Physiological and morpho-anatomical analyses of hyperhydric Arabidopsis thaliana influenced by media components

Nurashikin Kemat,Richard G.F. Visser,Frans A. Krens The Korean Society of Plant Biotechnology 2023 JOURNAL OF PLANT BIOTECHNOLOGY Vol.50 No.1

Hyperhydricity is a physiological anomaly that significantly affects the growth and proliferation rate of crops cultivated by tissue culture techniques. To better understand the mechanisms that govern hyperhydricity incidence, we examined the effects of several media components, particularly cytokinin and gelling agents. These elements were found to be influential in both in vitro propagation and the development of hyperhydricity. Our study revealed that Arabidopsis thaliana seedlings had a greater manifestation of hyperhydricity symptoms when exposed to high cytokinin concentrations compared with the control. The presence of gelrite led to the manifestation of hyperhydric symptoms by elevated water build-up in the apoplast. The phenomenon of stomata closure was observed in the hyperhydric leaves, resulting in an increased ability to retain water and a decrease in the transpiration rates when compared to their respective control leaves. Additionally, histological examinations of the cross sections of hyperhydric leaves revealed an irregular cellular arrangement and large intercellular spaces. Furthermore, hyperhydric seedlings displayed impaired cuticular development in comparison to their normal seedlings.

Shoot multiplication kinetics and hyperhydric status of regenerated shoots of gladiolus in agar-solidified and matrix-supported liquid cultures

Gupta, S. Dutta,Prasad, V.S.S. The Korean Society of Plant Biotechnology 2010 Plant biotechnology reports Vol.4 No.1

In vitro shoot regeneration of gladiolus in three different culture systems, viz., semi-solid agar (AS), membrane raft (MR), and duroplast foam liquid (DF) cultures was evaluated following the kinetics of shoot multiplication and hyperhydricity at optimized growth regulator combinations. Compared to the AS system, matrixsupported liquid cultures enhanced shoot multiplication. The peak of shoot multiplication rate was attained at 18 days of incubation in the MR and DF systems, whereas the maximum rate in the AS system was attained at 21 days. An early decline in acceleration trend was observed in liquid cultures than the AS culture. The hyperhydric status of the regenerated shoots in the different culture systems was assessed in terms of stomatal attributes and antioxidative status. Stomatal behavior appeared to be normal in the AS and MR systems. However, structural anomaly of stomata such as large, round shaped guard cells with damage in bordering regions of stomatal pores was pronounced in the DF system along with a relatively higher $K^+$ ion concentration than in the AS and MR systems. Antioxidative status of regenerated shoots was comparable in the AS and MR systems, while a higher incidence of oxidative damages of lipid membrane as evidenced from malondialdehyde and ascorbate content was observed in the DF system. Higher oxidative stress in the DF system was also apparent by elevated activities of superoxide dismutase, ascorbate peroxidase, and catalase. Among the three culture systems, liquid culture with MR resulted in maximum shoot multiplication with little or no symptoms of hyperhydricity. Shoots in the DF system were more prone to hyperhydricity than those in the AS and MR systems. The use of matrix support such as membrane raft as an interface between liquid medium and propagating tissue could be an effective means for rapid and efficient mass propagation with little or no symptoms of hyperhydricity.

마디 절편의 현탁배양에 의한 카사바의 미세증식

윤실,조덕이,소웅영,Yoon, Sil,Cho, Duck-Yee,Soh, Woong Young 한국식물생명공학회 2000 식물생명공학회지 Vol.27 No.3

