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1.
An efficient method of repetitive somatic embryogenesis and plant regeneration was established in Coriandrum sativum L. Embryogenic callus was induced from cotyledon and hypocotyl segments on Murashige and Skoog (MS) medium with 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), upon subculturing on medium having same level of 2,4-D at an interval of 3 weeks developed somatic embryos, which progressed to cotyledonary stage through early developmental stages of somatic embryogenesis. The transfer of somatic embryos at an early cotyledonary and cotyledonary stage in clumps in succession to fresh 4.52 μM 2,4-D supplemented medium developed embryos in a cyclic manner. Upon transferal to embryogenic clumps (cotyledonary embryos) to modified MS medium (4 g l−1 KNO3, 0.29 g l−1 NH4NO3, 3 mg l−1 thiamine HCl, 0.5 mg l−1 pyridoxine HCl, and 5 mg l−1 nicotinic acid), the embryos irrespective of the cycles underwent maturation and germination. Germinating embryos transferred to half-strength MS medium favored healthy growth of plantlets. The system of recurrent somatic embryogenesis in coriander offers a system for genes transfer and also scale-up production of modified plants.  相似文献   

2.
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3.
We present here a method for the regeneration of the prickly-pear (Opuntia ficus-indica (L.) Mill.) through somatic embryogenesis (SE). Direct SE was induced by cultivating shoot apices devoid of leaf primordia on semisolid Murashige and Skoog (MS) basal medium supplemented with 4-amino 3,5,6-trichloropicolinic acid (picloram) at 4 mg l−1. Somatic embryogenesis was influenced by the type, age, physiological and developmental stage of the explants, and also by light conditions, wounding, and the sucrose concentration in the induction medium. A histological analysis confirmed SE by revealing the presence of a closed vascular system in the developing embryos and the absence of a vascular connection between the somatic embryos and the explant.  相似文献   

4.
Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l−1 sucrose, 2.5 g l−1 gellan gum, 0.1 mg l−1 α-naphthaleneacetic acid (NAA), 0.1 mg l−1 6-benzyladenine (BA) and 50 mg l−1 gibberellic acid (GA3). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l−1 sucrose and 3 g l−1 gellan gum, without plant growth regulators (PGRs) or with 1 mg l−1 zeatin and 0.1 mg l−1 NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.  相似文献   

5.
Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l−1) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l−1) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l−1 ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l−1) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.  相似文献   

6.
Pineapple guava (Acca sellowiana) syn. Feijoa sellowiana, a Brazilian indigenous Myrtaceae is under domestication in South Brazil. Previous works showed that this species is responsive to somatic embryogenesis and recalcitrant to conventional methods of clonal propagation. In the present work it was evaluated the role of components of culture medium in the induction and development of somatic embryos. The technology of synthetic seeds was also evaluated. Zygotic embryos were inoculated in LPm medium supplemented with 8 mM glutamic acid and 8 mM l-glutamine, 2,4-dichlophenoxiacetic acid (20 μM) and myo-inositol. For conversion of somatic embryos and synthetic seeds it was tested the effect of 6-benzylaminopurine and gibberellic acid combined or not with activated charcoal. The highest values for embryogenetic induction (100%) and number of somatic embryos/explant (113) were observed in the LPm medium supplemented with Glu (8 mM), and 2,4-D. The culture medium supplemented with BA (0.5 μM) and GA3 (1 μM) and activated charcoal (1.5 g L−1) enhanced the conversion of somatic embryos to plantlets. Pre-germinated somatic embryos encapsulated in sodium alginate with BA (0.5 μM) and GA3 (1 μM) developed radicles. The use of synthetic seed was a requisite for the survival of plantlets.  相似文献   

7.
Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L−1 indole-3-acetic acid (IAA), 0.3 mg L−1 kinetin, 30 g L−1 sucrose and 8 g L−1 agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L−1 IAA, 0.5 mg L−1 zeatin, 30 g L−1 sucrose and 8 g L−1 agar; (3) 96.67% rooting on MS medium containing 30 g L−1 sucrose and 8 g L−1 agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.  相似文献   

8.
High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO3 on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l−1, 6-BA at 3.0 mg l−1, AgNO3 at 4.0 mg l−1 and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l−1, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.  相似文献   

