Optimisation of shoot bud surface sterilisation technique for Curcuma xanthorrhiza Roxb.

Boesenbergia rotunda, a medicinal herb under the Zingiberaceae family, has been proven to be the most prominent anticancer remedies. The conventional breeding of this plant is inapplicable as it is susceptible to rhizome soft rot and leaf spot diseases. The yellow rhizome also produces limited buds. Therefore it is necessary to propagate this plant through in vitro propagation to obtain abundant uniform planting materials. Unfortunately, high cost is incurred due to the high shoot bud explants contamination level. Hence, it is addressed in the present study to solve the contamination problem. Mercuric Chloride is well known to solve this problem, but it is not advisable to use, because of its poisoning and other hazardous effects. Moreover, explant sterilization technique should also accelerate the shoot response. To find an alternative, 6 different surface sterilization methods (SSM) were designed and evaluated on the explants where different combinations of sodium hypochlorite and ethanol (instead of mercuric chloride) were applied. The sterilized shoot bud explants were then cultured on Murashige & Skoog (MS) media with no additional vitamins or plant growth regulator under the light below 25oC. The contamination was recorded for 3 consecutive weeks along with visible shoot responses. SSM 5 showed minimum contamination and maximum visible shoot response, compared to other SSMs. Therefore, it is suggested that SSM5 could be used to conduct surface sterilization to avoid contamination problem.

Paphiopedilum gratrixianum (Masters) Rolfe is a relatively rare orchid species belonging to family Orchidaceae. Because of several factors including limited geographical range of growth, habitat destruction and mass exploitation, the orchid species’ natural growth rate is declining and under Critically Endangered (IUCN 3.1). So as to conserve this orchid species, the current study provides a protocol for in-vitro mass propagation using MS (Murashige & Skoog) basal media enhanced with the use of several PGRs (Plant Growth Regulators) for seed germination, growth and rooting at different doses, either single or in combination. Fastest seed germination (46.84 days) was favored by combination of MS+1.0 mg/l GA3+1.0 mg/l BAP. It also produced tallest plantlet (4.94 cm), longest (5.82 cm) and broadest leaves (0.557 cm) per plantlet at 90 days after seed germination. For PLB formation, the combination of MS+1.0 mg/l GA3+0.5 mg/l BAP was best suited (15.64 DAG) and treatment with MS+1.5 mg/l KIN for shoot proliferation (4.01) and highest number of leaves (6.61) per plantlets. Root initiation was fastest (7 DAI) in MS+1.5 mg/l IBA, while the number of roots per plantlet were highest (4.56) in the treatment containing MS+ 1.5 mg/l NAA. The same treatment also showed the largest root diameter (1.15 mm). Maximum root length (3.72 cm) was observed in MS+1.0 mg/l IBA. Amongst all the hardening media studied, the well rooted plantlets were best survived in media composed of vermiculite and charcoal in 1:1 ratio with a survival rate of 93.89%.

Plants have very potential compounds to live on earth as they supply 90% of human calorie intake, 80% of protein intake directly, and perhaps the most vital sources of medicine with a vast diversity of microorganisms. As such it’s important to know those microorganisms, their kinds, the features they possess, and the significant compounds/metabolites they can produce. So, this study is based on identifying such microorganisms. To achieve this aim, isolation of endophytes has been done to know their biochemical activities and properties. Various identification procedures have been followed to get pure endophytic strains without any contamination. Surface sterilization of the plant tissue is a must in this progress, various surface sterilization techniques have been tried and finally, for 4/5 plant tissues, sodium hypochlorite and ethanol were given the best result and for 1/5 with the addition of mercuric chloride were the standardized method for surface sterilization. About 30 different bacterial endophytes have been isolated from five kinds of medicinal plants. 4% sodium hypochlorite and 75% ethanol were found effective in sterilizing the surface of Psidium guajava, Cassia occidentalis, Calotropis procera, and Hibiscus rosa-sinensa. While Mangifera indica required an addition of 0.1% mercuric chloride. 19 strains isolated were Gram-positive, 11 Gram-negative (5 were Lactose fermenters and 6 were not), and most of which were bacilli. All isolates have shown different biochemical results, 25 showed a positive result for oxidase, and 28 gave a positive result for catalase. Most of the endophytes identified in this work are Bacillus spp. However, this study highlighted the significance of surface sterilisation and most importantly the presence of potential endophytes capable of producing novel bioactive compounds usable in pharmaceutical/medicinal application.

