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.     

Somatic embryogenesis in a broad spectrum of grape genotypes

Somatic embryogenesis is the preferred method for cell-to-plant regeneration of grapevine. In this study, we tested the embryogenic capacity of anther-derived calli from 59 grape genotypes, representing a diverse group of Vitis vinifera and hybrid varieties, and hybrids and accessions of non-vinifera Vitis species. Most genotypes were tested on two types of media: MST1 medium, which contained plant growth regulators (PGRs) 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), and MSE medium, which contained 2,4-D and 6-benzylaminopurine (BAP). Twenty-four of the grape genotypes produced embryogenic callus on one or both of these media, eighteen of which have not been reported to form somatic embryos before. The results also suggested that the various PGR combinations are differentially effective at inducing somatic embryos in various classes of grape genotypes. For example, seven of the eight V. vinifera conv. occidentalis varieties brought forth somatic embryos on MSE medium, and three out of four American Vitis genotypes produced somatic embryos on MST1 medium. We could not observe any apparent association between frequency of callus formation and embryogenic capacity of the anthers.     

High frequency embryogenic callus induction and plant regeneration from mature caryposis of big bluestem and little bluestem

Big bluestem (Andropogon gerardii Vitman) and little bluestem [Schizachyrium scoparium (Michaux) Nash.] are native to the North America and are important forage grasses and ornamental grasses. Both grasses are proposed as ideal biomass producers for cellulosic ethanol production. To apply genetic transformation, which is an important tool for incorporating desirable agronomic traits into plants to both species, however requires an efficient and reproducible regeneration protocol. We used mature caryopses from big and little bluestem as explants and tested the effect of various combinations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) (1, 2, 3, 4 or 5 mg l⁻¹) and kinetin (KT) (0, 0.1 or 0.2 mg l⁻¹) on embryogenic callus induction with LS as the basal medium. The highest percentage of embryogenic calli induction occurred on medium containing 2, 4-D alone at 2 mg l⁻¹ for ‘Bison’ and on medium containing 4 mg l⁻¹ 2, 4-D alone for ‘Bonilla’ big bluestem. For little bluestem, the highest percentage of embryogenic callus induction occurred on medium containing 3 mg l⁻¹ 2, 4-D plus 0.1 mg l⁻¹ kinetin, suggesting that addition of KT is beneficial. Shoot regeneration took place on LS basal medium without any plant growth regulator for both species, although the addition of KT increased both regeneration frequency and the number of shoots produced per callus. Rooting of shoots reaching about 2 cm long occurred readily with or without α-naphthaleneacetic acid (NAA). Rooted plantlets were all successfully established in the soil.     

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

Cryopreserved callus,a source of protoplasts for citrus improvement

Summary

The availability and maintenance of embryogenic callus is a major limitation for large-scale application of somatic hybridization for citrus breeding. The suitability of cryopreserved callus as source of protoplasts was evaluated. Sweet orange callus were frozen by slow cooling and stored for two years in liquid nitrogen. Cryopreserved callus were fast thawed and used as source of material for protoplast isolation, protoplast fusion and plant regeneration, in comparison with control non-cryopreserved callus. No differences were found in protoplast yield, quality, growth and regeneration capacity between both callus types. Protoplasts isolated from cryopreserved callus were also successfully used in somatic fusion assays. Plants regenerated from protoplasts of the two sources had the same phenotypic characters and no differences were detected by microsatellite analysis. Availability of cryopreserved callus facilitates the development of breeding programmes based on somatic hybridization, avoiding the risks and high labour needs associated with standard maintenance by periodical subcultures.     

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.     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

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.     

