High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides)

Rakesh Yadav,Pooja Arora,Dharmendar Kumar,Neeraj Dilbaghi,Ashok Chaudhury 한국식물생명공학회 2009 Plant biotechnology reports Vol.3 No.3

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l-1 KIN and 0.25 mg l-1 IAA. Nodal and internodal segments were found to be extraprolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l-1 KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l-1 IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future. Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with 0.25 mg l-1 KIN and 0.25 mg l-1 IAA. Nodal and internodal segments were found to be extraprolific over shoot apices during course of aseptic culture establishment, while 0.25 mg l-1 KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with 0.15 mg l-1 IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at 27 ± 2C for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.

A robust genome-editing method for wild plant species Nicotiana attenuata

강문영,안효민,Eva Rothe,Ian T. Baldwin,Kim Sang-Gyu 한국식물생명공학회 2020 Plant biotechnology reports Vol.14 No.5

CRISPR genome-editing techniques theoretically enable us to edit any genes in any plants. However, plant tissue culture is required for generating targeted mutants in plants, except in some model plant species such as Arabidopsis thaliana. To modify ecologically or agronomically important traits in plants using the CRISPR system, a robust plant regeneration method and efcient genome-editing tools must be developed. This study shows the entire process of genome editing and the regeneration process for wild tobacco, Nicotiana attenuata. We delivered T-DNA harboring Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) via Agrobacterium-mediated transformation into the hypocotyl cells of the wild tobacco and regenerated gene-edited plants. The efcacy of genome editing was measured in N. attenuata protoplasts in which SpCas9 and gRNA were transiently expressed. Light intensity (476.66–627.00 μW/cm2 , 20.69–52.21 μE) was optimized to enhance the emergence of plant shoots during callus induction, and the core step of dealing with plant tissues was recorded. In addition, we found that wounding the bottom part of mature plants was critical for root regeneration. By tracking mutation patterns and efciency at each regeneration step, we found that the mutation was induced early on in the tissue culture process and maintained throughout the regeneration process. Genome-editing techniques have opened the way to study the function of the genes in all plants, and this study will provide guidelines for editing a gene in the plant of interest.

'다유들깨'품종의 하배축에서 캘러스를 통한 고효율 식물재분화

서여월,손지희,강홍규,선현진,이효연,Ruyue Xu,Ji-Hi Son,Hong-Gyu Kang,Hyeon-Jin Sun,Hyo-Yeon Lee 한국식물생명공학회 2023 JOURNAL OF PLANT BIOTECHNOLOGY Vol.50 No.1

