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STUDIES ON VITRO REGENERATION AND GENETIC TRANSFORMATION IN CHILLI (Capsisum ammuumL.)
... Cefotaxime a semi-synthetic, broad spectrum and cephalosporin antibiotic, is a β-lactam antibiotic that inhibits cell wall synthesis in dividing bacterial cells and results in cell lysis. Enhancement of shoot regeneration frequency by cefotaxime has been reported in other plants such as finger millet [34], wheat [35], pearl millet [36], sorghum [37], chilli [38], rice [28] and sugarcane [39]. ...
... The growth of Agrobacterium was strongly inhibited in treatments with cefotaxime. Channappagoudar [38] reported that cefotaxime was the most effective antibiotics against LBA4404. Nonetheless, the results of this study revealed that timentin effectively removed the bacteria from cultured leaf explant of strawberry and at the same time induced more number of shoots per explant. ...
An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.
... However, there is an occurrence of cross pollination which leads to the formation of variants within the species. It is grown in the world on an area of 1.5 million ha with a production of about 10.60 million tonnes of green and dried chillies [2]. India ranks first in the world with an area of 7.75 lakh ha with an average yield of 1.6 metric tonne/ha [3]. ...
Climate change has become the major concern for the world today. Focusing on agriculture, climate change is already beginning to have a negative impact on world’s crop production levels. Global warming/climate change is causing a significant harm to the cultivation of crops including chillies around the world. Chilli is one of the important spice crops. Many studies have been done/conducted to look for the possible damage to chilli crop growth and development due to change in climate and global warming and also to find out the solution to these problems. This review article highlights about some of the consequences of global warming such as extreme temperature, drought, floods, soil acidity, soil salinity, etc. on chilli and the mitigation and adaptation techniques that are being used or can be used to make the plants more tolerant and productive under this changing climate.
Sweet pepper (Capsicum annum L.) under the family Solanaceae is an important vegetable crop, grown worldwide for its delicate taste, pleasant flavour and colour. Sweet pepper fruits are also known to have high nutritional values as they contain different pigments particularly, carotenoids, xanthophylls, different vitamins, dietary fibre and several essential minerals. Germplasm collection and conservation is the major step in improvement of sweet pepper. Promising germplasm may directly be introduced from other countries. Quality specifications such as size, shape, colour and non-pungency make the task of developing new hybrids/cultivars very challenging. Future thrust areas for research and developmental works for genetic improvement in sweet pepper include- Strengthening germplasm collection, tropicalization of the crop, development of multiple disease resistance, standardization of seed production techniques, creation of model plants through crop ideotype.
Capsicum or chili peppers were predominantly domesticated first in America and was introduced from there to rest of the world by Columbus. Capsicum breeding initially started as selection from wild species for different purposes and further improvement was based on the art of selection. With time, the breeding for crop improvement became more scientific and classical methods like mass selection, pedigree method, single-seed descent method, backcross, and hybridization are currently being utilized for capsicum improvement. Genetic diversity of capsicum is large, allowing alternatives to several new gene rearrangements. Capsicum fruits have high nutritional value, bringing benefits to consumer’s health. This fact has contributed to increase the market and consumption of capsicum in the world. Search for capsicum genotypes with increased yield, disease and abiotic stress resistance and improved quality is the goal in capsicum breeding programs. Lately, new strategies for improvement like mutation breeding, polyploidy, haploid breeding, embryo rescue, and utilization of molecular markers have been used in capsicum breeding. With continuous advancement in molecular technologies, it is becoming an essential tool which when combined with traditional selection and crossing techniques can result in significant progress in already established capsicum genetic breeding program.
Chilli (Capsicum annuum) is an important commercial and export-oriented crop in India. In this paper, an overview of chilli breeding research in India has been given.Germplasm sources resistant to Chili/pepper leaf curl virus (ChiLCV) Chili veinal mottle virus (ChiMoV), Cucumber mosaic virus (CMV), anthracnose, bacterial wilt, nematodes and powdery mildew have been identified and used in current breeding programs. The northeast region has emerged as having a novel diversity in the cultivated species of the genus Capsicum. Nuclear and cytoplasmic male sterility system have been developed and exploited at commercial scale to develop hybrids and their seeds. The developmental accomplishments including national releases of commercial chilli cultivars (open pollinated varieties and hybrids) and dissemination of their seed has been discussed. Changes in climate and society are explored as they relate to chilli production; research across multiple sectors is advocated to address these and other concerns to boost the chilli industry.
