Plant regeneration from cotyledonary explants of jute, Corchorus capsularis L.

A liquid culture protocol was developed to regenerate shoots from cotyledons of germinating seeds of jute (Corchorus capsularis L.). Reproducibility of the protocol was tested in three genotypes of jute. Highest bud differentiation rates into normal shoots (via shoot-forming explants) were obtained on modified Murashige and Skoog's liquid medium containing 2.7 μM α-naphthaleneacetic acid and 4.4 μM benzylaminopurine. Regenerated shoots were excised, and the best root formation could be induced in medium with 2.5 μM indole-3-butyric acid and 1.5% sucrose. Bud primordia were formed directly on the cut surface of the cotyledons. Scanning electron micrographs and histological studies confirmed the organogenic nature of the regenerated shoots. The physical condition of the culture medium and the age of the explants played crucial roles in the induction of shoot development using shoots; 2-day-old explants being optimal. Approximately 70% of the shoots were successfully established in soil after hardening. Received: 20 October 1997 / Revision received: 4 October 1998 / Accepted: 27 October 1998     

Characterization of a lignified secondary phloem fibre-deficient mutant of jute (Corchorus capsularis)

BACKGROUND AND AIMS: High lignin content of lignocellulose jute fibre does not favour its utilization in making finer fabrics and other value-added products. To aid the development of low-lignin jute fibre, this study aimed to identify a phloem fibre mutant with reduced lignin. METHODS: An x-ray-induced mutant line (CMU) of jute (Corchorus capsularis) was morphologically evaluated and the accession (CMU 013) with the most undulated phenotype was compared with its normal parent (JRC 212) for its growth, secondary fibre development and lignification of the fibre cell wall. KEY RESULTS: The normal and mutant plants showed similar leaf photosynthetic rates. The mutant grew more slowly, had shorter internodes and yielded much less fibre after retting. The fibre of the mutant contained 50 % less lignin but comparatively more cellulose than that of the normal type. Differentiation of primary and secondary vascular tissues throughout the CMU 013 stem was regular but it did not have secondary phloem fibre bundles as in JRC 212. Instead, a few thin-walled, less lignified fibre cells formed uni- or biseriate radial rows within the phloem wedges of the middle stem. The lower and earliest developed part of the mutant stem had no lignified fibre cells. This developmental deficiency in lignification of fibre cells was correlated to a similar deficiency in phenylalanine ammonia lyase activity, but not peroxidase activity, in the bark tissue along the stem axis. In spite of severe reduction in lignin synthesis in the phloem cells this mutant functioned normally and bred true. CONCLUSIONS: In view of the observations made, the mutant is designated as deficient lignified phloem fibre (dlpf). This mutant may be utilized to engineer low-lignin jute fibre strains and may also serve as a model to study the positional information that coordinates secondary wall thickening of fibre cells.     

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.     

Effect of urea on the germination and yield of jute (Corchorus capsularis)

Summary The observations on the effect of urea application on the germination and yield of jute under two different soil conditions are reported.     

Genetic variation and phenotypic plasticity of roots in two cultivated species of jute (Corchorus olitorus L. andC. capsularis L.)

1. 1.
   A number of varieties of cultivated juteGorchorus oHtorius L. andG. capsularis L. were tested in artificially created drought, waterlogging and control conditions.     
   
   

Inheritance of a male sterile complex mutant in white jute (Corchorus capsularis L.)

A perfectly monofactorially segregating male sterility mutant in jute, associated with a morphological marker (ribbon leaf, rl) was found to form a pleiotropic complex with several undesirable characters, which make it unsuitable for hybrid breeding, in spite of unimpaired viability. It is improbable that the unfavorable characters can be made harmless by selection for compensating genes.     

Alteration of reproductive effort by a monogenic,recessive mutation in jute (Corchorus capsularis L.)

