Heterochromatin and repetitive DNA frequency variation in regenerated plants of Helianthus annuus L.

Plant regeneration from cotyledons of seeds of a single progeny of a pure line of Helianthus annuus was studied in respect of the nuclear DNA contents of control and regenerated plants. Control plants were divided into two groups: those developed from seeds at the periphery of the inflorescence (showing a high basic 4C DNA content) and those from seeds developed in the middle of the inflorescence (showing a low basic 4C DNA content). It was observed that plants from peripheral seeds have a higher morphogenetic potential than those from central seeds. Cytophotometric analyses indicated that plants regenerated from cotyledons of both peripheral and central seeds show the same basic 4C DNA amount, which is higher that that observed in vivo in peripheral seeds. Molecular analysis by slot blotting and hybridization with different DNA families showed that the difference in nuclear DNA content between plants from peripheral and central seeds in vivo are mainly related to differences in the frequency of highly repeated, slow medium repeated (MR2), and ribosomal DNA families; by contrast, the increase in DNA amount in regenerated plants is mainly due to fast medium repeated sequences (MR1). Moreover, the frequency of kinetically isolated unique sequences was higher in peripheral seeds than in central ones and still higher in regenerated plants. Optical-density measurements of interphase nuclei showed an increase of heterochromatin in regenerated plants, suggesting that, whatever DNA is amplified in these plants, it remains condensed and probably inactive.Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 2, Paper No. 2069     

Effects of genotype,explant orientation,and wounding on shoot regeneration in tomato

Summary Effects of genotype and explant orientation on shoot regeneration from cotyledonary explants of tomato were studied using 10 commercially important cultivars. The explant orientation affected shoot regeneration in all the tested genotypes. Cotyledons placed in abaxial (lower surface facing down) orientation consistently produced better shoot regenerative response and produced greater numbers and taller shoots compared to those inoculated in adaxial (upper surface facing down) orientation. Genotypic variation in terms of shoot regeneration response, number of shoots, and shoot height was apparent. Wounding of cotyledonary explants increased shoot regeneration response. However, shoot height was much lower in shoots regenerated from wounded explants compared to those that originated from intact cotyledons. Shoots produced from wounded cotyledons were abnormal in their form and structure.     

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

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

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

Detection of protoplast-derived DNA tetraploid Lisianthus (Eustoma grandiflorum) plants by leaf and flower characteristics and by flow cytometry

Plants of lisianthus (Eustoma grandiflorum (Griesbach)Schinners=Lisianthus russellianus Hook.) were regenerated from protoplasts and grown in pots until flowering. Vegetative and floral characteristics were measured and compared with parent plants. Larger leaves and petals and longer guard cells, sepals and filaments were recorded from protoplast-derived plants suggestive of polyploidy. The nuclear DNA contents of protoplast-derived and parental plants were determined by flow cytometry. Protoplast-derived plants were confirmed as DNA tetraploid by flow cytometry with a DNA index of 1.95. Their nuclear DNA content was measured as 6.33±0.04 pg DNA per 2C nucleus compared with 3.26±0.10 pg DNA per 2C nucleus from parental plants. Polyploidisation induced during protoplast regeneration offers an alternative to that of colchicine treatment.     

Plant regeneration from immature cotyledon explant cultures of bean (P. coccineus L.)

A method for plant regeneration, via organogenesis and somatic embryogenesis, from P. coccineus is described. Immature cotyledons from plants regenerated and cloned in previous experiments were used. The highest percentage of regeneration (37.5%) was observed from the cotyledons of clone C7 on a modified Murashige & Skoog basal medium to which (2-isopentenyl)adenine (2iP, 10 mg l^-1) and 2-naphthoxyacetic acid (NOA, 0.05 mg l^-1) were added. On average, the same medium gave the highest percentage of regeneration also from the cotyledons of the 7 clones of P. coccineus used in the experiment. Newly regenerated plants were normally fertile. The procedure described may serve for induction of somaclonal variation for in vitro selection of valuable genotypes and for genetic transformation by A. tumefaciens.Abbreviations 6BA 6-benzylaminopurine - CCC chlorocholine chloride - IAA indole-3-acetic acid - 2iP (2-isopentenyl)adenine - NOA 2-naphthoxyacetic acid     

Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

High Efficiency Organogenesis in Sweet Pepper (Capsicum annuum L.) Tissues from Different Seedling Explants

In vitro plant regeneration was achieved from eightsweet pepper varieties (Capsicum annuum L.). The effect ofvarious explant types (cotyledons, leaves, cotyledonary nodes and shoot-tip from25-day-old seedlings and embryonic cotyledons, embryonic hypocotyls and woundedseedlings) on bud and shoot regeneration and shoot elongation was evaluated.Differences in ability for in vitro shoot regeneration andelongation depended on the variety and explant type. In general, highregeneration frequency was obtained from all varieties. Agridulcedisplayed the highest regeneration response: an average of 3.45 elongated shootsper explant using embryonic cotyledons. Elongated shoots were excised and rootedon Murashige and Skoog (MS) basal medium either without plant growth regulatorsor with 0.5 IBA (indole-3-butyric acid) or 0.05 NAA (-naphthaleneacetic acid). Plantlets weretransplanted to soil and acclimatised in the greenhouse showing normaldevelopment and growing to maturity bearing normal fruits with seeds.     

