Plant Cell, Tissue and Organ Culture最新文献

The cultivation of Vanda orchids in Indonesia faces challenges due to their slow growth, leading to a reliance on importing varieties from foreign countries. Our primary objectives were understanding the relationship between morphological characters and in vitro propagation success and establishing mass in vitro propagation technology from initiation to maturation stages. Additionally, we explored the influence of genotypes, explant types, and media during the initiation stage, alongside evaluating the media's role in the proliferation, regeneration, and maturation of plantlets. A well-defined protocol was developed for the culture induction, multiplication, rejuvenation, and development of Vanda orchids. Established correlations highlight the correlation between floral structure colors and the effectiveness of explant initiation. The optimal medium for culture initiation was Murashige & Skoog (MS) with the addition of 0.75 mg/l thidiazuron (TDZ) and 0.25 mg/l 6-benzyl amino purine (BAP), particularly effective by using rachis explants with the best response found in V. ‘Nilareta Agrihorti’, which exhibited organogenic callus and adventitious shoots within 58 days. During the proliferation stage, culturing 0.5 cm clumps on MS medium with 1 and 2 mg/l BAP resulted in a substantial 3.30 and 2.83 times increasing in clump weight, respectively, and a multiplication rate of 4.30 and 3.83, respectively. MS was added with 0.3 mg/l meta-topoline for shoot regeneration, achieving an average shoot growth percentage of up to 89% during the third subculture period. Half-strength MS medium was combined with 100 g/l banana extract, demonstrating superior plantlet performance during maturation. These findings represent a significant advancement in Vanda hybrid propagation technology, holding promise for cultivating and commercializing these unique orchid varieties in Indonesia and international market.

Papaya (Carica papaya L.) is widely grown in tropical and subtropical regions. The destructive disease caused by emerging strains of Papaya ringspot virus (PRSV) demands effective transgenic resistance to target atypical virus strains threatening the crop. Papaya transformations are mainly conducted on the explants of immature zygotic or somatic embryos, which are difficult to obtain and proceed, and are largely affected by seasonal factors. Here, we attempted to develop an efficient process for organogenesis using various tissues of ex-vitro or in-vitro grown papaya seedlings. Leaf lamina, hypocotyl and stem sections of seedlings of the papaya variety Sindhi were used for establishment of callus culture through 12 different callus induction treatments (CIT 1–12), with various combinations of plant growth regulators (PGRs). Our results revealed that CIT-11 and CIT-10 enhanced callus induction from ex-vitro leaf discs with midrib, with 86% and 80% efficiency respectively, superior to 53% of CIT-10 using in-vitro leaf discs with midrib. The expression of binary vector construct pSN-PRSV CP in Agrobacterium strain GV3101 was verified in Nicotiana benthamiana and papaya by RT-PCR analysis. Calli derived from leaf tissues (with midrib) of papaya, transformed with the binary vector were successfully regenerated on the shoot induction treatment SIT-13 (Gamborg B5 medium + 0.5 mg/L TDZ + 0.01 mg/L NAA) and were subsequently rooted on root induction treatment RIT-4 (Gamborg B5 medium + 1 mg/L IBA). The transformed explants were regenerated with an efficiency of 26%. The whole process is unique in term of explant selection, source of explant (ex-vitro grown papaya plants) and media formulations as, the leaf tissue from ex-vitro grown papaya showed highest callusing and regeneration efficiency overall.

