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A wounding method and liquid culture in Paphiopedilum delenatii propagation
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An innovative method for mass shoot propagation of Paphiopedilum delenatii ('Pink' slipper orchid), an endemic and endangered species of Vietnam, was established in this study. Nine-month-old seeds cultured on Knudson C medium was the optimal condition for in vitro germination. Seeds began swelling and became greenish in color after 30 to 40 days of culture. Then, rounded bodies, known as protocorms, were observed. Leaves and roots developed after 90-100 days of subculturing on the same medium. The combination of wounding and liquid culture was shown to be appropriate for highly frequent adventitious shoot formation. Shoot regeneration rate of 5.2 per wounded seedling could be obtained in MS liquid media supplemented with 1.0 mg l-1 TDZ via this method.
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... At present, regenerating Paphiopedilum plantlets through the division of axillary buds from the mother plant is time-consuming and inefficient [7]. Although there have been rapid developments in tissue culture over the last century, progress on tissue culture of Paphiopedilum is currently sporadic and unsuitable for commercial applications [7][8][9][10]. culture of Paphiopedilum is currently sporadic and unsuitable for commercial applications [7][8][9][10]. ...
... Although there have been rapid developments in tissue culture over the last century, progress on tissue culture of Paphiopedilum is currently sporadic and unsuitable for commercial applications [7][8][9][10]. culture of Paphiopedilum is currently sporadic and unsuitable for commercial applications [7][8][9][10]. Thus, large-scale propagation of Paphiopedilum is mainly achieved by asymbiotic seed germination [11]. ...
... This finding is consistent with previous work that showed P. Maudiae germination is greater in 1/2 MS media than in 3/4 MS or MS medium and that seeds browned and shoot buds did not form in high-salt media [29]. Nhut et al. also suggested that nutrition-poor Knudson C medium is suitable for germination of P. delenatii seed [8]. High salt concentrations in the medium may damage cells. ...
Paphiopedilum tigrinum is an endangered orchid with high ornamental value. However, seed germination and seedling regeneration in P. tigrinum is very difficult in vitro. Little is known about why P. tigrinum seedlings are difficult to propagate or how to improve the seed germination and seedling rates of this species. In this study, we investigated the developmental process of P. tigrinum from asymbiotic seed germination to seedling rooting by comparing it with P. appletoniantum, a much easier species for germination and seedling formation. We found that asymbiotic seed germination in P. tigrinum is limited by severe browning of the protocorm at the seed germination stage, and protocorm rooting at the differentiation stage was also proved to be difficult. The optimal medium for seed germination of P. tigrinum was a modified Harvais (mHa) medium supplemented with 0.5 mg·L−1 kinetin (Kin), 0.1 g·L−1 activated charcoal (AC) and 100 mL·L−1 coconut water (CW). At the protocorm differentiation stage, seedlings with 1–2 leaves were obtained on a 1/4 MS medium supplemented with 1.0 mg·L−1 6-benzylaminopurin (BA), 0.3 g·L−1 AC and 50–100 mL·L−1 CW after culturing for 120 day. At the seedling subculture stage, a 1/2 MS medium supplemented with 0.5–1.5 g·L−1 AC and 100 mL·L−1 CW was better for leaf and root growth of P. tigrinum. At the rooting stage, a 1/2 MS medium supplemented with 1.0 g·L−1 AC, 0.5 g·L−1 dolomite flour, 15 g·L−1 potato homogenate and 30 g·L−1 banana homogenate was most suitable for the growth and rooting of seedlings. This study has established an effective protocol for seed germination and seedling regeneration of P. tigrinum.
... Lin et al. [23] reported callus induction and plantlet formation via leaves derived-in vitro seedlings of P. callosum 'Oakhi' × P. lawrenceanum 'Tradition'. The rate of shoot regeneration (75 %) of P. delenatii were induced by liquid culture medium of wounding nodal segments derived-in vitro seedlings [10,22,23]. ...
... Nhut et al. [10] reported that non-shoot formation in P. delenatii shoot non-wounded treatment in all types of media treatment. The green and vigorous shoots (2.3 shoots) were obtained when wounded seedlings were cultured on solid MS medium adding 0.25 mg.L -1 thidiazuron (TDZ) combined with 0.5 mg.L -1 NAA. ...
... The basal media used in the Paphiopedilum micropropagation including Heller [13], MS [10,14,22], modified MS [15,25,26] and ½ MS [1,2,23,27], although no comparative experiment exists on the effect of media in the micropagation of Paphiopedilum spp. ...
