Mature Embryo Explants Research Articles

The Influence of Plant Growth Regulators on Callus Induction in Mature Rice Embryos (Oryza Sativa L) Amber Variety

Several nations across the globe depend profoundly on rice as the main food source. Several research works have been undertaken to improve rice yield and breeding, specifically concerning plant tissue culture. This paper aims to contrast the impact of several growth regulator levels on rice callus induction based on mature embryo explants. Over a four-week duration, mature rice plant embryos (Amber variant) were planted to produce calluses using the Skoog (MS) and Murashige supplements at different concentration levels, i.e., 2.0, 2.5, and 3.0 mg/L of 2,4-D and 0.2 and 0.3 mg/L of BA. The outcomes indicated that 3 mg/L 2,4-D and 0.2 mg/L BA levels led to optimal induction and fresh weight concerning callus. Further studies are required to enhance the media environment for potent in vitro callus induction to augment biotechnology endeavours.

Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of moroccan durum wheat

Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens.

Exploring potential of copper and silver nano particles to establish efficient callogenesis and regeneration system for wheat (Triticum aestivum L.)

ABSTRACT In vitro recalcitrance of wheat to regeneration is the major bottleneck for its improvement through callus-based genetic transformation. Nanotechnology is one of the most dynamic areas of research, which can transform agriculture and biotechnology to ensure food security on sustainable basis. Present study was designed to investigate effects of CuSO4, AgNO3 and their nanoparticles on tissue culture responses of mature embryo culture of wheat genotypes (AS-2002 and Wafaq-2001). Initially, MS-based callus induction and regeneration medium were optimized for both genotypes using various concentrations of auxin (2,4-D, IAA) and cytokinins (BAP, kinetin). The genotypes differed for embryogenic callus induction and regeneration potential. Genotype AS-2002 yielded maximum embryogenic calli in response to 3.0 mg/l 2,4-D, whereas Wafaq-2001 offered the highest embryogenic calli against 3.5 mg/l 2,4-D supplemented in the induction medium. Genotype AS-2002 showed maximum regeneration (59.33%) in response to regeneration protocol comprising 0.5 mg/l IAA, 0.3 mg/l BAP and 1.0 mg/l Kin, while Wafaq-2001 performed best in response to 0.5 mg/l IAA, 0.3 mg/l BAP and 1.5 mg/l Kin with 55.33% regeneration efficiency. The same optimized basal induction and regeneration medium for both genotypes were further used to study effects of CuSO4, AgNO3 and their nano-particles employing independent experiments. The optimized induction medium fortified with various concentrations of CuSO4 or CuNPs confirmed significant effects on frequency of embryogenic callus. Addition of either 0.020 mg/l or 0.025 mg/l CuSO4, or 0.015 mg/l CNPs showed comparable results for embryogenic callus induction and were statistically at par with embryogenic callus induction of 74.00%, 75.67% and 76.83%, respectively. Significantly higher regeneration was achieved from MS-based regeneration medium supplemented with 0.015 mg/l or 0.020 mg/l CuNPs than standard 0.025 mg/l CuSO4. In another study, the basal induction and regeneration medium were fortified with AgNO3 or AgNPs ranging from 1 to 7 mg/l along with basal regeneration media devoid of AgNO3 or AgNPs (control). The maximum embryogenic calli were witnessed from medium fortified with 3.0 mg/l or 4.0 mg/l AgNPs compared with control and rest of the treatments. The standardized regeneration medium fortified with 5.0 mg/l AgNO3 or 3.0 mg/l AgNPs showed pronounced effect on regeneration of wheat genotypes and offered maximum regeneration compared with control. The individual and combined effect of Cu and Ag nanoparticles along with control (basal regeneration media of each genotype) was also tested. Surprisingly, co-application of metallic NPs showed a significant increase in embryogenic callus formation of genotypes. Induction medium supplemented with 0.015 mg/l CuNPs + 4.0 mg/l AgNPs or 0.020 mg/l CuNPs + 2.0 mg/l AgNPs showed splendid results compared to control and other combination of Cu and Ag nanoparticles. The maximum regeneration was achieved by co-application of 0.015 mg/l CuNP and 4.0 mg/l AgNPs with 21% increment of regeneration over control. It is revealed that CuNPs and AgNPs are potential candidate to augment somatic embryogenesis and regeneration of mature embryo explants of wheat.

