High Frequency Plant Regeneration Research Articles

High-frequency plant regeneration via adventitious shoot formation from deembryonated cotyledon explants of Sesamum indicum L.

ABSTRACT Sesamum indicum L. was used as an important oil crop in the world. An efficient protocol for in vitro plant regeneration via adventitious shoot formation from deem-bryonated cotyledon explants isolated from mature seeds of sesame is developed. Optimal medium for direct adventitious shoot formation was Murashige and Skoog (MS) medium with 22.2 μM 6-benzylaminopurin (BA) and 5.7 μM indole-3-acetic acid (IAA). Abscisic acid (3.8 μM ABA) and AgNO3 (29.4 μM) were effective in enhancing the frequency of adventitious shoot formation. Preculture of cotyledon explants on high sucrose concentration (6–9%) for 2 wk and subsequent transfer to 3% sucrose enhanced the frequency of adventitious shoot induction. Root formation from the adventitious shoots was easily achieved on MS medium containing 2.7 μM of α-naphthalene acetic acid (NAA). Regenerated plantlets were acclimatized on sand and peat moss (1:1), showing 95% survival with subsequent flowering and seed set. We established the high-frequency plant rege...

High-frequency plant regeneration through adventitious shoot formation in castor (Ricinus communis L.)

An efficient plant regeneration protocol was established for castor (Ricinus communis L.). Hypocotyl tissue from zygotic embryo axis produced adventitious shoots when treated with either thidiazuron (TDZ, 1 μM) or 6-benzylaminopurine (BA, 20 μM). TDZ resulted in more than a threefold higher rate of shoot induction (a maximum of 24.2 shoots per explant) than BA (6.8 shoots). Our results also showed that the pretreatment of explants in the dark increased the number of shoots regenerated per explant by 82% and 36% with TDZ and BA, respectively. The elongation of hypocotyl tissue in the dark appears to be the primary cause of the increase. Comparable rates of rooting were achieved on the media supplemented with either indole-3-butyric acid (IBA, 84.3%) or 1-naphthaleneacetic acid (NAA, 87.4%) at 5 μM. However, IBA was more efficient in promoting root and shoot development, resulting in a higher rate of establishment (93.5%) in the soil, compared to the rate with NAA (39.5%). Histological analysis showed the adventitious induction of the shoot buds originated from the cortex of the hypocotyl tissue.

Plant regeneration from zygotic embryo-derived embryogenic cell suspension cultures of Ranunculus kazusensis

Culture conditions for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Ranunculus kazusensis are described. Zygotic embryos formed white nodular structures and pale-yellow calluses at a frequency of 84.9% when cultured on half-strength Schenk and Hildebrandt (SH) medium supplemented with 0.1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). However, the frequency of white nodular structure and off-white callus formation decreased with an increasing concentration of 2,4-D up to 10 mg l−1, when the frequency reached 25%. Cell suspension cultures were established from zygotic embryo-derived pale-yellow calluses using half-strength SH medium supplemented with 0.1 mg l−1 of 2,4-D. Upon plating onto half-strength SH basal medium, over 90% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted to potting soil and grown to maturity at a survival rate of over 90% in a growth chamber. The plant regeneration system established in this study can be applied to mass propagation and conservation of this species.

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.

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l−1 2,4-dichlorophenoxyacetic acid and 0.5–2.0 mg l−1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0–2.0 mg l−1 α-naphthaleneacetic acid (NAA) and 0.5–2.0 mg l−1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l−1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl−1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.

Regeneration via organogenesis in callus cultures of Argyrolobium roseum

A reproducible protocol has been developed for high frequency plant regeneration from immature embryos of Argyrolobium roseum Jaub & Spach, an important medicinal legume. Green nodular calli were initiated from immature embryos excised from 10-d-old pods in 70 % of cultures within 3 weeks when grown on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm-3 benzylaminopurine (BAP) + 0.25 mg dm-3 indole-3-acetic acid (IAA). Subsequent transfer of 5 mm2 callus pieces to MS medium supplemented with BAP (0.5 mg dm-3) alone or in combination with IAA (0.25 mg dm-3) facilitated regeneration of multiple shoots. Organogenic calli bearing multiple shoots when transferred to MS medium supplemented with BAP (0.5 mg dm-3) + IAA (0.25 mg dm-3) supported rapid shoot elongation. Shoot propagules subcultured to Gamborg's medium (B5) with 0.5 mg dm-3 indole-3-butyric acid (IBA) rooted with 80 % frequency and developed into phenotypically normal plants. Plantlets were successfully acclimatized in a sterile mixture of sand and garden soil (1:1) under greenhouse and thereafter transferred to field beds.