For the micropropagation, node explants of cassava were cultured in liquid MS medium with various concentrations of cytokinins on a rotary shaker (100 rpm) for 2 weeks. The adventitious roots and shoots from the explants were differentiated more efficiently in liquid medium than in solid. But root formation was not inhibited in medium with BAP and kinetin at low concentration (>0.05 mg 1/sup -1/), while in medium added with BAP and zeatin at high level (<0.25 mg 1/sup -1/), it was inhibited by callus forming on cut end of the cuttings. However, all of plantlets grown in liquid medium for more than 2 weeks showed symptoms of hyperhydricity. The plantlets grown in liquid medium were transferred into culture bottles filled with fine sand or artificial soil (pitmoss:perlite:vermiculite, 1:1:1 v/v) wetted with half strength of Knop's solution. After transplanted to culture bottles, some of vitriscent leaves were defoliated and new leaves were normally formed from shoot apex. Most of plantlets (>95%) were hardened-off successfully only in culture bottles with fine sand, and grew into 3-5 cm seedlings possessing 4-6 nodes after 4 weeks. Thus, the mass propagation of cassava on medium containing cytokinin could be established based on the suspension culture using node explants. 카사바 (Manihot esculenta Crantz, cv. MCol 22)의 마디 절편을 MS 기본배지에 3종류의 사이토키닌을 여러 농도로 첨가한 조건에서 2주동안 현탁배양한 결과, 고체배지에서 3주 배양된 것보다 훨씬 생장이 효과적이었다. 현탁배양된 유식물에서는 과수화현상이 나타났으나, 이들을 세사 (마사토 + 1/2농도의 크놉씨액)를 담은 유리배양병에 이식하여 생육시킨 결과, 유경에서 새 잎이 신장되면서 과수화를 벗어나 4주후에는 4~6개의 마디를 가진 유경길이 3~5 cm의 정상 유묘로 생장됨으로서, 목본식물인 카사바의 경우 마디절편의 현탁배양에 의한 대량증식의 가능성을 확인했다. 현탁배양시 저농도의 제아틴 (0.01-0.05 mg 1$^{-1}$) 처리는 BAP 최적조건에서 보다 2배나 길게 부정아 길이를 신장시켰다. 사이토키닌 처리로 부정아가 잘 발생된 유식물은 토양이식 작업을 간편하게 했으며, 토양 이식 후 부정근을 활발히 발생시켜 one step culture시스템 배양이 가능하게 했다. 과수화된 유식물은 기외에서 순화가 어려웠으나 유기물이 혼입되지 않은 세사를 담은 유리배양병에서는 비무균조건일지라도 95% 이상 순화될 수 있었다.

Proteomic Analysis Reveals the Dynamic Role of Silicon in Alleviation of Hyperhydricity in Carnation Grown In Vitro

Muneer, Sowbiya,Wei, Hao,Park, Yoo Gyeong,Jeong, Hai Kyoung,Jeong, Byoung Ryong MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.1

<P>The present study depicted the role of silicon in limiting the hyperhydricity in shoot cultures of carnation through proteomic analysis. Four-week-old healthy shoot cultures of carnation “Purple Beauty” were sub-cultured on Murashige and Skoog medium followed with four treatments, viz. control (–Si/–Hyperhydricity), hyperhydric with no silicon treatment (–Si/+Hyperhydricity), hyperhydric with silicon treatment (+Si/+Hyperhydricity), and only silicon treated with no hyperhydricity (+Si/–Hyperhydricity). Comparing to control morphological features of hyperhydric carnations showed significantly fragile, bushy and lustrous leaf nature, while Si supply restored these effects. Proteomic investigation revealed that approximately seventy protein spots were differentially expressed under Si and/or hyperhydric treatments and were either up- or downregulated in abundance depending on their functions. Most of the identified protein spots were related to stress responses, photosynthesis, and signal transduction. Proteomic results were further confirmed through immunoblots by selecting specific proteins such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), PsaA, and PsbA. Moreover, protein–protein interaction was also performed on differentially expressed protein spots using specific bioinformatic tools. In addition, stress markers were analyzed by histochemical localization of hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>) and singlet oxygen (O<SUB>2</SUB><SUP>1–</SUP>). In addition, the ultrastructure of chloroplasts in hyperhydric leaves significantly resulted in inefficiency of thylakoid lamella with the loss of grana but were recovered in silicon supplemented leaves. The proteomic study together with physiological analysis indicated that Si has a substantial role in upholding the hyperhydricity in in vitro grown carnation shoot cultures. </P>

Short Communications : Reduction of Shoot Hyperhydricity in Micropropagated Potato Plants via Antisense Inhibition of a chCu/ZnSOD Gene

Yong Hwa Lee,Hyun Soon Kim,Mi Sun Kim,Yoon Sik Kim,Hyouk Joung,Jae Heung Jeon 한국농화학회 2009 Applied Biological Chemistry (Appl Biol Chem) Vol.52 No.4

The occurrence of hyperhydricity in transgenic potato plants, which carry a chloroplastic Cu/ZnSOD) gene of the lily in either a sense or antisense orientation, was investigated during in vitro culturing. Hyperhydric symptoms included curled leaves with a succulent appearance that were mainly observed in Cu/ZnSOD) sense plants. By contrast, antisense plants showed a low frequency of hyperhydricity and a high rate of ex vitro survival. Therefore, a knockdown approach for a chCu/ZnSOD gene could he a practical method to reduce hyperhydricity in plant micropropagation.

