Hypocotyl Segments Research Articles

Restoration of Mature Etiolated Cucumber Hypocotyl Cell Wall Susceptibility to Expansin by Pretreatment with Fungal Pectinases and EGTA in Vitro

Mature plant cell walls lose their ability to expand and become unresponsive to expansin. This phenomenon is believed to be due to cross-linking of hemicellulose, pectin, or phenolic groups in the wall. By screening various hydrolytic enzymes, we found that pretreatment of nongrowing, heat-inactivated, basal cucumber (Cucumis sativus) hypocotyls with pectin lyase (Pel1) from Aspergillus japonicus could restore reconstituted exogenous expansin-induced extension in mature cell walls in vitro. Recombinant pectate lyase A (PelA) and polygalacturonase (PG) from Aspergillus spp. exhibited similar capacity to Pel1. Pel1, PelA, and PG also enhanced the reconstituted expansin-induced extension of the apical (elongating) segments of cucumber hypocotyls. However, the effective concentrations of PelA and PG for enhancing the reconstituted expansin-induced extension were greater in the apical segments than in the basal segments, whereas Pel1 behaved in the opposite manner. These data are consistent with distribution of more methyl-esterified pectin in cell walls of the apical segments and less esterified pectin in the basal segments. Associated with the degree of esterification of pectin, more calcium was found in cell walls of basal segments compared to apical segments. Pretreatment of the calcium chelator EGTA could also restore mature cell walls' susceptibility to expansin by removing calcium from mature cell walls. Because recombinant pectinases do not hydrolyze other wall polysaccharides, and endoglucanase, xylanase, and protease cannot restore the mature wall's extensibility, we can conclude that the pectin network, especially calcium-pectate bridges, may be the primary factor that determines cucumber hypocotyl mature cell walls' unresponsiveness to expansin.

Efficient regeneration from hypocotyl explants in three cotton cultivars

A high frequency in vitro shoot bud differentiation and multiple shoot production protocol from hypocotyl segments of 8 to 10-d-old seedlings of cotton has been developed. Murashige and Skoog (MS) basal medium with Nitsch and Nitsch vitamins was found to be optimal in shoot regeneration. A combination of 2 mg dm-3 thidiazuron and 0.05 mg dm-3 naphthaleneacetic acid was the most effective for shoot regeneration (76 %) and an average of 10.6 shoots per responding explant. Combination of the cytokinins benzylaminopurine and kinetin induced better regeneration response than their individual treatments. Supplementation of the culture medium with ethylene inhibitor silver nitrate and activated charcoal showed beneficial effects. Optimal rooting was obtained on half-strength MS medium supplemented with 1 mg dm-3 indolebutyric acid and activated charcoal. Scanning electron micrographs of in vitro cultured explants revealed that shoot primordia were formed de novo.

Effect of sugars on auxin-mediated LeEXPA2 gene expression

The paper deals with the effect of sugars on LeEXPA2 expression in tomato (Lycopersicon esculentum) hypocotyl segments, which is a well-studied system for analysis of cell elongation. We tested the interactions between sugars and different plant hormones classically known to be involved in plant growth. We found the induction of LeEXPA2 transcript accumulation to be positively affected by the presence of sucrose and other metabolizable sugars. The effect mediated by sorbitol and by a non-metabolizable glucose analogue (3-O-methyl glucose) is lower, while the structural analogue of sucrose, turanose, leads to any auxin-induced increase in LeEXPA2 transcript abundance.

