Characterization of leaf senescence and pod development in soybean explants

Excised soybean (Glycine max [L.] Merrill) cv Anoka leaf discs tend to remain green even after the corresponding intact leaves have turned yello on fruiting plants. We have found that explants which include a leaf along with a stem segment (below the node) and one or more pods (maintained on distilled H₂O) show similar but accelerated leaf yellowing and abscission compared with intact plants. In podded explants excised at pre-podfill, the leaves begin to yellow after 16 days, whereas those excised at late podfill begin to yellow after only 6 days. Although stomatal resistances remain low during the first light period after excision, they subsequently increase to levels above those in leaves of intact plants. Explants taken at mid to late podfill with one or more pods per node behave like intact plants in that pod load does not affect the time lag to leaf yellowing. Explant leaf yellowing and abscission are delayed by removal of the pods or seeds or by incubation in complete mineral nutrient solution or in 4.6 micromolar zeatin. Like chorophyll breakdown, protein loss is accelerated in the explants, but minerals or especially zeatin can retard the loss. Pods on explants show rates and patterns of color change (green to yellow to brown) similar to those of pods on intact plants. These changes start earlier in explants on water than in intact plants, but they can be delayed by adding zeatin. Seed dry weight increased in explants, almost as much as in intact plants. Explants appear to be good analogs of the corresponding parts of the intact plant, and they should prove useful for analyzing pod development and mechanisms of foliar senescence. Moreover, our data suggest that the flux of minerals and cytokinin from the roots could influence foliar senescence in soybeans, but increased stomatal resistance does not seem to cause foliar senescence.     

Indirect Organogenesis from the Leaf Explants of Medicinally Important Plant Curculigo orchioides Gaertn

Curculigo orchioides Gaertn is an important medicinal plant of commercial value. A protocol for indirect regeneration from the leaf explants was developed. The leaf explants on MS (Murashige & Skoog) basal medium fortified with 6-benzylaminopurine (9μM) gave rise to proliferating green callus. The calli regenerated shoots on subculturing in the MS medium with 1.1μM 6-benzylaminopurine which was the optimum concentration for multiplication. Rooting was observed in MS medium supplemented with α-napthaleneacetic acid (5.3μM), indole-3-butyric acid (1.2μM), and on MS basal medium free of plant growth regulators. The rooted plants were hardened and transferred to field with 80% survival.     

Development of a rapid and high frequency Agrobacterium rhizogenes mediated transformation protocol for Ocimum tenuiflorum

A rapid and efficient Agrobacterium rhizogenes mediated transformation system for Ocimum tenuiflorum L., a traditional Indian medicinal plant that occurs in red and green forma, was developed. The plant is a repertoire of several pharmaceutically and nutraceutically important metabolites. Three different types of explants i.e. leaves, hypocotyls and excised shoots, obtained from shoot cultures of in vitro germinated red and green forma plants were transformed using Agrobacterium rhizogenes strain ATCC 15834. The transformation efficiency was equal between similar explants of both forma. Transformation efficiency was best in leaves of 4 days while excised shoots and hypocotyls had 6 and 8 days respectively. Transformation frequency of green forma leaves was the highest (70.6%) among all explants. Excised shoots of green forma plants exhibited better transformation (58.3%) than the red forma excised shoots (42.59%). Red forma hypocotyl explants displayed marginally better (26.27%) transformation frequency than green hypocotyl explants (21.14%). Transformation with hairy root was confirmed by the presence of rolC gene through PCR amplification and Southern hybridization. The development of hairy root-based transgenic system for O. tenuiflorum will pave the way for in vitro production of important secondary metabolites.     

Factors influencing the regeneration capacity of oilseed rape and cauliflower in transformation experiments

The efficiency ofAgrobacterium-based transformation technique in oilseed rape and cauliflower was influenced by cultivar specificity, donor plant age and explant type. Marked differences in demands for plant hormone contents in the regeneration medium were recorded already among different types of nontransformed explants. The highest regeneration capacity was recorded with stem and leaf segments isolated from one-month-old aseptically grown plants. The regeneration was markedly species-dependent. Regeneration of transformed plants from stem segments and thin layers isolated from field-grown oilseed rape plants (at the most 2% of regenerating explants) and from oilseed rape hypocotyls (0.8% of regenerating explants) and cauliflower (1.2% of explant regenerated transformed shoots) was achieved after disarmedAgrobacterium treatment. Hypersensitive reaction of explants could be prevented by using prolongedin vitro precultivation and delayed application of the selective agent.     