The aim of this study was to the isolate the endophytic bacteria, optimize its isolation procedure. Ethanol, sodium hypochlorite, and mercuric chloride at various concentrations and duration were employed to optimize the surface sterilization for the isolation of endophytes from Phaseolus vulgaris, Pisum sativum, and Hordeum vulgare. A total of 21 endophytic bacteria have been isolated from three plants. Combination of 2% sodium hypochlorite, 70% ethanol, and 0.1% mercuric chloride was found effective for the surface sterilization of Phaseolus vulgaris and Pisum sativum. In case of Hordeum vulgare, 70% ethanol and 2% sodium hypochlorite was found suitable for the surface sterilization. Ethanol, sodium hypochlorite, and mercuric chloride were found effective decontaminating agents in optimum condition.

An efficient protocol has been developed for a rapid callus induction in Leptadenia pyrotechnica. Nodal, internodal and pod explants from mature plant of L. pyrotechnica were cultured after surface sterilization on Murashige and Skoog medium supplemented with plant growth regulators (PGRs) cytokinins and auxins individually and with various combinations. Nodal segments proved the best explants (90% callus induction) compared with internodal and pod explants (5% and no callus induction respectively). Different treatments were employed for surface sterilization of explants revealing that combination of sodium hypochlorite (NaOCl2) and mercuric chloride (MC) were found significant. Minimum contamination (7%) occurred at 30% NaOCl2 + 2 g/L MC, while 80% occurred at 30% NaOCl2 + 1 g/L MC. The nodal segments cultured on MS medium supplemented with 0.5 mg/L NAA that produced maximum callus (90%) within four weeks. It was followed by 60% callus induction at 5 mg/L NAA + 2.5 mg/L benzyl-adenine (BA) and 30% at 10 mg/L 2,4-D + 3 mg/L 2ip + 3 g/L activated charcoal (AC), while only 20% appeared at control (without hormone application). This is the first successful attempt to establish consistent callus formation protocols from nodal stem segments of L. pyrotechnica. This study may contribute in conservation management of this native plant species found especially in the deserts of Pakistan. Key words: Callus induction, Leptadenia pyrotechnica, plant growth regulators, cytokinins, auxins.

Begonia bimaensis Undaharta & Ardaka is a potential ornamental plant, and currently known only from one population in Sumbawa. Propagation programs, both conventional and in vitro culture are necessary to ensure its conservation. The aim of this research is to observe the effects of explant types and plant growth regulator combination (2,4-D and kinetin) in inducing callus from B. bimaensis leaf in vitro. Callus induction was initiated from three parts of leaf explant, namely petiole, leaf base, and leaf lamina. The explants were planted on Murashige & Skoog (MS) medium with addition of 2,4-D and kinetin. Concentrations of 2,4-D were 0, 0.5, and 1 ppm, while kinetin concentrations were 0, 1, and 2 ppm. Each treatment was replicated 10 times. Results showed that leaf base was the best explant used for callus induction. Medium D1K2 (MS + 1 ppm kinetin) showed the fastest time for callus induction that was at 20 days after planting. The highest percentage of callus production (100%) was found on D1K3 (MS + 2ppm kinetin); D2K2 (MS + 0.5ppm 2,4-D + 1 ppm kinetin); D2K3 (MS + 0.5ppm 2,4-D + 2ppm kinetin) and D3K2 (MS + 1ppm 2,4-D + 1ppm kinetin).

