High Frequency Plant Regeneration Research Articles

High-frequency Plant Regeneration from Cultured Flower Bud Receptacles of Allium hookeri L.

Allium hookeri L. (Alliaceae family) is an important ethnomedicinal plant native to the Himalayan region of Asia. The aim of this research was to establish a high-frequency plant regeneration system for in vitro propagation of A. hookeri. Among the tissue types examined, receptacle explants derived from immature flower buds showed the highest regeneration rate of shoots (<TEX>$93.33{\pm}4.63%$</TEX>), roots (<TEX>$76.67{\pm}7.85%$</TEX>), and calli (<TEX>$80.00{\pm}7.43%$</TEX>) when cultured on Gamborg B5 (B5) medium containing <TEX>$10{\mu}M$</TEX> 6-benzylaminopurine (BA) + <TEX>$1{\mu}M$</TEX> naphthalene acetic acid (NAA), <TEX>$0.5{\mu}M$</TEX> BA + <TEX>$5{\mu}M$</TEX> NAA, and <TEX>$1{\mu}M$</TEX> BA + <TEX>$10{\mu}M$</TEX> NAA, respectively. Shoot multiplication was superior when cultured in liquid rather than on solid medium and relatively high concentrations of BA, ranging from 5 to <TEX>$10{\mu}M$</TEX>. Efficient bulblet formation following root induction from shoot clumps was achieved with culture in liquid B5 medium containing 7% (w/v) sucrose. Regenerated bulblets were successfully acclimatized to ex vitro conditions with a greater than 95% survival rate. By this method, a maximum of 62 plantlets per receptacle could be propagated within 9 weeks of initial culture. The in vitro propagation system established in this study will promote A. hookeri biotechnology, including large-scale production of healthy and aseptic clones, preserving parental genotypes with desirable traits, and genetic manipulation to enhance medicinal value.

The effect of plant growth regulators on embryogenic callus induction and regeneration from coleoptile in rice

To achieve a repeatable tissue culture system is the first successful step in the genetic engineering of plants. For obtaining this objective in this study potential of coleoptile explant and tow genotype for regeneration were examined. Effect of different treatments on callus induction and regeneration from coleoptile explant was investigated. Significant difference was observed between two cultivars in callus induction and somatic embryogenesis. The plant regeneration was influenced by the genotype as well as composition of the medium. The high frequency plant regeneration was achieved from both cultivars in medium containing 2 mg/L kinetin plus 0.5 mg/L NAA and medium containing 2 mg/L BAP plus 0.5 mg/L kinetin. Regeneration percentage of Neda variety (32.39%) was higher than Nemat variety (20/95%).

Establishment of somatic embryogenesis and podophyllotoxin production in liquid shake cultures of Podophyllum hexandrum Royle

The objective of this study was to establish a simple and efficient method for high frequency of somatic embryogenesis and plant regeneration of Podophyllum hexandrum and podophyllotoxin (PTOX) production. Embryogenic callus (100mg FW) cultured on 0.75 strength MS medium with 4% sucrose and 3g/l PVP produced maximum number of somatic embryos (1089) after five weeks of culture under dark condition. When these somatic embryos were subcultured to the same MS medium with 1mg/l ABA, the embryos matured after 2 weeks of culture. The MS medium supplemented with1.0mg/l GA3 led to high frequency germination (91.1%). The accumulation of PTOX varied in somatic embryos, matured embryos and germinated embryos. Highest PTOX accumulation (2.8mg/l) was recorded in somatic embryos cultured in 0.75 strength MS medium with 8% sucrose. Regenerated plantlets were successfully acclimatized in the growth chamber. The protocol established in this study will be helpful for large scale production of podophyllotoxin.

Effects of CuSO4 and Uniconazole on Mature Embryo Culture in Japonica Rice

In order to establish the system of high frequency plant regeneration for japonica rice mature embryos, the effects of different concentrations of CuSO4 and uniconazole on in vitro culture of mature embryos were studied using three rice cultivars of Kongyu 131, Longjing 24, and Dongnong 425 as test materials. The results showed that callus induction and differentiation of japonica rice mature embryos were apparently improved on the medium with 10–15 μmol • L−1 CuSO4 and 0.50–1.00 mg • L−1 uniconazole. Induction and differentiation rates of different genotype rice mature embryos displayed different sensitivities to CuSO4 and uniconazole. For the callus induction frequency of three varieties, the optimal concentration of CuSO4 was 15.0 mol • L−1. When the concentration of CuSO4 was 15 μmol • L−1, the plantlet differentiation rates of Kongyu 131 and Dongnong 425 got to the highest, while the concentration of CuSO4 was 10 μmol • L−1 for Longjing 24. For the callus induction and plantlet differentiation rates of Kongyu 131 and Dongnong 425, the ideal concentration of uniconazole was 0.50 mg • L−1 and for Longjing 24 was 1.00 mg • L−1.