Gene transformation potential of commercial canola (Brassica napus L.) cultivars using cotyledon and hypocotyl explants

Canola (Brassica napus L.) is one of oil crops cultivated in many areas of Iran. Its molecular breeding and production of varieties with new characteristics using genetic engineering needs the establishment of efficient transformation methods in commercial varieties. In this research transformation potential of 8 commercial cultivars; Licord, SLM046, RGS003, Zarfam, Okapi, Sarigol, Modena and Opera adapted to different regions of Iran was studied using cotyledon and hypocotyl explants. Agrobacterium tumifaciens strain AGL0 containing the plasmid pCAMBIA3301 was used for transformation. Cotyledon and hypocotyl explants after inoculation with Agrobacterium were co-cultivated on MS medium containing 1 mg/l 2,4-D and 4.5 mg/l BAP, respectively. Cotyledonary explants after co-cultivation were transferred on selection MS medium, containing 4.5 mg/l BAP and 3 mg/l phosphinothricin. Hypocotyl explants
were transferred to selection MS medium containing 4 mg/l BAP, 2 mg/l Zeatin and 5 mg/l phosphinotricin. The regenerated plants were analyzed by PCR and histochemical GUS assay for gene transformation. The results showed that all of genotypes had gene transformation potential using hypocotyl segments, while this potential was limited to some cultivars using cotyledonary explants. Among transgenic plants regenerated from hypocotyls, Licord cultivar had the most transformation rate (15.26%) and Sarigol the least (0.2%). Also among regenerated plans from cotyledon, SLM046 cultivars had the most transformation frequency (4.7%), and Modena, Opera and Zarfam cultivars did not respond to transformation.

TDZ-induced plant regeneration in Astragalus cicer L.

We developed a regeneration protocol using thidiazuron (TDZ) with a high frequency in vitro root induction in Astragalus cicer. High in vitro germination ratio (75%) for hard-seeds of A. cicer was also achieved. For this, hypocotyl and cotyledon explants were cultured on Murashige and Skoog medium supplemented with different concentrations of TDZ. The highest frequency of shoot regeneration (53.3%) was achieved from hypocotyl segments through an initial callus growth stage on MS medium containing 0.25 mg/l TDZ. The shoots were cultured on the different strength (1/1, 3/4, 1/2 and 1/4) of basal Murashige and Skoog medium containing different concentrations of NAA. High rooting (100%) and survival (100%) were achieved using half strength MS medium supplemented with 0.25 and 0.50 mg/l NAA.

SOMATIC EMBRYOGENESIS IN CIMICIFUGA RACEMOSA

Since a long time Cimicifuga has been used for therapeutic purposes. The demand for the plant drug Cimicifugae rhizoma has been increased tremendously during the last years. This study aimed to develop a method for somatic embryogenesis in Cimicifuga. Hypocotyl segments were used as explants and cultivated for 2 to 18 weeks on a modified medium according to Murashige and Skoog containing 1 mg L -1 2,4 D. Embryogenic callus occurred after 2 weeks. After transferring this callus onto a hormone-free MS medium with 2.5 g L -1 activated charcoal, somatic embryos were formed and matured. Mature embryos were separated and continued growing on the hormone-free medium. After two months from 100 embryos 80 plantlets were formed but only about 44 of these plantlets could be transplanted after 6 months. The plantlets were cultivated till a size of 3 cm in vitro and than transplanted in a peat substrate for acclimatization. The acclimatized plantlets were transferred into the greenhouse. Plants of a size of 20 cm were planted in the open field. Since 3 years plants derived from somatic embryos have been cultivated in the field and evaluated concerning morphological and physiological properties.

Pyramiding of alleles with different rust resistance specificities in Linum usitatissimum L.

