Frequency Plant Regeneration Research Articles

Effect of promoter driving selectable marker on corn transformation

Identification of an appropriate selection agent and its corresponding selectable marker gene is one of the first steps in establishing a transformation protocol for a given plant species. As the promoter controls expression level of the genes, the promoter driving the selectable marker gene can affect transformation. However, investigations into the direct effect of promoters driving selectable marker on transformation are lacking in the literature though many reports of relative strengths of promoters driving reporter genes like GUS or CAT or GFP are available. In the present study, we have compared rice Actin1 and CaMV.35S (commonly used promoters in monocotyledonous plant transformation) promoters driving nptII for their effectiveness in paromomycin selection of transgenic corn events. To enable statistically meaningful analysis of the results, a large sample size of nearly 5,000 immature embryos (explants) was employed producing approximately 1,250 independent events from each of the two constructs in four independent experiments. The rate of appearance of resistant calli and percentage of resistant calli recovered was higher with P-Os.Actin1/nptII/nos3' as compared to P-CaMV.35S/nptII/nos3' in all four experiments. There was no appreciable difference either in the frequency of plant regeneration or in the morphological characteristics of plants recovered from the two constructs. Although the escape rate trended lower with P-Os.Actin1 as compared to P-CaMV.35S, the recovery of low copy events was significantly higher with P-CaMV.35S. The higher transformation frequency with P-Os.Actin1 could be related to the strength of this promoter as compared to P-CaMV.35S in the explants and/or calli. Based on these results, we infer that the promoter driving the selectable marker is an important factor to be considered while establishing a high throughput transformation protocol as it could not only influence the transformation frequency but also the copy number of the transgene in the recovered transgenics.

High Frequency Shoot Regeneration from Petal Explants of Chrysanthemum morifolium Ramat. in vitro

An efficient and novel method was developed to initiate multiple shoots from the petal which is indispensable for normal and transgenic plant of Chrysanthemum morifolium. The study indicates that high frequency of plant regeneration that would be crucial for the application of genetic transformation methods. The 7 mm petal explants were cultured on a Murashige and Skoog's (MS) basal nutrient medium containing plant growth regulators (cytokinin or auxin-cytokinin) with various combinations and concentrations for the study of callus formation and shoot induction. The highest callus formation (96%) in 4037 genotype cultured in MS medium supplemented with 2 mg L(-1) BA and 0.1 mg L(-1) NAA. The highest number of shoots was obtained from genotype 89 and 4037 in MS medium supplemented with 2 mg L(-1) BA and 0.1 mg L(-1) kinetin, 1 mg L(-1) kinetin and 0.1 mg L(-1) NAA, respectively. A significant difference in regeneration capacity was observed among five genotypes. Genotypes x growth regulators interaction caused considerable variation in the expression of regeneration responses, suggesting that determination of specific level of growth regulator concentration in the medium is necessary for a particular genotype to obtain optimum response. Strong seasonal variation in plantlet regeneration frequency was observed for every genotype. The elongated shoots were multiplied on a multiplication medium, rooted and acclimatized in a green house.

Embryogenesis and plant regeneration of hot pepper (Capsicum annuum L.) through isolated microspore culture

We report high frequencies of embryo production and plant regeneration through isolated microspore culture of hot pepper (Capsicum annuum L.). Microspores cultured in modified NLN medium (NLNS) divided and developed to embryos. Globular and heart-shaped embryos were observed from 3 weeks after the beginning of culture, and many embryos reached the cotyledonary stage after 4 weeks of culture. These cotyledonary embryos developed to plantlets after transfer to solid B5 basal medium. We also optimized conditions for embryo production by varying the pretreatment media, the carbon sources, and culture densities. Heat shock treatment in sucrose-starvation medium was more effective than in B5 medium. Direct comparisons of sucrose and maltose as carbon sources clearly demonstrated the superiority of sucrose compared to maltose, with the highest frequency of embryo production being obtained in 9% (w/v) sucrose. Microspore plating density was critical for efficient embryonic induction and development, with an optimal plating density of 8 x 10(4)-10 x 10(4)/ml. Under our optimized culture conditions, we obtained over 54 embryos, and an average of 5.5 cotyledonary embryos when 10 x 10(4) microspores were grown on an individual plate.

Production of multiple shoots and plant regeneration from leaf segments of fig tree (Ficus carica L.)