Endogenous Abscisic Acid and Indole-3-Acetic Acid and Somatic Embryogenesis in Cultured Leaf Explants of Pennisetum purpureum Schum. : Effects in Vivo and in Vitro of Glyphosate, Fluridone, and Paclobutrazol

Effects of application in vivo of glyphosate, fluridone, and paclobutrazol to glasshouse-grown donor plants of Pennisetum purpureum Schum. on endogenous levels of abscisic acid (ABA) and indole-3-acetic acid (IAA) in young leaves and on somatic embryogenesis in cultured leaf explants were studied. Treatment of plants with glyphosate (100 milligrams per liter) resulted in elevated levels of endogenous ABA and IAA in young leaves. In contrast, paclobutrazol (50% active ingredient; 200 milligrams per liter) did not alter the endogenous levels of ABA and IAA. Fluridone (100 milligrams per liter) markedly inhibited synthesis of ABA and leaf explants from fluridone-treated plants lost the capacity for somatic embryogenesis. Explants from glyphosate- or paclobutrazol-treated plants did not show any reduction in embryogenic capacity when compared with untreated control plants. Glyphosate and fluridone were also incorporated into the culture media at various concentrations (0 to 20 milligrams per liter) to study their effects in vitro on somatic embryogenesis in leaf explants from untreated, field-grown plants. Glyphosate was inhibitory to somatic embryogenesis but only at concentrations above 5 milligrams per liter. Fluridone inhibited somatic embryogenesis at all concentrations tested. Inhibition of somatic embryogenesis by fluridone, by either in vivo or in vitro application, could be overcome partially by (±)-ABA added to the culture medium. Exogenous application of (±)-ABA enhanced somatic embryogenesis and reduced the formation of nonembryogenic callus. Application of IAA or gibberellic acid (GA₃; >5 milligrams per liter) was inhibitory to somatic embryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass.     

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

The tobacco A-type cyclin, Nicta;CYCA3;2, at the nexus of cell division and differentiation

Although most of the components of the cell cycle machinery are conserved in all eukaryotes, plants differ strikingly from animals by the absence of a homolog of E-type cyclin, an important regulator involved in G1/S-checkpoint control in animals. By contrast, plants contain a complex range of A-type cyclins, with no fewer than 10 members in Arabidopsis. We previously identified the tobacco A-type cyclin Nicta;CYCA3;2 as an early G1/S-activated gene. Here, we show that antisense expression of Nicta;CYCA3;2 in tobacco plants induces defects in embryo formation and impairs callus formation from leaf explants. The green fluorescent protein (GFP)-Nicta;CYCA3;2 fusion protein was localized in the nucleoplasm. Transgenic tobacco plants overproducing GFP-Nicta;CYCA3;2 could not be regenerated from leaf disc transformation, whereas some transgenic Arabidopsis plants were obtained by the floral-dip transformation method. Arabidopsis plants that overproduce GFP-Nicta;CYCA3;2 showed reduced cell differentiation and endoreplication and a dramatically modified morphology. Calli regenerated from leaf explants of these transgenic Arabidopsis plants were defective in shoot and root regeneration. We propose that Nicta;CYCA3;2 has important functions, analogous to those of cyclin E in animals, in the control of plant cell division and differentiation.     

Age and orientation of the cotyledonary leaf explants determine the efficiency of de novo plant regeneration and Agrobacterium tumefaciens-mediated transformation in Jatropha curcas L.

Effects of age and orientation of the explant on callus induction and de novo shoot regeneration from cotyledonary leaf segments of Jatropha curcas were studied. The callus induction and shoot regeneration capacity of cotyledonary leaf segments were found significantly related to the age of the explants and their orientation in culture medium. The youngest explant, derived from the cotyledonary leaf of germinated seed induced the highest regeneration response as compared to one- and two-week-old explants. A gradient response with age of the explant was observed in percentage of callus induction, shoot regeneration from callus and the number of shoots per regenerating callus. The explants cultured with their abaxial side in medium showed significantly higher regeneration response. The youngest explant was found to be most amenable to Agrobacterium-mediated transformation as compared to older explants. The fact that callus induced from the edges of the explant followed by de novo shoot induction, and strong transient gus expression observed in the edges of the explant are significant for routine Agrobacterium-mediated transformation and generation of stable transgenic plants in J. curcas.     

Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation of Picrorhiza kurroa: an endangered medicinal herb of the alpine Himalayas

Picrorhiza kurroa Royle ex. Benth. is a medicinal herb of immense therapeutic value with restricted geographic distribution. Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation was developed for this plant. Multiple shoot bud induction was achieved from leaf explants cultured in Gamborg??s B₅ medium containing 3?% (w/v) sucrose, 3?mg/l kinetin and 1?mg/l indole-3-butyric acid. More than 90?% of leaf explants formed shoot buds leading to whole plant regeneration. An Agrobacterium-mediated genetic transformation protocol was developed using A. tumefaciens strain GV3101 harboring binary vector pCAMBIA1302 containing the green fluorescent protein and hygromycin phosphotransferase genes. Leaf explants precultured for 2?d were the most suitable for co-cultivation with Agrobacterium and transformation efficiency was enhanced with 200???M acetosyringone. Putative transformants were selected using media containing 15?mg/l hygromycin. Transformation was verified by detection of the green fluorescent protein using fluorescence microscopy and by polymerase chain reaction. Approximately 56?% of the explants were transformed with an average of 3.4?±?0.4 transgenic plantlets per explant. An efficient regeneration and transformation protocol thus developed enabling a fresh perspective of metabolic engineering in P. kurroa using an Agrobacterium-mediated transformation. This is the first report of direct organogenesis from leaf explants and genetic transformation of P. kurroa.     

High frequency organogenesis in hypocotyl,cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.     

The effect of 2,4-dichlorophenoxyacetic acid and donor plant environment on plant regeneration from immature inflorescence-derived callus of Lolium perenne L. and Lolium multiflorum L.

Callus cultures were obtained from immature inflorescences of perennial ryegrass (Lolium perenne) and Italian ryegrass (Lolium multiflorum). Inflorescence segments were cultured on Murashige and Skoog medium (MS) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The response in culture with regard to compact callus induction, embryogenesis and plant regeneration was determined for different varieties. The in vitro response was compared for explants from field-grown plants and explants from greenhouse-grown plants. The effect of different 2,4-D concentrations on the in vitro response was also investigated in one L. perenne variety and one L. multiflorum variety. The percentage of explants that formed compact callus and embryogenic callus differed strongly with the cultivar. There was no consistent effect of the growth conditions of the donor plants or the 2,4-D concentration of the medium on this response. Green plants were regenerated from all the cultivars tested. Explants from field-grown plants showed a higher tendency to form albino shoots than explants from greenhouse-grown plants. In the L. perenne variety tested higher 2,4-D concentrations (up to 15 mg/l) resulted in a lower regeneration frequency of green shoots and a higher regeneration frequency of albino shoots (up to 12.5 mg/l). In the L. multiflorum variety tested the effect of 2,4-D on regeneration was less pronounced.     

Improvement of adventitious shoot formation from carnation leaf explants

Adventitious shoot formation from leaf explants of carnation (Dianthus caryophyllus L.) was investigated. The two leaves from one node of in vitro-grown plants showed different shoot-forming potential, depending on the order in which the leaves were removed from the stem. The leaf removed second formed more shoots and also had a large amount of adhering stem tissue. Explants with equal amounts of adhering stem tissue were obtained by making two incisions through the fused leaf bases, prior to their removal, resulting in an improved shoot formation. The procedure developed for leaf explants from in vitro-grown plants was also applied to leaf explants from greenhousegrown plants. Shoot formation from leaf explants taken from greenhouse-grown plants was further improved by cutting the leaf explant longitudinally into two parts.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid     

In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

Polyploidy has played a significant role in the evolutionary history of plants and is a valuable tool for obtaining useful characteristics. Because of the novelty of polyploids, comparison of their in vitro culture responses with diploids would be notable. In this study, leaf explants from diploid, autotetraploid and mixoploid plants of Cichorium intybus L. were cultured in vitro on the similar media and under same conditions. The ploidy level of the obtained calluses and regenerants were determined by flow cytometry analysis. The callogenic response of leaf explants cultured on the callus induction medium did not depend on the ploidy level of their parental plants. According to the flow cytometry analysis, the increased ploidy levels (4x) and (8x) were observed in the callus cultures with diploid and tetraploid origin, respectively. A considerable difference was observed between the ploidy level of mixoploid plants and their calluses, indicating the dominance of diploid cells in the callus tissue. The results showed that polyploidy led to the loss of organogenic potential as the tetraploid origin calluses failed to regenerate, while the diploid origin calluses successfully regenerated to whole plants.     

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri T_L-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.     