Somatic Embryogenesis and Organogenesis for Regeneration of Endangered Multipurpose Desert Plant &amp;lt;i&amp;gt;Leptadenia pyrotechnica&amp;lt;/i&amp;gt; Forsk. Decne in the Kingdom of Bahrain

Leptadenia pyrotechnica is an important multipurpose endangered plant in the Kingdom of Bahrain with restricted distribution. Nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of indole acetic acid (IAA) and 6-benzylaminopurine (BAP). Initially, 80% and 60% explants responded in direct shoot and callus initiation response respectively in presence of 8.88 μM BAP with 5.71 μM IAA in modified MS media after two weeks of culture. The highest frequency of plant regeneration was observed in presence of 8.88 μM BAP with 1.14 μM IAA following organogenic pathway of differentiation. Hundred percent callus proliferation was observed while initial callus developed in presence of 4.44 μM BAP with 2.85 μM IAA and was transferred in media containing 4.44 μM, 6.66 μM BAP with 2.85 μM IAA and 13.32 μM BAP with 5.71 μM IAA. The callus derived plants were regenerated following the pathway of indirect somatic embryogenesis. The induction of somatic embryogenesis and plant regeneration from callus was also observed in modified MS media supplemented with 4.44 μM BAP and 2.85 μM IAA. The plant regeneration protocol we developed for Leptadenia pyrotechnica will be very beneficial for biodiversity conservation and environment protection of Bahrain. Moreover, the present paper reports for the first time specifically the somatic embryogenesis in this multipurpose desert plant Leptadenia pyrotechnica.

Optimization of Regeneration Protocol of Rice from Embryo Derived Callus

An efficient and reproducible protocol for in vitro regeneration is required to achieve high frequency transformation from transformed calli. We report here high frequency plant regeneration from mature embryonic calli of two BINA rice cultivars Binasail and Binadhan-4. Embryonic calllus initiated on MS basal medium supplemented with 2 mg/l 2, 4-D. Several media with different combinations of growth regulators were tried. Maximum shoot regeneration frequency (63.33%) was observed in Binadhan-4 on MS medium supplemented with 5 mg/l Kinetin + 0.5 mg/l NAA. Maximum root regeneration frequency (70.00%) was observed in Binadhan-4 on MS medium supplemented with 6 mg/l Kinetin + 0.5 mg/l NAA. Well developed plantlets were hardened and transferred to the glasshouse.DOI: http://dx.doi.org/10.3329/pa.v18i2.17461 Progress. Agric. 18(2): 25 - 33, 2007

테프그라스 조직배양을 통한 캘러스 형성 및 식물체 재분화 효율

Teff grass is a warm season C4 annual grass that is used for dry hay, silage and haylage. We have developed a high-frequency plant regeneration system for teff grass via callus culture using mature seeds. It was revealed that mature seeds cultured on MS medium supplemented with 2 mg/l 2,4-D, 0.5 g/L proline, 0.5 g/L casamino acid and 3 g/L Gelrite under light condition produced the highest percentage of callus formation (91.9%). Addition of cytokinins (BA) at 0.0~0.5 mg/L to media containing 2 mg/l 2,4-D enhanced callus growth. The most suitable medium for plant regeneration from dehydrated calli was MS agar medium supplemented with 0.1 mg/l NAA, 1 mg/l BA, 0.5 g/L proline, 0.5 g/L casamino acid 3 g/L Gelrite which induced the highest percentage of calli forming shoots (47.0%). The shoots were rooted at the highest rate (100%) when transferred onto 1/2 MS medium and acclimated in greenhouse conditions.