A method for increased plant regeneration from immature F1 and BC1 embryos of Cucurbita maxima Duch. x C. pepo L. hybrids

Plants have been regenerated from abnormal embryos with spongy cotyledons and albino sectors, derived from Cucurbita maxima and C. pepo F₁ and BC₁ hybrids. Shoot regeneration was induced directly from the cotyledons without an intervening callus phase on the medium without hormones. On the rooting medium, shoots continued to proliferate, which allowed for further multiplication in vitro. The number of plants obtained varied with genotype and ranged up to 65 plants per embryo.     

Prolific direct plant regeneration from cotyledons of white clover

Summary A facile procedure has been developed to regenerate white clover (Trifolium repens L.) plants, rapidly and directly from cotyledon explants of 3 day old seedlings. Scanning electron microscopy and histological sectioning demonstrated that shoot meristems developed from individual epidermal cells on the adaxial surface of the cotyledonary stalk, proximal to the site of excision. Initial cell divisions occurred after 2 days of culture and regenerated plants were transferred to soil within 6–8 weeks. Regenerated plants were normal, flowered and set seed. The highest shoot regeneration frequency (an average of 20 shoots per cotyledon) was obtained using an MS based medium containing 1.0 mg 1^-1 6-benzylaminopurine and 0.05 mg 1^-1 -napthaleneacetic acid. A similar regeneration frequency was obtained from cotyledon explants taken from eight different white clover cultivars.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - MS Murashige and Skoog medium     

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate     

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differences in hormonal requirements, the various explants showed preferential growth in different basal media.     

Responsive regions for direct organogenesis in sunflower cotyledons

Regeneration efficiency from three different regions of cotyledonary explants was examined in six sunflower inbred lines. Proximal, middle and distal regions from seedling cotyledons were cultured on regeneration medium supplemented with growth regulators. Plant regeneration by direct organogenesis was observed after four weeks. Significant differences among inbred lines were found for regeneration percentage and average number of shoots per total explants. Also a decreasing regeneration capacity was observed from proximal to distal sections for all inbred lines. Regeneration ability from cotyledonary explants in this species is strongly influenced by the genotype and by the region from which the explant was obtained. The distance to the cotyledonary node plays a preponderant role in the expression of shoot forming capacity. Shoot differentiation via seedling cotyledons depends upon the presence of the proximal region of cotyledon regardless of the genotype.     

Maturation,germination, and conversion of norway spruce (Picea abies L.) somatic embryos to plants

Summary Quantitative data are presented on the efficiency of three stages of plant regeneration from somatic embryos of Norway spruce (Picea abies L.): 1) Maturation, the development of immature embryos to the cotyledonary stage; 2) Germination, primary root growth; and 3) Conversion, plantlet survival and continued growth in nonaxenic conditions. Maturation frequency was calculated relative to the number of immature somatic embryos induced to develop on the basal medium of von Arnold and Eriksson (1981). The average number of immature somatic embryos was 700 per gram of embryogenic callus, on medium supplemented with ABA and IBA (1 μM each). Maturation was the least efficient stage of regeneration; an average of 3% of the embryos induced to develop reached the cotyledonary stage. Mean germination frequencies were improved on treatments which avoided immersion of the radicle in medium solidified with agar. Whereas, 27% of the somatic embryos germinated when radicles were immersed in agar medium, 45% germinated when placed on the surface of the medium, and 56% germinated when cotyledons were immersed in agar medium and the culture vessel inverted. Twenty-nine percent of the somatic embryos germinatedin vitro were converted to plants. Under greenhouse conditions these plants set dormant buds, subsequently survived overwintering (to −5°C), and renewed vegetative growth synchronously with seedlings grown under the same conditions. Our results verified long-term (2 year) growth and development potential of conifer somatic embryo plants.     

