Immature Cotyledons Research Articles

Production of herbicide-resistant cowpea (Vigna unguiculata L.) transformed with the bar gene

Plant genetic engineering has opened new avenues to modify crops and has provided a powerful tool for crop improvement. The present study reports the development of regeneration and genetic transformation protocol for the Turkish cowpea cultivar Akkiz. The immature cotyledons were cultured on Murashige and Skoog (MS) medium containing 0.25-0.75 mg L-1 6-benzylaminopurine with or without 0.25 mg L-1 a-naphthalene acetic acid. Shoot regeneration varied 44.4%-83.3% with 2.1-5.0 shoots per explant. Regenerated shoots were rooted in MS medium containing 0.50 mg L-1 indole-3-butyric acid and acclimatized in the greenhouse, where they flowered and set seeds. Immature cotyledons were transformed with Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pRGG containing an herbicide tolerance gene (bar) along with a uida (GUS) gene under 35S promoter. Phosphinothricin was used as a selectable marker at a concentration of 2.5 mg L-1. Putative transformants were screened by the histochemical GUS assay. Furthermore, molecular analysis revealed the presence of the introduced gene in the genome of cowpea cultivar Akkiz. The selected transgenic plants showed a resistance to Basta® nonselective herbicide at up to 10 mL L-1 of water. Putative transgenic plants retained their pigmentation and continued to grow in the greenhouse.

Induction of somatic embryogenesis in gum arabic tree [Acacia senegal (L.) Willd

Factors affecting somatic embryogenesis from immature cotyledon of gum arabic tree [Acacia senegal (L.) Willd.] were investigated. Induction of somatic embryogenesis was influenced by plant growth regulator concentrations and addition of amino acids in medium. Best induction of somatic embryogenesis was obtained on MS medium supplemented with 0.45μM 2, 4-D, 2.32μM Kin and 15mM L-glutamine. L-glutamine plays a significant role in the maturation of somatic embryos and most of embryos attained maturity only on L-glutamine (15mM) containing medium. Maximum percent (75.0 ± 2.5) germination of somatic embryos was recorded on medium containing 0.22μM BAP.

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l−1 citric acid, 100 µM acetosyringone, and 100 mg l−1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l−1 hygromycin and the embryos were germinated in basal medium containing 20 mg l−1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.

Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

A cDNA encoding γ-tocopherol methyltransferase from Brassica napus (BnTMT) was overexpressed in soybean [Glycine max (L.) Merr.] under the control of seed-specific promoter of Arabidopsis fatty acid elongase 1 (FAE1) or soybean glycinin G1. Two and three transgenic plants were selected, respectively, after Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) and Southern blots confirmed that BnTMT was single-copy integrated into the genome of transgenic plants. RT-PCR analysis showed that the expression of BnTMT was higher in the immature cotyledons than in the mature cotyledons, while no expression was detected in the leaves. Moreover, the expression level under the control of FAE1 was higher than that of G1. HPLC analysis indicated that the seed-specific expression of BnTMT resulted in 11.1-fold and 18.9-fold increase in α- and β-tocopherol content, respectively, in T2 seed. These results suggested that introducing BnTMT into soybean can be used to increase the vitamin E composition in seeds.

SOMATIC EMBRYOGENESIS OF CASSAVA (MANIHOT ESCULENTA CRANTZ) VAR. KM297

Somatic embryos of cassava var. KM297 received from pieces of in vitro immature leaf lobes or cotyledon of somatic embryos, were induced on the MS medium supplemented with 8mg/l picloram after 13 days inoculation in the dark condition. Different states of embryo were obtained after 10 days cultured on MS medium supplemented with 0.1 mg/l BA and 0.01mg/l NAA, in the light condition. Role of endogenous AIA and Zeatin of the globular state of embryos was studied.

Somatic embryogenesis in immature cotyledons of Manchurian ash (Fraxinus mandshurica Rupr.)

Somatic embryogenesis was obtained from immature cotyledon explants that were cultured on half-strength Murashige and Skoog (MS) salts and vitamins with 5.4 μM naphthaleneacetic acid (NAA) and 0.2 μM thidiazuron (TDZ) plus a 4 × 4 factorial combination of 0, 9.8, 24.6, or 49.2 μM indole-3-butyric acid (IBA) and 0, 8.9, 22.2, or 44.4 μM 6-benzyladenine (BA). The addition of 44.4 μM BA improved the percentage of cotyledon explants that produced somatic embryos to >20%, if 9.8 or 24.6 μM IBA was also present. Somatic embryogenesis was >30% when seeds were harvested on 31 July or 15 August. The addition of 50 or 70 g l−1 sucrose enhanced embryogenesis. Histological examination showed that somatic embryos originated from epidermis cells of zygotic embryos. A peak germination rate (69%) was attained when somatic embryos were desiccated for 10 min before they produced green cotyledons and elongating shoot tips. Of the germinated embryos from this desiccation treatment, 65.6% also grew roots and therefore converted into plants. Chilling somatic embryos at 4°C for 15 days resulted in the highest germination rate (69.4%), which was significantly higher than those without chilling treatment (27.6%). However <10% of the chilled germinated embryos formed roots and grew into plants. Plantlets from somatic embryos were transplanted into a 2 vermiculite: 1 sphagnum peat medium, where they had a survival rate of 80.8%, and had no morphological abnormalities.

Antioxidant enzymes activities during secondary somatic embryogenesis in Persian walnut ( Juglans regia L. )

Somatic embryogenesis was achieved from immature cotyledon explants of Persian walnut (Juglans regia L.) cave. Chandler on DKW medium. Secondary somatic embryogenesis, the process by which adventitious embryos are formed from primary somatic embryos, is frequent during somatic embryogenesis in Persian walnut. It has certain advantages compared with primary somatic embryogenesis such as high multiplication rate, independence to the explant source and repeatability. Proteins and activities of antioxidant enzymes including catalase (CAT), peroxidase (POX), superoxide dismutase (SOD) and polyphenol oxidase (PPO) were evaluated in two types of secondary embryogenic calli and secondary somatic embryos in Persian walnut. In dark brown calli from which their secondary somatic embryos were separated, CAT, POX and POX had the highest activities while the activity of SOD and also protein content showed the lowest level. High SOD activity, protein content and low activities of CAT, POX and PPO were determined in secondary somatic embryos and light brown calli containing secondary somatic embryos. According to the obtained results, some proteins and antioxidant enzymes have been regarded as markers for secondary somatic embryogenesis. Key words : Persian walnut, secondary somatic embryogenesis, antioxidant enzymes.

Evaluation of soybean cultivars on the embryogenic and organogenic potential

The first step in plant transformation using somatic embryogenesis or organogenesis for plant regeneration in vitro has been the identification of genotypes with the best totipotency. Thus, the objective of this experiment was to evaluate the totipotency of ten cultivars and one soybean line from the Coodetec Gene Bank. The somatic embryos developed from immature cotyledons measuring from 4 to 6 mm in length, and the organogenesis was evaluated from mature soybean seeds. The cultivars CD 201, CD 216 and CD 220 were the most promising genotypes to produce somatic embryos, whereas the cultivar CD 217 had the highest quantity of differentiated explants, which represents the best regeneration efficiency for participating in organogenic systems. In the experiment with somatic embryogenesis, time to plant regeneration was ten months, but the plants were regenerated after only four months in the experiment with organogenesis.

Histological evidence of morphogenesis from various explants of Jatropha curcas L.

Jatropha curcas, a non-domesticated energy plant, has emerged as a source of biodiesel as it does not compete with the edible oil supplies. Realizing its importance, in vitro regeneration methods have been established to meet the demand of large scale supply of superior clones. However, no precise histological analysis has been conducted to document the in vitro morphogenesis in J. curcas. Here, we present a detailed histological description of the initiation of growth and morphogenesis from callus induced from immature cotyledon and embryonal axis of J. curcas. Microscopic observations revealed that the cells of sub-epidermis in immature cotyledon became meristematic and divided extensively to result in the formation of meristemoids within 3–4 weeks of culture. The meristemoid cells are compact, small, and exhibited densely stained cytoplasm compared to the loosely packed large non-meristematic cells. Presence of meristemoids in different developmental stages above the cambial zone, and the absence of vascular connection to the cotyledon explant led us to conclude that the origin of the meristemoids was adventitious in nature and regeneration is through organogenesis of morphogenic callus. On the other hand, callus derived from immature embryonal axis followed two types of regeneration—one type of shoot regeneration was via organogenesis while the second type was through multiplication of the pre-existing meristems. It was noticed that under similar experimental conditions, the process of organogenesis varied from explant to explant. The present study contributes to adequate knowledge and understanding the process of in vitro shoot morphogenesis in J. curcas.

Somatic embryogenesis and Agrobacterium-mediated transformation of Japanese apricot ( Prunus mume) using immature cotyledons

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.

Direct somatic embryogenesis and plant regeneration from immature explants of chickpea

A protocol for plant regeneration via somatic embryogenesis was developed in two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri. Somatic embryos were induced from immature cotyledons on Murashige and Skoog's (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), &alpha;-naphthaleneacetic acid (NAA) and picloram alone or in combination with 0.5 - 2.0 mg dm-3 N6-benzylaminopurine (BA) or kinetin (KIN). NAA was better for somatic embryo induction compared to other auxins. The well formed, cotyledonary shaped embryos germinated into plantlets with 36.6 % frequency on MS medium supplemented with 2.0 mg dm-3 BA + 0.5 mg dm-3 abscisic acid (ABA). The frequency of embryogenesis and plantlet regeneration was higher in cv. ICCV-10 as compared to cv. Annigeri. Regenerated plants were transferred to soil (40 % survival) and grown to maturity. Histological studies of explants at various developmental stages of somatic embryogenesis reveled that somatic embryos developed directly from the cotyledon cells and they were single cell origin.

Regeneration of soybean (Glycine max L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

We describe here a simple and efficient system of soybean (Glycine max L. Merrill) regeneration through direct somatic embryogenesis by using immature embryonic shoot tips (IEST) as explants. The cultivar Kaohsiung 10 (cv. K10) used in this study did not show embryogenic response either from mature seed-derived explants (cotyledon, embryonic tip, leaf, shoot and root) or immature cotyledons. However, it showed a high percentage (55.8%) of somatic embryo (SEm) formation from the IEST excised 2–3 wk after flowering, thus indicating the crucial roles of type and age of explants. The IEST put forth primary SEm after 2 mo of culturing on Murashige and Skoog (MS) medium supplemented with 6% sucrose, 164.8 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM asparagine and 684 µM glutamine. Subsequently, secondary SEm were developed 1 mo after culturing on MS medium containing 123.6 µM 2,4-D and 3% sucrose. Cotyledonary embryos were induced on MS medium supplemented with 0.5% activated charcoal after 1 mo. The embryos were desiccated for 72–96 h on sterile Petri dishes and regenerated on hormone-free MS medium. Plantlets with well-developed shoots and roots were obtained within 5–6 mo of culturing of IEST. The SEm-derived plants were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration of soybean through somatic embryogenesis are discussed. To our knowledge, this is the first report to employ IEST as explants for successful direct somatic embryogenesis in soybean.

Induksi dan Proliferasi Kalus Embriogenik pada Beberapa Genotipe Kedelai

The successful of gene transformation on crop plants depends on the ability of explant to develop somatic embryos. The effect of medium composition on induction and proliferation of embryonic callus was analyzed on several soybean genotypes, including low irradiance (LI) tolerant genotype (Ceneng, Pangrango C6-30-10 and C6-76-10) and two LI sensitive genotypes (Godeg and Slamet). Immature cotyledons (14 DAA) were cultured in induction medium including MSIA (MS, vitamin B5, 30% sucrose, 0.2% gelrite, 10 mg L -1 2,4-D and 10 mg L -1 NAA) and MSIB (MS, vitamin B5, 30% sucrose, 0.2% gelrite, and 40 mg L -1 2,4-D). Embryonic calli was subcultered one month after initiation onto proliferation medium including MSIIA (MS, vit. B5, 30% sucrose, 0.2% gelrite, 5 mg L -1 2,4-D and 5 mg L -1 NAA) and MSIIB (MS, vitamin B5, 30% sucrose, 0.2% gelrite, and 20 mg L -1 2,4-D). The result showed that percentage of callused explant was 76-94% and the highest initiation obtained on Pangrango genotypes. The average of calli diameter at 1 month after initiation was 0.5-1.2 cm. Calli which was obtained on MSIA medium showed yellow, tranparent, and friable, whereas calli was obtained on MSIB medium showed yellow-brown, transparent, and friable. Increasing calli diameter and structure were obtained on proliferation medium. The combination of 2,4-D and NAA on MSIA and MSIIA both are induction and proliferation medium respectively were better than MSIB and MSIIB which were contain only 2,4-D. Ceneng genotype showed best performance of somatic embryogenesis than others, and Slamet genotype showed lowest response both on callus induction and proliferation. Keywords: immature cotyledons, embryonic callus, NAA, 2,4

Identification of QTLs controlling somatic embryogenesis using RI population of cultivar × weedy soybean

Quantitative trait loci (QTLs) controlling ability of somatic embryogenesis were identified in soybean. A frame map with 204-point markers was developed using an RI population consisting of 117 F11 lines derived from a cross between cultivar ‘Keburi’ and a weedy soybean ‘Masshokutou Kou 502’. The parents differed greatly in their abilities of somatic embryogenesis using immature cotyledons as explants. The ability of somatic embryogenesis was evaluated in five different experiments: the F11 (evaluated in 1998) and F15 (2002) generations cultured on basal media supplemented with 40 mg l−1 2,4-D (2,4-D1998 and 2,4-D2002), F14 (2001) generation on medium with 40 mg l−1 2,4-D and high sucrose concentration [2,4-D2001 (30 g l−1 sucrose)], and the F11 (1998) and F12 (1999) generations on medium with 10 mg l−1 NAA (NAA1998 and NAA1999). The RILs showed wide and continuous variations in each of the five experiments. In the composite interval mapping analysis, 2 QTLs were found in group 8 (D1b + W, LOD = 5.42, r2 = 37.5) in the experiment of 2,4-D1998 and in group 6 (C2, LOD = 6.03, r2 = 26.0) in the experiment of 2,4-D2001 (high concentration sucrose). In both QTLs, alleles of ‘Masshokutou Kou 502’ with high ability of somatic embryogenesis contributed to the QTLs. For the other three experiments, no QTL was detected in the criteria of LOD >3.0, suggesting the presence of minor genes.

Somatic embryogenesis in apomictBoechera holboellii

In this study we establish an efficient method for the regeneration for Boechera holboellii via somatic embryogenesis. Immature cotyledons from siliques of 4-6 month-old plants were cultured on MS medium supplemented with plant growth regulators (BA - 6-benzylaminopurine; NAA - alpha-naphthaleneacetic acid; TDZ - 1-phelyl-3-(1,2,3-thiadiazol-5-yl) urea: 2,4 D-2,4-dichlorophenoxy-acetic acid). A high frequency of embryogenic callus was produced after two weeks in culture. The somatic embryos were obtained with a frequency of 10% of explants on MS medium supplemented with 1.34 muM NAA + 8.87 muM BA and 2.68 muM NAA + 17.74 muM BA within 3 weeks in culture. The alternative regime of MS medium supplemented with 1.34 muM NAA + 4.44 muM BA produced somatic embryos at a frequency of 38%.

Somatic embryogenesis and plant regeneration of northern red oak (Quercus rubra L.)

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.

Induction of Somatic Embryogenesis from Immature Cotyledons in ‘Tombul’ Hazelnut

Açıkta tozlanan „Tombul‟ fındık çeşidinde, 6-benziladenin‟ in BA , 2,4- diklorofenoksiasetik asit 2,4-D ve Naftalenasetik asit NAA ile kombinasyonlarının ve kotiledon olgunlaşma düzeyinin somatik embriyo oluşumu üzerine etkileri incelenmiştir. Olgunlaşmamış kotiledon eksplantları hasattan 45 gün 1 Temmuz ve 30 gün 15 Temmuz önce alınmıştır. En yüksek somatik embriyo oluşumu %66.7 ve eksplant başına en yüksek somatik embriyo sayısı 5.5 1.0 mg·L-1BA, 0.5 mg·L-1 NAA ve 250 mg·L-1 L-glutamin ilave edilmiş ½ makro element içeren Murashige ve Skoog MS temel besin ortamından elde edilmiştir. Somatik embriyo oluşumu üzerine hasattan 45 gün önce alınan kotiledonların 30 gün önce alınanlara göre daha etkili olduğu belirlenmiştir

Green Fluorescent Protein (GFP) in Vector Systems Played Sense Role of Epigenetic in Plants

AbstractThe green fluorescent protein (GFP) of jellyfish (Aequorea victoria) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. However, sGFP(S65T) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(S65T) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(S65T) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system.

Plant Regeneration from Callus Derived from Immature Embryo Cotyledons of Prunus mume

A regeneration protocol for Prunus mume sieb.et Zucc was developed through indirect organogenesis. Immature cotyledons were excised from the open-pollinated seeds of two cultivars and cultured on a modified MS medium supplemented with various combinations of plant growth regulators. Shoot-organogenic calli were induced on half-strength MS medium supplemented either with combinations of 2.2 μm benzyladenine (BA), 5.4 to 10.8 μm 1-naphthaleneacetic acid (NAA), and 0 to 5.0 μm indolebutyric acid (IBA) or with combinations of 2.2 μm BA, 4.5 to 9.0 μm dichlophenoxyacetic acid (2,4-D), and 0 to 5.0 μm IBA. High frequencies of shoot regeneration (81.5% and 91.3% in P. mume cvs. Lv'e and Xuemei, respectively) were obtained from smooth-white nodular calli cultured on a half-strength MS medium supplemented with 2.2 μm BA, 2.2 μM thidiazuron, and 1.0 μm IBA. A high rate of rooting (90.2% and 88.9% in ‘Lv'e’ and ‘Xuemei’, respectively) occurred when shoots were cultured on WPM supplemented with 5.0 μm IBA. Chemical names used: benzyladenine (BA), thidiazuron (TDZ), indolebutyric acid (IBA), dichlophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA).

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”