Frequency Of Callus Research Articles

An efficient plant regeneration system through callus for Pseudarthria viscida (L.) Wright and Arn., a rare ethnomedicinal herb

The purpose of this study was to develop a protocol to induce high frequency of callus and subsequent plantlet regeneration for Pseudarthria viscida; an important medicinal plant. The cotyledonary node and young leaf pieces (1 × 0.5cm, length × breadth) were used as explants for callus induction and subsequent shoot regeneration and adventitious roots induction from the shoots. The best results were achieved on the following media: (1) 96% callus induction from cotyledonary node explants on MS medium supplemented with 1.5mgl(-1) 2, 4 dichlorophenoxyacetic acid (2, 4-D) and 0.5mgl(-1) 1-naphthalene acetic acid (NAA), (2) 97% shoot regeneration from cotyledonary node derived calli with an average of 44.9 shoots per explant on MS medium fortified with 3.0mgl(-1) N6-benzylaminopurine (BA) and 1mgl(-1) NAA,37 (3) 98% rooting with an average number of 3.3 roots per shoot on MS medium containing indole-3-butyric acid (IBA) or NAA (0.5-4mgl(-1)) after 45days. The plantlets were transferred to the field after acclimatization. Of the 40 plantlets transplanted to the soil, 29 survived (72.5%).

The effect of auxin 2,4-D and cytokinin 2-ip on direct somatic embryogenesis formation of Coffea arabica L. leaf explant

One of the propagation technique for coffee plant production is tissue culture. Tissue culture technique for Coffea arabica L. faces some problems, mainly in the planlet formation regenerated from explants. The objective of this experiment was to examine the effect 2,4-D and 2-ip combination on the formation of direct somatic embryogenesis of Coffea arabica L. in leaves explant. Auxin (2,4-D) and cytokinin (2-ip) concentrations of, respectively, 1; 5 µM and 5; 10; 15; 20 were used as treatments. This research was conducted using completely randomized design with 10 replications. Observation to induce somatic embryos was done by quantitatively on number of callus from explant and number of embryogenic callus. Beside that, observation by qualitative descriptive was also done on deve lopment of embryogenesis. The results showed that Arabica coffee leaves explant of AS 2K clones could be induced in all medium combination except 5µM 2,4-D and 20µM 2-ip combination. Arabica coffee leaves explant of S 795, Sigararutang and AS 1 varieties could be induced in all medium combination. The highest frequency of callus formation was found in AS 2K, Sigararutang and AS 1 varieties on medium containing 1µM 2,4-D in combination with 10µM 2-ip, whereas for the S 795 variety on medium containing 5µM 2,4-D in combination with 10µM 2-ip. The highest frequency of embriogenic callus in all Arabica coffee variety could be reached on medium containing 5µM 2,4-D in combination with 15µM 2-ip. Key words : Coffea arabica L., somatic embryogenesis, 2,4-D, 2-ip, tissue culture, leaves, callus embryogenic.

English

Callus induction from cotyledon explants was studied in indigenous chick pea ( Cicer arietinum L.) cultivars KK-1 and Hassan-2K on MS and B 5 media containing different combinations and concentrations of growth regulators. Different MS and B 5 callusing media containing varying level of 2, 4-D (2 and 4 mg/l), NAA (0.50 and 1 mg/l), BAP (5 and 10 μM) and their combinations were tested for callus induction response. Percent callus and callus fresh weight (g) were recorded after two and four weeks of culture for both genotypes. For KK-1 cultivar, the maximum callus frequency (71 and 97%) followed by (65 and 96%) were observed on 4 mg/l 2,4-D+5 μM BAP in MS and 4 mg/l 2,4-D in B 5 media, respectively after two and four weeks of culture. Similarly, the highest callus fresh weight (0.411 and 0.787 g) were also recorded for MS+4 mg/l 2,4-D+5 μM BAP in contrast to B 5 where the highest callus weight (0.401 and 0.693 g) was achieved on 4 mg/l 2,4-D only. In Hassan -2K, the highest callus % (68 and 96) and fresh weight (0.572 and 0.821 g) were recorded on MS+4 mg/l 2,4-D+0.50 mg/l NAA after two and four weeks of culture, respectively. In B 5 medium, 2,4-D+BAP combination produced average callus induction response for both cultivars. Key words : Callus induction, chick pea cotyledons, growth regulators.