A reproducible and high frequency plant regeneration from mature axillary node explants of Gymnema sylvestre (Gurmur)—An important antidiabetic endangered medicinal plant

Gymnema sylvestre is an important medicinal plant used in different systems of medicine as a remedy for the treatment of diabetes. The present study describes an efficient and rapid protocol for large scale in vitro plant regeneration from mature axillary node explants of G. sylvestre. The axillary node explants were cultured on MS medium supplemented with different concentrations of BAP and KIN (0.5–3.0mg/l) for shoot bud induction. In order to enhance the shoot bud multiplication, regenerated shoot buds were further subcultured onto MS medium fortified with different concentrations of BAP (0.5–3.0mg/l) in combination with 0.5mg/l of NAA/IBA/IAA/KIN. The highest frequency (84.22%) of multiple shoot bud regeneration with maximum number of shoots (14.20shoots/explant) was noticed on MS medium supplemented with 1.0mg/l BAP and 0.5mg/l KIN combination. In another experiment, in vitro derived shoot buds were cultured on different concentrations of GA3 (0.5–2.0mg/l) and various concentrations of BAP (0.5–3.0mg/l) in combination with KIN (0.5mg/l) and GA3 (1.0mg/l) for shoot bud multiplications as well as elongation. For large scale plant production, in vitro derived axillary buds were cultured on MS medium fortified with BAP (1.0mg/l)+KIN (0.5mg/l)+GA3 (1.0mg/l) combination, in which about 418.72shoots/explant were obtained after five subcultures on the same media composition. Elongated shoots (>2cm) were dissected out from the in vitro proliferated shoot clumps and were cultured on half-strength MS medium containing different concentrations of various auxins (IAA, IBA and NAA) (0.5–2.0mg/l) for root induction. Highest frequency of rooting (78%) was noticed on half-strength MS medium augmented with 2.0mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and subsequently they were established in the greenhouse. The present in vitro regeneration protocol would facilitate an alternative method for fast and large scale propagation of this endangered antidiabetic medicinal plant.

Direct Organogenesis in Brassica juncea var. NRCDR-2 and Analysis of Genetic Uniformity Using RAPD Markers

A high frequency plant regeneration protocol has been developed in Brassica juncea var. NRCDR-2 via direct organogenesis from cotyledonary petiole explants. Cotyledonary petiole explants were procured from 5-day-old in vitro germinated seedlings and cultured on MS medium supplemented with various combinations and concentrations of cytokinins (BAP, Kn) and auxins (IAA, NAA). The highest average number of shoots per explant (6.73) was obtained on MS medium supplemented with 1.0 mg/l BAP, 0.1 mg/l NAA and 10 mg/l AgNO3 with 93.34 percent explant regeneration. The highest frequency of rooting (100 %) was obtained on MS medium supplemented with 0.3 mg/l IAA within 10–12 days of transfer to the rooting medium. Regenerated plantlets were obtained after the two months of the initiation of culture. The rooted plants were successfully acclimatized to the green house conditions. Genetic stability of in vitro regenerated plants was studied by using random amplified polymorphic DNA markers and genetic uniformity in all the regenerants was confirmed.

High-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis

This study reports high-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis. Numerous green globular structures were directly formed on the surfaces of cotyledons and radicles from 2-week-old immature zygotic embryos at a frequency of 42.1 % when cultured on Murashige and Skoog (MS) medium supplemented with 2 mg l−1 of α-naphthaleneacetic acid (NAA) and 1 mg l−1 of 6-benzyladenine (BA). In comparison, white globular structures and pale-yellow calluses were formed simultaneously at a frequency of 28.3 % when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The pale-yellow calluses were transferred to MS liquid medium supplemented with 2,4-D to establish embryogenic cell suspension cultures consisting of round, isodiametric cells that formed cell aggregates. Upon plating of these cell aggregates on half-strength MS medium without growth regulators under light conditions, cell aggregates gave rise to numerous globular embryos at a frequency of 56 %. Of the globular embryos, 15 % were successfully converted into cotyledonary embryos when cultured on half-strength MS medium under light conditions. The plant regeneration system of H. cordata established in this study will be useful for the selection, genetic transformation, and mass proliferation of elite clones with medicinal potential.

TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in &amp;lt;i&amp;gt;Stevia rebaudiana&amp;lt;/i&amp;gt; Bertoni: An Important Medicinal Plant and a Natural Sweetener

An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana Bert. Nodal segments containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 1.00 to 9.00 μM. Maximum multiple shoots (96%) were obtained in MS medium supplemented with 1.0 μM TDZ with an average of 60 shoots per culture, having an average shoot length of 6.0 cm. The best in vitro root induction (89%) was achieved on half strength MS medium without any growth regulator with an average of 24 roots per culture and root length of7 cm. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 95% in the indoor grow room. High-performance liquid chromatography was used to assess the stability in chemical profile and quantification of stevioside and rebaudioside A content of in vitro propagated S. rebaudiana plants and compared with their mother plant at the peak vegetative stage. Our results show no significant differences (p in vitro propagated plants. Furthermore, fully developed in vitro propagated S. rebaudiana plants were also compared with mother plant for their gas and water vapour exchange characteristics and leaf anatomy. The results show that in vitro propagated and hardened plants of S. rebaudiana are morphologically as well as functionally comparable to each other and to their mother plant.