9.
The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10−4, 6.7 × 10−4 and 13.4 × 10−4 M). The liquid medium with 0.1 mg l−1 2,4-d and 6.7 × 10−4 M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10−4 to 6.7 × 10−4 M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10−4 to 13.4 × 10−4 M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l−1 2,4-d and 6.7 × 10−4 M glutamine (118 and 91 mg of proteins g−1 DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g−1 DW for “Jihel” cultivar and 182 mg of sugars g−1 DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l−1 BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l−1, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.  相似文献   

10.
This study describes a successful method of somatic embryogenesis and genetic transformation using immature cotyledons of Prunus mume. Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somatic embryogenesis and genetic transformation protocols. Somatic embryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of MS basic medium supplemented with 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM 6-benzyladenine (BA). They were cultured for 30 days and then transferred to somatic embryo propagation medium containing 0.1 μM α-naphthaleneacetic acid (NAA) and 5 μM BA. It appeared that the developmental stage of the immature cotyledons used as explants was the most important factor for somatic embryogenesis; higher frequencies of somatic embryogenesis were observed when the immature cotyledons were less than 5 mm in length regardless of cultivars. For genetic transformation, the immature cotyledons were inoculated with Agrobacterium tumefaciens EHA101 harbouring a binary plasmid vector with neomycin phosphotransferase II and an intron-interrupted β-glucuronidase gene under the control of cauliflower mosaic virus 35S promoter, and three transgenic plant lines were obtained from inoculated “Sirakaga” immature cotyledons. Transgenic somatic embryos and shoots were selected using 25 mg l−1 kanamycin. Integration of transgenes in the genome of GUS-positive putative transgenic shoots was confirmed by PCR and Southern blot analyses.  相似文献   

11.
The objective of this study is to establish plant regeneration system with the seed of the new Chinese selection “E-126”of centipedegrass [Eremochloa ophiuroides (Munro.) Hack] as explant. In present study, the following results were obtained: (1) The medium formulation most suitable for calluses induction was identified to be MS with 1.0 mg l−1 2,4-D + 30 g l−1 mannitol + 50 ml l−1 coconut milk and the ratio of calluses induction was 96.0%, including 5.2% of yellow granule calluses induction. The above medium formulation was adopted for subculture. (2) The rate of shoot regeneration from yellow granular calluses was 98.0% by MS optimum medium formulation with 2.0 mg l−1 KT + 50 ml l−1 coconut milk. The differentiated rate retained as high as 88.0% even after 5 times of subculture and 18.6% after 15 times of subculture. The optimum medium formulation for shoot growth was identified to be MS medium plus 2.0 mg l−1 BAP, 0.8 mg l−1 NAA and 50 ml l−1 coconut milk. (3) The optimum medium for shoot rooting was identified to be MS medium with 0.6 mg l−1 NAA + 50 ml l−1 coconut milk, and the rooting rate to be 98.0%. The survival rate of transplanted plantlets from tubes to basin with soil was 92.0%. In conclusion, the plant regeneration system was successfully developed in this study, which may provide basic reference for screening of somaclonal variants and genetic transformation of centipedegrass.  相似文献   

12.
An efficient plant propagation system through somatic embryogenesis was established in Cymbopogon pendulus, an aromatic grass followed by analysis of genetic status of regenerants using ISSR markers. Optimum embryogenic callus induction was observed on MS basal medium supplemented with 13.57 μM 2,4-dicholorophenoxyacetic acid (2,4-D) with 8.88 μM N6-benzyladenine (BA). Subsequent culturing of embryogenic calli on MS medium containing 4.52 μM 2,4-D and 8.88–13.32 μM BA gave maximum number of somatic embryos. Addition of coconut water (CW) promoted induction, growth and differentiation of callus and somatic embryogenesis. Further development of embryos into plantlets was achieved on MS medium supplemented with lower concentration of biotin and calcium pantothenate (CaP) along with BA (4.44–13.32 μM) and kinetin (2.32–4.65 μM). The root meristems were established on half strength MS medium containing 2% sucrose and 2.46–9.84 μM Indole3-butyric acid (IBA) and successfully established in soil with 77.8% survival rate in field condition. Thirteen randomly selected regenerated clones were screened using six ISSR primers. Nine clones produced similar monomorphic amplification profiles while remaining clones showed minor variation with absence of certain parental bands and appearance of unique band. Majority of the regenerants maintained genetic fidelity with the generation of few variants as evidenced from similarity matrix estimates using Nei Li's coefficient of similarity data.  相似文献   