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (Passiflora edulis Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.

Efficient propagation of an important medicinal plant Boesenbergia rotunda by shoot derived callus

A protocol for high frequency shoot organogenesis and plant establishment from shoot base derived callus was developed for Boesenbergia rotunda (L.) Mansf. Kulturpfl. The cultures produced callus in MS medium supplemented with various concentrations of dicamba and 2,4-D. Nevertheless, embryogenic callus only emerged on 2, 4-D augmented MS medium. In addition, the success of callus induction was achieved from meristem derived callus producing 100% of embryogenic callus. Multiple shoot induction was achieved from the surface of the callus after transferring onto MS medium supplemented with BAP or kinetin (shoot induction medium). The highest frequency of callus producing shoots (49.8%) was achieved on MS medium containing 2.0 mg/L BAP. Plantlets with well-developed shoots and roots on MS medium supplemented with 2 g/L activated charcoal were acclimatized and successfully grown in the greenhouse. Key words: Boesenbergia rotunda, plant cell cultures, medicinal plant, organogenesis, Zingiberaceae, micropropagation.

바이오에너지용 수수 품종의 재분화율 증진을 위한 배지와 생장조절제 효과

본 연구는 수수의 성숙 종자로부터 캘러스유기 및 재분화 효율 조사하기위하여 수행되었다. 배발생 캘러스 형성을 살펴보기 위해 생장조절제 효과를 조사하였다. 수수 성숙종자로부터 캘러스 유기에 적합한 생장조절제 종류와 농도는 2 mg/L 2,4-D인 것으로 나타났다. 배발생 캘러스의 고효율 빈도는 B5 배지에 2 mg/L 2,4-D을 첨가하였을 때 가장 양호한 것으로 나타났다. 배발생 캘러스로부터 식물체 재분화는 BAP와 IBA를 넣은 MS 배지가 가장 적절한 것으로 나타났다. BAP 농도가 높을수록 신초재분화가 활발하였다. 따라서, 본 연구 결과로부터 수수 기내배양시 신초와 발근 형성에 효과적인 생장조절제 조합은 0.5 <TEX>$mg\;l^{-1}$</TEX> BAP와 0.25 <TEX>$mg\;l^{-1}$</TEX> IBA인 것으로 나타났다. This study was carried out to optimize the embryogenic callus induction and plant regeneration from mature seeds of Sorghum bicolor. The effect of growth regulators was investigated on formation of embryogenic callus. The highest frequency of embryogenic callus was observed when the mature seeds were cultured on B5 medium supplemented with 2 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D). The highest frequency of plant regeneration from embryogenic callus was observed on MS medium with 0.5 <TEX>$mg\;l^{-1}$</TEX> 6-benzyl amino purine (BAP) and 0.25 <TEX>$mg\;l^{-1}$</TEX> indole-3-butyric acid (IBA) to optimize the shoot regeneration. High concentration of BAP (1 <TEX>$mg\;l^{-1}$</TEX>) supplemented with IBA (0.25 <TEX>$mg\;l^{-1}$</TEX>) was effective combination for shoot multiplication. MS medium supplemented with 1 <TEX>$mg\;l^{-1}$</TEX> IBA was found to be the most effective for inducing roots. Normal rooted plantlets were transferred to the greenhouse for hardening with over 90% survival rate. Hence, this reproducible protocol could be useful for mass propagation and genetic transformation of S. bicolor.

Effects of sorbitol on expression of genes involved in regeneration of upland rice (Oryza sativa L.)