High frequency of multiple shoots and plant regeneration has been obtained from the leaf segments of fig tree (Ficus carica L.). Budbreak from dormant buds is highly dependent upon cultivar, so we chose cv. Seungjung Dauphine because it shows an excellent degree of budbreak. Tissue-browning can be an important limiting factor duringin vitro culture. This phenomenon could be substantially delayed or reduced by treating the tissues with 0.5 mM phloroglucinol, thus oxidizing the phenolic substances exuded from the segments. Wounded leaf explants cultured on MS medium supplemented with TDZ in combination with IBA produced more multiple shoots than did other combinations of auxin and cytokinin. For example, 2 mg L-1 IBA along with either 0.5 or 1.0 mg L-1 TDZ resulted in 8.1 or 10.8 multiple shoots per explant, respectively. We achieved a frequency of approximately 90% when tissues were first maintained under darkness in the culture medium for one week before being transferred to the light. Regenerated shoots rooted best in a full-strength MS basal medium.In vitro regenerated plant-lets were then successfully transferred to greenhouse conditions. Here, we have demonstrated a regeneration protocol that is suitable for use in conservation as well as genetic transformation studies of figs and related species.

들깨의 자엽절편배양을 통한 고효율 식물체 재분화

들깨의 조직배양에서 배지의 생장조절제 조성은 0.1 mgyL의 NAA와 2.0 mg/L의 BA가 혼용된 배지에서 신초형성률이 24.7%로 가장 높게 나타났다. 신초형성에 적합한 배양부위는 자엽이었으며 품종별 신초형성율은 <TEX>$3.0{\sim}27.3%$</TEX>로 품종간 차이가 컸고, '만백들깨'의 자엽 배양에서 27.3%의 가장 높은 재분화율을 나타내었다. 조직부위별로는 공시된 모든 품종에서 배축보다는 자엽 조직에서 신초형성율이 높았으며, 신초의 분화형태는 배형성 (6.1%)보다 기관형성 (22.8%)을 경유하는 것이 많았다. A reliable and effective tissue culture system was established for Perilla frutescens (perilla) using different cultivar, explant source, and growth regulator composition in medium. MS medium supplemented with 2.0 mg/L BA and 0.1 mg/L NAA was maximum at shoot induction. Cotyledon explants formed more shoots than hypocotyl explants. The frequency of plant regeneration through organogenesis was higher (22.8%) than that (6.1%) of somatic embryogenesis. Five genotypes of perilla were screened for the feasibility of shoot regeneration, cotyledon explant of 'Manbaek' showed the highest shoot induction at a frequency of 27.3% among the tested cultivars.

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l−1 thidiazuron (TDZ) and 1 mg l−1 α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation.

High Frequency Somatic Embryogenic Callus Induction and Plant Regeneration from Various Indica Rice Genotypes

The paper evaluated the behavior of in vitro culture responses from a diverse set of Indica rice (Oryza sativa L.) genotypes. Significant differences were found in embryogenic callus induction frequency, callus growth and plant regeneration frequency when mature embryos of 11 cultivars, breeding lines and land races were compared. Genotype as well as plant growth regulator influenced the plant regeneration frequency. Callus induction frequency was not correlated with callus growth as well as plant regeneration frequency. The regenerated plants could grow to normal, fertile plants after they were successfully established in soil.

오차드그래스의 품종이 캘러스배양과 식물체 재분화에 미치는 영향

오차드그래스의 최적 조직배양조건을 확립하기 위하여 8가지 품종의 성숙종자로부터 배발생 캘러스 유도 및 캘러스로부터의 식물체 재분화 효율을 조사하였다. 4주 동안 배양한 종자로부터 캘러스 유도율은 Frontier, Roughrider, 93E, Frode, Potomac, Ambassador, Hapsung 2, Amba 순으로 높은 캘러스 유도율을 보였고 식물체로의 재분화율은 Roughrider, Frontier, Potomac, Frode, Amba, Hapsung2, 93E, Ambassador 순으로 나타났다. 전체적으로는 캘러스 유도율이 높은 품종이 식물체 재분화율도 높은 경향을 나타내었다. 본 연구를 통하여 밝혀진 캘러스 유도와 식물체 재분화율이 우수한 품종은 분자육종을 통한 신품종 오차드그래스의 개발에 유용하게 이용될 수 있을 것이다. In order to investigate the effects of genetic variations of orchardgrass in tissue culture response, calli were induced from mature seeds of eight varieties, 'Hapsung 2', '93E', 'Amba', 'Ambassdor', 'Frode', 'Frontier', 'Potomac' and 'Roughrider', and plant regeneration frequency was compared. Significant differences were observed among the varieties in both callus induction and plant regeneration. Callus induction rate of viable seeds varied from 24.3% to 71.7%. Plant regeneration frequency ranged from 76.6% to 29.7%. 'Roughrider' varieties showed higher regenerability with the frequency of 76.6%. These results can be used not only to provide additional improvements in the plant regeneration frequency from transgenic callus, but also useful for molecular breeding of orchardgrass through genetic transformation.