仙客来体细胞胚发生和发育过程中淀粉粒的变化

 以仙客来（Cyclamen persicum Mill.）开花植株的新生叶片为外植体，诱导筛选胚性愈伤组织，并使其进一步发育成体细胞胚。组织切片观察结果表明：胚性愈伤组织由胚性和非胚性细胞组成，胚性细胞多以胚性细胞团的形式存在，胚性细胞团起源于诱导的胚性决定细胞。体细胞胚起源于单个胚性细胞，经多细胞原胚、球形胚、心形胚和鱼雷胚等时期发育成完整植株。在体细胞胚的发生发育过程中，淀粉粒出现4次积累高峰，分别为胚性细胞、球形胚、早期鱼雷胚和成熟胚发育成完整的小植株时期，淀粉代谢与体细胞胚发生、发育及小植株的形态建成密切相关。     

Morphohistological examination on somatic embryogenesis of Musa acuminata cv. Mas (AA)

Somatic embryogenesis is a routine method in obtaining mass-production of plantlets especially in Musa spp. Somatic embryogenesis is a process characterized by a series of morphological changes leading to plant regeneration. Therefore it is important to identify stages in somatic embryogenesis through morphological and histological characteristics. In this study, stages of somatic embryogenesis through morphohistological study could differentiate between embryogenic callus consisting of somatic embryos and non-embryogenic callus. In Musa spp., globular to torpedo shaped somatic embryos resulted in good suspension cultures followed by recovery of somatic embryos during developmental stage. The morphological observations were supported by histological sections of structures at all particular stages. Histological examination also revealed the vascular connection in germinated somatic embryos which later turned into a complete plantlet.     

Direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis (Girard) Kuntze

The potentialities of direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis var. Golden Diamond invitro were investigated. Young whole leaf and cut leaf explants when cultured on MS basal medium supplemented with each of the growth regulators N⁶-benzyladenine (BA) (0.44–2.2 μM) or thidiazuron (TDZ) (4.54 μM) alone or in combination with a fixed concentration of α-naphthalene acetic acid (NAA) (1.07 μM) produced somatic embryos directly. More than 90% of the leaf explants produced white, globular somatic embryos on BA (2.2 μM) and NAA (1.07 μM) supplemented MS basal medium within 1 week of inoculation. Most of the embryos matured further and converted after 8 weeks of culture on the same medium. Histological observation showed that the somatic embryos originated from single cells of epidermal layer of leaf. Histological evidence of formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. After conversion the plantlets were further placed on MS medium containing 0.44 μM BA and 4.5 μM IBA for better shoot and root growth. About 90% of the plantlets transferred to the mixture of soil:perlite:vermiculite (1:1:1) in small plastic pots acclimatized successfully. Of these 85.5% plants survived after transferring into earthen pots containing a mixture of soil, coarse sand and cattle manure (1:1:1) under greenhouse or shady open condition.     

High frequency in vitro embryogenic callus induction and plant regeneration from indiangrass mature caryposis

Indiangrass [Sorghastrum nutans (L.) Nash.] is native to the North America and is an important component of the original tall grass prairie. It is also an important ornamental and forage grass. Recently, it has been proposed as an ideal biomass producer for cellulosic ethanol production. Genetic transformation is an important tool for introducing important agronomic traits into plants, but an efficient and reproducible in vitro regeneration protocol is a prerequisite for successful genetic transformation. In this report, we used mature caryopses as explants and tested the effect of various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (1–5) and kinetin (KT) (0, 0.1, and 0.2) on embryogenic callus induction using LS basal medium. Caryopses cultured on media supplemented with 2,4-D alone generally outperformed those cultured on media supplemented with both 2,4-D and kinetin for embryogenic callus induction. The best treatment is LS basal medium supplemented with 3 mg l⁻¹ 2,4-D. LS basal medium supplemented with KT of 0, 0.5, 1, 2 or 5 mg l⁻¹ were tested for regeneration efficiency which was shown to increase as the KT concentration increased. The quality of the shoots produced on the medium containing KT at 5 mg l⁻¹, which produced the highest regeneration frequency appeared to be lower as leaves become vitrified. Shoots were moved to a rooting medium containing either 0 or 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooted plantlets were then transferred to soil-containing pots and were placed in a mist room for 1 week before they are transferred to a normal greenhouse where they all survived. The reported regeneration protocol is very efficient and highly reproducible in spite of the heterogeneous nature of the tested cultivar; thus it should be suitable for genetic transformation.     