Effect of Crinipellis perniciosa Infection on Abscission of Cacao Cotyledons, Reserve Mobilization and Dry Matter Partitioning

Infection by Crinipellis perniciosa on cacao seedlings prevented the abscission of cotyledons which normally abscise within 32–61 days from seed sowing. The early growth and development of control and artificially inoculated seedlings was investigated for root dry mass (RDM), shoot dry mass (SDM), shoot‐to‐root ratio (SRR), total dry mass (TDM), cotyledonary dry mass (CDM) and cotyledonary water content (CWC). Cotyledonary reserve mobilization was estimated at 69 and 56% at 5 weeks and 79 and 64% at 8 weeks in control and infected seedlings, respectively. Infection induced the enlargement of tissues but was not accompanied by an increase in dry mass. Infection both delayed and reduced the utilization of cotyledonary food reserves resulting in slower accumulation of RDM, SDM and TDM culminating in a reduction of growth by a factor of 1.6 at 8 weeks. However, the SRR of inoculated plants was similar to that of control plants, suggesting that the balance between the root and shoot systems exists which is unchanged by the effect of the pathogen. CWC was decreased upon senescence to an average of 65% at abscission. The prevention of cotyledonary abscission in infected seedlings is suggested to be due in part to the CDM remaining above a critical level of 20% of the initial CDM. The implication of this response in relation to the infection biology of the pathogen is discussed.     

Effect of age of seedling and phytohormones on micropropagation of indica rice (Oryza sativa L.) from Meristem Culture.

An efficient protocol forin vitro micropropagation of seven indica rice varieties was developed from meristem culture. Meristem (leaf base) was isolated from different age of seedlings and cultured on MS medium without hormones and supplemented with different concentrations of NAA and BAP. Regeneration of plantlets from meristem was observed within five days of culture. The meristem isolated from 4-day old seedlings gave highest regeneration on hormone free MS medium. Histological study of meristem (leaf base) from 4-day old seedlings confirmed the presence of meristematic cells. Regenerated plants were multiplied on MS medium supplemented with 0.05 mg/L NAA and 5 mg/L BAP. An average of five plants were obtained from single regenerated meristem. The plants regenerated from meristem showed morphological uniformity.     

Regeneration and transformation in adult plants of Campanula species

Adult plants are known for recalcitrance when it comes to adventitious organ formation and regeneration. Methods used for regeneration in explants from seedlings of Campanula carpatica failed to work for explants from adult plants of the same species. The present investigation generated efficient regeneration methods for mature specimens of four species of Campanula, C. carpatica, C. haylodgensis, C. portenschlagiana and C. poscharskyana. Petiole explants from dark-grown in vitro shoot cultures grown from nodal cuttings of adult plants regenerated successfully (95%), while explants from light-grown in vitro shoot cultures and greenhouse-grown plants regenerated at 12% and zero percentage, respectively. Dark-treatment, along with media manipulation with plant growth regulators, further enhanced regenerative capacity of the explants. A MS-based medium containing 10mg l ⁻¹ TDZ and 0.25 mg l⁻¹ NAA was the most efficient regeneration medium. Transgenic shoots from C. carpatica (3%) and C. haylodgensis (1%) and transgenic callus from all species were produced using Agrobacterium tumefaciens, and transformation was confirmed by histochemical and Southern blot analyses. Protocols developed in this study may be useful for achieving efficient regeneration and transformation of recalcitrant adult plants.     

Organogenesis and embryogenesis from diverse explants in pigeonpea (Cajanus cajan L.)

Seed and seedling expiants of pigeonpea were evaluated for organogenesis and somatic embryogenesis. De novo plant regeneration through organogenesis was obtained from mature cotyledons, primary leaves and roots of seedlings. Production of multiple shoots from the cotyledonary node was observed in cultures of whole seeds on 6-benzylaminopurine enriched medium. Somatic embryos were induced from immature cotyledons and embryonal axes, however, well-developed plants could not be derived from these embryos. The regenerants obtained through organogenesis were transferred to the field and grown to maturity.     

Multiple shoots induction in wild cotton (<Emphasis Type="Italic">Gossypium bickii</Emphasis>) through organogenesis and the analysis of genetic homogeneity of the regenerated plants

Fertile plants were regenerated via organogenesis from cultures of Gossypium bickii Prokhanov cotyledonary nodes devoid of cotyledons. Cotyledonary nodes from 7-day-old seedlings yielded the maximum number of shoots (9.12 shoots per explant) using MSB medium [MS medium (Murashige & Skoog 1962) and B5 (Gamborg et al. 1968) vitamins] supplemented with the combination of 4 mg/L BAP (N6-benzyladinine) and 0.1 mg/L TDZ (thidiazouron), and the subculture was made on a medium containing 2 mg/L BAP. Elongation of multiple shoots was obtained on MSB medium containing 0.05 mg/L GA₃ (gibberellic acid). For rooting, a modified half-strength MS medium was utilized. Some regenerants were transferred to a greenhouse, and most of them were morphologically normal and fertile. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants. Flow cytometric analyses and chromosome counting suggested that the regenerants and their offsprings were diploid. DNA content of regenerants and their offsprings was homogeneous and similar to that of seed-derived plants with unchanged chromosome number (2n = 26). Random amplified polymorphic DNA (RAPD) analysis confirmed the genetic homogeneity of the regenerants and their offsprings with the diploid parent.