Regeneration capabilities of Capsicum annuum cotyledons and leaves were compared. Adventitious buds, leaf like structures and callus formation occurred on both types of explants, roots on cotyledons only. Murashige and Skoog basal medium containing 1.0 mg dm-3 indole-3-acetic acid (IAA) and 1.0; 5.0; 10.0; 15.0 mg dm-3 6-benzylaminopurine (BAP) were used. Adventitious buds were induced more frequently on cotyledons than on leaves. The middle part of cotyledon explants and the distal part of the leaf explants seem to be the best.
Shoot-tip and axillary shoot explants were used for in vitro regeneration and mass multiplication of two Capsicum annuum L. cultivars, Meiteimorok and Haomorok. Shoot-tip explants excised from in vitro-raised seedlings were used for multiple shoot bud induction in MS medium supplemented with BAP alone or in combination with IAA. Maximum number of shoot buds were obtained on MS medium containing 22.2 µM BAP or 44.4 µM BAP in the cultivar Meiteimorok and in medium containing 22.2 µM BAP in the cultivar Haomorok after four weeks of culture. Rooting and elongation of the regenerated shoot buds were achieved in medium containing 2.46 µM IBA or 4.90 µM IBA iMeiteimorok' and in 2.85 µM IAA or 5.71 µM IAA and 2.46 µM IBA or 4.90 µM IBA iHaomorok'. Axillary shoots were induced in the rooted plantlets by decapitating the plantlets. About 4-6 axillary shoots developed in both cultivars within two weeks of decapitation. The axillary shoots with a few leaf primordia were subsequently cultured in medium containing 22.2 µM BAP or 44.4 µM BAP for shoot multiplication and the regenerated shoots were rooted in medium supplemented with IAA or IBA. The rooted plantlets were further decapitated for mass multiplication.
The objective of this research was to define an in vitro regeneration and transformation system for bell pepper (Capsicum annuum L.) using six cultivars and one Guatemalan wild accession. The wild accession exhibited the best regeneration response. Only occasional elongation of shoot buds in 'Yolo Wonder L' was achieved by culture in the dark on a medium containing 10 mg/l BA and l mg/l IAA. Transformed shoot buds and leaf-like structures were obtained, showing beta- glucuronidase activity predominantly in the vascular and perivascular tissues, with no indication of contaminating Agrobacterium in the tissues. Attempts to regenerate whole transgenic plants from transformed shoot buds were unsuccessful.
Transgenic peanut (Arachis hypogaea L.) plants have been produced using an Agrobacterium-mediated transformation system. Zygotic embryo axes from mature seed were cocultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector that contained the genes for the scorable marker B-glucuronidase (GUS) and the selectable marker neomycin phosphotransferase II. Nine percent of the germinated seedlings were GUS+. Polymerase chain reaction analysis confirmed that GUS+ shoots and T1 progeny contained T-DNA. Molecular characterization of one primary transformant and its T1 and T2 progeny plants established that T-DNA was integrated into the host genome.
A reproducible system for production of transgenic plants via Agrobacterium mediated transformation of pigeonpea (Cajanus cajan L.) was developed. Shoot apices and cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain LBA4404 . The strain harbours a binary vector carrying the reporter gene (β-glucuronidase (uidA) and the marker gene neomycin phosphotransferase (nptII). Cocultivated explants were cultured on shoot regeneration medium with 2mg l-1 BAP and kanamycin (50 μg mL-1 for selection (MS1). Approximately 45-62% of the explants produced putatively transformed shoots on the selection medium. Multiple shoots were repeatedly selected on 0.5 mg l-1 BAP and 25 μg ml-1 kanamycin medium(MS2) The elongated shoots were subsequently rooted on a medium supplemented with 25 μg ml-1 kanamycin sulfate (MS3). The transgenic plants were later established in pots. Although transformation was achieved with both cotyledonary node and shoot apices, cotyledonary nodes responded better with 62% of the explants producing GUS positive shoots after selection on MS2 medium. The presence of uidA and nptII genes in the transgenic plants was verified by PCR analysis. Integration of T-DNA into the genome of transgenic plants was further confirmed by Southern blot analysis.