Paphiopedilum orchids are one of the most popular and rare orchid genera sold and exhibited as pot plants and cut flowers. Their wild populations are under the threat of extinction as a result of over-collection and loss of suitable habitats. Reduction in their commercial value through large-scale propagation in vitro is a preferable option to reduce pressure from illegal collection, to attempt at meeting commercial needs and to re-establish these threatened orchid species back into the wild. Although they are commercially propagated via seed germination in vitro, Paphiopedilum are considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. This paper aims to provide the most important techniques on Paphiopedilum propagation mainly including plant, cell, tissue and organ culture techniques applied to in vitro propagation of Paphiopedilum and to emphasize the importance of further improving tissue culture protocols from ex vitro-derived explants of mature plants.
... Cho đến nay, hầu hết các nghiên cứu thường sử dụng cây con có nguồn gốc từ hạt để làm vật liệu cho các nghiên cứu nhân nhanh bằng cách kích thích tạo chồi bên (Stewart, Button, 1975;Arditti, Ernst, 1993;Huang et al., 2001;Chyuam et al., 2010, Patcharawadee et al., 2011; tuy nhiên, phương pháp này tạo ra những cây con không đồng nhất về mặt di truyền. Ngoài ra, trên đối tượng lan Hài Hồng (Paphiopedilum delenatii) một số phương pháp nghiên cứu được sử dụng như phương pháp gây vết thương và phương pháp kéo dài đốt thân in vitro để tăng hệ số nhân chồi (Nhut et al., 2005(Nhut et al., , 2007; Vũ Quốc Luận và đồng tác giả (2014) đã sử dụng thành công phương pháp cắt đốt và trồng trực tiếp ra vườn ươm cây lan Hài Hồng. Năm 2013, Trần Thị Bích Hạnh và đồng tác giả đã khảo sát ảnh hưởng của chất điều hòa sinh trưởng và mật độ mẫu cấy của lan Kim Hài lên sự sinh trưởng và phát triển của chồi; tuy nhiên trong nghiên cứu này tác giả không chú trọng vào nghiên cứu tạo chồi bên. ...
... villosum) thu nhận từ vườn ươm của Viện Nghiên cứu Khoa học Tây Nguyên được khử trùng và tách lấy hạt gieo trên môi trường Knudson C (Knudson, 1946) trong 3 tháng. Sau đó, chồi non được cấy chuyển sang môi trường MS (Murashige, Skoog, 1962) bổ sung 2,0 mg/L N 6 -Benzyladenine (BA) (Nhut et al., 2005) để tạo những chồi có kích thước đồng đều (1,5 cm) để sử dụng làm vật liệu trong nghiên cứu này. ...
Paphiopedilum villosum is a beautiful orchid species and has high value in trade; however, this is one of the most difficult to propagate orchids. So far, there has been very little publication on micropropagation. In this study, the effects of plant growth regulators on shoot regeneration from stem node culture of elongated P. villosum shoots in the darkness were investigated. Shoots (1.5 cm) were elongated and produced individual stem nodes under darkness condition for 3 months. Stem nodes were cultured on SH medium and supplemented with individually BA, KIN or TDZ to investigate shoot regeneration. The shoot multiplication rate was also recorded in this study. The highest stem node was observed in the dark with 5.25 cm in the height and the number of stem nodes were 3 stem nodes/shoot. The isolated stem node was cultured on SH medium supplemented with 30 g/L sucrose, 8 g/L agar, 1 g/L charcoal and different concentrations of BA, KIN, TDZ. The results observed after 3 months showed that the best shoot regeneration rate (85%) and highest shoot multiplication coefficient (6.6 shoots/stem node) was obtained when shoots derived from stem node were cultured on SH medium supplemented with 0.5 mg/L TDZ, 30 g/L sucrose, 8 g/L agar and 1 g/L charcoal. Those shoots obtained in the above treatments were subcultured on MS medium supplemented with 0.3 mg/L NAA for rooting and gave the highest number of roots (6.6 roots/shoot) after 1 month; and these plantlets were acclimatized in Taiwan sphagnum moss and transferred into greenhouse with the best survival rate (100%) after 3 months.
... Quá trình nảy mầm và phát triển của hạt có thể bị ảnh hưởng rất lớn bởi các yếu tố như tuổi của hạt, việc xử lý hạt trước khi nuôi cấy, môi trường cũng như điều kiện, phương pháp nuôi cấy [4]. Đã có một số công trình nghiên cứu trên các loài Lan hài ở Việt Nam như đánh giá sự đa dạng di truyền của các loài Lan hài [6], [7], nghiên cứu phương pháp nhân giống ở một số loài như hài Hằng [8], hài Đỏ [9], hài Hồng [10], [11], hài Vân, hài Tam đảo [12]. Tuy nhiên, sự thành công của việc nhân giống tùy thuộc vào từng loài và mới chỉ thành công trên một số loài Lan hài nhất đinh. ...