Efficient somatic embryogenesis and Agrobacterium-mediated transformation of pothos (Epipremnum aureum) ‘Jade’

Leaf and petiole explants of monocotyledonous pothos (Epipremnum aureum) ‘Jade’ were cultured on Murashige and Skoog basal medium supplemented with N-(2-chloro-4-pyridl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4–8 weeks of culture; 9.1 μM TDZ with 1.1 μM NAA induced 61.1 % leaf discs and 94.4 % of petiole segments to produce plantlets through embryo conversion. Using this established regeneration method and an enhanced green fluorescent protein (GFP) gene (egfp) as a reporter marker, an Agrobacterium-mediated transformation procedure was developed. Leaf discs and petiole segments were inoculated with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pLC902 that contains novel bi-directional duplex promoters driving the egfp gene and hygromycin phosphotransferase gene (hpt), respectively. The explants were co-cultivated with strain EHA105 for 3, 5, and 7 days, respectively prior to selective culture with 25 mg l^?1 hygromycin. A 5-day co-cultivation led to 100 % of leaf discs to show transient GFP expression and 23.8 % of the discs to produce stable GFP-expressing somatic embryos. A 7-day co-cultivation of petiole explants resulted in the corresponding responses at 100 and 14.3 %, respectively. A total of 237 transgenic plants were obtained, and GFP fluorescence was observed in all plant organs. Regular PCR and quantitative real-time PCR analyses confirmed the presence of 1 or 2 copies of the egfp gene in analyzed plants. The highly efficient regeneration and transformation systems established in this study may enable genetic improvement of this vegetatively propagated species through biotechnological means.     

Effects of phosphogypsum on the growth of potato plants overexpressing the StDREB1 transcription factor

We developed an efficient system for agrobacterial transformation of plum (Prunus domestica L.) leaf explants using the PMI/mannose and GFP selection system. The cultivar ‘Startovaya’ was transformed using Agrobacterium tumefaciens strain CBE21 carrying the vector pNOV35SGFP. Leaf explants were placed onto a nutrient medium containing various concentrations and combinations of mannose and sucrose to develop an efficient selection system. Nine independent transgenic lines of plum plants were obtained on a regeneration medium containing 20 g/L sucrose and 15 g/L mannose. The highest transformation frequency (1.40?%) was produced using a delayed selection strategy. Starting from the 1st days after transformation and ending by regeneration of shoots from the transgenic callus, selection of transgenic cells was monitored by GFP fluorescence that allowed avoiding formation of escapes. Integration of the manA and gfp transgenes was confirmed by PCR and Southern blotting. The described transformation protocol using a positive PMI/mannose system is an alternative selection system for production of transgenic plum plants without genes of antibiotic and herbicide resistance, and the use of leaf explants enables retention of cultivar traits of plum plants.     

Adventitious shoot regeneration from Sinningia speciosa leaf discs in vitro and stability of ploidy level in subcultures

Summary Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N⁶ benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26.     

Somatic embryogenesis in clonal neem, <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss. and analysis for <Emphasis Type="Italic">in vitro</Emphasis> azadirachtin production

Summary Efficient and highly reproducible induction of somatic embryogenesis was obtained in four out of seven selected clones of neem, Azadirachta indica A. Juss. This was achieved either directly from root and nodal explants or indirectly from callus cultures initiated from leaf explants excised from 1-yr-old axenic plants. Direct induction of somatic embryogenesis was achieved both from nodal and root segments within 8 wk of culture on MS1 medium without growth regulators. However, the addition of 2.3–4.5 μM thidiazuron and 0.5 μM 2,4-dichlorophenoxyacetic acid into the medium were necessary to induce somatic embryogenesis via callus phase from leaf explants. Repetitive embryogenesis was observed within 3–4 wk following transfer of somatic embryos to a plant growth regulator-free medium. When somatic embryos of nodal and root segments were left on the induction medium without subculturing, approximately 15% of the somatic embryos developed into whole plantlets after passing through a series of developmental stages. Plantlets thus produced were hardy, lush green, and acclimatized casily under greenhouse conditions. However, somatic embryos derived from leaf explants showed low conversion rates (<5%). HPLC analysis revealed no detectable levels of azadirachtin in somatic embryos.     

Apical correlative effects in leaf epinasty of tomato

The influence of the stem apex on leaf curvature was investigated using debudded tomato (Lycopersicon esculentum Mill. cv Anahu) plants and petiole explants, consisting of a section of petiole attached to a section of stem.