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.     

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 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.     

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 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.     

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.     

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.     

Endogenous and exogenous polyamines in the organogenesis in Curcuma longa L.

The present work evaluated the development of different Curcuma longa L. explants (leaves basis, root tips and ancillary buds from rhizome) stimulated by exogenous polyamines, combined with naphtalen-acetic acid (NAA) or with 6-benzyl-aminopurine (BAP), to produce callus and its subsequent differentiation. The explants, isolated from field plants, were previously subjected to a basic cleaning method and were inoculated onto Murashige and Skoog culture medium (MS) [Murashige, T.S., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15, 473–497] supplemented with NAA (2.0 mg L⁻¹). Buds were subjected to different treatments, with or without 5.0 and 10.0 mmol L⁻¹ exogenous polyamines (mixture of putrescine:spermine:spermidine, 1:1:1) combined with NAA. The calluses obtained were transferred into the same medium, supplemented with the mixture of polyamines combined with BAP, in order to induce plant differentiation. For C. longa, buds were the most efficient explants for callus induction (p < 0.05). The application of exogenous polyamines (5.0 and 10.0 mmol L⁻¹) produced the most developed callus, with numerous roots. The medium supplemented with 10 mmol L⁻¹ polyamine mixture, combined with BAP, induced good regeneration, producing vigorous plants and excellent shoot formation.Polyamines addition promoted the formation of callus, roots and leaves, representing an important factor in the determination of indirect organogenesis in C. longa L., and putrescine content may be considered a valuable marker of the differentiation process in this specie, as well as the enzyme peroxidase.     

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.     

Plant regeneration of Carica papaya L. through somatic embryogenesis in response to light quality,gelling agent and phloridzin

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%.     

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.     

Improvements in somatic embryogenesis protocol in Feijoa (Acca sellowiana (Berg) Burret): Induction,conversion and synthetic seeds

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.     

Cryopreservation of coriander (Coriandrum sativum L.) somatic embryos using sucrose preculture and air desiccation

An efficient protocol for cryopreservation of somatic embryos of Coriandrum sativum, an important spice and medicinal herb, was developed. The successful cryopreservation procedure utilized embryo clumps (ECs) comprised of 3–4 somatic embryos at the globular or heart-shape stage. These ECs were precultured for 3 days on medium supplemented with 100 g/L sucrose, desiccated under the current of sterile air for 100 min, then sealed in cryovials and plunged directly into liquid nitrogen. Preliminary incubation on sucrose-enriched medium (100 g/L) improved both desiccation- and cryo-tolerance of ECs compared to medium with normal sucrose content (30 g/L) and enhanced embryo formation after cryopreservation. The regrowth after cryopreservation and average number of new embryos developed from cryopreserved ECs were retained at the level of the untreated control (98% and 13 embryos per clump, respectively). Both normal and abnormal plants were produced from control and cryopreserved cultures, indicating that appearance of abnormalities was not related to cryopreservation. The regenerants with normal phenotype showed the same peaks of relative DNA content regardless of cryopreservation. The results suggest that simple desiccation method is effective for cryopreservation of coriander somatic embryos with subsequent regeneration.     

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.     

Embryogenesis and plant regeneration from anther culture in loquat (Eriobotrya japonica L.)

The object of this study was to induce embryogenesis and establish plant regeneration system for anther culture in loquat (Eriobotrya japonica L.). Cold pretreatment was a key factor, and supplement of 2,4-D in the media was absolutely necessary for induction of calluses from cultured loquat anthers. The best response of anthers to in vitro culture was obtained when a 48-h cold pretreatment was employed to flower buds at 4 °C in darkness. Genotype was a decisive factor for embryo differentiation. When anther-derived calluses of three loquat cultivars, i.e., cv. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’, were transferred to embryo differentiation medium, embryos were induced only for cv. ‘Dawuxing’ on MS medium containing 3% sucrose, 0.23 μM ZT in combination with 0.05 μM NAA + 0.05 μM IBA or 0.11 μM NAA + 0.10 μM IBA, and the differentiation rates were 3.33% and 10.00%, respectively. The results of histological studies showed that embryos developed through typical globular, heart, torpedo and cotyledon stages after 4 weeks of culture. The treatment designed to mature the embryos on medium containing 3% of sucrose at 4 °C under darkness for 4 weeks was effective for subsequent embryo germination and plant conversion, which gave rise to 72.5% plant recovery. Cytological studies showed that 26 plantlets were haploids (n = 17) and the remaining 4 plantlets were diploids for the 30 regenerants tested.     

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 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.     

Development of a regeneration protocol through indirect organogenesis in prickly pear cactus (Opuntia ficus-indica (L.) Mill)

An indirect organogenesis regeneration protocol for Opuntia ficus-indica (L.) Mill var “Blanco sin Espinas” is described. One centimeter square cladode explants sections from previously micropropagated prickly pear plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 20 different combinations of 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyladenine (BA). The best calli induction and regeneration response were observed when 2.26 μM 2,4-D and 2.21 μM BA combination was applied to the nopal explants. Regenerating calli was capable of forming new buds when transferred to MS basal medium supplemented with 0.5 μM BA (proliferation medium). Shoot elongation and rooting were achieved on MS medium without plant growth regulators. Excellent acclimatization to greenhouse conditions was observed for all transferred plantlets. By this procedure no morphological differences were observed between the regenerated and mother plants. This protocol may be also utilized to carry out plant regeneration after genetic transformation, in order to develop transformed plants without the presence of chimeric zones.     

花烛体细胞胚胎发生及植株再生研究

 以花烛(Anthurium andraeanum Lind. ) 盆花品种无菌苗叶片、叶柄、根段为外植体, 对花烛体细胞胚胎发生及植株再生进行了研究, 建立了花烛体细胞胚胎发生体系。结果表明, 花烛叶片较其他外植体更适宜体细胞胚的诱导; 胚性愈伤组织及胚性结构诱导最适培养基为改良MS + 2, 4-D 2.0～3.0 mg·L ^{- 1} + KT 0.5 mg·L ^{- 1} +蔗糖2% +葡萄糖1% , pH 5.5, 暗培养40 d, 诱导率可达90.3%; 体胚发育适宜培养基为改良MS + 6-BA 0.5 mg·L ^{- 1} +蔗糖3% , 光强20 μmol·m ^-2 · s^-1 , 培养20 d后体胚萌发率达6219%; 萌发的体胚在发育培养基上继续培养30 d后均能发育成完整植株。对不同阶段培养材料的组织结构及超微结构的观察证明了花烛体胚发生过程。     

Somatic embryogenesis in pepper anther culture: The effect of incubation treatments and different media

The frequency of obtained androgenic plants depends highly on the genotype; therefore the low rate of haploid recovery limits the utility of anther culture in pepper breeding. The need for incubation treatment and adequate nutrition media supplemented with plant growth regulators, especially auxins, are suggested as important factors to obtain somatic haploid embryos in pepper anther culture. The effect of three incubation treatments of the androgenic potential in pepper anther culture on MS, N, LS, NN and CP medium are summarised, and the results demonstrate that:

•

by incubating treatment in cold conditions (at 7 °C) in darkness for 7 days, and then transferring the explants to light conditions (12-h photoperiod at 25 °C) for 4 weeks, on LS and NN mediums, anthers produced callus;