In vitro responses in Passiflora species with different chromosome numbers, ploidy levels and nuclear 2C values: revisiting and providing new insights

Tissue culture in Passiflora has emerged as a strategy to propagate species with agronomic relevance, which is the main focus of most in vitro studies. Different morphogenic responses have been obtained under the same environmental in vitro conditions, mainly for species of the subgenus Passiflora with distinct 2n chromosome numbers. The aims of this study were to verify and compare the in vitro responses in Passiflora species with distinct 2n chromosome numbers, ploidy levels and nuclear 2C values. Under the same in vitro conditions, only friable calli occurred from mature zygotic embryo explants of Passiflora coriacea (2n = 2x = 12 chromosomes, 2C = 1.00 pg), Passiflora lindeniana (2n = 4x = 24, 2C = 2.42 pg) and Passiflora contracta (2n = 8x = 48, 2C = 4.78 pg). In contrast, plantlets were regenerated from Passiflora foetida (2n = 20, 2C = 1.04 pg) and Passiflora miniata (2n = 18, 2C = 3.40 pg) via indirect organogenesis and indirect somatic embryogenesis, respectively. By now, from mature zygotic embryo explants, de novo shoot organogenesis and somatic embryos have been recovered for Passiflora species with 2n = 18 chromosomes and relative high nuclear 2C value (more than 2C = 2.93 pg—Passiflora cincinata), and only de novo shoot organogenesis for P. foetida with 2n = 20 chromosomes and relative low 2C value (2C = 1.04 pg). Therefore, in a taxonomic and evolutive context, this study showed that the in vitro morphogenic pathways pretty varied between the Passiflora species with distinct karyotype features.

Efficient In Vitro Somatic Embryogenesis and Plant Regeneration from Mature and Immature Embryos of Wheat ( Triticum aestivum L.)

ABSTRACT An efficient regeneration system is a pre-requisite for the application of genetic transformation and functional genomics study of important plants. In this study, the effect of different factors (plant growth regulators, casein hydrolysate, aspartic acid and ascorbic acid) on in vitro embryogenesis and regeneration of Arta, Bahar and Zagros cultivars from mature and immature explants were investigated. Immature and mature embryos were dissected from disinfected seeds 20-25 days after pollination and imbedded mature seeds, respectively, and cultured on MS (Murashige and Skoog) medium supplemented with different compounds. The results showed that immature embryos expose high capacity of embryogenesis and regeneration in comparison with mature embryos. There were significant differences between cultivars in terms of the percentage of callus induction and regeneration. Plant growth regulators had significant effect on percentage of callus induction in mature explants and percentage of regeneration from both explants. In immature explants, the highest percentage of regeneration (65%) was achieved with the Arta cultivar calli derived from MS medium supplemented with 1mg/L 2,4-D, 2 mg/L Picloram and 200 mg/L casein hydrolysate, and subcultured on MS medium. Also, the highest percentage of regeneration (52.38%) from mature embryo explants was achieved in the Arta cultivar with callus induction on MS medium supplemented with 1 mg/L 2,4-D, 2 mg/L Picloram and 200 mg/L casein hydrolysate and regeneration on MS medium containing 0.05 mg/L NAA.

