Plant Regeneration Medium Research Articles

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.

Callus Induction and Plant Regeneration in Eleven Perennial Ryegrass Cultivars

ABSTRACTPerennial ryegrass (Lolium perenne L.) is an important grass species as turf and forage. The optimal combination of 2,4-D and BAP in the medium for callus induction and the optimal kinetin concentration in the medium for plant regeneration were investigated. Cultivar variation in the callus induction and plant regeneration of eleven perennial ryegrass was studied using the optimized culture medium. The results showed that the highest callus induction rate with the best callus quality assurance was obtained on MS medium containing 5 mg l−1 2,4-D and 0.05 mg l−1 BAP. MS medium containing 3 mg l−1 BAP, 1 mg l−1 NAA and 1 mg l−1 kinetin was most suitable for plant regeneration. Callus induction and plant regeneration of the eleven cultivars of perennial ryegrass were varied greatly, and higher overall plant regeneration rates were observed in cultivars Barlennium, Pace, Emerald and Premier.

Low temperature storage of suspension culture-derived grapevine somatic embryos and regeneration of plants

A method of clonal germplams preservation utilizing dehydrated somatic embryos and cool temperature storage conditions was demonstrated. Somatic embryos of grapevine (Vitis vinifera L) Autumn Seedless and Chardonnay were produced from suspension cultures. After washing twice with sterile water mature somatic embryos were blot-dried and placed on sterile filter paper in an open Petri dish in a laminar flow hood until they reached about 25% of their initial weight. Approximately 300 dried embryos were placed in each sterile 90×15 mm Petri dish, which was tightly sealed with two layers of ParafilmTM. Sealed dishes were stored in the dark at 4°C in a standard refrigerator. Samples of 25–60 individual dehydrated somatic embryos were periodically tested for viability by placing them on solidified MS medium for germination and plant regeneration. After 42 mo. of dehydrated storage, 90% of the somatic embryos regenerated into plants. To further test utility, of this storage method dehydrated embryos stored for 12 and 26 mo. were shipped from Florida to Washington where 75 and 87.5% regenerated into plants, respectively. Cool temperature storage of dehydrated somatic embryos is a simple and inexpensive method of clonal, germplasm preservation when compared to alternatives such as cryopreservation.

Plant Regeneration from Protocorm-derived Callus of Five Paphiopedilum Hybrids

Callus was induced from protocorms of five Paphiopedilum hybrids (Paph. 03-1, Paph. 03-4, Paph. 03-5, Paph. 03-6, and Paph. 03-7) on callus induction medium [MS inorganics (412.5 mg NH4NO3 instead of 1650 mg and 475 mg KNO3 instead of 1900 mg) and vitamins plus (per liter) 0.1 g myo-inositol, 30 g sucrose, and 2.5 g Gelrite; pH 5.5] containing various concentrations and combinations of thidiazuron (TDZ; 4.5 and 45 μm) and 2,4-D (4.5 and 45 μm). Callus formation was greatest for protocorms of Paph. 03-1, Paph. 03-4, Paph. 03-6, and Paph. 03-7. Among the most competent hybrids, callus formation was greatest among protocorms induced in medium containing 4.5 μm 2,4-D and 4.5 to 45 μm TDZ. Induced calli were transferred to 100 × 15 mm petri dishes containing 25 mL of PLB and plant regeneration medium (similar to callus induction medium) containing various concentrations of either benzyladenine (BA; 0.5, 5, or 10 μm), TDZ (0.25, 2.5, or 5 μm) or no growth regulator (control). PLB and plant formation was greatest on medium containing BA.

Influence of potassium dihydrogen phosphate on callus induction and plant regeneration in rice (Oryza sativaL.)

The effect of potassium dihydrogen phosphate (KH 2 PO 4 ) on callus induction and plant regeneration from callus cultures of indica rice ( Oryza sativa cultivar Pusa Basmati-1) was investigated. Callus was induced on Murashige and Skoog (MS) medium supplemented with 11.31 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and subsequent plant regeneration was obtained on MS medium containing 2.68 μM α-naphthaleneacetic acid (NAA) and 8.87 μM 6-benzylaminopurine (BAP). Both the induction and the regeneration media were supplemented with different levels of KH 2 PO 4 (0 to 12.50 mM). The level of KH 2 PO 4 in the induction medium influenced the percent water content of the callus. KH 2 PO 4 was found to be essential for plant regeneration as no shoot regeneration occurred in the absence of KH 2 PO 4 . Highest number of shoots per explant was obtained when KH 2 PO 4 level in the callus induction and plant regeneration medium was 0.625 mM (half of MS level) and 1.25 mM (normal MS level) respectively. By making amendme...