In vitro genetic transformation of chile pepper, Capsicum annuum var. New Mexico 6-4, was achieved. Seeds of `New Mexico 6-4' were grown aseptically on Murashige and Skoog medium. Seedlings 22 days old were wounded on the hypocotyl region using a sterile hypodermic needle. A. rhizogenes strain K 599 harboring the plasmid p35S GUS Intron was inoculated on the wound site. Three days later the seedlings were transferred onto MS media with antibiotics (Cefotaxime, Carbenicillin, Amoxicillin, Clavulanic acid, and Kanamycin). New roots were seen to initiate from the wound site 15 to 20 days after inoculation. The roots were morphologically identified as “hairy roots.” Glucuronidase ( Gus ) assay performed 40 days after inoculation on randomly chosen roots that had grown into the selection medium, showed that 6/25 (24%) of the inoculated seedlings had roots that showed intense blue coloration. Presence of an intron makes it impossible for the bacteria to express the reporter gene. The seedlings that had transformed roots had a different morphology with wrinkled leaves and short internodes. The pattern of expression of the introduced gene varied greatly. Some positive tissues had the root tips alone being blue; a few had the vascular tissues and the root tips blue; and others had the vascular tissues, the surrounding parenchyma cells, root tips, and the root hairs turn very dark blue. The transformed roots did not need to grow into the selection media to be Gus positive. Isolated roots cultured on MS media supplemented with 0.2 mg/L IAA were maintained for 120 days and continued to express the reporter gene. Currently, methods to regenerate transformed shoots from roots are being tested. The “hairy root” transformation system in pepper could have application in the testing of root-expressible constructs for transgene expression assays.
Stable genetic transformation of Arabidopsis thaliana was achieved by simple in planta inoculation of Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBl121. The transformation procedure, which we call in planta transformation, involves severing of apical shoots at their bases, inoculation with Agrobacterium at the severed sites, and in planta generation of shoots from the severed sites. On average, 5.5% of the newly formed shoots produced transformed progenies. These progenies (T2 generation) contained T‐DNA in the genome as examined by assaying the T‐DNA encoded β‐glucuronidase and kanamycin resistance and by genomic Southern blot analysis, the copy number of the T‐DNAs in the Arabidopsis genome being single (33%) or multiple. The genetic behavior of the transformants examined at the T3 and T4 generations or with the F2 progenies of the outcrosses between transformants and wild‐type plants showed that most of the inserted T‐DNA are inherited in a Mendelian fashion. This procedure provides a new approach for simple and efficient transformation of A. thaliana, obviating the need for plant regeneration from tissue explants in vitro.
Capsicum (cv. arka gaurav) anthers were cultured on MS, B5, NN and RO media containing sucrose (3%), kinetin (0.5-5.0 μM) and 2,4-D (0.5 or 20.0 μM) or IAA (5.0 μM). callusing response of anthers averaged over different hormonal treatments and incubating environments was significantly higher in B5 (45.5%) followed by RO (40.1%), NN (34.7%) and MS (29.8%). The response was found to be inversely related to NH4+/NO3- ratio (0.08, 0.22, 0.49 and 0.52 respectively) in the medium but not sensitive to total N concentration. Reducing NH4+/NO3- ratio in MS or NN to the level of B5 without altering total N content significantly increased the per cent callusing in them. Conversely, increasing proportion of NH4+ in RO medium inhibited callusing. Complete replacement of NH4+ with NO3- as N source severely inhibited callusing indicating that a reduced form of N was essential. Callusing response was insensitive to a wide range of N concentration in the basal medium as long as NH4+/NO3 ratio was maintained.
An efficient medium for multiplication of Prunus cerasifera, J. F. Ehrh. cv. 'Atropurpurea', Pissardi plum, consisted of Linsmaier-Skoog basal medium (LS) supplemented with 3% sucrose, 10mg 1-1 N4-(Δ2-isopentenyl) adenine (2iP), and 162 mg 1-1 phloroglucinol (Phl). Phl in the medium significantly enhanced growth (P < 0.1 %) over cultures maintained on LS medium with 10 mg 1-1 2iP and no Phl. Plantlets were rooted on a half-strength Murashige-Skoog (MS) medium supplemented with 0.2 mg 1-1 indole-3-butyric acid (IBA).