Tinospora cordifolia (Willd.) Hook.f. & Thomson is an ethnomedicinal plant belonging to the family Menispermaceae, known for its multi-disciplinary use. The present study is focused on selecting an elite plant chemotype through phytocompound quantification and antimicrobial activity assay against multi-drug-resistant (MDR) urinary-tract-infecting (UTI) pathogens, being a growing concern for mortality and antimicrobial resistance nowadays. Twelve accessions of this plant were collected from different agro-climatic zones of West Bengal, and crude extraction was done by solvent optimization. Sample TC 07 resulted in maximum crude extract yield in methanol. All twelve sample extracts showed antimicrobial efficacy against eight pathogens producing significant inhibition zones, while TC 07 resulting maximum inhibition zone of 16.37 ± 0.12 mm against Staphylococcus aureus. Extracts were evaluated through high-performance thin-layer chromatography, which resulted in optimum amount of berberine (5.05 ± 0.05 mg/g) and palmatine (3.00 ± 0.11 mg/g) in TC 07, significantly higher compared to other accessions. Therefore, comparing the data, TC 07 was considered as the elite chemotype and introduced in in vitro culture for conservation. The maximum number of shoot (16.39 ± 0.09) production was obtained in MS medium containing 2.0 mg/l 6-Benzylaminopurine and 1.0 mg/l meta-Topolin. The plant was acclimatized in ex-vitro condition and confirmed for optimum bioactive compound production through high-performance liquid chromatography. The cytogenetic stability of the regenerated plantlets was ensured using chromosome number determination, and through start codon targeted (SCoT) polymorphism. Therefore, this study provides a simple, validated and reproducible method for optimal berberine and palmatine production and controlling MDR-UTI pathogens.

The frequently occurring browning of plant tissue cultures is a considerable problem in causing economic losses. However, the mechanism that causes browning of plant tissues is still controvesial and technologies to effectively prevent browning of plant tissues are still scarce. In the present work, two callus lines derived from grapevine shoot tips (ST-callus) and fruit flesh (F-callus) were used to investigate the relationship between callus-associated endophytes and callus browning. We observed the transfer of browning effects from grapevine brown calli to normal calli, in a contact cocultivation experiment, then detected the emergence of endophytic bacteria from brown calli when the calli were incubated in a microbial culture medium, and the isolates were identified as genus Bacilus. The inoculation of pure cultured B. sp. strain ST-B1 into normal calli at different concentrations caused gradient callus browning, validating the callus browning-causing (CBC) endophyte. In addition, the moderate inhibition of endophytic bacteria in grapevine calli by culturing the calli in ampicillin-containing media reduced the incidence and severity of callus oxidative browning. The experiments were performed separately on two types of grapevine calli, ST-callus (derived from the tip of grapevine shoots), and F-callus (derived from the pulp of grape berries), and gave similar results. The DNA sequence amplicon approach showed that the CBC endophytic bacteria were found in both the normal and brown calli, which differed greatly in their relative abundances. And compared to the normal calli, brown calli greatly reduced the diversity of bacterial endophytes, while the diversity of fungal endophytes between normal and browning calli showed no obvious difference. The work demonstrated that callus-associated endophytes are involved in causing oxidative browning of plant cells, and suggested technologies to minimize the occurrence of the oxidative browning during plant tissue culture practices.

Doubled haploidy (DH) technology has been utilized in cultivated tetraploid potato (Solanum tuberosum L. ssp. tuberosum) to accelerate crop improvement; however very little work has been done with the diploid species. Experiments were undertaken to improve microspore embryogenic response in the diploid germplasm. Several factors influencing embryogenic responses were evaluated. An increase in calcium nitrate, a reduction in the plant growth regulators 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA), as well as an incubation temperature of 28 °C resulted in an increase in callus production and, in some cases, embryo-like structures. Validation of the modified protocol was conducted with both diploid and tetraploid potato germplasm with responses from both diploid and tetraploid. Monoploid and di-haploid plants were also regenerated from these microspore-derived calli.