Regeneração in vitro de anteras de arroz irrigado (Oryza sativa L.) e mapeamento de QTL associado

A baixa taxa de regeneração in vitro de alguns cultivares de arroz da subespécie indica limita a utilização de técnicas de cultura de anteras como ferramenta para o desenvolvimento de novos cultivares. A identificação de regiões genômicas associadas à formação de calos e regeneração de plantas, a partir do cultivo de anteras, poderá permitir a transferência do caráter por seleção assistida. Duas populações de retrocruzamento foram utilizadas para o mapeamento genético e estudo destes caracteres, sendo Taipei 309// BRS 7 "Taim"/Taipei 309 (53 genótipos) e BRS 7 "Taim"// BRS 7 "Taim"/Taipei 309 (74 genótipos). Duzentas anteras imaturas de cada genótipo utilizado no estudo foram inoculadas em meio NL líquido e, após 40 dias, os calos formados foram transferidos para o meio MS sólido para regeneração de plantas. Todas as plantas doadoras de anteras foram usadas na extração de DNA genômico e sete primers RAPD foram utilizados na geração de marcadores para a construção dos mapas de ligação dos retrocruzamentos estudados. A taxa de formação de calos variou de 2,27 a 3,36% e a taxa de regeneração de plantas 1,38 a 1,82%, não se diferenciando significativamente. Seis grupos de ligação foram obtidos, três em cada população. Um QTL com LOD 3,10 foi detectado para o caráter formação de calos na população Taipei 309 // BRS 7 "Taim" / Taipei 309. Nenhum QTL foi detectado para a característica regeneração de plantas.

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2).

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH4NO3 and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6-7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.

돌나물 엽절편으로부텨 캘러스 형성 및 식물체 분화에 미치는 생장조절물질의 영향

돌나물 (Sedum samentosum)의 기내 식물체 재분화 체계를 확립하기 위하여, 생장조절물질을 다르게 조합한MS배지에 잎절편을 치상한 후,캘러스 유기 및 식물체 분야에 미치는 생장조절물질의 영향을 조사하였다. 캘러스 유기는 3.0mg/L 2,4-D와 1.0mg/L BA첨가 배지에서 가장 높은 캘러스 유기율을 보였으나, NAA와 BA 혼합첨가 배지에서는 캘러스 형성율이 0∼3%이하로 극히 낮았다. 식물체 분화는 NAA 단독첨가 배지에서는 전혀 분화되지 않았으며,0.2mg/L NAA와 3.0mg/L BA첨가배지에서 가장 양호하였다. 캘러스를 식물체 분화배지에 계대배양 후 50일까지는 신초수 증가와 함께 (17.2개/캘러스),식물체의 기형이나 잎비대 현상도 적어 식물체 분화배지로 가장 적합하였다. 분화 식물체는 생장조절물질을 첨가하지 않은 MS기본배지로 계대배양하였을 때 발근 및 생육이 양호하였다. To establish the system of in uitro plant regeneration, the leaf segments of Sedum sarmentosum were cultured on MS media supplemented with different levels of 2,4-D, NAA and BA. The callus induction and growth showed a good response on MS medium supplemented with 3.0mg/L 2,4-D and 1.0mg/L BA, but a few callus induced on medium containing NAA and BA. In plant regeneration, combination of BA and NAA promoted shoot organogenesis from callus, and the highest frequency was obtained on MS medium supplemented with 0.2mg/L NAA and 3.0mg/L BA. When calli were transferred to the plant regeneration medium containing 0.2mg/L NAA and 3.0mg/L BA, healthy shoots without hyperhydricity were continuously induced (17.2 plantlets per callus) after 50 days of culture. When regenerated plantlets were transferred onto hormone-free MS medium, rooting was easily achieved from all of them.