Sorbitol promotes plantlet regeneration frequency (PRF) of rice calli. To better understand this phenomenon, calli of upland rice cv. HD65 were induced on medium containing 10 g sorbitol/L, and subcultured and regenerated on media containing 20 and 40 g sorbitol/L, respectively. Using complementary DNA (cDNA)-amplified fragment length polymorphism (AFLP) analysis, we investigated differential gene expression between sorbitol-supplemented (SC) and sorbitol-free calli (NC) during the regeneration stage. From >4,000 transcript-derived fragments (TDFs), we identified 118 differentially expressed TDFs between SC and NC. Of these, 37 were cloned and sequenced, and 12 were further confirmed by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Database searches indicated that 10 TDFs showed no significant homology to genes with known or putative functions, although they were previously reported to be expressed in rice calli. These cDNAs might encode novel proteins related to plantlet regeneration. Of all the 27 well-known TDFs, 35.1% were highly expressed in SC, and had roles in signal transduction (13.5%), transcription and translation (13.5%), and regulation (8.1%). Four TDFs were related to plant growth and regeneration: TDF5, TDF17, TDF21, and TDF26, which showed homologies to a nucleoside diphosphate kinase (NDPK), a cyclin, a receptor-like protein kinase precursor, and TCP-1, respectively. Furthermore, 35.1% were related to metabolism, 27.0% to energy, and 8.1% to defense. These results suggest that, in addition to its roles as a carbon source and an osmotic regulator, sorbitol may also act as a chemical signal to directly or indirectly alter gene expression. Sorbitol exerts its effects via complex transcriptional networks to enhance PRF of calli.

Development of an Efficient &lt;i&gt;Agrobacterium&lt;/i&gt; Mediated Transformation Protocol for Sri Lankan Rice Variety - Bg 250

Experiments were conducted to establish an efficient and simple protocol for regeneration and <em>Agrobacterium</em> mediated genetic transformation of an agronomically important <em>indica</em> rice variety Bg 250. Callus induction was achieved on modified N6B5 medium supplemented with 2,4 &ndash;D (2.0 mg/L), BAP (1.0 mg/L) and NAA (1.0 mg/L). The maximum callusing frequency of 90% was observed after 21 days followed by 4 days of incubation on callus induction medium under dark. The highly prolific, nodular, compact yellowish large calli produced after 25 days were first checked for regeneration ability. Ninety five percent of regeneration frequency was observed with N₆B₅ medium supplemented with 3.0 mg/L BAP and 1.5 mg/L NAA. Therefore, embryonic calli induced after 25 days were used for genetic transformation in subsequent experiments. <em>Agrobacterium tumefaciens</em> strain GV 3101 was transformed with pCAMBIA 1303 binary vector which contains hygromycin marker and GUS reporter gene. The transformed colonies were selected on 50 mg/L kanamycin and 25 mg/L rifampicin and confirmed by colony PCR. The PCR positive colonies were used to transform Bg 250 rice calli. The maximum transformation efficiency of 20% was obtained using 500 mg/L cefotaxime as a bacteriostatic agent to inhibit growth of <em>Agrobacterium</em>. 100 &mu;M acetosyringone in co-cultivation medium and cocultivation for 3 days were the optimum conditions for maximum transformation. The expression of GUS gene revealed that the calli were successfully transformed. <br /><br /><strong>Key words: </strong><em>Agrobacterium</em>; Bg 250; Callus induction; Rice transformation. <br /><br />DOI: 10.4038/tar.v22i1.2669<br /><br /><em>Tropical Agricultural Research </em>Vol. 22 (1): 45-53 (2010)

Plant Regeneration Through Indirect Somatic Embryogenesis in Coelogyne Cristata Orchid

A newly efficient protocol has been established for high frequency somatic embryogenesis through callus culture of Coelogyne cristata. The best frequency of callusing was obtained from leaf segments (3-5 mm) cultured on the MS medium supplemented with 2 mg?L–1 2,4-D and 2 mg?L–1 BA combination. A negative effect of coconut water was ob-served on the callus induction medium. When callus lines no. 4, 6 and 8 induced from leaf segments were sub-cultured separately on ½ MS and MS media containing AC (1-3 g?L–1), formation of somatic embryos was found. However, percentages of embryo formation and the number of embryos per explants were strongly affected by media and callus lines used. The effect of 1/2 MS media is definitely better than MS medium for somatic embryogenesis from the selected lines of leaf derived callus. Among the callus lines, line no. 4 is the best for somatic embryogenesis followed by line no. 6 and 8. The somatic embryos converted into healthy plants with well developed shoots on the same media. The plant-lets were transferred to 1/2 MS medium containing 1 g?L–1 AC for plant regeneration until 8 weeks of culture and successfully acclimatized in the greenhouse.