Florida is home to 106 native orchid species, of which 77 are listed as endangered or threatened by the State of Florida. The Institute for Regional Conservation (IRC) has classified 62 of these species as either critically imperiled, imperiled, or rare in South Florida. Because of lack of endosperm, orchid germination rates are very low in nature, as they depend on an obligate relationship with mycorrhizal fungi for nutrients. Most orchid seeds can be germinated in vitro without the need for specific mycorrhizal fungi. This study aims are to establish a fast and efficient protocol for in vitro seed germination using different nutrient media and plant growth regulator (PGR) combinations, and to optimize seedling acclimatization protocols using different greenhouse media. To determine germination preferences, three different in vitro seed germination media were tested supplemented with PGRs, including 1) Murashige & Skoog (MS) (control), 2) MS supplemented with 1.5 mg/L 6-benzylaminopurine (BAP), and 3) MS supplemented with 1 mg/L BAP and 0.5 mg/L 1-Naphthaleneacetic acid (NAA) on Cyrtopodium punctatum, a state-listed endangered, IRC critically imperiled epiphytic orchid. There was no significant difference amongst the treatments after 2 and 6 weeks of seed sowing culture. To understand post-culture survivorship, two epiphytic and one terrestrial native species (Trichocentrum undulatum, Encyclia tampensis, and Oncidium ensatum) were chosen for the acclimatization study with two commercially available potting substrates (coir, sphagnum). To measure seedling growth rates, phenotypic measurements [leaf number, leaf length, root length, plantlet height, light intensity, pH, and electrical conductivity (EC)] and Soil Plant Analysis Development (SPAD) and Normalized Difference Vegetation Index (NDVI) values were recorded monthly for five months. All media/PGR combinations resulted in an initial high percentage of stage I growth but inhibited Protocorm-like bodies (PLBs) development, suggesting more research is necessary to determine later improvements or detriments to MS basal media with BAP and NAA. Comparing acclimatization media with the three species of orchid chosen for this experiment, neither O. ensatum nor E. tampensis showed a marked preference for sphagnum moss or coir. However, T. undulatum did perform better with coir compared with sphagnum. This research will help botanical gardens and commercial plant tissue culture laboratories to have a better understanding on selection of PGR combinations for in vitro cell culture and acclimatization media on increasing the viability and plant health and decreasing the mortality of endangered plants.

Phalaenopsis – known as moth orchids – are the most popular orchids cultivated indoors as decorative house plants. This makes propagation and cultivation of Phalaenopsis important for commercial growers. Enhancements to the micropropagation of Phalaenopsis would have pronounced economic benefits through reduced losses and wastage. We examined the effects of several nutrient media and specific plant growth regulators (PGRs) belonging to the gibberellic acid and cytokinin groups on the in vitro germination of Phalaenopsis seeds, utilizing a single group pretest-posttest model. The effects of several nutrient media such as: Knudson C (KCM), Lindemann (LM), Orchimax (-OM), Orchimax + activated charcoal (+OM), Murashige & Skoog (MS), as well as various PGRs such as 6-Benzylaminopurine (6BA), 6-Furfurylaminopurine (KIN), Adenin hemisulfate (AHS), Thidiazuron (TDZ), 2-Isopentenyl adenine (2iP), and Gibberellic acid (GA3), on the process of germination were also investigated. The explants obtained from the germinating seedlings were subjected to direct organogenesis, and the optimal PGR and tissue fragments were determined. The +OM medium facilitated the shortest germination period (in days). An inverse relationship between the concentration of TDZ and the percentage of germination in the context of the employed PGRs was observed. Apart from TDZ, the remaining PGRs exhibited a positive correlation with concentration. However, no significant difference in germination was observed in comparison to the control. The findings of direct organogenesis investigations revealed that the medium that exhibited the highest productivity was enriched with 5.0 ppm of 6BA. The media containing TDZ exhibited a reduced level of efficiency. Particularly, the group treated with 1.0 ppm of TDZ exhibited reduced efficacy compared to the control group. All concentrations of cytokinin in root elongation stage exhibited a favorable impact in comparison to the control. The variance between these PGRs was not statistically significant.