A procedure for plant regeneration from cell suspension cultures through somatic embryogenesis is described for Sapindus trifoliatus, a commercially and medicinally important tree. Callus was induced from leaf disc on agar-solidified MS medium with 5.0 mg l⁻¹ 2, 4-D and 0.01 mg l⁻¹ Kin. Embryogenic cell suspension cultures were established by placing leaf-derived friable calli in PGR-free full-strength MS liquid medium with 3% sucrose. The growth of cell suspension culture was significantly affected by the strength of the MS mineral solution and L-glutamine. Plating of the suspension on semisolid MS medium resulted in the formation of globular structures. These embryogenic globular structures differentiated into secondary globular structures or somatic embryos on a semisolid MS medium. The differentiation of globular structures and different stages of somatic embryos (from globular to cotyledonary) was enhanced by the addition of 200 mg l⁻¹ L-glutamine in the medium. Sucrose at relatively high concentrations (5%) or ABA (0.01 mg l⁻¹) promoted somatic embryo maturation. The highest percentage (about 90%) of germination of somatic embryo and plantlet conversion was achieved on a half-strength MS medium containing 2% sucrose. The plants were hardened and established in soil with a 90% survival rate

Dendrobium heyneanum Lindl. or Heyne’s Dendrobium is an endemic epiphytic orchid of Western Ghats, with a height of approximately 3 - 4 inches. The present study aimed to establish a conservation strategy using in vitro regeneration methods for this endangered taxon. Mature pods of the D. heyneanum were collected from the field, and aseptically inoculated on various nutrient media. Asymbiotic seed germination was most successful on half-strength macro-MS media, yielding an 86.70% germination rate within 12 days. Different morphogenic stages (I-VI) were observed, with 20.84% of seeds producing young seedlings with roots on half-strength macro-MS media. The micropropagation protocol of D. heyneanum was established by using the protocorms (Stage IV) from the asymbiotic germinated seeds, with the highest frequency (90.20%) observed in 1.0 mg/L Kinetin (KN). In vitro flower buds were observed at 0.5 mg/L and 1.0 mg/L 6-Benzyl amino purine (BAP) and callus induction at 2.0 mg/L of BAP. The synergistic effect of KN combined with auxins - α- Naphthaleneacetic acid (NAA), Indole- 3- acetic acid (IAA), and Indole- 3- butyric acid (IBA) plantlets were assessed, with KN + IAA (1.0 mg/L) inducing pseudobulb elongation (0.92 cm) and rooting (0.74 cm). The plantlets were subsequently acclimatized and hardened on pots containing cocopeat and brick pieces resulting in a survival rate of 52.73%. This study presents a comprehensive protocol for in vitro propagation of Dendrobium heyneanum Lindl., offering a viable method for ex-situ conservation efforts.

Lilium fargesii Franch is a slow-growing species endemic to China with small-sized bulbs; however, the factors affecting bulblet enlargement remain unclear. Here, we show that 60 g·L^− 1 sucrose and darkness are optimal conditions to increase the weight and expansion of bulblets. Both the weight gain and expansion rates of bulblets were further promoted by exogenous indole-3-butyric acid (IBA), the gibberellin (GA) inhibitor paclobutrazol (PBZ), and the jasmonic acid (JA) inhibitor diethyldithiocarbamic acid (DIECA), and were inhibited by exogenous benzylaminopurine (6-BAP) and methyl jasmonate (MeJA) under a 16-h light/8-h dark photoperiod. Exogenous application of GA₃, 6-BAP, and MeJA, but not IBA, increased internal hormone contents; the inhibitors kynurenine (Kyn), PBZ, and DIECA decreased internal hormone levels, whereas the cytokinin inhibitor lovastatin had no effect. The weight gain and expansion rates of bulblets were promoted by high internal IBA and 6-BAP contents and low internal MeJA content. The roots number of bulblets was promoted by decreased IBA and increased GA₃, and was inhibited by increased IBA and deceased 6-BAP. Endogenous GA₃ or 6-BAP levels influenced the proliferation and total weight of daughter bulblets but not the formation of new leaves. Bulblets enlargement was positively associated with the proliferation and total weight of daughter bulblets and negatively associated with internal MeJA content. The number of new leaves was significantly negatively correlated with the weight gain, expansion, and roots of bulblets. These findings provide valuable insights for the in vitro preservation of L. fargesii to facilitate the protection and utilization of wild germplasm resources.

