment of genetic stability in regenerants

Adventitious shoot regeneration from leaves of blackberry cultivar Čačanska Bestrna was examined using 30 different combinations of treatment. Young, fully expanded leaves taken from in vitro proliferating shoots were cultured on Murashige and Skoog (MS) medium containing either N ⁶-benzylaminopurine (BAP) (2.0 mg l⁻¹) or thidiazuron (TDZ) (1.0 and 2.0 mg l⁻¹) alone, or either of them combined with indol-3-butyric acid (IBA), α-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) at different concentrations (0.1, 1.0 and 2.0 mg l⁻¹). Indirect shoot formation was observed in 12 different treatments, though the efficacy varied greatly among types and concentrations of plant growth regulators. TDZ at 1.0 mg l⁻¹, applied either alone or combined with IBA, was significantly more effective than BAP in inducing shoot regeneration. The highest regeneration rate (41.66%) was obtained on medium containing TDZ alone. Cytological, flow cytometry and isozyme analyses were used for screening of genetic stability/instability in regenerants. Cytological study, based on chromosome counts in root tip meristems, and flow cytometry analysis indicated that adventitious shoots of Čačanska Bestrna are tetraploid with 2n = 4x = 28 as well as those derived from axillary buds. However, a study conducted on the peroxidase patterns of the different blackberry regenerating lines showed differences between some lines and control plants (in vivo plants and micropropagated plants). These differences were visible with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as hydrogenous donor for peroxidase detection.     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

Genetic transformation and overexpression of a rice <Emphasis Type="Italic">Hd3a</Emphasis> induces early flowering in <Emphasis Type="Italic">Saussurea involucrata</Emphasis> Kar. et Kir. ex Maxim

Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol for S. involucrata using Agrobacterium tumefaciens. Leaf explants 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 as a reporter gene. Following co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l⁻¹ benzyladenine (BA), 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l⁻¹ hygromycin, and 500 mg l⁻¹ cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA, 0.25 mg l⁻¹ gibberellic acid (GA3), 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots rooted on MS media supplemented with 0.2 mg l⁻¹ NAA, 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata.     

Plant regeneration of the mining ecotype <Emphasis Type="Italic">Sedum alfredii</Emphasis> and cadmium hyperaccumulation in regenerated plants

An efficient micropropagation system for mining ecotype Sedum alfredii Hance, a newly identified Zn/Cd hyperaccumulator, was developed. Frequency of callus induction reached up to 70% from leaves incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg l⁻¹ 6-benzyladenine (BA), and 83% from internodal stem segments grown on MS medium with 0.1 mg l⁻¹ 2,4-D and 0.1 mg l⁻¹ BA. Callus proliferated rapidly on MS medium containing 0.2 mg l⁻¹ 2,4-D and 0.05 mg l⁻¹ thidiazuron. The highest number of adventitious buds per callus (17.3) and frequency of shoot regeneration (93%) were obtained when calli were grown on MS medium supplemented with 2.0 mg l⁻¹ BA and 0.3 mg l⁻¹ α-naphthalene acetic acid (NAA). Elongation of shoots was achieved when these were incubated on MS medium containing 3.0 mg l⁻¹ gibberellic acid. Induction of roots was highest (21.4 roots per shoot) when shoots were transferred to MS medium containing 2.0 mg l⁻¹ indole 3-butyric acid rather than either indole 3-acetic acid or NAA. When these in vitro plants were acclimatized and transferred to the greenhouse, and grown in hydroponic solutions containing 200 μM cadmium (Cd), they exhibited high efficiency of Cd transport, from roots to shoots, and hyperaccumulation of Cd.     

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.     

Adventitious bud regeneration from leaf explants of <Emphasis Type="Italic">Platanus occidentalis</Emphasis> L. and genetic stability assessment