High Frequency of Plant Regeneration through Cyclic Secondary Somatic Embryogenesis in Panax ginseng.

Somatic embryogenesis is one of good examples of the basic research for plant embryo development as well as an important technique for plant biotechnology such as medicinally important plants. Single embryos develop into normal plantlets with shoots and roots. Therefore, direct single embryogenesis derived from single cells is highly important for normal plant regeneration. Here we demonstrate that the cyclic secondary somatic embryogenesis in Panax ginseng Meyer is a permanent source of embryogenic material that can be used for genetic manipulations. Secondary somatic embryos were originated directly from the primary somatic embryos on hormone-free Murashige and Skoog medium, and proliferated further in a cyclic manner. EM medium (one third of modified MS medium [MS medium containing half amount of NH4NO3 and KNO3] with 2% to 3% sucrose) favored further development of proliferated secondary somatic embryos into plantlets with root system. The plantlets developed into plants with well-developed taproots in half-strength Schenk and Hildebrandt basal medium supplemented with 0.5% activated charcoal.

Effect of plant growth regulator combination and culture period on in vitro regeneration of spinach (Spinacia oleracea L.)

The objective of this study was to develop an efficient system for the regeneration of spinach plants (Spinacia oleracea L.) by investigating the factors influencing callus and shoot induction. All plant growth regulator (PGR) combinations tested induced callus with high frequency (73–100 %), and the combination of 5 μM α-naphthaleneacetic acid (NAA), 10 μM 6-benzyladenine (BA) and 0.1 μM gibberellic acid (GA3) had the most significant effect on callus growth in term of weight (120.98 ± 22.56 mg). A high auxin-containing medium induced competent callus for shoot formation, while high cytokinin-containing media enhanced callus growth and made callus incompetent for shoot regeneration. Longer periods of callus induction in a high auxin-containing medium were required to form competent callus and led to a high regeneration capacity. The PGR combination shift from a high auxin to cytokinin ratio (ACR) to a low ACR resulted in highly efficient regeneration. Among the regeneration systems tested, the combination of 10 μM NAA and 0.3 μM GA3 for callus induction for 6 weeks followed by 2 μM NAA and 5 μM BA resulted in the highest plant regeneration frequency (83.33 ± 6.43 %) and the highest number of plantlets per explant (7.93 ± 1.24). Somatic embryos at cotyledonary stage and plantlets were transferred to PGR-free medium to establish whole plants. Regenerated female plants grew well to maturity in the greenhouse (77.17 ± 9.80 %) and produced seeds (175.21 ± 28.01 firm seeds per plant).

EfficientIn vitroplant regeneration through leaf base derived callus cultures of abiotic stress sensitive popular AsianIndicarice cultivar IR 64 (Oryza sativaL.)

A simple and efficient protocol has been developed for high frequency plant regeneration through callus cultures derived from leaf bases of abiotic stress sensitive Asian indica rice variety IR 64. Leaf base segments (4-5 mm diameter) were obtained from 6-day-old dark grown seedlings germinated on halfstrength Murashige and Skoog medium and cultured on MS medium supplemented with different concentrations of 2,4-Dichlorophenoxyacetic acid (2.2-18 μM) and Kinetin (0.2-1.7 μM). Among the various combinations, 13.5 μM 2,4-D and 1.3 μM Kn resulted in high callus induction frequency (87.5%) with a maximum fresh weight of 0.22 g per segment. The regeneration frequency was 75.5% with multiple shoots within 3 weeks of transfer on MS medium supplemented with 13.3 μM 6-benzylamino purine and 8 μM Naphthaleneacetic acid. The shoots readily rooted on half-strength MS medium without any hormonal supplements. In vitro regenerated plantlets with multiple shoots and roots were transferred to sterile soil and vermiculite mix and maintained in shade house for 30 days. Complete plantlets were then transferred to nursery and acclimatized to the external environment until seed set. RAPD profile reveals monomorphism and thus confirming the genetic stability of the regenerated plants. This method has the potential for both direct as well as indirect method of transformation for the production of genetically modified plants.