본 연구는 종유용 들깨 품종인 다유들깨'의 유식물체에서 캘러스 유도를 통한 고효율의 재분화 체계를 구축하기 위해 수행되었고 이미 보고된 바 있는'남천들깨'와 함께 연구를 진행하였다. 캘러스는 잎, 자엽, 하배축 중 0.1 mg/L NAA와 0.5 mg/L BA가 첨가된 배지에서 배양된 다유들깨의 하배축에서 가장 건강한 캘러스가 형성되었다. 암상태와 장일조건에서 각각 캘러스를 유도한 후 신초 재분화를 유도했을 때 모든 조건에서 남천들깨보다 다유들깨가 재분화율이 월등하게 높았다. 또한 0.1 mg/L NAA와 0.5 mg/L BA 배지의 암상태와 장일조건에서 다유들깨 하배축의 신초 재분화율은 각각 86.7%와 84.4%로 두 조건 간 차이는 낮은 것으로 조사되었지만 전체적으로 암조건에 비해 장일조건에서 유도된 캘러스의 재분화 빈도가 높았다. 본 연구에서 다유들깨의 하배축으로부터 고효율의 재분화 조건을 확립하기 위해 다양한 식물생장호르몬 조합실험을 수행한 결과 NAA 없이 0.5 mg/L BA 만 첨가된 배지에서 가장 높은 90%의 재분화율을 보여 주었으며 이 중 정상적인 식물체가 70.5% 와 비정상적인 식물체가 19.3%로 조사되었고 NAA가 첨가되거나 농도가 높아질수록 비정상 식물체의 출현율이 높아졌다. 정상적으로 재분화된 신초는 1/2 MS 배지로 옮긴 후 10~15일 후에 뿌리가 관찰되었고 30일 후에는 완전한 식물체로 성장하였다. 본 연구에서 확립된 다유들깨 하배축을 이용한 재분화체계는 지금까지 보고된 다른 들깨 품종들의 재분화 체계에 비해 재분화 효율이 높았으며 향후 들깨에서 조직배양과 형질전환에 의존하는 유전자편집 등의 분자육종 분야에 유용하게 이용될 수 있을 것으로 기대된다. This study was conducted to establish an efficient plant regeneration system in 'Dayu', a Korean variety of Perilla frutescens developed for seed oil production, in conjunction with the previously studied variety 'Namcheon'. The healthiest callus was formed on the hypocotyl explants cultured on a medium containing 0.1 mg/L NAA and 0.5 mg/L BA, outperforming the leaf and cotyledon samples. In both dark and long-day conditions, Dayu consistently exhibited significantly higher shoot regeneration rates compared with Namcheon. The highest shoot regeneration rates in Dayu were observed from the hypocotyl explants cultured on 0.1 mg/L NAA and 0.5 mg/L BA media, with shoot regeneration rates of 84.4% and 86.7% under dark and long-day conditions, respectively. Various combinations of plant growth regulators were tested to establish the optimal shoot regeneration conditions for Dayu hypocotyl explants. The results demonstrated that the highest shoot regeneration rate (90%) was achieved when 0.5 mg/L of BA was added to the medium without NAA. Among the regenerated shoots, 70.5% were normal plants, while 19.3% were abnormal. The addition of NAA or an increase in its concentration led to a higher occurrence of abnormal plants. After the regenerated shoots were transferred to 1/2 MS medium, roots were observed within 10-15 days. By day 30, they had developed into complete plants. The results obtained from the regeneration experiments with the perilla variety Dayu can valuably inform molecular breeding reliant on transformation techniques such as genome-editing and genetic modification technology.

금잔디 포복경으로부터 식물체 재분화에 있어서 식물생장조절물질과 배지첨가물질의 영향

김경희,이병현,김용구,허성현,배은지,이광수,박남창 한국잔디학회 2011 Weed & Turfgrass Science Vol.25 No.2

To optimize tissue culture conditions for genetic transformation of Zoysia matrella (L.) Merr., we investigated the effects of different plant growth regulators and medium supplements on plant regeneration using stolon explants excised from mature plant grown in a green house. Plant regeneration frequency was 33.3% when stolon tissues with a node were cultured on the regeneration medium supplemented with 0.5 mg/L 2,4-D and 1 mg/L of kinetin. Comparing the basal media tested, MS medium showed higher plant regeneration performance than N6 or SH medium. Addition of 5 mg/L AgNO_3 with 10 mg/L cysteine improved frequency of plant regeneration up to 40%. Among different carbon sources, 3% sucrose was found to show the best for regeneration frequency. This rapid and efficient plant regeneration system would be useful for using genetic transformation experiments of manilagrass without intervening callus-mediated regeneration. 금잔디(Zoysia matrella (L.) Merr.)의 포복경 조직으로부터 식물체 재분화를 위한 최적 조직배양조건을 확립하기위하여 온실에서 재배한 식물체의 포복경 절편체로부터식물체 재분화에 미치는 몇 가지 요인을 조사하였다. 포복경 조직으로부터 식물체 재분화에 있어서 기본배지로는MS배지가 N6배지나 SH배지에 비해 높은 효율을 보였으며, 배지에 첨가되는 식물생장조절물질로는 0.5 mg/L 2,4-D와 1 mg/L of kinetin을 첨가했을 때 33.3%로 가장 높은재분화율을 보였다. 또한 배지에 첨가되는 탄소원 종류로는 sucrose를 3% (v/v) 농도로 첨가해 주었을 때 재분화효율이 향상되었으며, 재분화배지에 첨가되는 항산화물질로는 5 mg/L AgNO_3와 10 mg/L cysteine을 동시에 첨가해주었을 때 40%의 가장 높은 식물체 재분화 효율을 나타내어 재분화효율이 향상되었다. 본 연구를 통하여 확립한금잔디의 포복경 재분화 시스템은 캘러스 배양기간 없이단기간 내에 재분화 식물체를 높은 효율로 얻을 수 있음으로써 유전자 형질전환을 통한 잔디의 분자육종에 있어서 유용하게 사용되어질 수 있을 것이다.

