Over the course of the study fromMarch to December 2023, the aim was to identify the optimal type and concentration ofPGRs for effective callus formation and embryo development in this p
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Phi et al (2017) researched the micropropagation of Paramignya trimera and investigated that WPM medium was the most suitable for rapid in vitro propagation
In a comparative study of growth media for callus formation, explants from 0.5 cm in vitro shoot segments were cultured in dark conditions on WPM medium supplemented with TDZ at 1.0 mg/L and 2,4-D at 3.0 mg/L, resulting in a remarkable callus formation rate of 86.67% after 12 weeks The callus exhibited rapid growth and a green appearance In contrast, individual growth regulators yielded lower callus formation rates, with TDZ at 1.0 mg/L achieving 70%, IBA at 3.0 mg/L resulting in 68.33%, and 2,4-D at 3.0 mg/L producing 63.33%.
In their 2023 study, Tran Trung Hieu et al developed an in vitro propagation process for Ð trimera, noting that explants typically lose leaves after 8-12 weeks of culture—a common issue in many woody species linked to ethylene production This ethylene negatively impacts callus proliferation, embryogenesis, and shoot regeneration To mitigate this, the researchers incorporated silver thiosulfate (STS) into the medium, effectively inhibiting ethylene activity and enhancing shoot height and tissue morphogenesis They also explored callus formation from leaf explants on WPM medium enriched with growth regulators BA, IBA, and 2,4-D, determining that the optimal results were achieved with WPM medium supplemented with STS.
BA 5 mg/l, and IBA 5 mg/l giving the highest callus formation (47%) from mature leaf samples after 12 weeks of culture Most of the callus is spongy white at the cut surface at both ends of the leaf's main veins, then spreads to the entire main vein surface Of the callus formed, 21% of the leaf samples produced firm, glossy, opaque white callus blocks at the cross-section of the leaf blade These callus blocks continue to grow and form small clusters shaped like an embryo (or bud) on the surface of the callus.
In 2018, Ngo Thi Xuyen et al studied the impact of 0.1% HgCl2 disinfection time on the survival rates of P trimera samples, finding that the optimal disinfection duration was 8 minutes for leaf samples, resulting in a 76.67% survival rate, while stem samples required 12 minutes, yielding a 33.33% survival rate Additionally, the research examined the influence of growth regulators NAA and TDZ on callus formation in leaf samples The results indicated that leaf samples did not respond to MS medium with NAA, remaining green with no growth However, in MS medium with 0.6 mg/l TDZ, callus formation reached a peak of 76.19% after 60 days, with most calluses exhibiting a spongy texture and dark yellow coloration.
In recent years, research team PIB (Plant Integrative Biology) has conducted surveys on a number of factors affecting callus formation Nguyen Thi My Duyen
In 2019, research focused on callus formation in media enriched with varying concentrations of 2,4-D (0.2 to 1.0 mg/L) and NAA (1.0 and 2.0 mg/L) alongside BA (0.0, 0.2, and 0.5 mg/L) Findings revealed a minimal callus formation rate when using only 2,4-D, peaking at 68.5% with the addition of 2 mg/L NAA and 0.2 or 0.5 mg/L BA Notably, increasing NAA concentration from 1.0 to 2.0 mg/L resulted in a 2.47-fold increase in callus formation Building on this, Tran Thi Nguyet Nga (2021) further explored callus induction on WPM medium, identifying optimal results with 1.0 or 2.0 mg/L NAA combined with 0.2 mg/L BA.
Cao Thi Cam Huong (2022) conducted research on the callus formation rates of samples cultured on MT and WPM media, supplemented with varying concentrations of NAA (1.0 mg/L, 2.0 mg/L, 3.0 mg/L, and 4.0 mg/L) The study found that both media resulted in a high callus formation rate of 75%.
Leaf samples from one-year-old P trimera plants cultured on WPM medium with 0.2 mg/L BA and 2 mg/L NAA achieved a 100% callusing rate after 6 weeks, resulting in nodular and hard callus tissue capable of generating seedlings This study highlights the effective formation of somatic embryogenesis from the callus of P trimera.
In 2021, Tran Thi Nguyet Nga conducted a study on somatic embryogenesis using leaf callus from the P trimera plant, cultured on WPM medium enriched with NAA (0.5 and 1.0 mg/L) and BA (0.2 and 0.5 mg/L) The findings revealed that a combination of 1.0 mg/L BA and 1.0 mg/L NAA resulted in 75% of the explants developing somatic embryos within four weeks of culture.