Transgenic flax plants expressing flax rust resistance specificities conferred by L2 and L10 alleles of L locus were produced through Agrobacterium tumefaciens-mediated transformation of hypocotyl segments or anther culture derived-calli. Transgenic plants were characterized by PCR amplification and Southern hybridization. Homozygous transgenic lines containing a single locus of effective transgene were isolated by consecutive progeny analyses of transgenic plants. In addition, homozygous lines were directly obtained from transformation of haploid cells followed by spontaneous or artificial chromosome doubling when anther culture derived-calli were used as the explants. All transgenic plants containing L2 transgene expressed L2 flax rust resistance specificity and had unambiguous infection type (IT) “0” (immune) reactions to flax rust race 22, which is virulent to endogenous L6 and K1 genes present in the untransformed Linola™1084. Transgenic plants containing L10 transgene exhibited various levels of resistance to flax rust race 191. Five plants had IT “fleck” (immune) reactions, similar to the reactions with the L10 rust differential line, Bolley Golden Selection. The enhanced resistance to rust race 191 in these transgenic plants was attributed to the expression of L10 rust resistance specificity. Further evaluation of the transgenic plants containing L2 or L10 transgene to flax rust race 258 and race 247 respectively showed that the endogenous rust resistance specificities were not modified. The implication of this study in producing transgenic flax plants with multiple resistance specificities and for crop improvement using molecular breeding strategy is discussed.

Effect of Sodium Chloride on Establishment of Callus and Organogenesis in Brassica napus L.

In order to produce salt tolerant canola (Brassica napus L.) plants hypocotyl segments of its that were excised from 7-days-old-seedlings cultured in MS medium with various concentrations of PGRs (NAA, IBA, 2, 4-D, KN and BA) and sodium chloride. The best media for induction and growth of callus (6 mg L(-1) 2, 4-D and 2 mg L(-1) BA), production of adventitious shoots (1 mg L(-1) IBA and 1 mg L(-1) BA) and roots (1 mg L(-1) NAA and 0.5 mg L(-1) KN) were determined. Then explants cultured in these media plus 0-273.53 mM L(-1) sodium chloride. Explants in these media established and in some of concentrations of sodium chloride (68.37-102.56 mM L(-1)) produced calli and adventitious shoots and roots better than the same media but free from sodium chloride. This finding suggests that somaclonal variation can yield stable-tolerant canola plants.

Cell wall water content has a direct effect on extensibility in growing hypocotyls of sunflower (Helianthus annuus L.)

It has been proposed that spacing between cellulose microfibrils within plant cell walls may be an important determinant of their mechanical properties. A consequence of this hypothesis is that the water content of cell walls may alter their extensibility and that low water potentials may directly reduce growth rates by reducing cell wall spacing. This paper describes a number of experiments in which the water potential of frozen and thawed growing hypocotyls of sunflower (Helianthus annuus L.) were altered using solutions of high molecular weight polyethylene glycol (PEG) or Dextran while their extension under constant stress was monitored using a creep extensiometer (frozen and thawed tissue was used to avoid confounding effects of turgor or active responses to the treatments). Clear reductions in extensibility were observed using both PEG and Dextran, with effects observed in hypocotyl segments treated with PEG 35 000 solutions with osmotic pressures of > or =0.21 MPa suggesting that the relatively mild stresses required to reduce water potentials of plants in vivo by 0.21 MPa may be sufficient to reduce growth rates via a direct effect on wall extensibility. It is noted, therefore, that the water binding capacity of plant cell walls may be of ecophysiological importance. Measurements of cell walls of sunflower hypocotyls using scanning electron microscopy confirmed that treatment of hypocotyls with PEG solutions reduced wall thickness, supporting the hypothesis that the spatial constraint of movement of cellulose microfibrils affects the mechanical properties of the cell wall.

High-frequency shoot multiplication in Artemisia vulgaris L. using thidiazuron

An in vitro propagation system for Artemisia vulgaris L., a traditional medicinal plant, has been developed. The best organogenic response, including adventitious shoot number and elongation, was obtained when hypocotyl segments were cultured onto MS medium supplemented with 4.54 μM TDZ (N-phenyl-N′-(1,2,3-thidiazol-yl) urea). Up to 28 shoots formed per explant for an optimal duration of exposure of 48 days. Regenerated shoots formed roots when subcultured onto a medium containing 8.56 μM IAA (indole-3-acetic acid). Healthy plantlets were transferred to a garden soil:farmyard soil:sand (2:1:1) mixture for acclimatization, which was successful, and subsequent maturity was achieved under greenhouse conditions over a six-month period. The survival rate of the plantlets varied under acclimatization. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of A. vulgaris. This optimized protocol has been successfully employed for genetic transformation studies in A. vulgaris, which are currently underway in our laboratory.