Regeneration of well-developed shootlets from cotyledonary explants of chilli 'Pusa Jwala' and 'G4' was obtained in MS medium supplemented with (9-18 μM) zeatin in combination with GA3 (2.89 μM). The elongated shoots could be rooted successfully on IBA containing MS medium. Cotyledonary explants of 'Pusa Jwala' were transformed with Agrobacterium tumefaciens st C58 containing binary vector pGV1040 harbouring the two reporter genes npt II and GUS. The transformed shoots were selected on regeneration medium containing kanamycin (100 mg 1-1) and cefotaxime (500 mg 1-1) and subsequently rooted in the presence of kanamycin (75 mg 1-1). The presence of the transgene was confirmed through histochemical staining of GUS, polymerase chain reaction (PCR) and Southern hybridization analysis of npt II gene.
The effective protocol for plant regeneration and genetic transformation, which can be used for the improvement of hot pepper (Capsicum annuum L.) quality, was established. High frequency of plant regeneration was observed when cotyledon segments were cultured on MS medium added with NAA (0.1 mg-L -1) and zeatin (2.0 mg-L-1) or IBA (10.0 mg-L -1) and BAP (1.0 mg-L-1). An addition of 5 μM AgNO 3 to these media improved the regeneration frequency up to 8%. Verification of presumed transgenic plants that survived in selection media was done by the amplification of selectable marker genes (ADA, NPTII, bar) by polymerase chain reaction (PCR). Successful expression of the ADA (adenosine deaminase) gene was detected by Northern blot and enzyme activity, suggesting a potential application of ADA gene as a selectable marker in genetic transformation system. Herbicide resistant gene, bar, was also introduced into hot pepper, and progenies of the transgenic plants were obtained. Stable inheritance (3:1 ratio) of bar gene was confirmed by PCR analysis and herbicide treatments of 5,000 mg-L'1. This highly efficient transformation system can be further used for the improvement of hot pepper quality using other useful genes.
We have examined the toxicity of over twenty antibiotics to protoplast-derived cells of Nicotiana plumbaginifolia. The least toxic antibiotics are the betalactams: ampicillin, carbenicillin and the cephalosporins can be used to provide broad spectrum antimicrobial activity without significant toxicity to plant cells. Similar broad spectrum activity can also be obtained by combining rifampicin and trimethoprim. Other antibiotics which may be useful are erythromycin and colistin. The aminoglycosides are not recommended.
Leaf protoplasts were isolated from axenic shoot cultures of four varieties of Capsicum annuum (Americano, Dulce Italiano Florida Gynat and Nigrum) and a wild species C. chinense. Protoplasts of both species, cultured in KM8P medium and using agarose bead culture, entered division with the exception of the variety Nigrum. Cell colonies formed callus in agar-solified MS medium supplemented with zeatin and for C. annuum v. Dulce Italiano shoots were regenerated when protoplast-derived calli were transferred to MS medium with 6-BAP. Excised shoots were rooted on MS medium which lacked phytohormones.
In vitro plant regeneration has been obtained from Capsicum annuum cvs. Pico and Piquillo. Shootbuds were induced from hypocotyl and cotyledon segments after 15-20 days of culture on MS basal medium supplemented with IAA and BAP or Zeatin. Shoot-buds grew into rosettes that rooted in MS plus NAA (0.1 mg/l) and IBA (0.05 mg/l) after 15 days. The small plantlets were successfully transferred to pots with a mixture of peat and perlite and maintained under greenhouse conditions. Elongation took place when the plantlets were growing in the greenhouse.
Organogenesis of shoots of bell pepper (Capsicum annuum L.) was achieved in fourteen cultivars on Murashige and Skoog's medium (MS medium) supplemented only with 0.4% (w/v) Gellan gum (pH 5.8). Mature seeds of cv. Shinsakigake-2 were sown on filter paper that had been wetted with sterilized water and precultured for zero to five days in under 16 hr of light per day at 25 °C. Explants, consisting of the proximal part of the hypocotyl and the radicle, were excised from the seeds and formed adventitious buds around the cut surfaces of elongated hypocotyls after four weeks of culture. When explants were subcultured on MS medium, 57% of the explants that had produced adventitious buds extended shoots after an additional three weeks of culture. Shoots were rooted on MS medium after two further weeks of culture. Chromosome numbers of all 30 regenerated plants that weexamined were normal (2n=24). The morphology of the mature plants was also normal and they set normally shaped fruits with mature seeds. Regenerated whole plants were also obtained in the case of 13 other cultivars by applying this simple procedure.