Establishment of efficient regeneration protocol from leaf explants of Iris pumila shoots cultured in vitro

An efficient plant regeneration protocol via somatic embyogenesis by leaf base culture of in vitro grown Iris pumila shoots was developed. Induction of embryogenic calli was achieved on MS media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (4.5 μM, each) and some additives (L-proline, casein hydrolysate, adenine sulphate and tyrosine). Further differentiation of embryogenic calli was achieved on MS hormone-free media, and on media supplemented with either BAP (4.5 μM) or BAP + zeatin (4.5 and 0.2 μM, respectively), which allowed somatic embryos, as well as shoot-like structures to form. Fully developed somatic embryos germinated on an MS hormone-free medium. An anatomical study confirmed that shoot-like structures represented early germinating stages of somatic embryos. Acclimatization of plants derived from somatic embryos was 64% after 1 year and no morphological variation was observed.     

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

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%.     

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.     

酿酒葡萄‘神索’体胚发生及再生体系遗传稳定性分析

 以酿酒葡萄‘神索’未成熟合子胚为外植体, 通过植物生长调节剂、光照、温度等因素的控制, 研究了葡萄体胚的产生、保存及植株再生。结果表明, 以NN为基本培养基, 诱导胚性愈伤组织的适宜植物生长调节剂水平是1.0 mg·L ^{- 1} 2,4-D; 诱导体胚的生长调节剂水平是1.0 mg·L ^-1 NAA + 0.5 mg·L ^{- 1} BA, 体胚诱导率为37.5%; 5℃微光条件, 适宜体胚的保存; 体胚成熟及植株再生的植物生长调节剂水平是0.05 mg·L ^{- 1} NAA + 0.5 mg·L ^{- 1} BA, 成苗率为42.1%。利用流式细胞仪并结合染色体计数对体胚及再生植株细胞核DNA含量及染色体鉴定表明, 体胚细胞染色体存在一定的变异, 而体胚再生植株倍性稳定, 细胞核DNA含量及染色体数与供体母株一致。     

Somatic embryogenesis from floral tissues of feijoa (Feijoa sellowiana Berg)

The objectives of the present work were to study the embryogenic competence of floral tissues of Feijoa sellowiana and to investigate the influence of plant growth regulators on somatic embryo induction and development in order to establish a somatic embryogenesis protocol starting from somatic tissues. Petals, stamens and ovaries of floral buds were cultivated onto LPm basal medium supplemented with different levels of 2,4-D, Picloram, 2-iP, Kin and BAP. The highest embryogenic callus induction was obtained with Picloram (10 μM) and Kin (1 μM). Rates of embryogenic calluses induction in stamens and petals were significantly affected by PGRs. Embryogenic calluses were transferred to the same medium, supplemented with gradually reduced levels of PGRs-free medium. After 60 days in suspension cultures with 2,4-D (1 μM) and 2-iP (1 μM) calluses were transferred to PGR-free medium. After 30 days it was observed the development of globular somatic embryos on the surface of 18% of friable calluses previously induced with Picloram (10 μM) and Kin (1 μM). Only embryogenic calluses derived from stamens gave rise to this morphogenetic pattern.Torpedo and cotyledonary somatic embryos transferred to PGR-free culture medium were converted to complete plantlets. This is the first report of somatic embryogenesis in this species starting from somatic tissues.     

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.     

Effects of the antiauxin PCIB on microspore embryogenesis and plant regeneration in Brassica rapa

An efficient protocol to improve microspore embryogenesis and plant regeneration in Brassica rapa was established. The antiauxin p-chlorophenoxyisobutyric acid (PCIB) was used to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. All the 4 tested genotypes responded positively to PCIB. The optimum concentration of PCIB application was found to be 40 μM in NLN-13 medium, which resulted in a 3.4- to 6.2-fold increase in the number of embryos (8.27–19.2 embryos per bud) and a 9.6-fold increase (21.33%) in the plant regeneration frequency in comparison with the controls. Heat-shock treatment by incubation at 35 °C for 1 day was more efficient in inducing embryogenesis in the 2 tested genotypes. The embryos, produced in NLN medium supplemented with 40 μM PCIB and transferred at the 21-day-old followed by a treatment at 4 °C for 5 days, reached the highest direct plant regeneration rate of 58.00%.