Inheritance and resistance to insects in CryIA(c) transgenic cabbage

AbstractUsing hypocotyl segments of aseptic seedlings of cabbage (Brassica oleracea var. capitata) as explants, regenerated plants with kanamycin resistance were obtained mediated by Agrobacterium tumefaciens (strain LBA4404). The transformed plants with the CryIA(c) (Bt) gene were confirmed by Southern blotting analysis, indicating the integration of the transgene into the cabbage genome. The majority of the transgenic plants had only a single copy of the inserted CryIA(c) gene. Leaf section bioassays showed that resistance against larvae of diamondback moth in CryIA(c) transgenic cabbage was significantly enhanced. The inheritance patterns of the transgene in T1 offspring of transgenic cabbage were investigated using polymerase chain reaction (PCR) analysis and a kanamycin resistance test on the leaves of young seedlings. The results showed that dominant gene loci, CryIA(c) or neomycin phosphotransferase gene (NPTII), followed Mendelian inheritance, with a ratio of 3:1 segregation in T1 populations.

Micropropagation of Enterolobium cyclocarpum (Jacq.) Griseb

Shoot multiplication was achieved from cotyledon and hypocotyl segments of in vitro germinated seedlings of Enterolobium cyxclocarpum on Murashige and Skoog's medium supplemented with BA (0.5-2.0 mg/L) and IAA (0.1-0.3 mg/L). Among the different concentrations and combination tested, BA (1.0 mg/L) in combination with IAA (0.2 mg/L) stimulated the best response in terms of shoot bud formation from cotyledon expiants in which a maximum of 60% culture responded, with a maximum of 2.7 ± 0.3 shoot buds per expiant with a mean shoot length of 2.0 ±0.2 cm. A maximum of 80% of the hypocotyl expiants exhibited such organogenic response when cultured on MS medium supplemented with 1.5 mg/L BA and 0.2 mg/L IAA with a mean shoot bud number of 4.1 ± 0.3 per expiant and a mean shoot length of 2.7 ±0.1 cm. Elongated shoots could be rooted on the half-strength MS basal medium supplemented with either IAA or IBA. Cotyledonary node expiants showed poor response.

In Vitro Propagation Of Four Watsonia Species

The genus Watsonia has a number of species with potential to be developed as new ornamental crop plants, but to date there are no reports on in vitro propagation of any member of this genus. Seeds from four Watsonia species, Watsonia gladioloides, Watsonia lepida, Watsonia laccata, and Watsonia vanderspuyiae were decontaminated and germinated on one-tenth strength MS media without hormones or sucrose. Shoots were induced from seedling hypocotyl segments when both an auxin [α-naphthaleneacetic acid (NAA)] and cytokinin [N6−benzylaminopurine (BA)] were present in the medium, while root and leaf explants failed to produce shoots. Multiplication of axillary shoots was greatest when only BA (0.5 mg l−1) was added to the medium. Shoot explants propagated in a ‘liquid-shake’ culture exhibited greater growth rates than those on agar-solidified medium, but shoot production varied between species. Meristemoids were induced in all species, but no significant trend was found between growth index (GI) and meristemoid formation, suggesting that reduction in GI may not necessarily be a prerequisite for producing meristemoids. Corm formation was inconsistent and storage organs could only be induced in one of the four species, W. vanderspuyiae. This occurred best at 25°C with 3% sucrose and an agar level of 15%. Indole-3-acetic acid (IAA) and NAA at 1 mg l−1 significantly increased mean number of roots per shoot explant on all four species, while indole-3-butyric acid (IBA) was more effective when applied at 0.1 mg l−1. Plantlet survival ex vitro was negatively affected when NAA and 2,4-dichlorophenoxyacetic acid (2,4-D) were used to root shoot explants for all species. In W. laccata, all auxin treatments [IAA, IBA, NAA, phenylacetic acid (PAA), and 2,4-D] at a concentration of 1 mg l−1 significantly reduced ex vitro survival of plantlets. Successful micropropagation of Watsonia is an important step in the further development of this genus as a horticultural crop.