Optimizing embryo and shoot tip derived callus production and high frequency plant regeneration in the model grass Brachypodium distachyon (L.) P. Beauv

Brachypodium distachyon has been recently proposed as a new model system for monocot functional genomics because of its unique properties such as small size, short generation time, self-fertile, chromosome base number five and small genome size. In this study, we have standardized a protocol to obtain 100% green plant regeneration from embryo and shoot tip explants. The optimum callus induction in terms of percentage of cultures responding and callus growth was observed on Murashige and Skoog (MS) medium supplemented with 4 mg/l 2, 4 dichlorophenoxy acetic acid (2, 4-D) and 0.5 mg/l kinetin (Kn). On this medium, 88% and 100% explants responded from embryo and shoot tip explants, respectively. Comparatively, shoot tip explants produced better response than embryo explants. The callus multiplied and maintained on MS medium supplemented with 2, 4-D (4 mg/l) and Kn (0.5 mg/l) for about 10 months without decline in growth rate. The callus subcultured on MS medium supplemented with thidiazuron (TDZ; 0.5–7 mg/l) or 6-benzyladenine (BA; 0.5–7 mg/l) alone or in combination with naphthalene acetic acid (NAA; 0.5 mg/l) for regeneration. Maximum shoot regeneration was observed on MS medium supplemented with 5 mg/l TDZ and 0.5 mg/l NAA. On this medium, 87% and 96% cultures responded with mean shoot number 10.2 and 16.6 from embryo and shoot derived callus, respectively. The shoots were rooted on MS medium supplemented with either NAA (2–8 mg/l) or indole-3-butyric acid (IBA; 2–8 mg/l). The optimum response (98%) was observed on MS medium supplemented with 4 mg/l IBA with 4.5 roots per shoot. The rooted plantlets were successfully transplanted with 90% success. The acclimatized plants produced flowers and viable seeds.

갈대(Phragmites communis Trinius.)의 종자배양에 있어서 식물생장조절물질이 캘러스 유도와 식물체 재분화에 미치는 영향

갈대의 성숙종자로부터 최적 조직배양조건을 확립하기 위하여 종자로부터 캘러스 유도 및 식물체 재분화 체계를 확립하였다. 종자로부터 캘러스 유도시 첨가되는 auxin으로는 2,4-D가 가장 효율적이었으며, 1.0 mg/L 2,4-D와 0.5 mg/L BA가 첨가된 MS 배지에서 가장 높은 빈도로 캘러스가 유도되었다. 캘러스로부터 식물체 재분화는 cytokinin류인 BA와 kinetin이 첨가되지 않은 MS 배지에서 59.6%로 높은 재분화 효율을 나타내었다. 본 연구를 통하여 확립된 성숙종자로부터의 고효율 재분화 시스템은 유전자 형질전환기술을 이용한 신품종 갈대 개발에 유용하게 이용 될 수 있을 것이다. In order to develop an efficient, reliable and reproducible tissue culture system for reed (Phragmites communis Trinius), an efficient plant regeneration system via callus induction was established using mature seeds as explants. MS medium containing 1 mg/L 2,4-D and 0.5 mg/L BA was optimal for callus induction from mature seeds. The highest frequency (88.7%) of callus formation was obtained in 1.0 mg/L 2,4-D. The highest plant regeneration frequency (59.6%) was found when the embryogenic calli were subcultured on MS medium supplemented with 100 mg/L myo-inositol, whereas, adding of plant growth regulators was not so promising in this case. Our result would be useful for development of transgenic reed plants.

Siberian Wildrye Grass의 성숙종자 유래의 캘러스로부터 식물체 재분화

Ki-Won Lee, Ochirbat Chinzorig, Gi Jun Choi, Sei Hyung Yoon, Ki-Yong Kim,Hee Chung Ji and Sang-Hoon LeeABSTRACTSuccess in molecular breeding for better adapted varieties to environmental stresses depend upon the concerted efforts by various research including tissue culture, transformation, genetics and breeding. In order to optimize tissue culture conditions of Siberian wildrye grass, the effects of plant growth regulators on callus induction and plant regeneration were investigated with mature seeds. The highest callus induction frequency was observed when the mature seeds were cultured on MS medium supplemented with 5mg/L 2,4-D. The highest plant regeneration frequency was observed when callus was transferred to N6 medium supplemented with 1mg/L 2,4-D and 3mg/L BA. Regenerated plants were grown normally when shoots were transplanted to the soil. A short tissue culture period and regeneration system would be beneficial for molecular breeding of Siberian wildrye grass by the production of transgenic plant.(Key words:Siberian wildrye grass, Callus, Plant regeneration, Plant growth regulators)

Rapid direct adventitious shoot organogenesis and plant regeneration from mature seed explants of Phellodendron amurense Rupr.