In 2022, Cao Thi Cam Huong conducted a study on the effects of mineral mediums and growth regulator substances (GPRs) on callus induction and cloned embryos of P trimera The research utilized three mineral mediums (MS, MT, and WPM), each supplemented with 500 mg/L malt extract and varying concentrations of BA (3.0 mg/L and 4.0 mg/L) over a six-week culture period The findings revealed that the MT medium, enriched with 500 mg/L malt extract and 4 mg/L BA, achieved the highest somatic embryo induction rate of 83.8%, with the embryos developing into globular and heart-shaped stages.
MATERIALS AND METHODS - -+5-<S2<<2<+zcserrrreerrrrree 17 3.1 Places and duration for conducting eXperiImenifS -++++£++s<c++ecezs+ 17 3.2 Equipment and tool ce lý 3.5 MIA(GTHBÌSs:ssc6cáxssco na sg51616011210581338EHXEESAELSSSLRSEESSEECSESEENSEXEXREESSLESSXSSEEEEHSEESSELRXSEESSSRSSEEASESE 17 2;3ằ L; Blỉlop16al:Trial6TiaèSeesesessasaeseseeibsbeisusssllikl3eixessssereitEoksSilSésuEtieEgotssedficgptsrfehgiEEk 17
RESULTS AND DISCUSSION sissssssssesssssassssssonsssrsssseacxvoeanssnassnsvezneesenes 22
Efficacy of sterilizing P trimera seed explants with Javel Solution DB 4.2 Influence of kinds of explants and NAA, BA concentrations on callus formation 23 4.3 Examination of somatic embryo induction - 55-5555 *S+<£++c£+ezzcrseeeres 30 4.3.1 Examination of somatic embryo induction from zygotic embryo
A study demonstrated the effectiveness of Sodium hypochlorite (NaClO), or Javel solution, in sterilizing P trimera seed explants The main goal of this sterilization method is to eliminate bacterial and fungal infections while preserving the explants' growth potential and viability Experimental results indicated that modifying the concentration of Javel solution and the duration of exposure had a significant impact on the cleanliness and germination rates of the explants, successfully lowering infection rates.
Table 4 1 Efficacy of sterilizing P trimera seed explants with Javel Solution
Rate of Rate of clean Javel ; ơ Rate of Time bacterial living a Treatment ; (%, ; ; germination
In the analysis, average values sharing identical letters are not significantly different A double asterisk (**) denotes a statistically significant difference with a p-value less than 0.01, while a single asterisk (*) indicates significance at a p-value under 0.05 Conversely, "ns" signifies no significant difference, with a p-value exceeding 0.05 The data is processed using the formula asin(x/10) × 180/3.14, with respect to Time (A).
Data from Tables 4.1 reveal that higher Javel concentrations and extended exposure times significantly reduce bacterial infection rates while enhancing the rates of clean explants and germination This highlights the critical need for optimizing sterilization conditions However, an increase in Javel concentration to 25% negatively impacts the germination rate, indicating the necessity of balancing concentration levels for effective results.
22 exposure time to achieve effective sterilization without harming explant viability After
After 28 days of culture, the majority of seed samples retained their green color with minimal damage, indicating that Javel solution is an effective sterilant for P trimera seeds while being mindful of environmental and human health Transitioning from Mercury (II) chloride to Sodium hypochlorite significantly lowers health and environmental risks, all while preserving high sterilization efficiency, thus presenting innovative options for managing plant explants.
Table 4.1 reveals that a 20% Javel solution applied for 15 minutes resulted in a 46.25% bacterial infection rate and a germination rate of 37.24% However, increasing the exposure time to 20 minutes reduced bacterial contamination to 22.50%, while clean living explants rose to 70% and the germination rate improved to 44.79% Elevating the Javel concentration to 25% further decreased contamination rates but negatively impacted germination, highlighting the detrimental effects of high NaClO concentrations on seed viability due to their strong oxidizing properties, which can damage plant cell structures, including chlorophyll, complicating explant recovery and increasing mortality.
The Javel solution proves to be an effective method for sterilizing P trimera seed explants, but achieving optimal sterilization efficiency requires a precise balance between the concentration of the disinfectant and the duration of exposure to prevent damage to the explants.
4.2 Influence of kinds of explants and NAA, BA concentrations on callus formation
The results indicate that the addition of NAA and BA to the cultivation medium significantly enhances callus formation from various explants, including cotyledons, hypocotyls, radicle roots, and zygotic embryos The observed increase in callus formation over time underscores the necessity of growth regulators like NAA and BA in stimulating this crucial step for plant regeneration from explants Previous research has consistently demonstrated the effectiveness of NAA and BA in promoting callus development.
BA plays a crucial role in promoting cell division and development in plant cells, reinforcing its significance in plant tissue culture The variation in callus formation rates among different explants and NAA concentrations highlights the intricate interplay between explant type, growth regulators, and the cultivation medium, underscoring the need for a comprehensive understanding to enhance the regeneration process.