Peace포플러의 기내 배양시 발생하는 과수화 식물체의 조직적 특성

강효진,문흥규,박소영,김판기,Kang, Hyo-Jin,Moon, Heung-Kyu,Park, So-Young,Kim, Pan-Gi 한국식물생명공학회 2004 식물생명공학회지 Vol.31 No.2

목본류 생리학 연구에 있어서 중요한 수종인 Peace 포플러의 기초 연구자료를 얻고자 본 실험을 실시하였다. 액아 마디를 BA 0.2 mg/L처리된 MS 배지에 배양하여 shoot의 증식시 나타나는 과수화 된 shoot를 정상 shoot와 비교하였다. 과수화 된 shoot는 진한 녹색을 띠고, 잎은 두껍고 휘어져 있으며 투명하였다. 또 줄기는 정상적인 줄기보다 두껍고 길이가 짧았다. 조직학적 관찰 결과 과수화 된 잎은 책상조직의 발달이 부진하였으며 정상적인 잎에 비하여 책상조직과 해면조직 그리고 표피 세포 모두 불규칙하고 세포간극의 크기가 큰 것으로 나타났다. 또한 과수화 된 잎의 기공은 크기가 커서 단위면적 당 밀도가 낮았으며 공변세포와 그 주변 세포들이 변형되어 있었다. 과수화된 shoot의 직경은 정상적인 것보다 큰 것으로 나타났는데 이것은 피층과 수층 세포의 비대에 따른 것으로 관찰되었다. 한편 과수화 된 줄기는 정상 줄기에 비해 목질화 발달이 미약하여 형성층 구별이 어려웠다. We investigated the anatomical aspects of vitrification in peace poplar. Comparisons were made with regard to characteristics occurring between hyperhydric and normal shoots in shoot proliferation cultures on MS medium containing 0.2 mg/L BA. Compared with normal plants, hyperhydric plants had thick, curled, and dark green leaves. Hyperhydric stems were thicker and shorter than those of normal stems. When examined under the microscopes, the mesophyll palisade cells of hyperhydric leaves were vacuolated, whereas those of normal leaves contained normal and enriched vacuole with cytoplasm. Generally, the hyperhydric leaves showed poorly developed palisade parenchyma, and revealed irregular and bigger sized intercellular structures in both palisade and spongy parenchyma as well as epidermis cells compare to those of normal leaves. In addition, the hyperhydric leaves had lower stomatal density and bigger sized cell. Vascular tissues of hyperhydic stems were less differentiated because of poorly lignified xylem tissue. The greatly expanded cortical cells and pith appeared to be the main cause of thick stems as compared with normal stems.

Reduction of Shoot Hyperhydricity in Micropropagated Potato Plants via Antisense Inhibition of a chCu/ZnSOD Gene

Lee, Yong-Hwa,Kim, Hyun-Soon,Kim, Mi-Sun,Kim, Yoon-Sik,Joung, Hyouk,Jeon, Jae-Heung The Korean Society for Applied Biological Chemistr 2009 Applied Biological Chemistry (Appl Biol Chem) Vol.52 No.4

The occurrence of hyperhydricity in transgenic potato plants, which carry a chloroplastic Cu/ZnSOD gene of the lily in either a sense or antisense orientation, was investigated during in vitro culturing. Hyperhydric symptoms included curled leaves with a succulent appearance that were mainly observed in Cu/ZnSOD sense plants. By contrast, antisense plants showed a low frequency of hyperhydricity and a high rate of ex vitro survival. Therefore, a knockdown approach for a chCu/ZnSOD gene could be a practical method to reduce hyperhydricity in plant micropropagation.

Effects of Plant Growth Regulators and Light Intensity on the Micropropagation of Date Palm (Phoenix dactylifera L.) cv. Mejhoul

Reda Meziani,Fatima Jaiti,Mouaad Amine Mazri,Mohamed Anjarne,Mustapha Ait Chitt,Jamal El Fadile,Chakib Alem 한국작물학회 2015 Journal of crop science and biotechnology Vol.18 No.5