Identification of a novel elite genotype for in vitro culture and genetic transformation of cotton

Hypocotyls of cotton (Gossypium hirsutum L.) cultivars cv. YZ-1, Coker 312 and Coker 201 were inoculated on Murashige and Skoog callus induction medium. YZ-1 exhibited a very high regeneration potential, with 81.9 % of the explants inoculated differentiated into embryogenic callus within 8–10 weeks. During the process of callus maintenance (subculture for 1 to 3 years), the total embryos number in Coker 312 and Coker 201 calli dropped sharply, and the percentage of embryo germination decreased. On the contrary, the callus of YZ-1 consistently maintains a high frequency of plant regeneration after long-time subculture. Transgenic kanamycin-resistant calli of Coker 201 partially lost the ability of somatic embryogenesis and plant regeneration. The stress produced by the transformation procedure slightly affected somatic embryogenesis and plant regeneration of YZ-1, which showed minimum loss of plant regeneration ability.

Genotypic variability in callus induction and plant regeneration through somatic embryogenesis of five deepwater rice (Oryza sativa L.) cultivars of Bangladesh

Experiments were carried out to induce embryogenic callus and plant regeneration from five different deepwater rice cultivars. Using mature embryos as explant, all the cultivars demonstrated high callus induction and plant regeneration frequencies. When coleoptile and root segments used as explants, they had low frequencies of embryogenesis. Depending on different genotypes, the best plant regeneration was obtained on LS (Linsmaier and Skoog, 1965) based medium supplemented with 2 mg l -1 BAP + 1.5 mg l -1 2,4-D. Large variabilities in callus growth and plant regeneration potential were revealed among the cultivars tested. Cultivar HA-8 formed a high frequency (78%) of callus than that of other cultivars. In contrast cv. HA-1 produced the highest percentage (72%) of plant regeneration. The callus growth potential was not correlated with the plant regeneration potential. Coleoptiles and root segments produced calli, which did not develop any shoot bud in regeneration media. Moreover, the calli turned blackish, watery and translucent after 25-28 days of culture. It is clear that mature seed scutellum (MSS) is the best explant for callus induction and plant regeneration.

Influence of antibiotic cefotaxime on somatic embryogenesis and plant regeneration in indica rice

An antibiotic, cefotaxime (Omnatax) has been found to promote somatic embryogenesis and subsequent plant regeneration in vitro in indica-type basmati rice cultures. Response was highly genotype specific. The number, mass and morphology of the calli formed on the scutellar tissues were dependent on the growth medium (with or without cefotaxime). The embryogenic nature of nodular calli was confirmed through histological analysis and their plant regeneration ability. The calli of variety Pusa basmati 1 grown on medium supplemented with cefotaxime (100 mg/L) exhibited up to 70.5% plant regeneration as compared to control (51.51%). Plants regenerated from emryogenic calli were phenotypically normal and identical to seed-derived plants and exhibited normal fertility. A limited humidity and an optimal aeration of the culture tubes further enhanced the frequency of somatic embryogenesis and plant regeneration.

Plant Regeneration from Immature Inflorescence Culture and Genetic Transformation of Wheatgrass ( Agropyron cristatum × A. desertorum cv. Hycrest-Mengnong)

The plants of hybrid wheatgrass ( A. cristatum × A. desertorum cv. Hycrest-Mengnong) were directly induced from embryogenic callus regenerated from immature inflorescence. Immature inflorescence was cultured on improved MS medium containing 2.0-3.0 mg L −1 2,4-D to regenerate callus. The calli were then transferred to hormone-free MS medium for differentiation and 1/2 MS medium for rooting. Results showed that callus initiation frequency was 83.4% and plant regeneration frequency was 59.6%. Phosphinothricin acetyltransferase ( bar) gene was transformed into the hybrid wheatgrass by particle bombardment. Resistant callus was obtained using selecting agent, herbicide glufosinate of 0.5 mg L −1, and some transgenic plants were recovered in vitro. The transgenic plants were identified by PCR and Southern blot analysis and these plants developed normally in the glufosinate medium, whereas the nontransgenic plants did not. The results demonstrated that bar cDNA integrated into the genomic DNA of the transgenic plants. The transgenic frequencies of bar gene were 1.1%.