Explants from 13-d old pepper (Capsicum annuum, L. cv. Early California Wonder) seedlings were cultured in Murashige and Skoog (MS) medium supplemented with different levels of 1-naphthalene acetic acid (NAA) and 6-benzylamino purine (BAP). Multiple shoot-buds proliferated from the cut surfaces of cotyledon, shoot-tip and hypocotyl explants in one month. The best NAA to BAP combinations (mg/l: mg/l) for multiple shoot-bud regeneration of the above three explant types were 0.1 ∶ 5.0, 0.0 ∶ 5.0, and 0.1 ∶ 10.0, respectively. Root explants did not express any new morphogenetic response in all hormonal combinations tested. Regenerated shoot-buds were excised from the explants and cultured in 1/2X or 1X MS medium supplemented with different levels of Indole-3-acetic acid (IAA) or NAA. When cultured in full strength MS medium with 0.5 mg/l IAA or 0.4 mg/l NAA, 70% of the buds rooted in one month. Plantlets were established successfully ex vitro under greenhouse mist and grown to maturity.
In vitro shoot regeneration ability of 17 (7 Italian and 10 Hungarian) bell pepper genotypes was investigated using excised cotyledons and rooted hypocotyls as explants. Most of the Italian genotypes and two of the Hungarian genotypes responded well, producing shoots from rooted hypocotyls. Only two genotypes (one Italian and one Hungarian) gave a weak response using cotyledons. For direct shoot induction in these explants, in addition to the methods cited in the relevant papers, a new method was applied using thidiazuron as a cytokinin. Shoots were successfully regenerated from cotyledons of two Italian and two Hungarian genotypes using thidiazuron which were considered to be non responsive to the usual methods.
A protocol for protoplast isolation was developed and tested with five Capsicum genotypes representing two cultivated species, C. annuum and C. chinense. Key variables included growth conditions for source plants and the concentration of mannitol used as osmoticum. Protoplasts isolated from each of the genotypes became infected when inoculated via electroporation with viral RNA from either pepper mottle potyvirus, tobacco etch potyvirus or cucumber mosaic cucumovirus.
Pepper (cv. New Mexico - 6 and Rajur Hirapur) plants were regenerated from immature zygotic embryos via direct somatic embryogenesis. Somatic embryos were formed directly, without any intervening callus, on the zygotic embryo apex, embryo axis and cotyledons on Murashige and Skoog's (MS) medium containing 2,4-D (418 μM), thidiazuron (10 μM) and a high concentration of sucrose (6-10%). The best response was observed on MS medium containing 2,4-D (9 μM), coconut water (10%) and high sucrose (8%). The entire process of induction and maturation of the embryos was completed on the same medium. Histological examination indicated that secondary embryogenesis also occurred directly from the primary somatic embryos. Differentiation of embryos was nonsynchronous, and some embryos were swollen and distorted with fasciation. More than 70% of the mature normal somatic embryos germinated readily on MS medium containing GA3 or TDZ, alone and in combination, and following transfer to pots developed into normal plants.
In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 μmol m-(2) s-(-1)) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation (≥ 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10-14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L(-1) for 3 h and then subculturing on MS medium without growth regulators.
The present study was carried out to analyze the dihydrodipicolinate synthase (dhdps) gene promoter activity by tracing the GUS expression in tissues and in organs of Arabidopsis thaliana by in planta transformation. The Agrobacterium construct pBI101 used in the studies consists of the reporter gene gus under the control of Arabidopsis thaliana dhdps promoter with 3’ nos controlling sequences and nptll gene under the control of nos promoter and nos terminator. GUS expression in transformed Arabidopsis thaliana was found to be cell type-specific and expressed mainly in the fast growing tissues, where the protein synthesis is high. The histochemical analysis results indicate that the GUS expression was mainly observed in root meristem (elongation zone), emerging lateral roots and in the leaf vascular tissues. In reproductive organs, the GUS expression was observed in anthers, pollen grains and young immature embryos. Southern blot analysis results of T2, progeny showed the presence of a single integration locus for both the nptll and dhdps promoter.The segregation analysis results showed that the kanamycin resistance gene has not followed the normal Mendelian inheritance.This might be due to the methylation of the nptll gene in some of the transformants.