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.

High Frequency Plant Regeneration of a Dessert Banana Cv. Mehersagar for Commercial Exploitation

High Frequency Plant Regeneration of a Dessert Banana Cv. Mehersagar for Commercial Exploitation

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.

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.

켄터키 블루그래스에 있어서 캘러스 배양 및 식물체 재분화에 미치는 요인의 영향

In order to optimize tissue culture conditions of Kentucky bluegrass(Poa pratensis L.), effects of culture medium supplements, media and cultivars on embryogenic callus induction and regeneration of plants were investigated. MS medium containing 3mg/L 2,4-D and 0.1mg/L BA was optimal for embryogenic callus induction from mature seeds. The highest plant regeneration frequency(57.7%) was observed when the embryogenic calli were cultured on N6 medium supplemented with 1mg/L 2,4-D and 3mg/L BA. Among several basic media, MS and N6 medium were optimal for callus induction and plant regeneration, respectively. Genotype was an important factor in plant regenerability. ‘Newport’ showed to have higher regeneration frequency of 53.4%. Regenerated plants were grown normally when shoots transplanted to the soil. A short tissue culture period and high-frequency regeneration system would be beneficial for molecular breeding of Kentucky bluegrass through genetic transformation.

High frequency in vitro regeneration of Lathyrus sativus L.

A simple and efficient protocol for high frequency plant regeneration of a grain legume grasspea (Lathyrus sativus L.) is described. Of different explant types tested epicotyl segments were most responsive. Murashige and Skoog's (1962) medium augmented with 17.76 µM 6-benzyladenine + 10.74 µM α-naphthaleneacetic acid showed the highest percentage of direct shoot regeneration. Among cultivars IC-120487 showed the highest regeneration frequency (80 %) with maximum shoot numbers (8.2 shoots per explant) and maximum average shoot length (4.1 cm). About 78 % of the regenerated shoots were rooted in half-strength MS medium containing 2.85 µM indole-3-acetic acid. After primary hardening the plantlets were established in soil with a survival rate of 75 %.

High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.)

A high-frequency plantlet regeneration protocol was developed for black pepper (Piper nigrum L.) through cyclic secondary somatic embryogenesis. Secondary embryos formed from the radicular end of the primary somatic embryos which were originally derived from micropylar tissues of germinating seeds on growth regulator-free SH medium in the absence of light. The process of secondary embryogenesis continued in a cyclic manner from the root pole of newly formed embryos resulting in clumps of somatic embryos. Strength of the medium and sucrose concentration influenced the process of secondary embryogenesis and fresh weight of somatic embryo clumps. Full-strength SH medium supplemented with 1.5% sucrose produced significantly higher fresh weight and numbers of secondary somatic embryos while 3.0 and 4.5% sucrose in the medium favored further development of proliferated embryos into plantlets. Ontogeny of secondary embryos was established by histological analysis. Secondary embryogenic potential was influenced by the developmental stage of the explanted somatic embryo and stages up to "torpedo" were more suitable. A single-flask system was standardized for proliferation, maturation, germination and conversion of secondary somatic embryos in suspension cultures. The system of cyclic secondary somatic embryogenesis in black pepper described here represents a permanent source of embryogenic material that can be used for genetic manipulations of this crop species.

Factors Enhancing Induction of High Frequency Plant Regeneration from Somatic Embryos of Indica Rice (Oryza sativa L.)

Factors Enhancing Induction of High Frequency Plant Regeneration from Somatic Embryos of Indica Rice (Oryza sativa L.)