13.
Camellia reticulata L. plantlets were regenerated by direct and indirect somatic embryogenesis from immature zygotic embryos. Initial explants (cotyledon sections and embryonic axes) produced somatic embryos without intermediate callus tissue when grown on Murashige and Skoog’s basal medium with 30 gl-1 sucrose and no growth regulators; the somatic embryos completed their development in 4-6 weeks in the same medium. Embryogénie competence was increased by 0.5 and 1 mg l-1 IBA. Histological observation showed the embryos to originate from epidermal and subepidermal cells of the cotyledon and hypocotyl explants. Secondary somatic embryos developed directly from the cotyledons and hypocotyl region of primary somatic embryos by a process that was morphologically very similar to that occurring on zygotic explants. Direct repetitive embryogenesis was maintained by this system. Up to 40% germination occurred when mature somatic embryos were isolated and incubated in medium supplemented with 1 mgl-1 GA3 + 1 mgl-1 IAA. Indirect somatic embryogenesis was induced in callus differentiated on cotyledon explants after three months’ culture in media containing IBA or NAA and/or BAP, embryogenic capacity being retained by callus subcultured on 0.5 mg l-1 IBA + 1 mg l-1 BAP.  相似文献   

14.
Difficulties to develop an easy and reproducible protocol to get healthy and well formed plants from somatic embryos of papaya (Carica papaya L.) had included low germination, callus production at the base of the embryo radicle and the occurrence of hyperhydric plantlets among others, and by consequence unsuccessful transfer to the field. With the aim of improving a propagation method, the effects of light quality, gelling agent and phloridzin concentration on the germination of somatic embryos of hermaphrodite C. papaya L. var. Maradol were studied. Somatic embryos were grown on half strength MS medium, with the addition of Chen vitamins [Chen, M.H., Wang, P.J., Maeda, E., 1987. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants. Plant Cell Rep. 6, 348–351], solidified with three distinct gelling agents: Sigma® Agar–Agar, Difco® Bacto agar and Phytagel®; supplemented with phloridzin and exposed to different light qualities: blue (54 μmol m−2 s−1), red (65 μmol m−2 s−1), gro-lux (68 μmol m−2 s−1), red + blue, white (32 μmol m−2 s−1) and wide spectrum (49 μmol m−2 s−1) during a period of 4 weeks. Results show that light quality and gelling agent had important effects on germination and plant growth, while 3.0 mg L−1 phloridzin had an important role on germination as well as in root development. Somatic embryos exposed to white light, culture medium solidified with 3.0 mg L−1 phytagel and 3.0 mg L−1 phloridzin showed longer roots. Meanwhile, germination and plant length were promoted on an improved culture medium solidified with 7.5 g L−1 Difco® Bacto agar, 3.0 mg L−1 phloridzin and exposed to gro-lux lamps. Under these conditions, 70% of somatic embryos germinated and developed normal roots without hyperhydricity. The regenerated plantlets with well developed roots and shoots were successfully transferred to a greenhouse with a survival rate of 95%.  相似文献   

15.
An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l−1 thidiazuron (TDZ), 0.8 mg l−1 adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l−1 indole-3-butyric acid (IBA), 0.5 mg l−1 6-benzyladenine (BA) and 2.0 mg l−1 kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l−1 IBA. All plantlets acclimatized well in the greenhouse.  相似文献   

16.
The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l−1 BAP and 0.1 mg l−1 NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l−1 NAA and 3.0–5.0 mg l−1 BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l−1 BAP and 0.1 mg l−1 NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l−1 NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.  相似文献   