Surface sterilization is a vital step in preparation of healthy and viable explants in tissue culture. Most surface contaminants can be eliminated by surface sterilization with a suitable sterilizing agent. The study aimed to present an effective disinfection method for Clinacanthus nutans shoot regeneration using nodal segments. A total of four different sterilization approaches were conducted by treating nodal explants with various concentrations of sterilizing agent. Sterilizing agents used were Rhizophora apiculata Pyroligneous acid (PA), sodium hypochlorite (Clorox) thiophanate-methyl (fungicide), and Mercuric chloride (HgCl2). Nodal explant then was cultured on plant growth regulator-free Murashige and Skoog (MS) basal medium. This study sterilizing agents revealed that PA showed strong bactericidal activity. However, it led to a high number of fungal contaminations. The pyroligneous acid did not exhibit a strong potential as a disinfectant for C. nutans nodal explant. Overall, HgCl2 exhibits the best reduction in fungal contamination and gives a significant result with thiophanate-methyl fungicide. Surface sterilization with mercuric chloride (0.2%) for 1 hour was the optimum concentration and duration, which resulted in the highest percentage of nodal explant survival and viability. All viable nodal segments developed into shoots. It had been concluded that the best surface sterilization agent was HgCl2.

BACKGROUND: Amelanchier alnifolia is an attractive small fruit difficult to propagate by traditional methods. Moreover, this species can be susceptible to dormancy after transplanting to the soil. OBJECTIVE: The aim of this work was to evaluate factors that contribute to effective in vitro rooting and acclimatization of micropropagated shoots of Amelanchier alnifolia. METHODS: Different auxins, media concentrations, and sprays containing plant growth regulators were tested. The experimental data were treated by analysis of variance. RESULTS: 1-naphthalene acetic acid (NAA) was superior for rooting compared to indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). Cultivation of shoots on Murashige & Skoog (MS) medium with a half-strength concentration (1/2 MS) led to higher rooting frequencies than on full-strength MS medium. Addition of 1.5 mg l–1 spermidine to cultivation medium did not significantly improve rooting. Gibberellic acid (GA4+7) alone or in combination with 6-benzylaminopurine (BAP) did not effectively break post-rooting dormancy. CONCLUSION: The greatest number of actively growing plants was recorded after rooting the shoots on 1/2 MS medium with 1 mg l–1 NAA followed by a spray treatment with 1 mg l–1 BAP. These results are directly applicable for improving rooting efficiency and acclimatization of micropropagated Amelanchier spp. plantlets.

A study on rapid propagation of cassava through tissue culture was conducted with three elite cassava genotypes: Slicass 6, Slicass 11 and Cocoa from Sierra-Leone. They showed slow growth in Murashige & Skoog (MS) basal medium which was proven to be optimal for a vast number of cassava accessions. Prior to mutation induction, a large population needs to be produced for mutagen susceptibility test and for mutant population development. The ultimate objective of this study was to investigate the effects of plant growth regulators on the shoot development of three cassava genotypes. In vivo shoot tips were sterilized and sub-cultured on MS media supplemented with six combinations of plant growth regulators (PGRs) at different concentrations. The results showed that from all media used, the MS medium with 1.0 mg/L α-naphthalene acetic acid (NAA) showed the best response for rooting (5.50), fresh weight (0.29 g), root number (10.00) and plantlet height (3.81 cm), while 0.1 mg/L 6-benzylaminopurine (BAP) was found to be more favourable to shoot development of leaves (6.38). The highest plant height and fresh weight were 3.81 cm and 0.29 g, respectively for Cocoa at 1.0 mg/L α-naphthalene acetic acid (NAA), 10.00 roots for Slicass 6 at 1.0 mg/L, 6.37 leaf numbers for Slicass 11 at 0.1 mg/L 6-benzylaminopurine (BAP) and 5.6 at 1.0 and 1.5 mg/L α-naphthalene acetic acid (NAA). These observations indicate that a supplement of 0.1 mg/L 6-benzylaminopurine (BAP) in MS medium can be useful in propagation of recalcitrant cassava and low concentration of α-naphthalene acetic acid (NAA) will be beneficial in root induction prior to acclimatization with promotion in recovery of the ex vitro plants before field assessment. Key words: Cassava, Manihot esculenta, propagation, shoot tip, 6-benzylaminopurine (BAP), α-naphthalene acetic acid (NAA).  