Indirect organogenesis in milkweed ( Calotropis procera ) from mature zygotic embryo explants

Indirect organogenesis in milkweed ( Calotropis procera ) from mature zygotic embryo explants

In vitro morphogenic responses of African yam bean (Sphenostylis stenocarpa (Hochst ex. A. Rich.) Harms) accessions to plant growth regulators

Morphogenic responses of two accessions of African yam bean to different concentrations of plant growth regulator supplements to Murashige and Skoog basal medium was investigated to develop a more efficient regeneration system. Mature embryo explants were cultured on growth regulator-free and BAP + NAA supplemented media. Nodal cuttings excised from 4-week old shoots of the regenerated embryos were cultured on media containing varying concentrations and combinations of 6-benzyl aminopurine (BAP), kinetin and α-naphthalene acetic acid (NAA). Growth regulator-free medium favored embryo regeneration and growth over supplemented media and both enhanced shoot regeneration and rooting, but could not induce multiple shoot formation on embryo explants. Multiple shoots were produced by nodal explants and the highest average number of shoots (5.3 ± 2.3), leaves (7.7 ± 3.6), roots (3.7 ± 2.9) and root length (3.1 ± 0.0 cm) were obtained on a medium with 0.6 mg l−1 BAP + 0.03 mg l−1 NAA for accession TSs154, while in TSs5, highest number of shoots (3.2 ± 2.5) and leaves (5.9 ± 1.5) were induced by 2.0 mg l−1 Kinetin + 0.05 mg l−1 NAA. Such differential morphogenic responses to culture media underline the genotypic control of in vitro propagation of this crop. Embryo and nodal explants rooted directly on shoot regeneration media, and regenerated plantlets were successfully acclimatized. The efficient regeneration system obtained will enhance genetic improvement of African yam bean by facilitating molecular genetic transformation for advanced breeding.

Somatic embryogenesis and plant regeneration from zygotic embryo explants of onion

ABSTRACT: The study was undertaken to examine the effect of two synthetic auxins on somatic embryogenesis and plant regeneration from mature embryo explants of 16 onion cultivars. Cotyledons were removed from the embryos and remaining portions were cultured on MS medium supplemented with 1.0 mg/L 2,4-D and 5.0 mg/L picloram alone or in combination (1.0 mg/L 2,4-D + 2.5 mg/L picloram) to produce embryogenic callus. MS medium supplemented with 5.0 mg/L picloram was found to be the best one for both embryogenic callus induction (85%) and callus diameter (3.8 mm). Of the 16 cultivars studied, Yeoeuijuhwang exhibited the lowest frequency of embryogenic callus induction (50.5%), whereas all the other 15 cultivars showed more than 60% embryogenic callus induction. Scanning electron micrograph (SEM) analysis of embryogenic callus showed all stages of somatic embryos such as globular, scuetellar and coleoptilar. Plant regeneration was significantly affected by the composition of embryogenic callus induction medium. The greatest frequency of somatic embryo conversion was obtained from embryogenic callus developed in MS medium with 2,4-D (70.1%) followed by picloram (38.9%) and 2,4-D + picloram (34.5%). The germinated plantlets were further developed on the half-strength MS medium containing 3% sucrose and were acclimatized in the culture room with 98% survival rate.

Somatic embryogenesis and plant regeneration from zygotic embryo explants of onion

We investigated the effect of two synthetic auxins on somatic embryogenesis and plant regeneration from mature embryo explants of 16 onion cultivars. Cotyledons were removed from the embryos and remaining portions were cultured on MS medium supplemented with 1.0 mg L -1 2,4-D, 5.0 mg L -1 picloram or 1.0 mg L -1 2,4-D and 2.5 mg L -1 picloram. Among the media studied, MS medium supplemented with 5.0 mg L -1 picloram was found to be best for both embryogenic callus induction (85%) and callus growth (3.8 mm). Of the 16 cultivars studied, ‘Yeoeuijuhwang’ exhibited the lowest frequency of embryogenic callus induction (50.5%), whereas all the other 15 cultivars showed more than 60% embryogenic callus induction in all studied media. SEM analysis of embryogenic callus showed all stages of somatic embryos such as globular, scuetellar and coleoptilar. Plant regeneration was significantly affected by the embryogenic callus induction media. The greatest frequency of somatic embryo conversion was obtained from embryogenic callus develop in MS medium with 2,4-D (70.1%) followed by picloram (38.9%) and 2,4-D + picloram (34.5%). The germinated plantlets further developed on the half-strength MS medium containing 3% sucrose and were acclimatized in the culture room with 98% survival rate.