A protocol has been developed to obtain whole plants from apical shoot meristems of red pepper (Capsicum annuum L. cv. Bhivapuri), susceptible to viral infections. The meristems (∼ 0.8 mm long), from aseptically grown seedlings (one-month-old), cultured on filter paper bridge in liquid Murashige and Skoog medium supplemented with 2 mg/l benzylaminopurine produced multiple shoots (5–7 per explant). The differentiated shoots developed further upon transfer to agar-solidified medium. Complete plantlets were obtained after rooting of shoots on MS medium fortified with 1 mg/l naphthaleneacetic acid.
Sweet pepper has been considered recalcitrant to genetic transformation. As a first step for the development of a transformation system for pepper, we investigated the suitability of five Agrobacterium tumefaciens strains for inducing tumours in nine different varieties. A. tumefaciens wild-type strains A281, Ach5, C58, 42CNBP and 1102 were used for inoculation of plants of the nine pepper cultivars Lamuyo, Dulce Italiano, Yolo Wonder, Negral, Luesia, Toledo, Piquillo, Agridulce and Yatsufusa. Different responses to bacterial infection were found among the pepper varieties tested. 'Yatsufusa' and 'Piquillo' had a low response to Agrobacterium infection while for the other seven varieties at least one and usually two strains produced tumours at 80-100% frequency. Furthermore, C58 and 1102 strains showed significantly greater virulence and also induced more tumours per wound than Ach5, 42 CNBP and A281. To investigate any correlation between Agrobacterium virulence and transformation ability, sweet pepper cotyledon explants were co-cultivated in vitro with disarmed derivatives of C58 and A281 carrying the binary plasmid pBin19-sgfp. It was shown that genotypes with poor tumourigenic responses also were recalcitrant to genetic transformation in vitro. For those cultivars highly responsive to Agrobacterium infection, the C58 disarmed strain induced 6-14-fold higher stable transformation frequency than the A281 derivative, suggesting that C58 disarmed derivatives would be the most appropriate to genetically transform these sweet pepper cultivars.
Conditions for plant regeneration from explants of tomato (Lycopersicon esculentum) cv. UC82B were studied for optimizing transformation procedure. The best regeneration rate was obtained from cotyledon explants
from 8–10-d-old seedlings on a modified Murashige and Skoog medium (1962) with 0·5 mg dm−3 zeatin and 0·5 mg dm−3 indolylacetic acid. Tomato cultivars (UC82B, Castone, Fl Ferline, Monalbo) and a Lycopersicon peruvkmum ‘CMV sel. INRA’ were
studied. The cultivarUC82B and the wild Lycopersicon species showed an efficient shoot regeneration potential.
Early events in the transformation of tomato cotyledons were analysed using an Agrobacterium tumefaciens strain carrying a binary vector with an nptII (pnos) gene and a reporter GUS-intron (p35S) chimeric gene. Two days after infection, GUS activity appeared specifically
at the cut surface. Subepidermal cells were more susceptible to transformation than epidermal cells. When selection for kanamycin
resistance was applied 2 d after inoculation, transformed cells were efficiently recovered. Preculture with feeder cells stimulated
cell transformation, but reducedregeneration capacity from transformed cells. The optimal transformation rate was observed
witha time of preculture of 1 and 2 d. Transformation events for two tomato cultivars (UC82B and Monalbo) occurred at the
same rate as 55% of the inoculated explants developed kanamycin resistant calli. However, transformed plants were obtained
at different rates of 8% and 14% for cv. Monalbo and cv. UC82B.
An Agrobacterium tumefaciens -mediated transformation procedure was developed for the non tuber-bearing, diploid wild potato species Solanum brevidens, and the tetraploid S. tuberosum cv. Pito. Cointegrative transformation vectors pGV2260 and pGV3850, and binary vector pGUS-INT were employed in transformation of S. brevidens and Pito, respectively. Leaf and stem explants of S. brevidens and microtuber discs of Pito were precultured 24 h, and cocultivated 48 h on solidified callus induction medium with a 24-h liquid culture of A. tumefaciens C58C1 diluted 1:10 with liquid MS medium. Explants were rinsed with cefotaxime solution (500 mg/1) to remove Agrobacterium, grown without the selective agent kanamycin on solified callus induction media for 14 days, and then exposed to kanamycin selection for the first seven days on callus induction medium and subsequently on shoot regeneration medium. Shoots regenerated faster from stem explants than from leaf explants. Up to 49% and 57% of the transformed leaf explants of S. brevidens and microtuber discs of Pito, respectively, produced transformed shoots.