The aim of this work is to develop a method of plant regeneration from leaf explants of Platanus occidentalis L. successfully. Woody plant medium (HortScience 16:453–459, 1981) and Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium were used as induced and rooted basal medium, respectively. The effects of combinations of 6-BA, IBA, NAA and KT with different concentrations on adventitious bud regeneration from P. occidentalis leaf explants were compared. The results showed that the highest shoot regeneration frequency (90%) and maximum number (13.72 ± 0.44) of shoots per explant was recorded on WPM medium supplemented with 22.20 mmol l⁻¹ 6-BA and 0.49 mmol l⁻¹ IBA. A 40-day-old explants were much more productive for shoot formation than others in this study. The regenerated shoots were cultured on MS medium supplemented with 1.33 mmol l⁻¹ 6-BA, 0.16 mmol l⁻¹ NAA and 2% (w/v) adenine, after 2-week shoots were transferred to 1/2 MS medium supplemented with 0.49 mmol l⁻¹ IBA for rooting. Hardened plantlets via acclimatization were transferred to pots and transplanted to the soil finally. To ascertain whether tissue culture had effects on the genetic stability of plantlets regenerated, the genetic diversity was assessed using RAPD marker. A total of 96 bands ranging from 0.5 to 2.2 kb with an average of 6.4 bands per primer, were obtained using 15 primers. Amplified products exhibited few of polymorphic patterns across all the plants of P. occidentalis and the overall frequency of detection of somaclonal polymorphisms was lower than 0.0104%. Yuehua Sun, Yanling Zhao, and Xiaojuan Wang contributed equally to this work.     

In vitro propagation of four threatened <Emphasis Type="Italic">Paphiopedilum</Emphasis> species (Orchidaceae)

The effects of seed maturity, media type, carbon source, and organic nutrient additives on seed germination, protocorm development, and plant growth of Paphiopedilum villosum var. densissimum Z. J. Liu et S. C. Chen were investigated. Micropropagation frequency was enhanced through the use of 200-day-old seed, Knudson C (KC) medium, and the presence of both glucose and coconut milk in the medium. The effects of various plant growth regulators on the frequency of shoot organogenesis in four Paphiopedilum species were also investigated. Explants of P. villosum var. densissimum and P. insigne (Lindl.) Stein incubated in the presence of 5 mg l⁻¹ 6-benzyladenine (BA) with 0.5 mg l⁻¹ α-naphthalene acetic acid (NAA) and 0.2 mg l⁻¹ BA with 0.1 mg l⁻¹ NAA, respectively, showed a twofold increase in the frequency of shoot organogenesis. For explants of P. bellatulum (Rchb. f.) Stein and P. armeniacum S. C. Chen et F. Y. Liu, the combination of 5.5 mg l⁻¹ BA with 0.5 mg l⁻¹ NAA and 4 mg l⁻¹ BA with 0.1 mg l⁻¹ NAA, respectively, resulted in the highest frequencies of shoot organogenesis.     

Cryoconservation and regeneration of axillary shoot meristems of <Emphasis Type="Italic">Indigofera tinctoria</Emphasis> (L.) by encapsulation–dehydration technique

In vitro axillary shoot proliferation was achieved from single-node explants of Indigofera tinctoria on a well-defined medium, Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ N ⁶-benzyl adenine (BA) and 0.1 mg l⁻¹ indole-3-acetic acid. Axillary shoot meristems from cultures derived from up to three subcultures were used in the encapsulation–dehydration technique. Preconditioned, calcium alginate-encapsulated, and precultured axillary shoot meristems were subjected to different lengths of desiccation in a laminar flow cabinet. Maximum survival and regeneration rates of 56.7% and 62.2%, respectively, were obtained in half-strength (half the macro- and micronutrients and full-strength vitamins) MS medium supplemented with 0.5 mg l⁻¹ gibberellic acid and 0.2 mg l⁻¹ BA after 4 h of desiccation, during which the moisture content was reduced to 16.0%. According to the analysis of six random amplified polymorphic DNA markers, plantlets derived from cultures initiated with cryopreserved plant material were genetically identical to those derived from nonfrozen (control) tissues.     

Plant regeneration via somatic embryogenesis of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis> in temporary immersion system (TIS)

The present study describes a protocol for plant regeneration via somatic embryogenesis in temporary immersion system (TIS) for Camptotheca acuminata. Somatic embryos were induced by culturing hypocotyl segments from 14-day-old in vitro grown C. acuminata seedlings in TIS. Hypocotyl segments were placed in culture vessels modified with a mechanical device to support the fixation of explants. Cultures were maintained under a 16 h photoperiod with a light intensity of 60 μmol m⁻² s⁻¹ PPF at 25 ± 1°C. After 16 weeks of incubation embryogenic calli were formed above the edge of the mechanical device in the basal Murashige and Skoog (MS) medium containing 35 g l⁻¹ sucrose and without hormonal supplementation. For plantlet regeneration, somatic embryos at cotyledonary stage were cultured in three different concentrations of 6-benzylamino-purine (0.5, 1.0 and 1.5 mg l⁻¹ BAP) and in plant growth regulator (PGR) free medium. In general, 0.5 mg l⁻¹ BAP was found to be the most effective concentration for growth and development of Camptotheca embryos in TIS. Conversion of somatic embryos into plantlets was also successfully achieved on sterile substrates moistened with 0.5 mg l⁻¹ BAP. Plantlets derived from cotyledonary embryos were rooted in vitro with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) before transfer to ex vitro conditions.     