Effects of static magnetic field pretreatment with and without PEG 6000 or NaCl exposure on wheat biochemical parameters

In this study, the static magnetic field (SMF) flux of 2.9–4.7 mT was applied to mature embryo explants of Tekirdag and Selimiye wheat cultivars (Triticum aestivum L.) under tissue culture conditions at three different pretreatment times of 0, 2.2, and 19.8 s, at the rate of 1 m/s with and without 60 g/L PEG 6000 or 100 mM NaCl. Changes in different biochemical parameters were investigated. SMF pretreatment with and without 60 g/L PEG 6000 or 100 mM NaCl increased chlorophyll and carotenoid contents, FRAP values, and antioxidant enzyme (SOD, CAT, POX, and APX) activities in all experimental groups except for the activities of Mn-SOD in the cv. Selimiye root samples. Among SOD isozymes, Fe-SOD was affected by SMF stronger than other izozymes. The combinations of SMF pretreatment with and without 60 g/L PEG 6000 or 100 mM NaCl differently affected SOD isozyme activities besides its effects on other biochemical parameters.

Optimum Protocol for Shoot Formation in karandafi Red Sorghum (<i>Sorghum bicolor</i> (L.) Moench) through Somatic Embryogenesis Using Mature Embryo

In this study, an optimum protocol for shoot formation through somatic embryogenesis using mature embryo explants was developed. Calli were initiated on Murashige and Skoog (MS) media supplemented with varying concentration of 2,4-Dichlorophenoxyl acetic acid (2,4-D) ranging from 1.5 mg/l-4.0 mg/l alone or in combination with 0.5 mg/l Kinetin (KN). Significance difference (p 0.05) was observed among the different concentrations of hormone used for callus induction. The highest percentage callus formation was obtained from the media fortified with 4 mg/l 2, 4-D for mature embryo obtained from imbibed seed while for the preconditioned mature embryo, the media supplemented with 2 m/l 2,4-D + 0.5 mg/l kinetin (KN) recorded more percentage callus formation compared to what was obtained from the media supplemented with 2.5 mg/l2,4-D + 0.5 mg/l. More percentage shoot formation was obtained from the media supplemented with 1 mg/l 6-benzylaminopurine (BA). Average number of shoot per callus was also more in the media fortified with 1 mg/l BA (2.25) but this was not significantly different from what was obtained from the media fortified with 2 mg/l BA+ 0.1 mg/l 2,4-D at 5% level of significance.

Plant regeneration via somatic embryogenesis and organogenesis in Verbascum speciosum Schard

Plant regeneration was achieved in Verbascum speciosum Schard. via organogenesis and somatic embryogenesis by culture of mature embryo explants. Two types of calli, embryogenic and non-embryogenic, were induced from mature embryo explants on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyl adenine (BA) and α-naphthalene acetic acid (NAA). In order to further proliferate the somatic embryoids, the yellow and friable embryogenic calli were transferred on MS medium containing 0.5 mg-1 charchol and 0.1 or 1 mg-1 2,4-dichlorophenoxy acetic acid (2,4-D) or into MS medium containing 60 g-1 sucrose, 50 mgl-1 casein hydrolysate (CH), 0.5 mg-1 kinetin (Kin), 5 mg-1 2,4-D and 0.5 mg-1 charchol. Among the 3 tested media, MS medium containing 0.1 mg-1 2,4-D and 0.5 mg-1 charchol was more effective for proliferation of embryonic calli. Somatic embryos were transferred to hormone free MS medium for maturation and shoot regeneration. In addition, shoots and roots regenerated from non-embryogenic calli in hormone free MS medium or containing NAA and BA. Shoot buds were obtained from non-embryogenic calli and they were transferred to MS medium supplemented with 1 mg-1 BA or Kin for further growth and multiplication. Regenerated plants then were potted and maintained in the greenhouse.