The effect of the explant position on the donor plant, illumination and explant pretreatment with high cytokinin concentrations on the induction, proliferation and development of somatic embryos from young, fully expanded leaves of chilli pepper (Capsicum annuum L.) was investigated. Explants were cultured either directly on a solid Murashige and Skoog medium supplemented with 9μM 2,4-dichlorophenoxyacetic acid+12.9μM 6-benzyladenine or incubated for 24h in a liquid MS medium containing the cytokinin at a tenfold concentration (129μM) and then transferred to the solid MS medium. Globular embryo proliferation depended on the leaf position on the donor plant: fewer embryos were derived from the third leaf (counting from the base of the shoot) than from the first two leaves. The initial pretreatment of pepper explants with increased 6-benzyladenine concentrations significantly reduced the overall proliferation of somatic embryos without affecting the percentage of globular embryos which further developed into the torpedo-shape stage and germinated. Depending on the leaf position, somatic embryo induction was significantly affected by the initial culture incubation under illumination or in darkness. Heart- and torpedo-shaped embryos could be observed only on callus pieces initially incubated for 3 weeks in darkness.
Leaf and cotyledon pieces, shoot apices and hypocotyl segments of sunflower (Helianthis annuus L.) were grown on Murashige and Skoog's medium supplemented with several concentrations of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and/or of the cytokinin 6-benzylaminopurine (6-BAP). Responses differed according to the explant type and the hormonal treatment. 6-BAP was able to induce callus from the different explants. 2,4-D alone produced poorly developed, nodular callus whereas 6-BAP alone induced abundant growth of compact, green callus. Several of the calli induced by 6-BAP regenerated many shoots, some of which flowered in vitro.
Cotyledon and hypocotyl explants of 3 varieties of Capsicum were excised from aseptically germinated seedlings and cultured on a basal nutrient medium supplemented with auxins, cytokinins and auxin-cytokinin combinations. Regeneration of shoot buds was achieved on basal medium supplemented with indoleacetic acid (IAA) and benzyladenine (BA). Presence of 2,4-dichlorophenoxyacetic acid (2,4-D) in the medium promoted friable callus development, but had an inhibitory influence on shoot bud formation. Rooting occurred in media containing IAA or naphthaleneacetic acid (NAA). Shoot buds regenerated from the explants developed roots on the same medium and grew into whole plants and were eventually transferred to soil.
Fertile transgenic sweet pepper (Capsicum annuum var. grossum) plants were regenerated at relatively high rate from various explants that were cocultivated with Agrobacterium tumefaciens strain GV3111-SE harbouring a plasmid that contains the cucumber mosaic virus coat protein (CMV-CP) gene. The rate of plant regeneration was found to depend on the types of explants cultured and the media used. Young leaves were most effective for bud induction and subsequent plant elongation while hypocotyls were the most inefficient. Southern analysis of DNA isolated from putative transgenic plants revealed that 3 out of 5 R1 plant lines reacted positively with the CMV-CP gene. Western blot analysis of CMV-CP containing R1 plants showed that two of them accumulated significant levels of the foreign gene product while the other two expressed it only to low levels. Thus, like many other dicotyledonous plant species, sweet pepper can be transformed by A. tumefaciens and regenerated into healthy, fertile plants that express foreign genes.
Summary Axenic shoot cultures ofCapsicum annuum cv.California Wonder were used as the source for isolation of protoplasts from mesophyll cells. Protoplasts underwent sustained mitotic activity and proliferated to form callus masses on NT or DPD medium enriched with 2,4-D, NAA and BAP each at 1 mg/l level. The callus could be differentiated into whole plants on the differentiation media and plants floweredin vitro under long day conditions.
A vector molecule for the efficient transformation of higher plants has been constructed with several features that make it efficient to use. It utilizes the trans acting functions of the vir region of a co-resident Ti plasmid in Agrobacterium tumefaciens to transfer sequences bordered by left and right T-DNA border sequences into the nuclear genome of plants. The T-region contains a dominant selectable marker gene that confers high levels of resistance to kanamycin, and a lac alpha-complementing region from M13mp19 that contains several unique restriction sites for the positive selection of inserted DNA.