The present study aimed to determine the best combination of plant growth regulators (PGRs) and light intensity in order to improve organogenesis of date palm (Phoenix dactylifera L.) cultivar Mejhoul. Organogenic cultures obtained from shoot tip explants were cultured on half-strength Murashige and Skoog (MS/2) medium supplemented with various combinations of two auxins: 2-naphthoxyacetic acid (NOAA) and indole-3-acetic acid (IAA) at 0, 0.2, or 0.4 mg L-1and two cytokinins: kinetin and 6-(dimethylallylamino) purine (2iP) at 0, 0.4, or 0.8 mg L-1 under different light intensities. Our results revealed that after 3 months of culture, the combination of 0.2 mg L-1 NOAA, 0.2 mg L-1 IAA, 0.4 mg L-1 2iP, and 0.4 mg L-1 kinetin was the optimal for shoot proliferation (12.8 shoot buds per explant), with low levels of hyperhydricity (20.0%), tissue browning (20.0%), and precocious rooting (30.0%). The present study showed also that darkness and low light intensity (500 lux) significantly increased the incidence of precocious rooting while high light intensities (2,000 - 3,000 lux) decreased shoot bud proliferation and stimulated shoot elongation and greening. The optimal light intensity during the multiplication phase was 1,000 lux (13.2 shoot buds per explant, 15.0% greening, 25.0% precocious rooting). Shoot elongation and rooting occurred on PGR-free MS/2 medium then the plantlets were transferred to a greenhouse where 88.0% survived after 3 months. Our results are beneficial for the efficient and large-scale propagation of the elite and endangered date palm cultivar Mejhoul.

수박의 유묘 정단 배양시 유전적 차이가 기관 형성에 미치는 영향

이현기,백기엽,서영기,리왕영 한국식물생명공학회 1994 식물생명공학회지 Vol.21 No.4

The genotypic (2n, 3n, 4n) response of watermelon in vitro shoot tip culture was evaluated. Different genotypes had similar response in terms of shoot formation and growth. Shoot formation was better at lower concentration of 0.3 mg/L BA and higher concentration of 5-10.0 mg/L 2iP and kinetin, but growth of newly formed shoot was inhibited. With further subculture, kinetin did not promote shoot formation Better shoot formation was observed at 0.3-0.5 mg/L BA. Combination of 0.3 mg/L BA and 0.3-0.5 mg/L BA was effective in shoot multiplication, growth and induction of more internodes. Varrying levels of light intensity and agar concentration did not affect the performance of tetraploid plants. Higher light intensity and agar concentrations decreased the number of shoot formed in triploid plane. Growth in both genotype, however was inhibited. Higher light intensity was found to promote leaf senescence in all genotypes. All growth inhibitors decreased the number of shoots formed and slowed plant growth there by prolonging duration of cultures. Growth inhibitors were to observed to decrease incidence of hyperhydricity in culture. No difference in shoot formation was observed in each of the concentrations used in Ancymidol, TIBA, CCC and PP333. Shoot formation and growth was more inhibited in ABA treatments. Leaf expansion and growth was poor in all treatments. 배수성이 상이한 수박(Citrullus lanatus Thunb.) 2배체, 3배체 및 4배체의 유묘 정단을 배양해 본 결과 배수성 차이가 기관 형성에 큰 영향을 미치지 않았으나 시토키닌의 종류와 농도에 따라 기관 형성에는 차이가 심했다. BA 처리시에는 0.3 mg/L의 저농도에서, 2iP 나 kinetin 처리시에서 5∼10.0 mg/L 첨가 배지에서 신초형성이 양호하였으나 생장은 심히 억제되었다. 재생된 신초의 정단을 배양해 본 결과 kinetin은 신초 형성에 거의 효과가 없었고 BA 0.3∼0.5mg/L 첨가구에서 신초증식이 양호하였으며 IAA 0.3 mg/L와 혼합처리는 신초의 증식과 생장 및 마디수 증가에 효과적이었다. 기내에서 형성되는 신초는 부정아 및 액아 유래였으나 대부분은 액아가 발달하여 신초로 생장하였다. 광도와 아가 농도를 달리하여 배양해 본 결과 4배체는 광도와 아가 농도에 관계 없이 신초형성수가 거의 비슷하였으나 생장은 억제되었으며, 3배체는 광도와 아가의 농도가 증가할수록 신초형성수 및 생장이 감소하였다. 또한 광도가 높아질수록 측지의 발생이 양호하였으나 잎의 노화현상을 나타냈다. 생장억제제의 처리는 신초형성수 및 생장을 현저하게 감소시켜 계대배양 기간을 연장할 수 있었다. Ancymidol ; TIBA ; CCC ; PP333은 처리농도간 신초형성수에 차이를 나타내지 않았으며 억제제 중 ABA 처리구가 신초형성과 생장에 가장 억제적으로 작용하였다. 모든 억제제 처리구에서 잎의 전개와 발달이 불량하였다.

Short Communications : Reduction of Shoot Hyperhydricity in Micropropagated Potato Plants via Antisense Inhibition of a chCu/ZnSOD Gene

Yong Hwa Lee,Hyun Soon Kim,Mi Sun Kim,Yoon Sik Kim,Hyouk Joung,Jae Heung Jeon 한국응용생명화학회 2009 Journal of Applied Biological Chemistry (J. Appl. Vol.52 No.4