Liquid culture as a positive condition to induce and enhance quality and quantity of somatic embryogenesis of Lilium longiflorum

Abstract The objective of this study was to establish a simplest and efficient method for high frequency of somatic embryogenesis and plant regeneration of Lilium longiflorum ‘Easter Lily’. Embryogenic cell formation was obtained from friable calli cultured in either agar-solidified or liquid MS media at different volumes containing 1.0 mg l−1 α-naphthaleneacetic acid (NAA) and 0.2 mg l−1 thidiazuron (TDZ). However, the number of somatic embryos derived from embryogenic calli cultured in liquid medium, especially at a volume of 20 ml, were shown to be more than in solidified medium, 170 rather than 28, and the forms of somatic embryos in different developmental steps (globular-, heart- and cotyledon-shaped) were clearly distinguished under microscope. The submerged calli in liquid medium became necrotic due to the loss of respiration while the emerged calli underwent embryogenesis. Mature somatic embryos were transferred onto hormone-free 1/2 MS medium for germinating and developing into plantlets. About 78% of the embryos developed into plantlets with normal radicle and plumule. One-month-old regenerated plantlets were then transplanted in nursery and the survival rate was of 98%. After 6 months, more than 1500 plants originated from somatic embryos were shown to be more vigorous than plants obtained via direct organogenesis.

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25 degrees C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25-27 degrees C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.

Organogenesis and embryogenesis in several Hypericum perforatum genotypes

St John's wort (Hypericum perforatum) is a valuable plant used as a herbal remedy or in phytopharmaceutical drugs to treat a variety of physical ailments. Much research has been performed to study the biochemical production of secondary metabolites of in vitro cultured plants or organs. However, all of these studies have looked at the regeneration of plants from explants in only one genotype. In addition, no study has revealed the mechanism of plant regeneration in H. perforatum, i.e. organogenesis or somatic embryogenesis. We found that different genotypes Helos, Topas, Elixir, and Numi responded similarly to regeneration medium. The regeneration responses (i.e. callus, root, or shoot production) of identical explants from different genotypes were similar. However, the source of explant material (leaves, hypocotyls, and roots) from the same genotype had significant effects on the response to media and plant regeneration frequency. Using scanning electron microscopy and light microscopy, the progress of organogenesis and embryogenesis under similar culture conditions was recorded. Root segments were the most responsive explants, producing the maximum number of shoots per explant of all the genotypes.

캘러스의 형태와 항산화물질 첨가가 톨 페스큐의 식물체 재분화와 형질전환효율에 미치는 영향

유용유전자 도입을 통한 신품종 톨 페스큐를 개발할 목적으로 Agrobacterium을 이용한 효율적인 식물체 재분화 및 형질전환에 미치는 몇 가지 요인을 조사하였다. 성숙종자로부터 유도된 캘러스를 형태에 따라 3가지 type으로 분류하였고 type II 캘러스는 유백색으로 녹색을 띠며 조직적으로 치밀한 상태이며 식물체로의 재분화효율이 52.6%로 가장 높게 나타났다. 또한 재분화 배지에 <TEX>$AgNO_3$</TEX>와 cysteine을 동시에 첨가해 준 경우 무첨가구에 비해 캘러스 유도율은 6.7%, 식물체 재분화율은 12% 씩 각각 증가하였다. 캘러스 type 별 형질전환 효율을 조사한 결과 type II 캘러스는 58.0%로 가장 높은 형질전환효율을 나타내었다. 형질전환체를 PCR 및 PCR-Southern blot 분석을 실시하여 본 결과 발현벡터의 T-DNA 영역이 형질전환 식물체의 genome에 성공적으로 도입되었음을 확인할 수 있었다. 본 연구를 통하여 확립된 효율적인 형질전환 시스템은 분자육종을 통한 신품종 톨페스큐의 개발에 유용하게 이용될 수 있을 것이다. An efficient transformation system for the production of transgenic plants has been developed for tall fescue (Festuca arundinacea Schreb.) via Agrobacterium-mediated transformation of seed-derived callus. From the point of morphogenetic capacity, three types of callus were selected. High frequency of plant regeneration was obtained by selection of type II callus, and the plant regeneration frequency was 52.6% when embryogenic callus were cultured on the regeneration medium. Supplementation of the media with 10 mg/L <TEX>$AgNO_3$</TEX> and 40 mg/L cysteine enhanced frequencies of plant regeneration up to 65.3%. The highest transformation efficiency was also obtained when type II callus were inoculated with Agrobacterium. Southern blot analysis of PCR products of transgenic plants demonstrated that transgenes were successfully integrated into the genome of tall fescue. Efficient regeneration system and transformation established in this study will be useful for molecular breeding of tall fescue through genetic transformation.