Plant regeneration from mesophyll protoplasts of pepper,Capsicum annuum
L. cv. California Wonder has been demonstrated via shoot organogenesis. Protoplasts isolated from fully expanded leaves of
3-week-old axenic shoots when cultured in TM medium supplemented with 1 mg l
−1
NAA, 1 mg l
−1
2,4-D, 0 5 mg l
−1
BAP (CM 1) resulted in divisions with a frequency ranging from 20–25 %. Antioxidant ascorbic acid and polyvinylpyrrolidone
(PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed
in TM medium containing 2 mg l
−1
NAA and 0.5 mg l
−1
BAP (CM II) and MS gelled medium containing 2 mg 1
−1
NAA and 0 5 mg 1
−1
BAP (CM III), respectively. Regeneration of plantlets was possible via caulogenesis. Microshoots, 2–5 percallus appeared on
MS gelled medium enriched with 0.5 mg l
−1
IAA, 2mg l
−1
GA and l0mg l
−1
BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mg l
−1
NAA and 0.5mg l
−1
BAP. Protoplasts isolated from cotyledons failed to divide and degenerated eventually.
Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 260C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.
The hypocotyl of 3-week-old seedlings of pepper (Capsicum annuum ‘T. Hatvani’) was cut into 6 equal-length segments from the root to the cotyledon. The segments were cultured on Murashige-Skoog medium with 2 mg/l 6-benzylaminopurine and 1 mg/l indoleacetic acid. The orientation of the segments as explants was identical to that of the whole seedling. The segments responded differently on the medium. The apical section produced only shoot buds, the middle segments formed mostly roots, and the basal segments developed abundant callus. No shoot primordia were obtained from the basal section. The hypocotyl segments that produced shoots were subsequently rooted and some of them developed into normal plantlets which could be grown to mature plants by the usual techniques.
Plant transformation vectors and methodologies have been improved to increase the efficiency of plant trans - formation and to achieve stable expression of transgenes in plan ts. Due to the simplicity of the transformation system and precise integration of transgenes, Agro- bacterium Ti plasmid -based vectors continue to offer the best system for plant transformation. Binary vectors have been improved by the incorporation of supe rvirulent vir genes, matrix attachment regions (MAR) and the inser- tion of introns in marker genes and reporter genes. With these improvements and with the use of acetosyringon e, transformation of monocotyledon ous plants using Agro- bacterium has almost become a routine process. The green fluorescent protein (GFP) gene has been extensively modified for plant codon preference, ER/plastid targeting and for greater solubility thereby making it a versatile vital reporter for transgenic plants. Significant progr ess is seen in developing transgenic plants devoid of antibiotic marker genes. Cotransformation of multiple T-DNAs, site-specific recombination strategies and deploy ment of Ac/Ds-based transposition have helped in the elimination of marker genes in transg enic plants. Positive selection strategies using ipt, xylose isomerase and phospho-mannose isomerase have been demonstrated to be useful in many crop plants. The development of BIBAC vectors, a demonstrated capability to transfer multiple genes of a pathway and successful T-DNA tagging in rice, signal the readiness with which transformation technologies can be deployed for the study of 'functional genomics' in plants. The particle bombardment system continues to find use in organelle transformation and tran sformation of plants that lack efficient regeneration systems. A detailed understanding of gene silencing has led to the design of vectors that minimize transgene silencing while ensuring desired levels of transgene expression. Efforts are underway to unde rstand the mechanism of T -DNA integration in plants so that 'knock out' mutagenesis and homology-based gene replacements can be achieved in plants. We review in this article the current status of transformation technologies. An overview of the status of deployment of plant transformation technologies in India is also presented. Plant transformation is performed using a wide range of tools such as Agrobacterium Ti plasmid vectors, microprojectile bombardment, microinjection, chemical (PEG) treatment of prot oplasts and electroporation of protoplasts. Though all methods have advantages that are unique to each of them, transformation using Agrobac- terium and microprojectile bombardment are currently the most extensively used methods 1 . Recent develop- ments in t hese two technologies have been reviewed together with the phenomenon of 'gene silencing' that has come to centrestage after a large number of trans - genic plants have been carefully evaluated for transgene expression in successive generations.