Lan hài Đốm (Paphiopedilum concolor Lindl. Pfitz) là loài Lan hài bản địa của miền Bắc Việt Nam. Hiện nay, loài này đã bị khai thác cạn kiệt ngoài tự nhiên nên việc phát triển phương pháp nhân giống in vitro để bảo tồn là rất cần thiết. Bài báo này trình bày kết quả nghiên cứu về một số yếu tố ảnh hưởng đến hiệu quả nhân giống in vitro Lan hài Đốm bao gồm môi trường khoáng phù hợp cho hạt nảy mầm, môi trường cho phát triển cây con từ hạt, môi trường phù hợp cho tái sinh chồi và sinh trưởng cây con in vitro. Kết quả cho thấy hạt Lan hài Đốm ở độ tuổi 190 ngày (sau thụ phấn) nuôi cấy trên môi trường ½MS hoặc ½VW cho tỷ lệ hạt nảy mầm cao là 82,5% và 80%, thời gian nảy mầm là 37,5 đến 40 ngày. Môi trường phù hợp cho biệt hóa và phát triển của hạt nảy mầm là môi trường P5 (VW + BAP 1,0 mg/L, NAA 0,5 mg/L, chuối 30 g/L, khoai tây 40 g/L) có tỷ lệ tạo chồi đạt cao nhất (50,1%). Môi trường phù hợp cho tái sinh chồi và sinh trưởng của cây con là môi trường VW bổ sung BAP 2,0 mg/L, NAA 0,5 mg/L, số chồi/mẫu đạt 2,0, số lá/chồi đạt 3,5, chiều dài lá đạt 1,35 cm, chồi to, lá xanh đậm và dày.
... In 180 DAP seeds, P. wardii and P. hangianum had the highest germination (65% and 73%, respectively), while the germination was reduced to 20% in 300 DAP seeds [7,8]. The germination of 270 DAP seeds of P. delenatii and P. callosum reached above 90%, while The germination of 300 DAP seeds of P. callosum was only 20% [9,10]. ...
Paphiopedilum armeniacum is a rare orchid native to China with high ornamental value. The germination of P. armeniacum seeds is difficult, especially for the mature seeds, which is the major limitation for their large-scale reproduction. This study explored the reasons for seed germination inhibition from the aspects of the important plant endogenous hormone—abscisic acid (ABA). The major endogenous hormone contents of seeds were determined at different developmental stages. The ABA content was 5.8 ng/g in 73 days after pollination (DAP) for the immature seeds, peaked at 14.6 ng/g in 129 DAP seeds, and dropped to 2.6 ng/g in the late mature stage of the 150 DAP seeds. The reduction of ABA content in the mature seed suggests a possible contribution to the increased expression of CYP707A, an ABA catabolism gene. The germination rate of the immature seeds was reduced to 9% from 69% when 5 μg/mL ABA was added to the Hyponex N026 germination medium. The result showed that ABA can inhibit the germination of P. armeniacum immature seeds. However, for the heavily lignified mature seeds, reduction in endogenous ABA level does not result in an increase in the germination rate. Lignin accumulation in the seed coat imposes the physical dormancy for P. armeniacum. In summary, the germination of P. armeniacum is regulated by both ABA and lignin accumulation.
Paphiopedilum spp. are high-value horticultural crops due to their wild populations are under the threat of extinction as a result of overcollection and loss of suitable habitats. They are commercially propagated via seed germination in vitro, Paphiopedilum is considered to be difficult to propagate in vitro, especially by plant regeneration from tissue culture. Therefore, it is necessary to find out adequate solutions to improve the culture techniques of these recalcitrant species. Furthermore, there have been few researches on breeding of Paphiopedilum spp. This chapter aimed to improve the shoot regeneration rate of Paphiopedilum callosum orchids by applying the wounding manipulation and shoot tip removal methods. The results of the study were successful in using wounding manipulation or shoot tip removal methods in combination with plant growth regulators in increasing the efficiency of shoot regeneration as well as improving the quality of Paphiopedilum callosum plants on the in vitro rooting medium supplemented with organic and increasing plant growth on peat moss substrate in the greenhouse.KeywordsWounding manipulationShoot tip removal
Paphiopedilum callosum
Nodal and internode explant culture is a simple and effective method in micropropagation. However, some plants have very short and not well-defined internodes, such as Paphiopedilum and Nepenthes. As a result, defined nodal and internodal explants are difficult to obtain for micropropagation purposes. Furthermore, the close clustering of leaves makes surface decontamination of explants difficult. Red LED, dark conditions, and gibberellic acid (GA3) have been reported to stimulate stem elongation under ex vitro and in vitro conditions. In this chapter, the effects of LED lights, different blue to red LED ratios, dark conditions, and GA3 are used to study the stem elongation of Paphiopedilum, Anthurium, and Nepenthes. The results of this study could increase the propagation efficiency in these plants.