17.
The effect of enhanced ultraviolet-B on allelopathic potential of Zanthoxylum bungeanum was investigated. A significant inhibitory effect on germination rate of crop seeds under bioassay was observed at 25 g l−1 and 50 g l−1 by extracts from Zanthoxylum leaf both treated with enhanced ultraviolet-B radiation and untreated control. Medicago sativa and lettuce were more sensitive than radish to the extract from Zanthoxylum leaf treated with enhanced UV-B radiation, as the germination rates of M. sativa and lettuce were significantly reduced compared to control at 25 g l−1 and 50 g l−1, and so did alfalfa at 12.5 g l−1. However, as for radish (Raphanus sativus) there was no significant reduction in germination rate at any concentration under bioassay compared to control. Content of UV-B absorbing compounds and total phenols in Zanthoxylum seedlings responded positively to enhanced UV-B radiation. The results suggest that the allelopathic potential of Z. bungeanum was generally improved under enhanced UV-B radiation.  相似文献   

18.
An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l−1 2,4-d plus 8.9 μmol l−1 BA, callus subculture on medium F (1/2MS with 4.5 μmol l−1 2,4-d, 2.7 μmol l−1 NAA and 0.44 μmol l−1 BA) and then on medium T (1/2MS with 4.5 μmol l−1 2,4-d, 2.7 μmol l−1 NAA and 2.2 μmol l−1 BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l−1GA3, 23.6 μmol l−1 AgNO3, 0.02% active charcoal and 4.5 μmol l−1 TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l−1, TDZ 9.1 μmol l−1 and GA3 8.7 μmol l−1), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l−1 NAA and 1.1 μmol l−1 BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.  相似文献   

19.
Abiotic factors affect the induction of PLBs and callus in hybrid Cymbidium Twilight Moon ‘Day Light’. The initiation and proliferation of new PLBs and callus could be achieved on NAA and kinetin, supplemented at 0.1 mg l−1 each, respectively, both within 45–60 days. Bacto agar was found to be the most suitable solidifying agent for PLB induction, although a higher shoot fresh weight was obtained on Gelrite; a pH 5.3 was optimal while pH 4.5 caused 100% explant necrosis; coconut water, when supplied at 10–20% (v/v) resulted in a significant increase in the number of PLBs formed per PLB segment (23.1 versus 14.6 in controls) while a massive (almost four-fold) increase in fresh top weight occurred when PLB explants were placed in liquid culture, as a result of hyperhydricity; Fe-EDTA (1 mg l−1) and activated charcoal (1 g l−1) stimulated total fresh weight and PLB formation in the presence of PGRs; PLB formation decreased but total fresh shoot weight increased with the addition of niacin or myo-inositol, both vitamins. Dark-grown PLB-induced plants were etiolated and had longer internodes and higher fresh weight than light-grown control plants at 45 μmol m−2 s−1; at 15 μmol m−2 s−1 shoots were slightly etiolated, fragile, and PLB formation was scarce. RAPD and mtDNA analysis of all resultant PLBs, callus or plants showed them to be genetically identical, with comparable chlorophyll contents. Despite the detection of cytological variation between different plant parts, little variation resulted from abiotic factor treatment.  相似文献   

20.
The data presented report on trials conducted during 24 months using the Portuguese olive cultivar ‘Galega vulgar’. The effectiveness of coconut water, BAP, or kinetin, as possible zeatin substitutes in olive micropropagation protocols, was investigated. In all stages of the micropropagation process, the mineral and vitamin formulation of olive medium (OM) was used. Regarding culture establishment the best results were achieved when 50 ml l−1 coconut water and 2.22 μM BAP were used as medium supplements. For the in vitro multiplication stage, the highest proliferation rates with an average of 3.4 new explants on each 30 days were achieved maintaining the coconut water concentration at 50 ml l−1 and increasing BAP up to 8.87 μM. The effects of IBA and activated charcoal on the in vitro root induction were also studied. Rooting rates of over 85% were obtained by basal immersion of the explants in IBA solution at 3 g l−1 for 10 s, followed by inoculation in the OM culture medium, added with 2 g l−1 of activated charcoal and without growth regulators. All in vitro rooted plants were transferred into Jiffy-Pots filled with vermiculite–perlite 3:1 (v/v) substrate. Those were subsequently wetted with the OM mineral solution, placed into polystyrene plates each one with 100 Jiffy-Pots capacity, which were transferred to traditional rooting mist benches, on a water-cooling equipped greenhouse. Such a simple acclimatization procedure allowed for 95% of plants survival.  相似文献   

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