Summary Reproductive effort (RE) of a day-length neutral mutant TCJ-5, its parent, and two other cultivars of jute (Corchorus capsularis) was estimated as reproductive biomass/aerial biomass. Plant height at flowering and aerial biomass were significantly higher in the mutant, while the reproductive biomass at 55 days after flowering was statistically equal. Therefore, the estimated RE was significantly lower in the mutant compared to the parent and other cultivars. The lower RE of the mutant was due to delayed initiation of flowering and additional vegetative growth in this period. The results show alteration of RE by a recessive mutation.     

Stimulation of shoot regeneration from jute cotyledons cultured with non-ionic surfactants and relationship to physico-chemical properties

Summary The effects have been studied of the non-ionic surfactants, Plutonic F-68, Tween 20 or Triton X-100, on shoot regeneration from cultured jute (Corchorus capsularis L.) cotyledons with attached petioles. This group of non-ionic surfactants was selected in order to determine a possible relationship between the physico-chemical properties of individual compounds and their observable effects on plant morphogenesis in cultured jute cotyledons. Supplementation of culture medium with 0.001–0.5% (w/v) Pluronic F-68 increased the mean percentage of cotyledons producing shoots and the mean number of shoots/cotyledon, with maximal responses at 0.5% (w/v). By contrast, Tween 20 produced maximal effects at 0.001% (v/v), with inhibition of shoot formation at 0.5% (v/v). In both cases, phenotypically normal plants were recovered which could be grown to maturity. Culture of cotyledons with 0.001% (v/v) Triton X-100 similarly increased both the percentage of cotyledons producing shoots and the number of shoots/cotyledon. However, these shoots did not develop into mature plants. Additionally, shoots did not regenerate from cotyledons cultured with Triton at 0.01–0.5% (v/v). These results demonstrate mat there is an apparent relationship between the hydrophilic-hydrophobic (HLB) balance of surfactants which determine their cell permeabilising properties and consequently, their effects on morphogenesis.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962)     

Plant regeneration from protoplasts of Panax ginseng (C.A. Meyer) through somatic embryogenesis

Summary Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22–25 × 10⁶ protoplast / g tissue were obtained following 5–6 h digestion with 2% Cellulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of 1×10⁵ protoplasts /ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus, which regenerated into plantlets.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberellic acid - MS Murashige and Skoog medium     

Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation

Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.     

Plant regeneration through somatic embryogenesis in root-derived callus of ginseng (Panax ginseng C. A. Meyer)

Summary Callus culture was initiated from expiants of mature root tissues of ginseng (Panax ginseng C.A. Meyer) on MS medium enriched with 2,4-D. The ageing callus produced numerous embryoids in this medium. Reculture of these embryoids in media (1/2 MS or B5) supplemented with benzyladenine and gibberellic acid resulted in profuse plantlet regeneration.     

Plant regeneration from protoplasts of Solanum species with potential agricultural value (S. hjertingii,S. polyadenium,S. capsicibaccatum)

Protoplasts have been isolated from three tuber-bearing Solanum species, S. hjertingii, S. polyadenium and S. capsicibaccatum, that are sexually incompatible with S. tuberosum, but possess potentially useful characters. For isolating protoplasts from leaves of in vitro shoot cultures of S. hjertingii and S. capsicibaccatum growth was improved by including silver thiosulfate in the medium. However, for S. polyadenium, leaves of pot-grown plants were the best source for protoplasts. Following protoplast division and culture, plants were regenerated from protoplasts of each of the species. The pattern of chromosome variation in regenerants was similar to that observed for other diploid and tetraploid Solanum species. The results indicate that it should be possible to introduce the potentially useful germplasm from these wild species into somatic hybrids with S. tuberosum by protoplast fusion.Abbreviations STS silver thiosulfate - BAP benzylaminopurine - GA₃ gibberellic acid - NAA naphthalene acetic acid - IAA indole-3-acetic acid     

First report of bacterial leaf blight of jute (Corchorus capsularis L.) caused by Xanthomonas campestris pv. capsularii in India

Xanthomonas campestris pv. capsularii causing blight on jute (Corchorus capsularis) leaves was reported for the first time in India. The symptom of the disease initially observed was appearance of small angular brown leaf spots and later as blighted areas on leaf lamina. The disease-causing pathogen was isolated and identified on the basis of its colony morphology, PCR, sequencing and subsequent BLASTn analysis.     