Research on induction and regeneration of callus initiated from seedling-derived young leaf segments of maize

The initiation,maintenance and differentiation of callus from seedling-derived leaf segments of six maize genotypes had been studied.It was revealed that the optimum concentration of 2,4-D for the leaf segments-induced callus was 3 mg/L.Callus induction and regeneration were dependent on the genotypes.Due to their better performance in callus induction and morphology,embryogenic callus of Qi319,Mo17 and Luyuan 92 were transformed with a GUS construct.The transient expression of GUS was performed by the histochemical staining assay.The results showed that the GUS+ frequency of young leaf segments-derived callus was high.This study provides technological reference for selection of leaf segment as an efficient explant for maize regeneration and genetic transformation.

English

A system for induction of callus and plant regeneration via&nbsp;somatic embryogenesisfrom root explants of&nbsp;Hevea brasiliensis&nbsp;Muell. Arg. clone&nbsp;Reyan&nbsp;87-6-62 was evaluated. The influence of plant growth regulators (PGRs)&nbsp;including 2,4-dichlorophenoxyacetic acid&nbsp;(2,4-D), 6-benzylaminopurine (6-BA)&nbsp;and&nbsp;kinetin&nbsp;(KT)&nbsp;on callus induction of root explants from&nbsp;in vitro&nbsp;plantlets&nbsp;were studied.&nbsp;The results showed that&nbsp;the highest induction frequency of embryogenic callus emerged on Murashige and Skoog (MS) medium&nbsp;supplemented with 1&nbsp;mg/l&nbsp;KT, 0.2&nbsp;mg/l&nbsp;6-BA without 2,4-D.&nbsp;Mean of 4 somatic embryos per embryogenic callus were obtained and approximately 11.8% of them developed into plantlets. The regenerated plantlets were successfully transplanted to sand bed. The plant regeneration system established in this study will facilitate&nbsp;mass propagation&nbsp;and&nbsp;may be applied to culture the roots of high-yielding&nbsp;rubber trees. &nbsp; Key words:&nbsp;Hevea brasiliensis&nbsp;Muell. Arg.,&nbsp;root explants, callus, somatic embryogenesis, regeneration.

Effect of different plant growth regulators on callus induction in Catalpa bungei

The present study describes a reliable method for callus induction of Catalpabungei plant. Calli were in vitro initiated from leaves, stem and petiole segments using DKW basal media. The effect of combinations of BA (6-benzyl adenine) and NAA (α-naphthalene acetic acid) on callus induction was investigated. It was found that growth regulators and their concentrations have obvious effects on the callus induction, the increment of callus index and callus physical appearance. Moreover, callus was formed at different times among three kinds of explants that is, leaves, stem segment and petiole. The best callus induction capabilities were obtained with low levels of NAA in combinations with BA. However, supplemented medium with high concentrations of NAA led to relatively low callus frequency and the callus obtained turned to brown color. Generally, the highest frequencies of callus formation from leaves, stem and petiole explants were observed in the medium containing 0.05 mg l-1 of NAA and 5.0 mg l-1 of BA. However, petiole is the most potential explant for callus induction. Key words: Catalpa bungei, tissue culture, plant growth regulators, callus.

THE EFFECTS OF PROLINE AND COCONUT WATER ON CALLUS INDUCTION OF CUCUMBER (CUCUMIS SATIVUS L.)

Callus was initiated from shoot tips and embryonic axes of cucumber (Cucumis sativus L.) in a Murashige and Skoog (MS) medium supplemented with 1 mg/L naphthalene acetic acid (NAA) and 0.5 mg/L kinetin in the presence of 0, 10 and 20 mM proline. Earlier callus induction was observed with embryonic axes (3 days after initiation), compared to shoot tips (7 days after initiation) with callusing frequency of 100%. Moreover, the average size of embryonic axis-derived calli was 1.3- to 1.9-fold larger than those derived from shoot tips at 2 weeks of culture. No effect of proline was found on percentage of callus induction of both explants. Addition of proline also did not affect the growth of callus from shoot tips. However, inhibitory effect was found on the growth of embryonic axis-derived calli (1.3-fold reduction in comparison with control (without proline)). When the effect of coconut water on callogenesis was evaluated on embryonic axes, callus formation was substantially retarded. However, coconut water significantly promoted hypocotyl elongation. Germination of seeds either in liquid MS medium supplemented with 0.1 mg/L thidiazuron (TDZ) or 0.1 mg/L kinetin prior to callus initiation slightly delayed callus induction but did not significantly affect callus size. However, at 2 weeks of culture kinetin significantly decreased the length of hypocotyls.