Wheat (Triticum aestivum L.) improvement via genetic transformation depends on an efficient regeneration system for recovery of transgenic events. This study reports a somatic embryogenesis-based regeneration system for two Kenyan wheat genotypes (Eagle 10 and Njoro bread wheat II) from mature embryos. The study investigated the efficiency of mercuric chloride, commercial bleach, and chlorine gas in surface sterilizing explants prior to in vitro culture. Callus induction and somatic embryogenesis were done by culturing wheat mature embryos in Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid (2,4-D) used either singly or in combination with 1-naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid (4-CPA) or 6-benzylaminopurine (BAP). Embryo germination and plantlet recovery were done by culturing embryogenic callus in MS medium without plant growth regulators (PGRs). Chlorine gas was significantly (p<0.001) the most effective in surface sterilization and maintenance of explant viability. All 2, 4-D concentrations tested (1, 2, 4 and 8 mg/l) induced embryogenic callus. A significantly higher callus induction rate and callus fresh weight were obtained when 2,4-D was used in combination with either NAA or 4-CPA than when it was used singly. Combining 2,4-D with BAP led to a significantly lower callus induction frequency. Somatic embryo germination was achieved in MS medium without plant growth regulators. These findings have the potential to inform future efforts in the application of modern biotechnology for accelerated wheat cultivar improvement. Key words: Wheat, somatic embryogenesis, mature embryos.

Surface sterilization is essential in explant preparation for in vitro cultivation to reduce contamination risk. This study focused on optimizing surface sterilizing technique to initiate bitter cassava callus culture (Rayong cultivar) for starch production based on the effects of plant growth regulator (PGR). Bitter cassava is widely used in the food and non-food industries thus, its demand is forecasted to be high in the next several years. Farmers faced many challenges in large-scale cassava plantations such as lack of infrastructure, cassava diseases and poor climatic conditions. Plant tissue culture is proposed due to its advantages such as high yield in a shorter time compared to the traditional method and the cultivation can be done outside the plant’s season. Soaking time in 70% ethanol (min), concentration of sodium hypochlorite (%) and soaking time in sodium hypochlorite (min) were selected for optimization of surface sterilizing condition of cassava explants (leaf and stem). Face Centered Central Composite Design (FCCCD) under Response Surface Methodology (RSM) in Design-Expert v9.0.6. was used for designing experiments and optimization purpose to minimize the percentage of contamination. Next, different concentrations of 2,4–dichlorophenoxyacetic acid (2,4-D) (5, 10, 15 and 20 mg/L) were supplemented to Murashige and Skoog (MS) medium to investigate their effect on callus initiation. Based on the results, soaking cassava explants for 5 minutes in 70% ethanol followed by 10 minutes in 5.75% in sodium hypochlorite gave the least percentage of contamination (16.67% for the leaf and 25% for the stem). The highest frequency of callus formation (41.67%) was achieved when using 5 mg/L of 2, 4-D from stem explant. The results from this study can serve as a starting point in establishing bitter cassava callus culture for starch production.