In vitro micro rhizome technology is a highly effective approach in combating seed-borne diseases and ensuring the production of healthy and high-quality planting material in ginger (Zingiber officinale Rosc.). To gauge the efficiency of micro rhizome production and their viability ex vitro, an experiment was conducted on several ginger varieties viz., IISR Varada, IISR Mahima, IISR Rejatha, and Karthika, at ICAR- Indian Institute of Spices Research, Kozhikode, Kerala, India. This experiment adhered to established protocols and standardized procedures. All four varieties exhibited varying rates of micro rhizome production after 180 days of culture. Among these, IISR Varada demonstrated the highest mean weight of cultured plant mass (96.0 ± 4.41 g), followed by Karthika (91.4 ± 5.72 g), IISR Rejatha (78.45 ± 5.59 g), and IISR Mahima (72.4 ± 3.56 g). IISR Rejatha exhibited the maximum number of micro rhizomes per bottle (11.35 ± 0.81) compared to IISR Mahima (10.8 ± 0.54), Karthika (9.8 ± 0.58), and IISR Varada (9.0 ± 0.63). The highest total weight of micro rhizome and mean weight of a single micro rhizome per bottle were recorded in IISR Varada (32.6 ± 1.92 g and 3.9 ± 0.29 g, respectively), followed by Karthika (27.1 ± 1.19 g and 2.9 ± 0.16 g, respectively), IISR Rejatha (27.0 ± 1.79 g and 2.5 ± 0.18 g, respectively) and IISR Mahima (24.5 ± 1.10 g and 2.4 ± 0.16 g, respectively). Besides, IISR Varada, followed by Karthika, emerged as the most promising varieties for micro rhizome production in terms of their multiplication rate. The evaluation extended to the first and second-generation progenies of micro rhizomes from IISR Varada. Results indicated the successful establishment of first-generation micro rhizomes in grow bags and second-generation micro rhizomes in the field, employing both direct planting and transplanting methods. Assessment of quality parameters revealed that the second-generation (V2) transplanted plants of micro rhizomes of IISR Varada exhibited the highest essential oil content 0.78%. The total phenolic content was highest in second-generation (V2) rhizomes directly planted in soil (23 mg GAE/g), whereas the first-generation micro rhizomes raised in grow bags registered the highest total flavonoid content (TFC) of 1.39 mg QE/g. Moreover, the genetic fidelity test conducted on the first and second generations (V1 and V2, respectively) of micro rhizome-derived plants, using molecular markers, exhibited a monomorphic banding pattern similar to that of the mother plant, confirming their genetic stability.

Artemisia alba Turra is an essential oil bearing plant, with Euro-Mediterranean and Southeastern European distribution. One of the distinctive characteristics of its essential oil is the variability of its terpenoid profile due to environmental, genetic and other factors. In the present work, tissue culture experiment has provided a model system of in vitro morphogenesis alteration. Auxin and cytokinin treatments were applied alone or in different combinations, leading to the development of directly rooting and root suppressed in vitro plantlets. Direct in vitro rooting was definitely related to obtaining biomass richest in flavonoid compounds (irrespectively of the PGR combination leading to obtaining this morphotype). The directly rooting morphotype was also distinguished by the highest flavones/flavonoles ratio as compared with the in situ and the rest of the in vitro samples. Underground parts of in vitro samples were shown to be significantly richer in caffeoylquinic acids as compared with aerial parts. An elevation of DCQA/CQA ratios in PGR treated plants as compared with the non-treated control could be estimated as an indication of stimulation of the esterification process with the purpose of coping with the stress of the impairment of the physiological state of normal shoot-to-root tissue formation. It was shown that in vitro morphogenesis moderation could be applied as a simple and reproducible protocol to alter polyphenolics production in tissue cultures of this plant species.