Plant growth regulators, adenine sulfate and carbohydrates regulate organogenesis and in vitro flowering of Anethum graveolens

In vitro regeneration protocol for Anethum graveolens (Apiaceae) was developed using leaf explants. MS basal medium used in experiments was augmented with various hormones for caulogenic and rhizogenic response. The optimum callus induction (100%) was obtained by leaf explants on MS media fortified with BA (0.5 mg l−1) singly and in combination with NAA (0.1 and 0.2 mg l−1). BA at 0.5 mg l−1, KN at 1.0 mg l−1 and NAA at 0.1 mg l−1 induced highest number of multiple shoots (10.0 ± 0.25) per explant and they also showed in vitro flowering within 3 weeks of culture. Influence of adenine sulfate on regeneration frequency of callus was evaluated. The highest frequency of rooting (100%) with 6.0 ± 0.25 roots per explants was obtained in one-fourth strength MS medium supplemented with 1/4 MS + IBA 0.5 mg l−1 within 4 weeks of transfer to the rooting medium. In vitro flowering (35%) was obtained with MS fortified with BA alone and also in combination with KN and NAA (5.3 ± 0.42 flowers per explants). In vitro flowering response was tested with different carbohydrates (fructose, glucose, mannose and sorbitol) and optimized. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. The proposed method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important species.

In vitro Clonal Propagation of Anthurium (Anthurium andraeanum L.) Using Callus Culture

High frequency of calli was obtained from leaf and spadix segments of Anthurium andraeanum L. when cultured on N6 medium containing 2.5 mg/l BAP and 0.2 mg/l 2,4-D in dark condition. The calli were maintained in dark condition on the same medium containing same hormonal supplements by sub-culturing up to three months but no shoot formation occurred during this period. Leaf and spadix segments derived calli were then tried for multiple shoot regeneration by culturing onto MS fortified with BAP and Kn singly or in combination with NAA in light condition. Best response towards multiple shoot regeneration was observed from both leaf and spadix segments derived calli on MS fortified with 1.0 mg/l BAP. In this combination an average of 18 shoot buds regenerated from leaf segment derived callus while 14 shoot buds regenerated from spadix segment derived callus. The number of shoot buds increased three to four-folds after subculturing in the same medium. Regenerated multiple shoots were excised and cultured onto half strength of MS with different concentrations of IBA and IAA for root induction. Best root development was obtained in half strength MS containing 1.0 mg/l IBA. About 85 per cent of the regenerated plantlets survived in natural conditions. Key words: Clonal propagation, In vitro, Anthurium andraeanum D.O.I. 10.3329/ptcb.v19i1.4961 Plant Tissue Cult. &amp; Biotech. 19(1): 61-69, 2009 (June)

Effect of Culture Media for Anther Culture of Indica Rice Varieties and Hybrids of Indica and Japonica