This chapter reports a protocol of polyploid induction of Paphiopedilum villosum using colchicine as a mutagen agent. Adventitious shoots of P. villosum were induced from nodal explants (from elongated in vitro shoots) by using cytokinins (BA, KIN, and TDZ). The shoot (1.5 cm in high) was exposure to different concentrations and durations of colchicine solutions and transferred into the in vitro rooting medium. The results showed that the treatment of 50 μM colchicine in 6 days gave a polyploidy induction rate of 19.88%. The flow cytometric analysis and chromosome counts of root tip squash were used for the analysis of tetraploids and mixoploids. The shoots derived from stem nodes treated with colchicine solution were effective for the production of polyploid plantlets for subsequent propagation of P. villosum and other endangered orchids.KeywordsChromosome countColchicine solutionFlow cytometricStem node
Paphiopedilum villosum
Polyploidy
Paphiopedilum spicerianum (P. spicerianum) is a rare orchid species with high ornamental value. Asymbiotic germination is the most efficient propagation method for conservation and commercial purposes because clonal propagation is very difficult and the separation of native species of Paphiopedilum through aseptic seeding is uncommon owing to their conservatism. However, a high protocorm developmental arresting rate during the asymbiotic germination is the major obstacle for seedling establishment. The fundamental understanding of embryo and protocorm developmental mechanisms will guide the development of an effective propagation method. The morphological and physiological characterization of the key developmental process of embryos and protocorms shows that the mature seeds of P. spicerianum consist of a spherical embryo without an endosperm. Seed coats become heavily lignified once the embryo is mature. Embryo cell size is relatively uniform, and significant structure polarity and cell size gradients occur at the early protocorm stage. The high level of auxin and cytokinin accumulation at the early stage of embryo development and protocorm stage may help to facilitate cell division. The transcriptome profiles of protocorms at three different developmental stages were compared to explore the regulatory mechanism of protocorm development. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that differentially expressed genes were implicated in secondary metabolite metabolism, plant hormone signal transduction and photosynthesis. The temporal expression patterns of candidate genes related to embryo and shoot development were analyzed to reveal their roles in protocorm development: in the early stage of protocorm development, embryonic development related genes such as SERKs and BBM1 were active, while in the late stage of protocorm, shoot apical meristem related genes such as WOX8, CLAVATA2, CUC2, and SCR were active.
The micropropagation of Paphiopedilum delenatii via gibberellic acid (GA3) treatment is reported. The juvenile axillary shoots of P. delenatii were isolated and cultured on Schenk and Hildebrandt (SH) medium supplemented with 5 concentrations of GA3 (1.0-5.0 mg l-1) for shoot elongation. Nodal explants were isolated from elongated shoots and placed on medium supplemented with N6-benzyladenine (BA), Kinetin (KIN), or Thidiazuron (TDZ) for shoot multiplication. The highest number of axillary shoots was recorded in SH medium supplemented with 0.5 mg l-1 TDZ after 60 days of culture. Vigorous and uniform plantlets with well-develop shoots and roots were planted in the greenhouse. The 3-year-old P. delenatii plants were treated with GA3 (50-500 mg l-1) for 4 consecutive months to stimulate inflorescence emergence. Moreover, GA3 also contributed positively to the early blooming of P. delenatii plants.
Thidiazuron, a synthetic phenylurea-type cytokinin, has previously been found to induce somatic embryogenesis and organogenesis in a wide range of plant species and to modulate the metabolism of endogenous auxins and cytokinins. In spite of these findings, the precise mode of action of TDZ remains undetermined. The current studies were undertaken to determine the fate of the TDZ molecule and the effects of TDZ exposure on auxin transport in plants. The fate of two radiolabelled versions of thidiazuron, [^14C-5-thidiazol]-TDZ and [^14C-U-phenyl]-TDZ, was investigated in sterile hypocotyl cultures of geranium (Pelargonium × hortorum Bailey). Radiolabelled TDZ was recovered from the tissue explants in ethanol-insoluble, ethanol-soluble and chloroform fractions as well as in acidic, basic and neutral eluants from Dowex resins. Hypocotyl sections that had been exposed to TDZ were found to accumulate more ^14C-IAA from the culture medium and to translocate the auxin over a greater distance within the tissues. These data provide the first evidence that the TDZ molecule remains intact in both a free and conjugated form within the plant tissues and provide some indication that TDZ-exposure enhances the accumulation and translocation of auxin within the tissues.
Plant regeneration through direct shoot bud formation from leaf culture of Paphiopedilum orchids
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