Transfer of a β-glucuronidase marker gene to triticale (xTriticosecale Wittmack) via particle bombardment (biolistic) method

Abstract This study was carried out to investigte the biotechnology laboratory of Department of Field Crops, Ankara University with the aim of investigating the optimum parameters of gene transfer to mature embryo and leaf segment explants of Triticale (x Triticosecale Wittmack) via particle bombardment (Biolistic) method. Mature embryo and leaf segment explants obtained from Triticale (x Triticosecale Wittmack) were bombardment with accelerated gold or tungsten particles coated with the plasmid pBI221.23 containing the b -glucuronidase (GUS: u idA ) marker gene. Different bombardment distances from the stopping plate (6, 9, 12, cm) and different rupture disk pressures (900, 1100, 1550 psi ) were used as physical parameters. Blue cells expressing the b -glucuronidase gene were detected by using a histochemical assay. The variation in the number of blue spots in bombarded explants was used to determine the gene transfer efficiency. The mature embryos of triticale were found to be more amenable for the direct delivery of foreign DNA by the particle bombardment technique than the leaf segments. Also the gold particles were more suitable than tungsten particles in the gene transfer efficiency. The highest gene transfer results were obtained in mature embryos when 6 cm bombardment distance and 1100 psi rupture disk pressure were used.

Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica

A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5 μM). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15 d followed by cycles of 8 h dark and 16 h light, under white fluorescent lamps with a light intensity of 3,000 lm/m2 and at temperature of 28 ± 2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9 mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.

High frequency plant regeneration from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture

An in vitro protocol for efficient plant regeneration has been developed from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture. Embryos with (endosperm-supported culture, ES) or without endosperm (non-endosperm-supported culture, NES) were excised from mature seeds and cultured on MS medium supplemented with various concentrations of 2,4-D (1–5 mg l−1) for callus induction. The percentage of callus induction from ES explants was significantly (P < 0.05) lower than that from NES. The highest frequency (97.6%) of callus induction was obtained from NES explants on MS medium containing 3 mg l−1 2,4-D. When the primary calli were maintained at a reduced concentration of 2,4-D (0.5 mg l−1) for 3 weeks, embryogenic calli were formed. The embryogenic calli were then transferred to MS medium supplemented with different concentrations of BA (1–5 mg l−1) and 500 mg l−1 casein hydrolysate (CH) for shoot regeneration. However, the capacity of plant regeneration from ES explant-derived calli was significantly (P < 0.05) higher than that from NES. The best response (81.3%) was observed from ES explant-derived calli on MS medium containing 2 mg l−1 BA. Regenerated plantlets with well-developed root systems were transferred to pots where they grew well, attained maturity and produced fertile seeds. This method could be employed for genetic manipulation studies.

Comparative analysis of the differential regeneration response of various genotypes of Triticum aestivum, Triticum durum and Triticum dicoccum

An efficient genotype independent, in vitro regeneration system was developed for nine popular Indian wheat cultivars, three each of Triticum aestivum L. viz., CPAN1676, HD2329 and PBW343, Triticum durum Desf. viz., PDW215, PDW233 and WH896, and Triticum dicoccum Schrank. Schubl. viz., DDK1001, DDK1025 and DDK1029, by manipulating the concentration and time of exposure to the growth regulator, thidiazuron (TDZ). A total of 18 (for immature inflorescence and embryo explant) and six (for mature embryo explant) different combinations of growth regulators were tried for callusing and regeneration, respectively. Media combination with low concentration of TDZ (2.2 μM) in combination to auxin and/or cytokinin (depending upon culture stage), was found to be effective for immature and mature explants. Compact, nodular and highly embryogenic calli were obtained by using immature embryo, immature inflorescence and mature embryo explants, and regeneration frequency up to 25 shoots/explant with an overall 80% regeneration was achieved. Comparable regeneration frequency was achieved for mature embryo explants. No separate hormone combination for rooting was required and plantlets ready to transfer to soil could be obtained in a short period of 8–10 weeks. This protocol can be used for raising transgenic plants for functional genomics analysis of agronomically important traits in the three species of wheat.