Optimization of Ionic and Chelated Iron and Its Interaction with Disodium Ethylenediaminetetraacetic Acid for Enhancement of Plant Regeneration in Rice (Oryza sativa L)

Interactive effects of Fe2(SO4)3 and Na2EDTA on callus induction and plant regeneration in indica rice (Oryza sativa cv Pusa Basmati-1) was investigated. Callus induction and subsequent plant regeneration from seed was obtained on MS medium supplemented with 11.31 µM 2,4-D and 2.68 µM NAA + 8.87 µM BAP respectively. Both the callus induction and the plant regeneration media were supplemented with different levels of Fe2(SO4)3, Na2EDTA and their combinations. Callus induction, its morphogenic potential, average number of regenerated plants as well as the appearance of the regenerants was influenced by the levels of Fe2(SO4)3 and Na2EDTA. Embryogenic callus could be induced in the presence of Fe2(SO4)3 / Na2EDTA. Iron was essential for plant regeneration. Non-chelated iron could induce callus formation as well as plant regeneration, yet the chelated form was more suitable. A higher level of Na2EDTA in the regeneration medium was detrimental. Differential requirement of Fe2(SO4)3 at induction and regeneration level was observed. Method of medium preparation affected the regeneration response. Nearly three fold increase in the number of regenerated plants was achieved with 0.05 mM Fe2(SO4)3 + 0.1 mM Na2EDTA at callus induction and 0.1 mM Fe2(SO4)3+ 0.1 mM Na2EDTA at plant regeneration and standard method of autoclaving.

Optimization of culture conditions for improved plant regeneration efficiency from wheat microspore culture

The production of haploid plants through microspore culture is a very important tool for plant breeding. However, progress in microspore culture for many species has been hampered by a number of factors that have resulted in low recovery of regenerated green plants. In this study, a series of experiments were conducted to increase the regeneration of haploid green plants from isolated wheat microspores. The use of different basal media and variations in media components resulted in the increased recovery (approximately double) of regenerated haploid wheat plants. Our findings demonstrate that CHB medium, in combination with 2,4-d, was a better medium for embryoids induction and plant regeneration than medium MC17 with either 2,4-d or PAA growth hormones. Wheat microspores cultured without ovary co-cultivation did not respond. Furthermore, high efficiency of microspore derived embryoids (up to 296 MDEs per 100 anthers) and green plant regeneration (up to 71 green plants per 100 anthers) were achieved by the use of gelrite instead of agarose as a gelling agent, and by the addition of media additives such as spent medium or MET.

Stevia 절편체 종류의 배양에 따른 식물체 재분화

This study was conducted to determine the optimal concentrations of plant hormones (2,4-D, picloram, dicamba, NAA, kinetin) and the suitable explants among seeds, hypocotyl, and cotyledons on calls formation and plant regeneration of stevia(Stevia rebaudiana Bertoni). The frequency of cellus formation was higher in the young leaf-explants then the older ones, and in the seeds then the hypocotyls and cotyledons on MS medium with 1mg/L 2,4-D. After transfer of seed-derived stevia callus producing embryogenic callus on plant-regeneration medium, the frequency of plant regeneration from callus was 23.8% in MS medium with 1mg/L NAA and 3mg/L kinetin.

이탈리안 라이그라스 X 톨페스큐 속간 교잡종의 약배양 기법을 이용한 식물체 생산

이탈리안 라이그라스 (Lolium multinorum Lam.)와 톨페스큐 (Festuca arundinacea Schreb.)의 속간 교잡종 3계통에 대한 약 배양을 실시하여 약 유래의 식물체를 생산하였다. MS 기본배지에 sucrose 30 g/<TEX>$\ell$</TEX>, NAA 2 mg/<TEX>$\ell$</TEX>, kinetin 1 mg/<TEX>$\ell$</TEX>을 첨가한 캘러스 유도배지에서 배양 20일만에 약 유래의 캘러스를 유도하였다. 캘러스 유도율은 평균 11.6%였으며, 캘러스의 크기는 평균 9.1 mg/callus/anther 였다. MS 기본배지에 sucrose 30 g/<TEX>$\ell$</TEX> , NAA 1 mg/<TEX>$\ell$</TEX>, kinetin 2 mg/<TEX>$\ell$</TEX>을 첨가한 재분화 배지에 캘러스를 치상하여 배양 50일만에 완전히 재분 화된 식물체를 얻었으며, 캘러스로부터 식물체로 재분화 되는 비율은 평균 27.1%였고, Hyb-1 계통의 식물체 재분화율이 30.2%로서 가장 높았다. Several regenerated plants were obtained from anther culture of the hybrid lines of Italian ryegrass (Lolium multiflorum Lam.) X Tall fescue (Festuca arundinacea Schreb.). When the anthers of the hybrid plants were incubated for 20 days on callus induction medium, MS medium containing 30 g/<TEX>$\ell$</TEX> of sucrose, 2 mg/<TEX>$\ell$</TEX> of NAA and 1 mg/<TEX>$\ell$</TEX> of kinetin, their calli were induced. The mean ratio of callus induction was 11.6 percent, and the mean of callus weight was 9.1 mg/callus/anther. When the calli of the hybrid plants were incubated for 50 days on plant regeneration medium, MS medium containing 30 g/<TEX>$\ell$</TEX> of sucrose, 1 mg/<TEX>$\ell$</TEX> of NAA and 2 mg/<TEX>$\ell$</TEX> of kinetin, the hybrid plants were regenerated. The mean ratio of plant regeneration was 27.1 percent and line Hyb-1 showed highest regenerabillity with the frequency of 30.2 percent.