Plant Regeneration from Sliced Mature Embryo Fragments of Wheat Cultivars

Kyung-Hee Kim,Moon-Seok Kang,Young-Up Kwon,Sang-Kyu Lee,Jung-Hun Moon,Sinae Han,Poo-Reum Oh,Byung-Moo Lee 韓國作物學會 2005 Korean journal of crop science Vol.50 No.5

Mature embryos were aseptically excised with a scalpel and sliced in fragments measuring 0.5 mm in diameter (sliced mature embryo fragment; 4 ~~ 5 fragments/one embryo). Sliced mature embryo fragments of six wheat cultivars were cultured to develop an efficient method of callus induction and plant regeneration. Callus derived from sliced mature embryo fragments showed a good capacity to embryogenesis and regeneration. Furthermore sliced mature embryo fragments decreased contamination from fungi and bacteria. The high efficiency of callus induction were obtained Keumkangmil and Bob-white. For plant regeneration, selected embryogenic calli were transferred to two types regeneration media. An average number of green spots per callus was 4 to 5 in regeneration media after about one week. Percentage of plant regeneration showed high in regeneration medium containing 0.1 mg/l 2,4-D and 5 mg/l zeatin. Especially, Keumkangmil (27.5~% ) and Bobwhite (33.3~% ) showed high regeneration efficiency. This regeneration system from sliced mature embryo fragments may provide an effective and convenient explant for plant transformation studies

Plant Regeneration from Sliced Mature Embryo Fragments of Wheat Cultivars

Kim Kyung-Hee,Kang Moon-Seok,Kwon Young-Up,Lee Sang-Kyu,Moon Jung-Hun,Han Sinae,Oh Poo-Reum,Lee Byung-Moo The Korean Society of Crop Science 2005 Korean journal of crop science Vol.50 No.5

Mature embryos were aseptically excised with a scalpel and sliced in fragments measuring 0.5 mm in diameter (sliced mature embryo fragment; 4 ${\~}$ 5 fragments/one embryo). Sliced mature embryo fragments of six wheat cultivars were cultured to develop an efficient method of callus induction and plant regeneration. Callus derived from sliced mature embryo fragments showed a good capacity to embryogenesis and regeneration. Furthermore sliced mature embryo fragments decreased contamination from fungi and bacteria. The high efficiency of callus induction were obtained Keumkangmil and Bob­white. For plant regeneration, selected embryogenic calli were transferred to two types regeneration media. An average number of green spots per callus was 4 to 5 in regeneration media after about one week. Percentage of plant regeneration showed high in regeneration medium containing 0.1 mg/l 2,4-D and 5 mg/l zeatin. Especially, Keumkangmil ($27.5\%$) and Bobwhite ($33.3\%$) showed high regeneration efficiency. This regeneration system from sliced mature embryo fragments may provide an effective and convenient explant for plant transformation studies.

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

Quyen Van Nguyen,선현진,부경환,이도성,이지현,임평옥,이효연,Key Zung Riu,이동선 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.1

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 lM a-naphthaleneacetic acid (NAA), 10 lM 6-benzyladenine (BA) and 0.1 lM 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 lM NAA and 0.3 lM GA3 for callus induction for 6 weeks followed by 2 lM NAA and 5 lM BA resulted in the highest plant regeneration frequency (83.33 ± 6.43 %) andthe 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).