Efficient Regeneration of the Eucommia ulmoides Oliver Plant from Hypocotyl Explant

A rapid and efficient method for the regeneration of Eucommia ulmoides Oliver, a rare Tertiary species surviving only in China, has been developed. Hypocotyl segments exscinded from 2 -week-old seedlings were chosen as explants. Multiple shoot buds were more significantly induced on medium containing MS basal medium supplemented with 10 μM BAP alone than on other media. The addition of NAA resulted in callus enlargement, but it did not promote the production of multiple shoot buds. However, the adventitious shoots became cirrhotic and stunted after subculturing on an identical medium supplemented with BAP alone. It was found that the addition of 10 μM GA3 to the medium was the most suitable for the stimulation of shoot elongation and further growth. Shoots longer than 2 cm cultured on the medium supplemented with 0.5 μM NAA and 0.1 μM ABA were highly responsive and produced the maximum number of roots per shoot. Regenerated plantlets with expanded leaves and well-developed roots could be successfully transferred to a pot containing vermiculite for acclimatization and then to a greenhouse. The regeneration protocol developed in this study provides the basis for E. ulmoides germplasm conservation, rapid propagation, and further genetic alteration for medicinal compound and EU-rubber production.

Micropropagation of Tilia amurensis via Repetitive Secondary Somatic Embryogenesis

A optimal procedure for plant production via repetitive secondary somatic embryogenesis in Tilia amurensis is described. Somatic embryos were induced directly from the culture of zygotic embryos on medium with 1.0 mg/l 2,4.-D. Repetitive secondary somatic embryos formed on the surface of the cotyledons and hypocotyls except for the radicles when explants of somatic embryos were cultured on medium with 4.0 mg/l 2,4-D. The highest frequency of secondary embryo-genesis was obtained in the cotyledons (90%) and hypocotyls(83.33%) on MS medium with 1.0 mg/L 2,4-D. The average number of secondary embryos per explant was 25.74 in cotyledon and 24.92 in hypocotyl. When the cotyledon and hypocotyl segments from somatic embryos at different developmental stages were cultured on MS medium with 1.0 mg/L 2,4-D, the highest frequency of secondary embryogenesis was obtained from late cotyledonary secondary embryos. Somatic embryos were transferred to MS basal medium and then they germinated within 2 to 4 weeks of culture. Germinated somatic embryos grew normally into plantlets on WPM medium, producing new shoots. The converted plantlets were acclimatized on artificial soil mixture. These results indicate that the repetitive secondary somatic embryogenesis in T amurensis can offer the possibility to use in vitro culture system for the micropropagation.

Direct Somatic Embryogenesis: A Highly Efficient Protocol for In Vitro Regeneration of Habanero Pepper (Capsicum chinense Jacq.)

To induce somatic embryogenesis in habanero pepper (Capsicum chinense Jacq.), the cultivar BVll-03, belonging to the red type, was used. Different explants were evaluated, as were different culture media, the composition of which varied in the content of plant growth regulators. Results showed the formation of somatic embryos from cotyledons, zygotic embryos, germinated zygotic embryos, hypocotyls, and cotyledonary leaves. Explants were cultured on Murashige and Skoog medium supplemented with 2,4-D (9.05 μm). The somatic embryos always formed directly from the explant, without callus formation, and the greatest efficiency was obtained when segments of hypocotyls were cultured, obtaining 175 ± 20 somatic embryos per explant. Only the somatic embryos obtained on Murashige and Skoog medium containing 2,4-D (9.05 μm) and treated with abscisic acid (ABA) (1.89 μm) before their transfer to the germination media (Murashige and Skoog + 1.1 μm GA3) emitted their radicule and expanded their cotyledonary leaves (60%), whereas the remaining embryos did not achieve germination because of different causes (abnormalities, delayed development). Not only is this protocol of somatic embryogenesis the first to be reported for this species (C. chinense Jacq.), but it is also the most efficient reported so far, within the Capsicum genus.