Pollen plants from anther culture in rice breeding allows selection of fine strains in early generations. Exploitation of anther culture technique in research is limited due to very low regeneration frequency of indica cultivars and the transfer of anther culture traits from japonica to indica varieties is important in improving these rice varieties. In this study the optimization of media requirements and culture conditions for high frequency callus induction and plant regeneration of several indica × japonica F1 hybrids were evaluated using improved anther culture media. Five indica rice varieties (BG 90-2, BG 379-2, BG 94-1, Dahanala and Suduru samba) and two japonica varieties (Hu Lo Tao and Chuan 4) were selected and F1 hybrids were taken with all possible crosses between them. Panicles were cold pre treated at 5°C for 7 days and anthers were cultured in agar solidified modified N6, B5 and Millers media. Calli were transferred to MS medium for plant regeneration. Best callus induction frequencies (0.8 - 29.4%) were obtained in N6 medium containing 5% (w/v) sucrose, than B5 (0.5 - 19.2%) and Millers (0.2 - 19.8%) media. The F1 hybrids were more responsive to anther culture than their parents. Highest callus induction frequency of 29.4% could observe in N6 medium for F1 hybrid Hu Lo Tao × BG 90-2. The green plant regeneration frequency of calli induced on N6 medium was higher than the other two media and the highest frequency of 41.0% was occurred in F1 hybrids of Hu Lo Tao × BG 90-2. Green plants were transferred to pots with 50 - 75% survival rate. Out of 99 plants survived, 28 plants had more than 5% spikelet fertility. Modified N6 medium had positive effect on anther culture performance and the F1 hybrid Hu Low Tao × Bg 90-2 had the best performance. Key words: anther culture; indica; japonica; modified media; plant regeneration; rice DOI: 10.4038/tare.v10i0.1866 Tropical Agricultural Research & Extension Vol.10 2007 17-22

Somatic embryogenesis and vegetative cutting capacity are under distinct genetic control in Coffea canephora Pierre

The purpose of the study was to evaluate the possible genetic effect on vegetative propagation of Coffea canephora. Diversity for somatic embryogenesis (SE) ability was observed not only among two groups of C. canephora Pierre (Congolese and Guinean), but also within these different genetic groups. The results therefore showed that, under given experimental conditions, SE ability is depending on genotype. Furthermore the detection of quantitative trait loci (QTLs) controlling the SE and cutting abilities of C. canephora was performed on a large number of clones including accessions from a core collection, three parental clones and their segregating progenies. On the one hand we detected eight QTLs determining SE. Six positive QTLs for SE ability, whatever the criteria used to quantify this ability, were localized on one single chromosome region of the consensus genetic map. Two negative QTLs for SE ability (frequency of micro calli without somatic embryo) were detected on another linkage group. Deep analysis of the six QTLs detected for SE ability came to the conclusion that they can be assimilated to one single QTL explaining 8.6–12.2% of the observed variation. On the other hand, two QTLs for average length of roots and length of the longest sprouts of cuttings were detected in two linkage groups. These QTLs detected for cutting ability are explaining 12–27% of the observed variation. These observations led to conclude that SE and cutting abilities of C. canephora Pierre appeared to be genetic dependent but through independent mechanisms.

Genetic transformation of Gentiana dahurica Fisch by Agrobacterium tumefaciens using zygotic embryo-derived callus

Gentiana dahurica Fisch is one of four important commercial Radix Gentianae stipulated by the Chinese Pharmacopoeia. We have established a rapid and effective regeneration system using zygotic embryo-derived callus of G.dahurica Fisch. Using this regeneration system, Agrobacteriumtumefaciens-mediated transformation of G. dahurica Fisch has been developed. Zygotic embryo-derived callus was infected with an A.tumefaciens strain (GV3130) harboring a pBI121 vector that contained an nptII selective marker gene. In a total of 60 zygotic embryo-derived calli assayed, frequency of calli with nptII gene PCR-positive transgenic plantlets is 5%. Transient glucuronidase (GUS) expression was observed from these transgenic plantlets. Frequency of GUS-positive transgenic plantlets (4/78) was approximately consistent with that of PCR assay (3/60). Our data indicate that we have successfully established a stable G. dahurica Fisch transformation system by A.tumefaciens. In general, this transformation system we have established might be useful for modifying physiological, medicinal and horticultural traits.

Comparison of in vitro regeneration pathways in Punica granatum L.

When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus. A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM NAA, and 6 μM giberrellic acid (GA3). However, adding 24 μM silver nitrate (AgNO3) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm) were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal. Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l−1 sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when these were transferred to MS medium was supplemented with 60 g l−1 sucrose.

Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige and Skoog (MS) medium supplemented with 2.0–5.0 mg dm−3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm−3 N6-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25 mg dm−3) and BA or kinetin (1–5 mg dm−3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established in the soil showed normal growth and fertility.