사과 ‘후지’의 잎 절편체로부터 신초 기관형성을 통한 식물체 재생에 적합한 배지조성

형남인,이윤경,권영주 한국식물생명공학회 2019 JOURNAL OF PLANT BIOTECHNOLOGY Vol.46 No.4

Plant regeneration protocols for adventitious shoot organogenesis from apple (Malus domestica ‘Fuji’) leaf explants were developed in the present study. The effects of different basal media, types and concentrations of carbon sources, and concentrations of plant growth regulators were evaluated to determine the optimal shoot regeneration conditions for ‘Fuji’ apple leaf explants. On different treatments involving combinations of basal media, LS and N6 media, and different types and concentrations of cytokinins, 6-benzyl-adenine (BA) and thidiazuron (TDZ), shoot regeneration rates were the highest in the N6 medium combined with BA. Among the plant growth regulator and carbon source combination treatments, 5.0 mg/L BA, and 0.1 mg/L α- naphthalene acetic acid (NAA) with 40 g/L sorbitol was the optimal combination for shoot regeneration. In addition, the optimal sorbitol concentrations for shoot regeneration were 40 g/L and 60 g/L. The highest regeneration (81.8%) was achieved using 40 g/L sorbitol. The regenerated shoots elongated and rooted on rooting medium, consisting of 1/4 MS medium with 0.2 mg/L indole-3-butyric acid (IBA). The plantlets were acclimatized and the regenerated plants exhibited normal phenotypes.

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

Nguyen, Quyen Van,Sun, Hyeon Jin,Boo, Kyung Hwan,Lee, Doseung,Lee, Ji-Hyun,Lim, Pyung Ok,Lee, Hyo Yeon,Riu, Key-Zung,Lee, Dong-Sun 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.1

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 ${\mu}M$ ${\alpha}$-naphthaleneacetic acid (NAA), 10 ${\mu}M$ 6-benzyladenine (BA) and 0.1 ${\mu}M$ gibberellic acid ($GA_3$) had the most significant effect on callus growth in term of weight ($120.98{\pm}22.56$ 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 ${\mu}M$ NAA and 0.3 ${\mu}M$ $GA_3$ for callus induction for 6 weeks followed by 2 ${\mu}M$ NAA and 5 ${\mu}M$ BA resulted in the highest plant regeneration frequency ($83.33{\pm}6.43%$) and the highest number of plantlets per explant ($7.93{\pm}1.24$). 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 ($77.17{\pm}9.80%$) and produced seeds ($175.21{\pm}28.01$ firm seeds per plant).

이탈리안 라이그래스의 성숙종자 유래 캘러스로부터 효율적인 식물체 재분화

우현숙,이상훈,이동기,김진수,원성혜,이병현 Plant molecular biology and biotechnology research 2004 Plant molecular biology and biotechnology research Vol.2004 No.-

As an initial step for future genetic manipulations to improve forage characteristics of Italian ryegrass (Lolium multiflorum Lam.), an efficient tissue culture system was established and the factors affecting plant regeneration were evaluated. MS medium containing 5 ㎎/L 2,4-D was optimal for embryogenic callus induction from mature seed and had a strong effect on successive plant regeneration. The plant regeneration frequency was observed at above 70% when embryogenic calli induced were transferred to N6 medium supplemented with 1 ㎎/L 2,4-D and 5 ㎎/L BA. Among several basic media tested, MS and M6 medium were optimal for callus induction and plant regeneration, respectively. Genotype was an important factor in plant regenerability. 'Jeanne' showed the highest regeneration frequency of 73%. A short tissue culture period and high-frequency regeneration system established in this study will be useful for molecular breeding of Italian ryegrass through genetic transformation.