Cryopreservation of embryogenic cell suspensions of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. (G) Don.

The present study describes the potential of in vitro grown adventitious roots of Hypericum perforatum L. commonly known as St. John’s wort at low nutrient and auxin levels in the liquid medium for micropropagation. Roots were regenerated from shoot-derived callus on MS medium containing 4.0 mg l⁻¹ Indole-3 acetic acid (IAA). IAA and Indole-3 butyric acid (IBA) were equally effective for the induction of roots from shoot cultures. Half strength MS medium containing 1.0 mg l⁻¹ IAA was most found suitable for culturing roots in liquid medium. A total biomass of 4.13 ± 0.67 g comprising 226 ± 34.4 shoots and shoot buds along with roots was obtained per culture starting with 200 mg roots inoculum. Pretreatment with kinetin (2.0 mg l⁻¹) enhanced the shoot multiplication. Shoots proliferated profusely from excised roots in static liquid medium supported with glass bead matrix. Growtek^™ vessel was found suitable and cost effective system for high throughput plantlet production. In vitro grown roots regardless of their source of origin were an excellent and easy to handle source of explant for aseptic production of plantlets without loosing the morphogenetic potential over the generations. The plants exhibited 84–99% similarity among themselves through RAPD. The in vitro shoots produced can either be multiplied or rooted perpetually, and alternatively they can also be explored for the in vitro production of hypericin and hyperforin.     

An elite protocol for accelerated quality-cloning in <Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus cv. Sciella

A novel, efficient, and simple protocol was developed on in vitro mass propagation and acclimatization of Gerbera jamesonii Bolus cv. Sciella, an ornamental plant with attractive flowers. Shoot tip was used as the primary explant for in vitro establishment in which Murashige and Skoog (MS) medium supplemented with a low level of NAA (0.5 mg l⁻¹) and BAP (1.5 mg l⁻¹) promoted earliest axillary bud initiation within 5 d in 91.6% of the inoculants. Five axillary buds were initiated from a single explant within 13 d after inoculation. A very high rate of shoot multiplication (14 shoots per inoculated axillary bud) and proliferation was achieved when MS medium was fortified with a relatively higher level of BAP (2 mg l⁻¹) and 60 mg l⁻¹ ADS within 27 d of multiple shoot culture. A maximum number of well-developed roots per plant was observed in MS medium with 0.5 mg l⁻¹ IAA in the next 26 d. In the easy low-cost acclimatization process of 20 d, a combination of sand, soil, cow urine, and tea leaves extract (1:1:1:1; v/v) ensured 95% survival rate. Sixty-one well-acclimatized plants were obtained from a single shoot tip within 86 d. The sustained multiple shoot culture for 15 mo paved the way toward the conservation of genetic resources as well as beneficial economics. The clonal fidelity study of micropropagated and sustained cultured clones using ISSR primers ensured the continuous supply of quality propagules retaining genetic uniformity. The in vitro-generated plants performed better over conventionally propagated plants in the field condition.     

Cytokinin-induced organogenesis in Lessertia (Sutherlandia) frutescens L. using hypocotyl and cotyledon explants affects yields of l-canavanine in shoots

A simple protocol for direct shoot organogenesis and plant regeneration in Lessertia frutescens using hypocotyl and cotyledon segments is reported. l-canavanine content in the derived shoots is also quantified. Media containing different concentrations and combinations of the cytokinins kinetin (K) and benzyladenine (BA) were tested for shoot induction potential. The best shoot regeneration rate (83%) was obtained from hypocotyl segments cultured in Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ K; these hypocotyls also produced the largest number of shoots per explant (3.5) and the longest shoots per explant (13.3 mm). The best shoot regeneration rate (46%) using cotyledons as explant material was obtained in MS medium supplemented with 1 mg l⁻¹ K and 1 mg l⁻¹ BA or with 5 mg l⁻¹ K and 0.5 mg l⁻¹ BA. The highest number of cotyledon-derived shoots (1.5) was obtained in MS medium containing 2 mg l⁻¹ K and 0.5 mg l⁻¹ BA, and the longest cotyledon-derived shoots (6.1 mm) were obtained in MS medium containing 1 mg l⁻¹ K and 0.5 mg l⁻¹ BA. Shoots derived from hypocotyls cultured on media containing 1 mg l⁻¹ K contained the highest quantity of l-canavanine (1.42 mg g⁻¹) relative to the control (0.52 mg g⁻¹). Shoots derived from cotyledons cultured on media containing 2 mg l⁻¹ K contained the highest quantity of l-canavanine (2.07 mg g⁻¹) compared to the control. Scanning electron microscopy revealed that shoots regenerated directly from the wounded epidermal tissue, although callus formation was observed in most cultures. Young shoot clusters proliferated into healthy adventitious shoots that were subsequently transferred directly onto rooting medium (MS medium containing 4 mg l⁻¹ indole-3-butyric acid), eliminating the need for an additional multiplication or elongation phase. The in vitro plants were successfully acclimatized in a growth chamber, achieving an 85% survival rate.     

