In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.     

In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [Cajanus cajan (L.) Millsp.] cv. JKR105

In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L⁻¹ 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L⁻¹ 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.     

In vitro plant regeneration and genetic transformation of Dichanthium annulatum

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.     

In vitro regeneration and Agrobacterium-mediated genetic transformation of Euonymus alatus

An in vitro plant regeneration method and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Euonymus alatus. More than 60% of cotyledon and 70% of hypocotyl sections from 10-day-old seedlings of E. alatus produced 2–4 shoots on woody plant medium (WPM) supplemented with 5.0 mg/l 6-benzylaminopurine (BA) plus 0.2 mg/l α-naphthalene acetic acid (NAA), and 77% of shoots produced roots on WPM medium with 0.3 mg/l NAA and 0.5 mg/l Indole-3-butyricacid (IBA). On infection with Agrobacterium tumefaciens strain EHA105 harboring a gusplus gene that contained a plant recognizable intron from the castor bean catalase gene to ensure plant-specific β-glucuronidase (GUS) expression, 16% of cotyledon and 15% of hypocotyl explants produced transgenic shoots using kanamycin as a selection agent, and 67% of these shoots rooted. Stable insertion of T-DNA into the host genome was determined with organ- and tissue-specific expression of the gusplus gene and further confirmed with a PCR-based molecular analysis.     

Potential of pigeon pea as a trap crop for control of fruit worm infestation and damage to okra

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In planta transformation of maize through inoculation of Agrobacterium into the silks

Integration of T-DNA into the maize genome as a result of treatment of silks with Agrobacterium cells, containing activated vir genes, was demonstrated. In planta treatment of maize (Zea mays L) was performed during flowering in field. Cell suspension of Agrobacterium tumefaiciens strain GV3101(pTd33), carrying activated vir genes, was applied onto the previously isolated silks, which were afterwards pollinated with the pollen of the same cultivar. Integration of T-DNA into maize genome was confirmed by PCR (the nptII and gus reporter genes) and hystochemical staining of the seedling tissues, obtained from the transformed seeds. Amplification of the nptII gene showed the presence of about 60.3% of PCR-positive plants out of the total number of kanamycin-resistant seedlings examined, or 6.8% of the total of number of seedlings.     

In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis.     

Response of pigeon pea cultivars to water stress

Decrease in soil water potential during vegetative and flowering stages of two cultivars of pipeon pea (Cajanus cajari) caused higher decrease in relative water content in cv. ICPL-151 than in cv. H-77-216. Both cultivars showed partial recovery during rehydration. Cv. H-77-216 also accumulated more proline and carbohydrates during stress and showed better drought tolerance than cv. ICPL-151.     

In planta soybean transformation technologies developed in China: Procedure, confirmation and field performance

Summary Soybean is a recalcitrant species for in vitro manipulation. Chinese scientists developed two in planta non-tissue culture soybean transformation procedures: (1) via the “pollen-tube-pathway” to introduce exogenous genomic total DNA of Glycine gracilis, the seed of which consisted of 50% protein, and (2) “ovarian injection” with exogenous plasmid DNA containing atrazine-resistant gene. A high yield and high seed protein (45.44%) cultivar, ‘Heisheng 101’, resulted from the first method, and atrazine-resistant F₁, F₂, and F₃ plants were obtained from the second method. Both exogenous single-genic Mendelian traits and multi-genic quantitative traits were transferrable with these simple and inexpensive procedures. However, some controversy exists with the acceptance of these novo procedures; mainly because instead of the standard Southern blotting, the RAPD and dot blotting techniques were used in the molecular confirmation of the transgenic status in the reported studies. This mini-review is dedicated to the memory of the late Dr. Guang-chu Yin (1936–1994), former Head of the Soybean Research Institute of Heilongiang, Academy of Agricultural Science, Harbin, China. Dr. Yin led the advancement of soybean biotechnology in China and pioneered the soybean pollen-tube-pathway transformation.     

In vitro regeneration in Allium species

An attempt to induce shoot regeneration from leaf disc explants from Allium sativum L., A. porrum L., and A. schoenoprasum L. and the induction of shoot regeneration from single flower-bud receptacles in A. porrum is presented. While the regeneration rate from leaf disc explants was low, an efficient method for propagating A. porrum in vitro was obtained by cultivating single flower-bud receptacles. The shoot regeneration ability was strongly controlled by the genotype. Up to 294 shoots per leek plant could be harvested. Simultaneously the same plant could be used for seed production and bulbil formation in vivo. The efficiency of the in vitro multiplication method described allows the integration of this procedure into breeding programmes of A. porrum Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - IAA 3-indole acetic acid - NAA 2-naphtalene acetic acid     

In vitro plant regeneration in the genus Cucumis

Plants were regenerated from cotyledonary and root explants of cucumber (Cucumis sativus L.) cultivars and breeding lines of diverse sex type, growth habit, and processing quality and from cotyledonary explants of muskmelon (C. melo L.). Somatic embryogenesis was induced on a medium consisting of Murashige and Skoog (MS) salts supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l α-naphthaleneacetic acid and 0.5 mg/l 6-benzylaminopurine. Embryos matured on the same medium without 2,4-D, and developed into normal plants on a hormone-free MS medium. Cucumber plants were also regenerated from cotyledonary protoplasts using a modified tomato protocol.     