Evaluation of Different Types of Daylily Explants for Somatic Embryogenesis

To improve somatic embryogenesis of Daylilies (Hemerocallis hybrid); six types of explants namely immature seeds, immature embryos, mature embryos, young inflorescence, ovary-sections and filaments of daylily flower buds were investigated as source of explants. Explants from field grown plants were surface sterilized and followed by culturing on MS medium supplemented with 1.5 mg/L NAA and 0.5 mg/L BAP for four weeks. Explants were scored for development of embryogenic tissue and formation of somatic embryos with/without the formation of an intermediate callus stage. Both mature and immature embryo explants produced direct development of embryogenic tissue followed by somatic embryos. Young inflorescence explants developed compact calli and produced roots around cut ends and showed no somatic embryogenesis. Ovary explants exhibited swelling and not produced embryogenesis. Production of embryogenic callus and formation of somatic embryos in filament explants depended on the sizes of flower buds. Explants from 0.5 to 1.2 mm size flower buds produced calli and formed somatic embryos while explants from sizes over 1.2 mm flower buds only non-embryogenic calli. Immature seeds failed to grow. The results indicated that immature and mature embryos and filaments from young flower buds responded better than other explants for developing somatic embryos in daylily.

Plant Regeneration From Mature Avena Tissue Explants

Successful plant transformation is dependent upon a reliable plant regeneration system. In cereals, immature embryos are the explants of choice but are inconvenient due to their temporal nature and maintenance requirements. The effects of three different auxins, 2,4-D, dicamba, and picloram, on two mature tissue explant types, mature embryo and hypocotyl, of three Avena genotypes, GAF/Park, Park, and 88Ab3073, were evaluated for callus formation, somatic embryogenesis (SE), and plant regeneration capabilities. Both mature embryo and hypocotyl explants were proliferated on modified Murashige and Skoog (MS) basal medium containing 5, 10, and 20 μM of each auxin and were regenerated on 1/2-MS medium without hormone. Although plant number was significantly correlated with SE number, it was not absolutely linked to a priori somatic embryogenesis, especially for 88AB3073 explants. While stimulating callus growth, both dicamba and picloram decreased embryogenicity and regenerability over that obtained with 2,4-D. Picloram may be useful for the isolation of SE-related genes as somatic embryogenesis in oat callus was strongly inhibited in the presence of picloram.

Plant regeneration from embryo-derived callus in Phaseolus vulgaris L. (common bean) and P. acutifolius A. Gray (tepary bean).

Regeneration-competent callus of Phaseolus vulgaris and P. acutifolius was obtained from mature embryo explants on a medium containing thidiazuron and indole-3-acetic acid. For the P. vulgaris genotype Xan-159, regeneration was achieved from cotyledon explants, but not from embryonic axis explants. Both explants could be used for the P. acutifolius genotype NI 574 but embryonic axes gave the best results. In-vitro-rooted plantlets of P. acutifolius could readily be established in the greenhouse. For P. vulgaris hardening problems with in-vitro-rooted plantlets could be overcome by means of in vitro grafting. The potential of the described procedure for obtaining transgenic P. vulgaris plants is discussed.

Agrobacterium-mediated transformation of Amaranthus hypochondriacus: light- and tissue-specific expression of a pea chlorophyll a/b-binding protein promoter.

Mature embryos of Amaranthus hypochondriacus (amaranth) were used to develop an in vitro culture system for plant regeneration and genetic transformation. Plants were regenerated from embryo-derived callus cultivated on Murashige and Skoog medium supplemented with 10 µM 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-2-methoxybenzoic acid and 10% coconut liquid endosperm. Transgenic plants were obtained by inoculation of mature embryo explants with a disarmed Agrobacterium strain containing the plasmid pGV2260(pEsc4), which carried the genes encoding neomycin phosphotransferase type II and β-glucuronidase. The presence of transgenes in the genome of transformed amaranth plants and their progeny was demonstrated by Southern blot hybridization. Tissue specific and light-inducible expression directed by a pea chlorophyll a/b-binding protein promoter was observed in transgenic amaranth plants and their progeny.