Plant Regeneration from In Vitro Cultured Hypocotyl Explants of Euonymus japonicus Cu zhi

Abstract Adventitious shoots were successfully regenerated from hypocotyl explants of in vitro cultures of Euonymus japonicus Cu zhi. Hypocotyl slices were cultured on Murashige and Skoog (MS) and B 5 basal medium supplemented with varied concentration of different plant growth-regulators, e.g., α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) in combination with 6-benzylaminopurine (6-BA) and kinetin. The study showed that shoots could be directly regenerated from hypocotyl explants without the intervening callus phase; MS medium was more suitable for adventitious shoots regeneration. The ability of hypocotyls segments to produce shoots varied depending upon their position on the seedlings. The highest regeneration rate was obtained with hypocotyl segments near to the cotyledon cultured on MS basal medium supplemented with 1.5 mg L −1 6-BA and 0.05 mg L −1 NAA (63.64%). The regenerated shoots were readily elongated on the same medium as used for multiplication and rooted on half-strength MS basal medium supplemented with 1.0 mg L −1 IBA and 100 mg L −1 activated carbon. After being transferred to greenhouse conditions, 96% of the plantlets were successfully acclimatized. This regeneration system is applied for genetic transformation now.

A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25+/-2 degrees C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.

Direct somatic embryogenesis from explants obtained from in vitro germinated embryonic axes of Camellia sinensis (L.) O. Kuntze

SummaryStudies on direct somatic embryogenesis in several types of explant from in vitro plantlets of tea cultivar TRI 2025 were undertaken to select those most suitable to induce cotyledonary-type somatic embryos. Mature zygotic embryonic axes were surface-sterilised and cultured on MS medium without growth regulators containing 0.6% (w/v) agar. Results showed that 65% of embryonic axes that converted into plantlets at the fifth week of culture had succulent leaves. Several types of explant (normal leaves, large and small succulent leaves, hypocotyl segments and root tips) were isolated from in vitro plantlets at the fifth week and cultured on half-strength MS medium containing 2 mg l–1 6-benzylaminopurine (BAP) and 0.2 mg l–1 naphthalene acetic acid (NAA). Morphological and histological observations on somatic embryogenesis were made. The results indicated that somatic embryos were produced at high frequency (25 – 50%) directly from the surface of hypocotyl segments (HS) and large succulent leaves (LSL) after 6 weeks of culture. Efficient somatic embryogenesis was induced in small succulent leaves (SSL) after 16 weeks. Most somatic embryos originated directly from the cortical tissues of HS or the upper epidermal layers of SSL or LSL. HS and SSL from in vitro plantlets gave the highest production of typical, firm somatic embryos for use in tea improvement programmes and for in vitro conservation of tea germplasm.

Histological study of embryo-like structures initiated from hypocotyl segments of flax (Linum usitatissimum L.)

Cultivation of flax hypocotyl segments on MS medium supplemented with auxin (2,4-D, NAA) and combination of auxin (NAA) and cytokinin (BAP, zeatin) resulted in production of callus on the cut ends of segments and prolonged cultivation in globular structures resembling early stages of somatic embryos. Embryo-like structures protruded on the surface directly from the subepidermal layers of hypocotyl segments. Despite these globular structures closely resembling somatic embryos, histological observations did not reveal their embryogenic character-organogenesis was the predominant developmental morphogenic pathway. Based on our experiments, as well as on critical revision of existing reports on flax somatic embryogenesis, we conclude, that there has not yet been convincing histological proof of somatic embyogenesis from flax hypocotyl segments.