<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of brown oak (<Emphasis Type="Italic">Quercus semecarpifolia</Emphasis> Sm.) from seedling explants

An efficient and reproducible method for the regeneration of multiple shoots of brown oak (Quercus semecarpifolia Sm.) has been developed in which a part of the petiolar tube containing a primary shoot is used as the explant. Explants derived from in vitro grown seedlings were cultured either on Murashige and Skoog or Woody Plant medium (WPM) containing different concentrations of benzyladenine (BAP) throughout the range of 1–20 μM. WPM supplemented with 20 μM BAP was found to be best for adventitious shoot induction and for the multiplication of individual shoots. In-vitro-produced shoots were rooted using a two-step method. Firstly, shoots were cultured on WPM containing indolebutyric acid (IBA) at either 50 or 100 μM for 24 or 48 h. Secondly, the shoots were transferred to plant-growth-regulator-free half-strength WPM. The second step not only considerably improved the rooting percentage but also minimized the formation of basal callus. The most effective first-step treatment was found to be 100 μM IBA for 24 h, which initiated rooting at a frequency of 100%. Well-rooted plants were transferred to plastic cups containing nonsterile, sieved soil and farmyard manure, hardened under greenhouse conditions, and then successfully established in pots. This procedure is suitable for use in large-scale production of plants and may have potential application in additional oak species.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

Induction and origin of adventitious shoots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

The chimeras between tuber mustard (Brassica juncea) and red cabbage (B. oleracea) were artificially synthesized in our previous study. Adventitious shoots were induced from nodal segments and leaf discs of TCC (LI-LII-LIII, LI -the outmost layer of shoot apical meristem; LII -the middle layer; LIII -the innermost layer. T = Tuber mustard, C = Red cabbage) chimeras. The origin of the shoots was analyzed by histology and molecular biology. As a result, the frequency of adventitious shoot induction rose with the increase of BA in MS medium in the area of the nodes. However, there was no different induction frequency of adventitious shoots from nodal segment bases in media with different BA concentrations. Most adventitious shoots (clustered shoots) arising from the node area were TTT (Tuber mustard- Tuber mustard- Tuber mustard) and only 4 shoots were chimeras, which indicated that more shoots originated from LI than from LII and LIII. All shoots from nodal segment bases were CCC (Red cabbage-Red cabbage- Red cabbage), indicating that the shoots originated from LII or LII and LIII. There were significant differences in the regeneration rate in the margin of the leaf discs among the three combinations of BA and NAA. Most adventitious shoots from the margin of leaf discs were CCC but 2 out of 70 were chimeras, which indicated that more shoots originated from LII or LII and LIII than from LI. All chimeras obtained by regeneration were different from the original explant donor in type in the present study. The origin of the adventitious shoots varied with the site of origin on the donor plant, and could be multicellular and multihistogenic.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.     

Genetic transformation and regeneration of transgenic plants from precultured cotyledon and hypocotyl explants of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis> Sm. using <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine (BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1.0 mg/l indole-3-butyric acid and 40 mg/l kanamycin. A strong β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective transformation and direct regeneration of E. tereticornis Sm.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> with thaumatin II gene to control fungal diseases

Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l⁻¹ kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l⁻¹ kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Fraxinus pennsylvanica</Emphasis> hypocotyls and plant regeneration

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were transformed in the presence of 100 μM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l⁻¹ was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with 13.3 μM 6-benzylaminopurine, 4.5 μM thidiazuron, 50 mg l⁻¹ adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse. This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash.     

Improvement of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in Hi-II maize (<Emphasis Type="Italic">Zea mays</Emphasis>) using standard binary vectors

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant l-cysteine alone or l-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.     

Establishment of an efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation of <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 10 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength MS basal medium supplemented with 10 mg l⁻¹ hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 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, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Pelecyphora aselliformis</Emphasis> erhenberg (cactaceae)

Summary In vitro propagation of Pelecyphora aselliformis, a Mexican cactus which is considered rare and is highly valued in the commercial market, was initiated using seeds as explants. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium containing 8.8 μM benzyladenine (BA) or 4.6 μM kinetin at pH 7.0. After 120 d, each explant gave rise to five shoots and this number of shoots increased 20–25% after subculture. The hyperhydricity was similar in both media, but callus formation was lower on the medium with BA. The shoot development, in terms of epicotyl length, and fresh and dry weight after 6 wk, was also recorded. The epicotyl length was similar on shoot-forming media but the quality of shoots was better on media containing BA. In about 1 yr, 500–600 well-defined shoots were obtained. The rooting of shoots was very slow and a vigorous radical system was observed after 1 yr of culture.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.