Thidiazuron (TDZ) was more effective than 6-benzyladenine (6-BA) for adventitious shoot induction from mature seeds of Phellodendron amurense Rupr. Explants of hypocotyl + cotyledon produced more adventitious shoots than explants of cotyledon alone. Water rinsing pretreatment of dehusked seeds, optimally for two or three days, shortened the adventitious shoot induction period to 5 days. Adventitious shoot induction frequency (91.8%) and shoot number per explant (38.4) were highest on Murashige and Skoog (1962) medium (MS) with 1.0 mg/L TDZ and 0.2 mg/L a-naphthaleneacetic acid. Histological investigations indicated that adventitious shoots were induced directly from hypocotyl endodermal cells. 6-BA was more effective than TDZ for adventitious shoot elongation. Rooting frequency was highest (85%) with 1.0 mg/L indole-3-butyric acid. The rapid and high frequency adventitious shoot induction and plant regeneration method that we developed may be useful in future studies on Agrobacterium-mediated genetic transformation of P. amurense. Key words: Phellodendron amurense, mature embryo, direct organogenesis, thidiazuron, ploidy analysis.

New basal media for half-anther culture of Anthurium andreanum Linden ex André cv. Tropical

A successful protocol for high frequency callus induction and plant regeneration from Anthurium andreanum Linden ex Andre cv. Tropical half-anthers is described. Different variables using Winarto and Teixeira and Murashige and Skoog basal media supplemented with several plant growth regulators [2,4-dichlorophenoxy acetic acid (0.1–1.0 mg/l), α-naphthalene acetic acid (0.01–0.2 mg/l), thidiazuron (0.5–2.0 mg/l), 6-benzylaminopurine (0.5–1.0 mg/l), and kinetin (0.5–1.0 mg/l)] were tested for their ability to induce high frequency callusing in half-anthers, indirect regeneration and rooting of shoots. Basal medium, as well as the combination and concentration of hormones applied, had a significant effect on callus formation, shoot regeneration and adventitious root formation. Winarto and Teixeira-1, an original basal medium containing 0.01 mg/l α-naphthalene acetic acid, 0.5 mg/l thidiazuron and 1.0 mg/l 6-benzylaminopurine was suitable for callus formation while an improved basal medium i.e., New Winarto–Teixeira-3 supplemented with 0.25 mg/l 2,4-dichlorophenoxy acetic acid, 0.02 mg/l α-naphthalene acetic acid, 1.5 mg/l thidiazuron and 0.75 mg/l 6-benzylaminopurine enhanced callus formation. High shoot regeneration and multiplication was also possible on New Winarto–Teixeira-3. Shoots formed a strong adventitious root system on New Winarto–Teixeira-3 containing 0.2 mg/l α-naphthalene acetic acid and 1.0 mg/l kinetin. Plantlets that varied in size and performance were successfully acclimatized and adapted to ex vitro conditions. Cytological analysis of 180 acclimatized-plantlets ex vitro revealed that 34 were haploid (n = 14–18), 15 aneuploid (n = 20–26), 126 diploid (n = 28–34) and 5 triploid (n = 45–57). The potential use of this protocol for developing half-anther culture of other Anthurium species or cultivars is discussed.

Efficient plant regeneration from cotyledonary node explants of Cucumis melo L.

An efficient regeneration system was developed using cotyledonary node of two inbred line of melon, Cucumis melo 'CM-15' and 'CM-23'. The induction of shoots was achieved on Murashige and Skoog (MS) solid medium supplemented with different combinations of 6-benzylaminopurine (BA) and indole- 3-acetic acid (IAA). The best shoot induction was observed when the explants were cultured on MS medium supplemented with BA (1.5 mg/L) and IAA (0.1 mg/L), and on MS medium supplemented with BA (1.5 mg/L) and IAA (0.5 mg/L) for 'CM-15' and 'CM-23', and inbred line CM-15 had the best regeneration rate. The shoot clumps were transferred to MS with BA (0.05 mg/L), resulting to the differentiation of most of the shoots initially into well developed shoots. Regenerated shoots were rooted on half-strength MS medium without plant growth regulators (PGRs). The rooted plants were established in soil with 100% success rate. This high frequency plant regeneration system provides improved technology to assist in genetic transformation of melon. Key words : Cucumis Melo L., cotyledonary node, shoots regeneration, plant regeneration.