Plant regeneration from protoplasts of a wild lettuce species (Lactuca saligna L.)

Protoplasts isolated from young, unexpanded leaves of the wild lettuce species, Lactuca saligna, divided to give colonies, when plated at low density in KP8 medium solidified with 1% (w/v) agarose. Sustained colony development was dependent upon the incorporation of the bead culture approach. Plant regeneration, via organogenesis, followed the transfer of colonies firstly to K8 medium and then to M/S medium supplemented with IAA and 6-BAP, both solidified with 1% (w/v) agarose. Excised shoots were rooted on agar-solidified M/S medium lacking phytohormones. The potential of this species as a source of race-non-specific mildew resistance for transfer into lettuce (L. sativa) via somatic hybridisation is discussed.Abbreviations 6-BAP 6-benzylamine purine - IAA indoleacetic acid - M/S Murashige and Skoog (1962) - fwt fresh weight     

Plant regeneration from seeds responds to phylogenetic relatedness and local adaptation in Mediterranean Romulea (Iridaceae) species

Seed germination is the most important transitional event between early stages in the life cycle of spermatophytes and understanding it is crucial to understand plant adaptation and evolution. However, so far seed germination of phylogenetically closely related species has been poorly investigated. To test the hypothises that phylogenetically related plant species have similar seed ecophysiological traits thereby reflecting certain habitat conditions as a result of local adaptation , we studied seed dormancy and germination in seven Mediterranean species in the genus Romulea (Iridaceae). Both the across‐species model and the model accounting for shared evolutionary history showed that cool temperatures (≤ 15°C) were the main factor that promoted seed germination. The absence of embryo growth before radicle emergence is consistent with a prompt germination response at cool temperatures. The range of temperature conditions for germination became wider after a period of warm stratification, denoting a weak primary dormancy. Altogether these results indicate that the studied species exhibit a Mediterranean germination syndrome, but with species‐specific germination requirements clustered in a way that follows the phylogenetic relatedness among those species. In addition, species with heavier seeds from humid habitats showed a wider range of conditions for germination at dispersal time than species from dry habitats possessing lighter seeds. We conclude that while phylogenetically related species showed very similar germination requirements, there are subtle ecologically meaningful differences, confirming the onset of adaptation to local ecological factors mediated by species relatedness.     

Plant regeneration and morphology during in-vitro organogenesis fromHeloniopsis orientalis (Thunb.) C. Tanaka

Heloniopsis orientalis (Liliaceae) is an important horticultural crop native to Korea. Under natural conditions, germination is poor and plant growth is delayed. Therefore, we have developed a vegetative propagation method to produce plants with vigorous growth characteristics via tissue culture. Leaf tissues were cultured on MS basal media supplemented with growth regulators 2,4-D, TDZ, BA, or zeatin. The regenerated shoots were then initiated directly from leaf expiants on an MS medium containing either 0.1 to 1.0 mg/L 2,4-D or 0.1 to 3.0 mg/L BA. Healthy plantlets with adventitious roots were formed on the medium supplemented with 0.1 mg/L BA. TDZ triggered callus initiation without caulogenesis or rhizogenesis, and callus formation was better on the half-strength MS medium than on the full-strength medium. After the plants were acclimatized for one month at 4°C, they were successfully transferred to soil. In addition, we used LM and SEM to investigate shoot morphogenesis at various stages of differentiation.     

Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae)

Key message

Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species.

Abstract

Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants.