Somatic embryogenesis and plant regeneration in Psidium guajava L. cv. Banarasi local

A protocol for plant regeneration by somatic embryogenesis was developed in guava cv. Banarasi local by using immature zygotic embryo explants. Best induction of somatic embryogenesis was achieved from 10-week-old zygotic embryos on MS medium supplemented with 2,4-d (4.52 μM) and 5% sucrose. Maximum number of somatic embryos was produced when zygotic embryo explants were transferred to growth regulator free full strength MS basal medium after 8 days treatment with 2,4-d. Full strength MS basal medium containing 5% sucrose was most favorable for maturation of somatic embryos. Highest frequency of conversion and normal plantlet production were recorded from elongated torpedo stages of somatic embryos on half strength MS medium containing 3% sucrose. Over 90% of rooted shoots survived acclimatization.     

In vitro somatic embryogenesis from Mangifera indica L. callus

The nucellus and globular adventitious proembryos were removed from 2-month-old fruits of mango (Mangifera indica L.) cultivars ‘Ono’ and ‘Chino’, and were cultured on sterile, solid Murashige and Skoog (MS) medium that had been modified as follows: half-strength major salts and chelated iron; 20% (v/v) coconut water (CW); 6% sucrose; 100 mg l^?1 ascorbic acid and 400 mg l^?1 glutamine. Embryogenic explants were sub-cultured after 4–6 weeks in liquid modified MS medium containing 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) instead of CW. Rapidly growing cultures were established and were sub-cultured monthly. Somatic embryogenesis was induced following sub-culture from MS medium with 2,4-D to MS without growth regulators and with or without activated charcoal (0.5%). Germination of somatic embryos appeared to be enhanced by 1 mg l^?1 benzyladenine (BA); however, most of the germinating embryos became embryogenic.     

Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L.

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.     

2,4-D对成熟与非成熟西洋参种胚体细胞胚发生的影响

以西洋参成熟种子及未成熟种子为试材,在无菌操作条件下剥取种胚,将其依次接种在不同2,4-D浓度的MS培养基中,在光照和暗培养条件下分别进行培养,研究2,4-D对西洋参种胚体细胞胚发生的影响.结果表明:成熟种胚在不同浓度2,4-D中皆可发生体细胞胚,最佳浓度为0.5 mg/L,光照可以促进体细胞胚发生;未成熟种胚不能形成体细胞胚.说明成熟种胚体细胞胚发生需要2,4-D诱导,未成熟种胚的表现与成熟种胚差异显著;西洋参种胚在发育控制上与人参存在明显不同.     

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

Summary

Studies 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.     

Somatic embryogenesis and plantlet regeneration from encapsulated embryos of Selinum tenuifolium

Summary

This paper reports, for the first time, somatic embryogenesis and synthetic seed production in Selinum tenuifolium Wall. Mature leaf explants inoculated in Murishige and Skoog (MS) medium supplemented with 3 µM 2,4-dichlorophenoxyacetic acid (2,4-D), containing 3% (w/v) sucrose and 0.7% (w/v) agar, induced 67% callus. Maximum production of globular structures, their differentiation into embryos and germination, occurred with a combination of 2 µM benzyladenine (BA) and 2 µM indole-3-butyric acid (IBA). To protect somatic embryos and produce synthetic seeds, gel capsules were standardised using a combination of sodium alginate and calcium nitrate concentrations. Gel capsules were most effective when formed with a combination of 3% (w/v) sodium alginate and 100 mM calcium nitrate for 30 min. The addition of MS medium to alginate capsules with 3% (w/v) sodium alginate, 3% (w/v) sucrose, 2 µM BA and 2 µM IBA significantly improved their germination rate to 77.8%, as well as their resulting shoot length (5.6 cm) and root length (7.2 cm), compared to controls (57.8%). Most plantlets (66%) survived under nursery condition. Storage at 4°C for different periods (10 d or 20 d) significantly (P < 0.05) reduced the percentage survival and germination of somatic embryos and artificial seeds compared to controls or 5 d storage.     

In vitro regeneration of Camellia reticulata by somatic embryogenesis

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.     

Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.)

The effect of sucrose concentration on callus induction followed by differentiation of embryogenic callus derived from petal explants of four carnation cultivars (Nelson, Sagres, Spirit and Impulse) was investigated. Embryogenic calli were produced on Murashige and Skoog [Murashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 154, 73–479] basal medium (MS) culture medium containing six concentrations of sucrose (3, 6, 9, 12, 15 and 18%, w/v) all supplemented with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 μM 6-benzyladenine (BA). Maximum frequency of embryogenic callus was obtained from the media containing 9 and 12% sucrose. Somatic embryos were induced on a hormone-free MS media containing the seven concentrations of sucrose. Development of somatic embryos was enhanced by increasing sucrose concentration from 1.5 to 12%, while it was reduced in higher concentrations of 15 and 18%. However, normal embryos were not developed in the media containing 1.5 and 3% sucrose. Ninety-five percent of somatic embryos were regenerated to form the entire plantlets when they transferred onto the half-strength hormone-free MS culture medium containing 3% sucrose. Plantlets were also continued to grow normally under greenhouse condition.     

褪黑素对‘无核白’葡萄体细胞胚的诱导作用

以欧洲葡萄‘无核白’（Vitis vinifera‘Thompson Seedless’）未开放的小花蕾为外植体,研究0、1.0、2.0、3.0 mg ? L-1褪黑素（Melatonin）对其体细胞胚的诱导效果。结果表明：‘无核白’小花蕾在 MS + 1.0 ~ 3.0 mg ? L-1 褪黑素 + 2.0 mg ? L-1 6-BA + 30 g ? L-1蔗糖 + 3 g ? L-1植物凝胶的培养基上继代培养30 d时愈伤组织诱导率较好,为73.67% ~ 89.10%,显著高于2,4-D处理,愈伤组织形成所用时间较2,4-D处理缩短了14 d。愈伤组织诱导120 d时,不同浓度褪黑素均出现体细胞胚,其中,以MS + 1.0 mg ? L-1褪黑素 + 2.0 mg ? L-1 6-BA + 30 g ? L-1蔗糖的培养基体细胞胚的发生率最高,180 d时达12.05%。体胚在 MS + 60 g ? L-1 蔗糖 + 0.5 g ? L-1活性炭的X6培养基中萌发30 d后,将萌发的子叶胚转移至MS + 0.2 mg ? L-1 6-BA + 0.1 mg ? L-1 NOA + 30 g ? L-1 蔗糖 + 0.5 g ? L-1活性炭的成苗培养基上,其中,1.0 mg ? L-1褪黑素诱导产生的体胚发育正常的数量较多,为14.84%。不同浓度褪黑素处理的体胚诱导率均于180 d后增长较快,且低浓度的褪黑素有利于体胚萌发与正常发育,2,4-D诱导的愈伤组织无体胚形成。     

Assessing the influence of subcultures and liquid medium during somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis Jacq.)

The current analysis describes an improved protocol for somatic embryogenesis and plant regeneration in oil palm (Elaeis guineensis) through liquid medium, and assesses the influence of successive subcultures during induction of calluses in three Brazilian oil palm varieties. Calluses were induced in a Murashige and Skoog (MS) medium with 450 Picloram, 0.5 g L^?1 glutamine, 2.5 g L^?1 activated charcoal, 30 g L^?1 sucrose, and solidified with 2.5 g L^?1 Phytagel. In a first experiment, the effect of continued subculture of explants every 30 days to fresh culture medium was determined. During a second experiment, part of the embryogenic calluses obtained were transferred to a liquid medium under agitation, consisting of MS with 5 µM picloram or 2,4-dichlorophenoxyacetic acid (2,4-D). After 210 days, the calluses were transferred to semi-solid media for differentiating somatic embryos. It was observed that continued subculture of explants monthly was a determinant in stimulating and improving the formation of embryogenic calluses. Embryogenic calluses in liquid medium with 2,4-D significantly improved the percentage of differentiated somatic embryos (up to 80.2%), with the largest amount of torpedo embryos (8.3 per callus). Regenerated plants with roots were individualised and transferred to a greenhouse, with close to 95% survival.     

Somatic embryogenesis in Cymbopogon pendulus and evaluation of clonal fidelity of regenerants using ISSR marker

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.     

菠萝叶基愈伤组织诱导体细胞胚

以菠萝[Ananascomosus(L.)Merr.]吸芽上的叶基部组织为材料,研究了2,4-D、NAA、BA、TDZ在菠萝愈伤组织诱导体细胞胚过程中的影响。结果表明,2,4-D在体细胞胚诱导过程中起着极为重要的作用,其适宜浓度为5mg/L,适当浓度(1mg/L)的BA在降低畸形胚比例和增加体细胞胚发生率方面有重要作用,但BA也会促进愈伤组织分化不定芽;低浓度的TDZ与BA效果相似,在不同生长调节物质处理组合中,MS+0.01mg/LTDZ+5mg/L2,4-D培养基的体细胞胚发生率最高(97%)。低浓度NAA(2￣5mg/L)利于非胚性愈伤组织的增殖,而高浓度的NAA虽能促进体细胞胚发生,但这种体细胞胚很难继续增殖,在萌发时只生根不发芽。成熟体细胞胚在MS+0.2%活性炭上30d时的转株率为22.7%。     

扁桃杧(Mangifera persiciformis)体胚发生及再生体系建立

以扁桃杧(Mangifera persiciformis Wu&Ming)未成熟珠心组织为外植体,建立体胚发生及再生体系,同时对幼苗进行茎尖染色体计数和胚根组织形态学观察。结果表明,在改良的B5基本培养基+2,4-D1.0mg·L-1+Gln400mg·L-1+6%蔗糖上培养4～5周后可诱导胚性愈伤组织分化。继代培养基与成熟培养基交替培养能有效降低胚性愈伤组织的褐化并保持旺盛的分化能力。培养3～4个月后,大部分体胚均能发育成熟,26.03%的体胚畸形。体胚在改良B5培养基+Gln400mg·L-1+4%蔗糖上的萌发率较低,仅为8.39%。次级体胚以直接体胚发生方式于萌发体胚的下胚轴产生。幼苗的生根不理想,生长极为缓慢;其茎尖染色体数目为2n=2x=40;胚根形态学上端内部维管组织解体,愈伤化,结构松散。     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

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.     

拟南芥新疆生态型体细胞胚胎发生的淀粉粒动态研究

以拟南芥新疆生态型无菌苗真叶和叶柄为外植体,MS 6-BA 0.1 mg/mL NAA 1 mg/mL 2,4-D 1 mg/mL培养基诱导体细胞胚胎发生,采用MS KT 1 mg/mL 2,4-D 0.2 mg/mL 为分化培养基,组织切片用PAS法研究体细胞胚发育过程中淀粉代谢的动态消长规律,结果表明:体细胞胚胎发育过程中出现二次淀粉积累高峰.     

Improved efficiency of somatic embryogenesis from zygotic embryos in Hyoscyamus niger by seed water-soaking

We describe an efficient procedure to obtain somatic embryos from mature zygotic embryos of Hyoscyamus niger (black henbane). It has several advantages over previous regeneration methods, which are: the use of mature seeds, an average 80% somatic embryogenesis rate and a high (eight-fold higher than the control) plant regeneration frequency. The critical step in this protocol was soaking of the seeds in sterile distilled water for 16 h, prior to excision and culture of zygotic embryos, on MS basal medium supplemented with 2% sucrose, 2 g/l myo-inositol, 0.5 g/l 2-(N-morpholino) ethanesulfonic acid and 1 mg/l α-naphthalene acetic acid (NAA). The regenerated somatic seedlings were fertile and were morphologically uniform. This procedure is simple, rapid and effective for high frequency of plant regeneration via somatic embryogenesis. Moreover, this new method should facilitate the development of strategies to routinely transform recalcitrant plant species, including henbane.     

Recurrent somatic embryogenesis and plant regeneration in Angelica glauca Edgew., a critically endangered medicinal plant of the Western Himalaya

Summary

Secondary somatic embryogenesis and plant regeneration from seedling explants of Angelica glauca, an endangered medicinal plant of the Himalaya, is reported for the first time. Callus was obtained from all the explants tested in the present study (i.e., epicotyls, hypocotyls, and cotyledonary nodes). The highest frequency of callus formation (95.8%) was observed using epicotyl explants on 4.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), whereas 70.8% of hypocotyl explants, and 58.3% of cotyledonary nodes produced callus. One-hundred percent embryogenic callus was induced from epicotyl explants in 2.0 µM 6-benzyladenine (BA) and 2.0 µM μnaphthaleneacetic acid (NAA), together with the maximum number of somatic embryos (34.2 embryos per explant). Cotyledonary nodes did not produce somatic embryos. Histological studies confirmed the induction of somatic embryogenesis. Somatic embryos germinated into plantlets upon transfer to half-strength Murashige and Skoog (MS) medium without added plant growth regulators. We observed 85% survival of these plantlets under field conditions. The development of secondary embryos was also observed when primary embryos were sub-cultured on full-strength MS medium containing 2.0 µM NAA plus 2.0 µM BA. This system of recurrent somatic embryogenesis provides a route for gene transfer and also for the large-scale production of this critically endangered medicinal plant.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

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.     

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.