식물생장조절물질과 배지첨가물질이 켄터키 블루그래스의 식물체 재분화에 미치는 영향

To optimize tissue culture responses for genetic transformation of Kentucky bluegrass, the effects of culture medium supplements on tissue culture responses were investigated with mature seeds of a cultivar `Newport` as explant tissues. The optimal concentration of 2,4-D (2.4-dichloro phenoxy acetic acid) for the induction of embryogenic callus from mature seed was 3 mg/L. Plant regeneration frequency was 54% when embryogenic callus was cultured on the regeneration medium supplemented with 1 mg/L 2,4-D and 3 mg/L of BA (6-benzyladenine). Addition of 1 g/L of casein hydrolysate and 500 mg/L of L-proline improved frequencies of embryogenic callus induction and plant regeneration up to 60.8% and 58.3%, respectively. Regenerated plants were grown normally when shoots transplanted to the soil. A rapid and efficient plant regeneration system established in this study. We suggest that the results may be useful for molecular breeding of Kentucky bluegrass through genetic transformation.

DNA marker를 이용한 벼의 조직배양 효율 재선

The purpose of this study is to improve the culturability of a indica type rice cultivar, IR 36, using DNA marker associated with the ability of plant regeneration in anther and seed culture. The varietal difference of ability of callus formation and plant regeneration was investigated in anther and seed culture of 8 rice cultivars. Three japonica rice cultivars showed to have better culturability than those of tongil and indica type genotypes. But two indica/japonica lines, `MGRI 079` and `MGRI 036`, which were selected to have good culturability in previous study showed the highest regenerability (20%) in anther culture of 8 rice cultivars. Thirty four lines were selected by marker screening using RZ400 for 100 lines derived from a cross . The frequency of callus formation of 30 lines was higher than those of `IR 36` in anther culture of the selected lines. The ability of plant regeneration of 15 lines was higher than that of `IR 36` in the seed culture of 34 lines. A promising line, , was selected to have better culturability in the anther and seed culture of the lines. The heading date and grain shape of the was similar to `IR 36`. In seed culture of 50 lines derived from a rice cross , 11 lines including showed to have higher regenerability compared with `IR 36`. The highest frequency of plant regeneration (11%) was obtained from in seed culture of the lines.

Application of nurse culture for plant regeneration from protoplasts of Lilium japonicum thunb

The regeneration of lily protoplasts isolated from suspension cells of Lilium japonicum was achieved by using the nurse culture method. The protoplasts divided only under the nurse culture application. The divided protoplasts grew into colonies and developed into visible calluses on a medium containing picloram. After the calluses were transferred to a hormone-free medium, plantlets formed from them. The highest frequency of plant regeneration was obtained on a medium containing 1 μM gibberellin 4 (GA4). The cleaved amplified polymorphie sequences (CAPS) method was used to confirm that the regenerants were not plants that had escaped from nurse cells. We were able to transplant the plantlets to soil in pots without acclimatization, and they showed normal growth.

Efficient plant regeneration from protoplasts of eggplant rootstock cultivar and its wild relatives

Protoplast culture and shoot regeneration from protoplast-derived calli were compared among different organs of Solanum integrifolium and three of its wild relatives (S. abutiloides, S. scabrum, and S. toxicarium). Leaves, cotyledons, and hypocotyls were used as sources of protoplast preparation. After one month of culture, a high frequency of visible colony formation was obtained from cotyledon protoplasts of S. integrifolium and S. scabrum, hypocotyl protoplasts of S. integrifolium and S. abutiloides, and leaf protoplasts of S. integrifolium and S. toxicarium. In addition, when the primary culture was started at a density of 2.5−5×104 protoplasts ml−1, the highest frequency of colony formation was obtained. Moreover, when the colonies were subcultured for 7 days on solid callus-proliferation medium before being transferred to shoot-induction medium, the plant regeneration frequency improved to between 91.8 and 98.8%.