In vitro regeneration and genetic transformation of the berberine-producing plant,Thalictrum flavum ssp. glaucum

Protocols have been developed for the in vitro regeneration and Agrobacterium -mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum . Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l⁻¹ sucrose, 2 mg l⁻¹ kinetin, and 3 g l⁻¹ Gelrite) containing 25 mg l⁻¹ hygromycin B as the selection agent and 250 mg l⁻¹ timentin to facilitate the elimination of Agrobacterium . Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l⁻¹ hygromycin B, 250 mg l⁻¹ timentin, and 3 g l⁻¹ Gelrite). Detection of the β -glucuronidase ( GUS ) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.     

Senescence of detached leaves in pigeon pea and chick pea: Regulation by developing pods

The senescence rate of the subtending leaves in deflowered and control plants of pigeon pea ICajanus cajan (L.) Millsp. cv., Prabhat] and chick pea ( Cicer arietinum L. cv. JG 62) were examined during the course of natural and induced senescence, at several stages of pod growth. The leaves from the top 5 nodes on the main axis in pigeon pea and the top 8 nodes on the main axis in chick pea were used throughout the experiments. The natural senescence was characterized in leaves taken directly from the field-growing plants. For the study of induced senescence, the leaves were excised from both control and deflowered plants at various stages of pod growth and placed in test tubes containing water under dark conditions. Senescence was assessed in terms of peroxidase activity and contents of tola] chlorophyll, soluble amino acids and total protein. During natural ageing in the field, the leaves from deflowered plants exhibited delayed senescence in both the species. In contrast, the rate of ageing during induced senescence was higher in the leaves of deflowered plants than in the controls. Although of the same chronological age when excised for induced senescence, the leaves of deflowered plants were evideatly metabolically different from the controls, due to the fact that deflowered plants did not support the development of pods. This difference probably determined the subsequent rate of induced senescence.     

In vitro regeneration and optimization of conditions for Agrobacterium mediated transformation in jute, Corchorus capsularis

Jute is a crop of commercial importance that is widely cultivated for its bast fiber production but susceptible to many diseases that results in major economic loss. New genes can be introduced into this plant through Agrobacterium mediated genetic transformation for its genetic improvement, which is dependent on the availability of suitable in vitro techniques. An efficient regeneration system has been developed for in vitro culture of jute (Corchorus capsularis) from the distal cut ends of cotyledonary petioles. High frequency shoot regeneration was obtained on Murashige and Skoog (MS) nutrient agar medium supplemented with 0.5 mg/l NAA, 0.5 mg/l BAP and 36 g/l sucrose. On transfer to soil, the regenerated plantlets survived and appeared to be morphologically similar to the normal seed-grown plants. They developed pods and set fertile seeds. Histological analysis revealed de novo origin of shoot buds in the in vitro cultured cotyledonary petioles. Parameters affecting transformation were optimized by assaying GUS activity in these regenerable tissues after cocultivation with Agrobacteria. These tissues appear to be susceptible for infection and transformation by Agrobacterium carrying uid (GUS INT) and nptII genes, as well as shoot multiplication. The cells at the cut end of the petioles were found competent to take up the DNA, which was monitored by transient GUS gene expression. EHA105 at 0.3 O.D and LBA4404 at 0.5 O.D were found to be compatible in giving optimal levels of transient GUS expression.     

In planta 2,3,5 truodobenzoic acid treatment promotes high frequency and routine in vitro regeneration of sugarbeet (Beta vulgaris L.) plants

Summary The effect ofin planta treatments with auxin inhibitors such as 2,3,5 triiodobenzoic acid (TIBA) on regeneration of plantsin vitro is not known. Here, we show the beneficial effect of preconditioning sugarbeet plants in the greenhouse with TIBA (3 mg/1) for efficientin vitro plant regeneration via a callus phase from cultured leaf explants. Without this treatment, no shoot developed on the control leaf-calluses. Several hundred plants were routinely regenerated using this protocol. More importantly, the number of shoots per explantcallus increased drastically over the subsequent subculture period. The most favorable media for callus induction contained a combination of an auxin and a cytokinin (0.1 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 N-6 benzylaminopurine) or a cytokinin alone (2.2 mg/1 thidiazuron). However, only the callus derived from leaves of TIBA-treated genotypes and induced on thidiazuron-medium produced numerous shoots. Histological studies showed the formation of meristematic zones only in the organogenic callus developed on thidiazuron-coutaining medium. The analysis of peroxidase activity showed that the activity was higher for the TIBA-treated plants than for the untreated control plants.     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp]

A requirement for generating transgenic pigeonpea [Cajanuscajan (L.) Millsp] plants is the development of a highly efficientin vitro regeneration procedure. This goal was achieved byusing germinated seedlings grown on B5 medium supplemented with 10 mgl^–1 6-benzylaminopurine, which induced differentiatingcallus formation in the cotyledonary node region. The calli were transferred onB5 medium with 0.2 mg l^–1 6-benzylaminopurine toobtain shoot induction. Elongated shoots were then further cultured on a B5hormone-free medium for rooting. Using this regeneration system transgenicpigeonpea plants were obtained both by particle bombardment andAgrobacterium tumefaciens-mediated gene transfer. Thepresence of the transgenes in the pigeonpea genome was confirmed by GUS assays,PCR and Southern hybridisation. The transgenic rooted plants were successfullytransferred to soil in the greenhouse. GUS and PCR assays of T1 progeniesconfirmed that the transgenes were stably transmitted to the next generation.This is the first report of successful use ofAgrobacteriumas well as particle bombardment for production of transgenic pigeonpea plants.     

Plumular meristem transformation system for chickpea: an efficient method to overcome recalcitrant tissue culture responses

Plant Cell, Tissue and Organ Culture (PCTOC) - Major biotic and abiotic stresses have led to the reduction of chickpea productivity, creating a strong barrier for its utilization as major food...