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl−1 casein hydrolyzate, 6.63 mg l−1 (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l−1 (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl−1 (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl−1myo-inositol, and 150 mgl−1 asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.

Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed-derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snow-drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin-containing medium, resistant callus was transferred to hygromycin-containing regeneration medium for plant regeneration. Twenty-six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy-three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.

Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed-derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snow-drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin-containing medium, resistant callus was transferred to hygromycin-containing regeneration medium for plant regeneration. Twenty-six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy-three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.

Agrobacterium-mediated genetic transformation of California poppy, Eschscholzia californica Cham., via somatic embryogenesis.

An efficient Agrobacterium-mediated protocol for the stable genetic transformation of Eschscholzia californica Cham. (California poppy) via somatic embryogenesis is reported. Excised cotyledons were co-cultivated with A. tumefaciens strain GV3101 carrying the pBI121 binary vector. Except for the co-cultivation medium, all formulations included 50 mg l-1 paromomycin as the selective agent and 200 mg l-1 timentin to eliminate the Agrobacterium. Four to five weeks after infection, paromomycin-resistant calli grew on 80% of explants in the presence of 2.0 mg l-1 1-naphthaleneacetic acid (NAA) and 0.1 mg l-1 6-benzylaminopurine (BAP). Calli were cultured on somatic embryogenesis induction medium containing 1.0 mg l-1 NAA and 0.5 mg l-1 BAP, and somatic embryos were visible on 30% of the paromomycin-resistant calli within 3-4 weeks. Three to four weeks after the somatic embryos were transferred to phytohormone-free plant regeneration medium, 32% converted to paromomycin-resistant plants. Detection of the neomycin phosphotransferase gene and high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all plant tissues confirmed the integrative transformation of the regenerated plants. The normal alkaloid profile of California poppy was unaffected by the transformation process; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regulation of the benzophenanthridine alkaloid pathway.

331 Effects of Explant Type, Sucrose Level and Callus Development Time on In Vitro Plant Regeneration of Sweetpotato

Several experiments were conducted to evaluate the influence of explant type, sucrose level, and callus development time on sweetpotato [Ipomoea batatas (L.) Lam] in vitro culture. Shoot tip, petiole, and leaf of Selection 75-96-1 was used as explants in Murashige and Skoog (MS) media with different plant growth regulators. Calli derived from shoot tip and petiole produced 42.1% and 10.3% somatic embryos, respectively, but the leaf failed to produce somatic embryos. The effect of sucrose level was determined using shoot tip as explants. Compared with 3% sucrose in the same plant growth regulators level medium during callus initiation and callus proliferation periods, 5% sucrose level suppressed root growth and improved shoot regeneration. The callus development time was measured by using shoot tips on callus initiation medium containing 1.5 mg/L alpha-Naphthaleneacetic acid (NAA) and 0.25 mg/L Kinetin (KIN) plus 5% sucrose. When explants were cultured for less than 6 weeks during callus initiation, then transferred onto plant regeneration medium, plant regeneration via organogenesis occurred; whereas, maintaining cultures for more than 12 weeks on the same callus initiation/proliferation medium, plant regeneration was favored via embryogenesis. Explant type and other factors affecting plant regeneration noted here could be applied to protoplast culture, somatic hybridization, and transformation in sweetpotato.

Development of transgenic rice pure lines by particle bombardment-mediated transformation and genetic analysis-based selection

Mature seed-derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β-glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)-containing medium, resistant callus was transferred to hygromycin (30 mg/l)-containing regeneration medium for plant regeneration. Twenty-three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin-containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment-mediated transformation and through genetic analysis-based selection.

Optimization of Different Media for Plant Regeneration from Callus Culture of Indica Rice (Oryza sativa) Genotype DM. 25

Optimization of Different Media for Plant Regeneration from Callus Culture of Indica Rice (Oryza sativa) Genotype DM. 25