The effect of co-cultivation and selection parameters on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l⁻¹) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l⁻¹ hygromycin for 10 days, 5 mg l⁻¹ hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l⁻¹ hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.     

Callus induction and shoot regeneration of <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L. cv. Wonder Bush and cv. Pole Sieva

We report the first protocol for callus induction and shoot regeneration for Phaseolus lunatus L. cv. Wonder Bush and cv. Pole Sieva. We used different explants viz., epicotyls, cotyledons and hypocotyls. The medium used was MS basal medium with thidiazuron (0.5 mg l⁻¹) and IAA (0.05 mg l⁻¹) for the induction of callus followed by BAP (1.0 mg l⁻¹) for the induction of shoots. Epicotyl explants showed the fastest response and the highest percentage of shoot regeneration. This protocol opens new biotechnological strategies to transfer economically important genes to this important crop species.     

In vitro propagation of <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> Hook using thidiazuron

A reliable in vitro propagation system was developed for the cut flower crop sandersonia (Sandersonia aurantiaca Hook). Explants comprised immature embryos from developing seeds and meristems from sprouting tubers. MS media supplemented with a range of thidiazuron (TDZ) concentrations lead to the formation of multiple shoot clusters. To overcome the deleterious effect of long-term exposure to TDZ concentrations, a multistage protocol of 4-weekly cycles was used. This comprised exposure to a high TDZ (3.0 mg l⁻¹) medium for a single cycle to induce proliferation, followed by one or two cycles of low TDZ (0.3 mg l⁻¹) media to allow for the development of shoot structures, and then a rest phase on growth-regulator free medium for one cycle prior to the return to a medium with high TDZ. Shoot clusters on growth-regulator free media formed minitubers (10–12 weeks), which sprouted, either in vitro, or ex vitro, with the resulting plants successfully acclimatised in the greenhouse.     

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.     

Fluoranthene influences endogenous abscisic acid level and primary photosynthetic processes in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) plants in vitro

The influence of increasing concentrations (0.1, 1.0 and 5.0 mg l⁻¹) of fluoranthene (FLT) on growth, endogenous abscisic acid (ABA) level and primary photosynthetic processes in 21-day-old pea plants (Pisum sativum L.) in vitro was investigated. Murashige and Skoog’s (MS) medium, with or without FLT, was enriched with indole-3-acetic acid (IAA; 0.1 mg l⁻¹) or a combination of IAA (0.1 mg l⁻¹) plus N⁶-benzyladenine (BA; 0.1 mg l⁻¹). The level of endogenous ABA significantly increased with increasing FLT concentrations in the presence of both IAA and IAA plus BA. An increased level of endogenous ABA was observed in plants treated with IAA alone. The growth of shoot, callus and the content of photosynthetic pigments (chlorophyll a and b, carotenoids), in both IAA- and IAA plus BA-treated plants, were significantly stimulated by FLT at its lowest concentration (0.1 mg l⁻¹) assayed in this study. However, FLT at higher concentrations (1.0 and 5.0 mg l⁻¹) significantly inhibited all these parameters. Chlorophyll fluorescence imaging showed that FLT only at the highest concentration (5.0 mg l⁻¹) in the presence of IAA (0.1 mg l⁻¹) significantly increased F₀, but decreased F_V/F_M and Φ_II.     

Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume

The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO₃ 475 mg l⁻¹, thiamine 1 mg l⁻¹, biotin 1 mg l⁻¹, calcium pantothenate 1 mg l⁻¹) containing 0.1 mg l⁻¹ 2,4,5-trichloroacetic acid and 0.1 mg l⁻¹ kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to ~12 mg l⁻¹ after 48 h of adding 100 μM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production.