톨 페스큐의 성숙종자로부터 효율적인 캘러스 배양 및 식물체 재분화

In an effort to optimize tissue culture conditions for genetic transformation of tall fescue (Festuca arundinacea Schreb.), an efficient plant regeneration system from seed-derived calli was established. MS medium containing 6 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.1 mg/L benzyladenine (BA) were optimal for embryogenic callus formation from mature seed and had a strong effect on successive plant regeneration. The plant regeneration frequency above 50% was observed when embryogenic calli induced in this medium were transferred to N6 medium supplemented with 1 mg/L 2,4-D and 3 mg/L BA. Among several basic media, MS and N6 medium were optimal for callus induction and plant regeneration, respectively. `Kentucky-31` showed to have high frequencies of embryogenic callus induction and plant regeneration up to 58.3 and 50%, respectively. Addition of sucrose to the regeneration medium as a carbon source increased regeneration frequency up to 55%. A short tissue culture period and high-frequency regeneration system established in this study will be useful for molecular breeding of tall fescue through genetic transformation.

High frequency of protocorm like bodies (PLBs) induction and plant regeneration from protocorm and leaf sections of Aerides crispum

An efficient and reproducible method for the large-scale propagation of Aerides crispum L. using protocorm and leaf sections has been developed. Protocorm and leaf sections were cultured on Murashige and Skoog (MS) medium supplemented with cytokinins [ N 6-benzyl adenine (BA), thidiazuron (TDZ), and kinetin (KN), 0.5, 1.0, 2.0 and 5.0 μM], auxins [α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), 0.5, 1.0, 2.0 and 5.0 μM] and coconut liquid endosperm (CW: 5, 10 and 15%). The explants developed protocorm like bodies (PLBs) within 5–8 weeks on the growth medium. BA supplemented medium was found best for the induction of PLBs and an optimum of 49.1 and 22.0 PLBs developed from protocorm and leaf sections on medium supplemented with 1 and 2 μM BA, respectively. Upon subculture on basal MS medium, the PLBs differentiated plantlets within 6–8 weeks. The resulting plantlets were successfully transferred to potting mixture and 85% of plantlets survived after green house transplantation. This simple protocol will be useful for large-scale propagation of A. crispum L.

Influence of picloram and thidiazuron on high frequency plant regeneration in elite cultivars of wheat with long‐term retention of morphogenecity using meristematic shoot segments

AbstractMeristematic shoot segments (MSSs) were used to develop an efficient and reproducible regeneration system via multiple shoot differentiation in two elite cultivars of winter wheal. Cylindrical basal shoot segments. 3 4 mm in length, were excised from seedlings derived from mature embryos that had been germinated directly on MS‐based medium containing growth regulators. These segments were further cultured on the same medium formulations. Out of 24 variations analysed, the combination of 2 mg1 4‐amino‐3.5,6‐trichloropicolinic acid (picloram) + 3 mg/1 thidiazuron (TDZ) was found to be the most effective in inducing clumps of multiple schools, buds with little callusing. Sectors of these clumps were (1) further multiplied by biweekly subculturing on 2 mg/l picloram + 2.5 mg/l TDZ leading lo highly differentiating green clusters of buds without loss of tnorphogenesis for more than 12 months so far. and without albinism, or (2) transferred to 0.1 nig I picloram + I mg/l TDZ for shoot development and elongation. The elongated shoots rooted on basal medium with 0.5 mg/l indole‐3‐butyric acid (IBA) were transferred to soil and showed normal growth and fertility. Both cultivars formed multiple schools ranging from 28 to 33 per responding MSS after 10 12 weeks.

Improved efficiency of somatic embryogenesis from zygotic embryos in Hyoscyamus niger by seed water-soaking

We describe an efficient procedure to obtain somatic embryos from mature zygotic embryos of Hyoscyamus niger (black henbane). It has several advantages over previous regeneration methods, which are: the use of mature seeds, an average 80% somatic embryogenesis rate and a high (eight-fold higher than the control) plant regeneration frequency. The critical step in this protocol was soaking of the seeds in sterile distilled water for 16 h, prior to excision and culture of zygotic embryos, on MS basal medium supplemented with 2% sucrose, 2 g/l myo-inositol, 0.5 g/l 2-( N-morpholino) ethanesulfonic acid and 1 mg/l α-naphthalene acetic acid (NAA). The regenerated somatic seedlings were fertile and were morphologically uniform. This procedure is simple, rapid and effective for high frequency of plant regeneration via somatic embryogenesis. Moreover, this new method should facilitate the development of strategies to routinely transform recalcitrant plant species, including henbane.