Table 4 2 Influence of types of explants and NAA, BA concentrations on callus induction
PGPs (mg/L) Rate of callus induction (%) Treatment Explant
NAA BA 4 weeks 6 weeks 8 weeks
In this study, average values sharing the same letter(s) indicate no significant difference, while a double asterisk (**) denotes a statistically significant difference with a p-value less than 0.01 A single asterisk (*) signifies a significant difference with a p-value below 0.05, and "ns" indicates no significant difference with a p-value greater than 0.05 The data were processed using the formula asin(/x/10) x 180/3.14 for accurate analysis of the explant results.
The study found that the rate of callus explants increased over 4, 6, and 8 weeks of culture, with no callus formation in treatments lacking growth regulators (C1, C4, C7, and C10) The highest callus formation rate was 93.75% for cotyledons and hypocotyls, 75% for zygotic embryo explants, and 56.25% for radicle explants, all cultured on WPM medium supplemented with 2 mg/L NAA and 0.2 mg/L BA (Treatments C6, C9, C3, and C12) Notably, the cotyledon-derived callus (treatment 6) exhibited a firm, glossy white appearance, primarily forming near the zygotic embryo site and developing tissue clusters resembling embryo blocks.
The callus morphology of propagules in treatment C9 is characterized by a pale yellow color and firmness, enabling embryo formation In contrast, the zygotic embryos from treatment C3 exhibit a callus tissue shape, presenting as small yellow-white blocks clustered together, resembling somatic embryo structures Additionally, the radicle callus in treatment C12 is described as pale yellow, shiny, firm, and spongy.
Figure 4 2 Callus induction from 4 types of explants after 8 weeks on WPM+ 2 mg/L NAA+0.2 mg/L BA medium (a) Zygotic embryo; (b) Cotyledons; (c)
The formation of calluses from explants is significantly affected by the type of explant and the concentration of NAA, with a notable interaction between these factors (p-value=0.006) When examining individual factors, zygotic embryos, cotyledons, hypocotyls, and radicle roots showed callus induction rates on WPM medium with 2 mg/L NAA and 0.2 mg/L BA that were 1.33, 1.5, 1.36, and 1.5 times higher, respectively, compared to the same explants on WPM medium containing 1 mg/L NAA and 0.2 mg/L BA.
BA (Treatments C2, C5, C8, and C11) time Regarding the concentration of NAA and
Without the addition of BA to the medium, explants failed to form callus and ultimately perished, highlighting the essential roles of BA and NAA in callus induction The inclusion of NAA, along with BA, significantly contributes to successful callus formation.
0.2 mg/L BA, the rate of callus explants increased the most at the concentration of 2 mg/L NAA in all 4 types of explants This result is similar to the results of Nguyen Thi
My Duyen (2019) and Cao Thi Cam Huong (2022) when surveying young leaf explants of the Ð trimera.
While all treatments did not affect the time required for callus formation, significant changes in the morphology and development of the callus were observed Factors influencing the culture led to alterations in callus morphology after 4 and 8 weeks After 2 weeks, the explants began forming calluses, which remained green after 4 weeks, predominantly developing at the cut site, with some samples appearing on the stem and surrounding areas By 8 weeks, distinct morphological changes in the callus were evident across the treatments.
Table 4 3 Callus morphology after 4 weeks and 8 weeks of culture
Morphological characteristics of the explant after culture Treatment
Cl Green gold sample, non-touch Green gold sample, non-touch
CONCLUSIONS AND RECOMMENDATION 39 3,1 CONCLUSIONS .0ic0cs.s-scoresnascasosssconsnsiionssenetcnsastubnnanasebasnanostessudastentenishentnantinnsnntsasanennen 39 5.2, RECOMMMENAIAUONS sercesercnsacomseransennnn T000 16050015130 54358489188010000104060001801005903038350881 39
Sterilization using a 20% Javel solution for 20 minutes resulted in a 70% survival rate and a 44.79% germination rate for seeds The highest callus induction rates were achieved in WPM medium supplemented with 2.0 mg/L NAA and 0.2 mg/L BA under darkness after 8 weeks, with zygotic embryos, cotyledons, and hypocotyls showing rates of 86.61% and 93.75%, while radicles had a rate of 56.25% The induced callus was characterized as nodular and firm, capable of producing embryos, with some calluses developing adventitious roots, showing a 55% rate for radicles and 86.61% for both hypocotyls and cotyledons.
Zygotic embryos cultured on WPM medium with 2.0 mg/L BA, 500 mg/L malt extract, and 5% sucrose developed into globular-shaped somatic embryos after 6 weeks However, they did not progress to fully formed embryos By 12 weeks, the zygotic embryos started to produce buds with leaves.
The study on somatic embryogenesis from callus explants derived from young leaves on WPM medium revealed that varying concentrations of TDZ (0.07 mg/L, 0.1 mg/L, 0.3 mg/L, and 0.7 mg/L) significantly influenced embryo formation rates After 8 weeks of culture, the globular-shaped somatic embryo formation rates ranged from 55.56% to 88.89% The optimal concentration was found to be 0.07 mg/L TDZ, yielding the highest rate of globular-shaped embryos at 88.89% Conversely, the highest occurrence of abnormal embryos, at 83.33%, was observed at a concentration of 0.3 mg/L TDZ.
Further research is recommended to advance the development of somatic embryos into complete seedlings.
Investigate the application of adventitious roots derived from callus of various in vitro explants for studying root biomass or secondary compounds produced by P.trimera roots.
In their 2008 study published in The Plant Journal, Atta et al explore the pluripotency of the xylem pericycle in Arabidopsis, demonstrating its crucial role in shoot regeneration from root and hypocotyl explants cultivated in vitro The findings highlight the potential of pericycle cells in plant tissue culture, offering insights into regenerative processes and applications in plant biology For more details, refer to the original article [here](https://doi.org/10.1111/j.1365-313X.2008.03716.x).
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In his 2022 graduation thesis at Nong Lam University, Ho Chi Minh City, T C H Cao evaluated the impact of mineral salts and various growth regulators on the formation of callus and somatic embryos in Paramignya trimera (Oliv.) Guillaum.
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The study by Kieu-Oanh et al (2022) investigates the phytochemical composition and potential bioactivities of essential oil extracted from the peels of Paramignya trimera, a plant native to Vietnam Published in the Journal of Essential Oil Bearing Plants, the research highlights the significant chemical constituents present in the essential oil and explores its various bioactive properties, contributing valuable insights into its potential applications in the fields of natural products and medicinal chemistry.
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In their 2018 study published in the Scientific Journal of Van Lang University, Ngo and Dang investigated the chemical components and bioactivities of Paramignya trimera (Oliv.) Guill leaf samples The research also focused on the morphogenesis of the plant from various explants, highlighting its potential applications in biotechnology and pharmacology.
Nguyen and Debergh (2005) focused on enhancing abnormal embryo structures in the nucellar culture of two orange varieties, Citrus aurantium and Citrus sinensis Their research was presented at the National Citrus, Mango and Pineapple Workshop, highlighting advancements in citrus cultivation techniques.
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In a study conducted by Nguyen et al (2018), the anti-cancer properties of the methanol root extract from Paramignya trimera, known as xao tam phan, were investigated using a three-dimensional model on the human breast cancer cell line MCF-7 The findings, published by Springer International Publishing, highlight the potential of this natural extract in cancer treatment research.
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Table 1: The rate of bacterial infection explants in disinfection treatments
Treatment Time Javel Rate of bacterial infection (%) Mean (%)
(mins) (%) Time Time Time Time
Table 2: Converted data on the rate of bacterial infection explants in disinfection treatments
Time Javel Rate of bacterial infection
Mean (mins) (%) Time 1 Time 2 Time 3 Time 4
20 25 260.5649890 0.640598137 12.92093617 18.43490571 14.48333273 Table 3: ANOVA table for the rate of bacterial infection explants in disinfection treatments
Source DF SeqSS Adj SS Adj MS F-Value P-Value
Table 4 Ranking test of the rate of bacterial infection explants in disinfection treatments
Table 5 Rate of clean living explant of sterilized explants in disinfection treatments
Time Javel Rate of clean living explant (%) Mean Treatment ;
A2 20 20 70.00 60.00 70.00 80.00 70.00 A3 15 25 70.00 65.00 80.00 65.00 70.00 A4 20 25 60.00 65.00 70.00 6500 65.00 Table 6 Converted data of the rate of clean living explant of sterilized explants in sterilized treatments.
Time Javel Rate of clean living explant Mean (mins) (%) Time | Time 2 Time 3 Time 4 (%)
Table 7 ANOVA table for the rate of clean living explant of sterilized explants in disinfection treatments
Source DF Adj SS Adj MS F-Value P-Value
Table 8 Ranking test of the rate of clean living explant of sterilized explants in sterilization treatments
Table 9 Percentage of germinated explants in sterilized treatments
Time Ty lệ nay mam (%) Mean
Table 10 Converted data of germination rate in sterilized treatments
Time Jave Ty lệ nay mam
Table 11 ANOVA table for the percentage of germinated explants in sterilized treatments
Source DF Seq SS Adj SS AdjMS_ F-Value P-Value Javel 1 2020.51 2020.51 2020.51 30.00 0.000
Table 12 Ranking test of germination rate in sterilization treatments