Background: Most orchid species in the Dendrobium genus are epiphytic. Dendrobium orchid (Dendrobium nobile L.) var. Sonia is a non-woody epiphytic orchid that bears a beautiful purple-coloured flower and grows in soilless medium. Dendrobium can be propagated vegetatively through divisions or Keikis. But this propagation method is extremely slow. Micropropagation is most suitable method for multiplication of dendrobium orchids. Surface sterilization is an important step in the preparation of vigorous and viable explants in micropropagation. Most of the surface contaminants can be abolished by surface sterilization with a suitable sterilizing agent. The aim of the investigation is to present an effective disinfection method for the aseptic culture of Dendrobium var. Sonia by using nodal segments. Methods: In the present investigation, two sterilant were employed namely mercuric chloride (HgCl2) and sodium hypochlorite (NaOCl) at different concentrations and durations. A total of nine combinations were tried for treating nodal explants in this investigation during 2022-2023. Explants were surface sterilized with 70% ethanol for 5 minutes followed by 3 to 4 washes with sterile distilled water. Thereafter, the explants were subjected to different surface sterilization treatments to establish contamination free cultures. Result: The study revealed that among the 9 treatments, minimum contamination (30.40%) was recorded in 0.1% HgCl2 for 10 minutes which helps in the maximum establishment of healthy culture (69.60%). Whereas, the lowest result for establishment was found in autoclaved distilled water with maximum contamination (100%). Maximum explant mortality (20.80%) due to sterilant was observed in NaOCl (2%) for 20 minutes.

Background A range of natural factors, invasive animals and human activity have been severely affecting stability of the ecosystem, resulting in the annihilation of plants habitats and so plant endangerment or even extinction. In Qatar, urgent action needs to be taken to stop decline of desert plant species as well as an effective strategy should be applied to reverse and save these wild endangered plants. Otherwise, they will be faced with the danger of their extinction in near future. Therefore, it is very important to have knowledge of protection measures, such as replanting and propagating through tissue culture technology, to protect the biodiversity in Qatar. Plant in-vitro culture systems have been used as an alternative approach to propagate and conserve a large number of rare and endangered plant species that show difficulties to be propagated using conventional methods of propagation. It was reported that standard culture environment could be effectively employed for short-term in-vitro conservation of different plant germplasm, through increasing intervals between subcultures especially in slow growing plant species. Objectives In the current study, conservation of rare and endangered desert plants using in-vitro culture were developed. Generally, these plants are not easy to be propagated by classical horticultural methods. Different techniques including micro-propagation, in vitro seed germination, and regeneration from callus were applied to propagate and conserve three endangered plant species in Qatar; Leptadenia pyrotechnica, Glossonema varians and Prosopis cineraria. Methods Collection of endangered plant species Location of the endangered plant species were identified and the plant parts- seeds, stem, shoots, roots, nodal cuttings or whole plant- depending on its type and availability, were collected. Surface sterilization of the collected material The plant materials were washed with tap water to remove dust and debris, then were soaked in 70% ethanol for 1–2 min, then were treated with sodium hypochlorite or Clorox for 10–20 min followed by rinsing 3–5 times with sterilized distilled water under aseptic conditions. In-vitro plants formation Organ culture using nodal sections of the plants were cultured on hormone-free MS medium (0.5X) for in vitro plants formation. For seeds were cultured on medium containing gibberellic acid (GA3) for efficient seeds germination. In this way, in vitro plants were established and multiplied to produce large number of healthy clones. In case seeds or nodal cuttings are not available, other plant parts like leaf or root were used as explants to initiate callus tissues. Results Seeds of Leptadenia pyrotechnica were collected, surface sterilized and germinated under aseptic condition using 0.5X MS media. In-vitro employing tissue culture via callus and shoot induction using different growth regulators was explored. Seedlings from in-vitro germination of the seed were used as explants. The results revealed that the highest callus production was obtained using 2.0 mg/L BAP. In addition, 0.5 mg/L and 2.0 mg/L NAA were good for callus initiation, compared to other hormones. Seedlings of Glossonema varians were collected were used as an explant for callus induction. Several plant growth regulator were used to initiate callus including 2, 4, D, NAA and BAP and their combinations. The results showed that the best plant growth regulators to induce callus were 1.5 mg/l IBA and 2 mg/l BAP. Prosopis cineraria, is a famous tree in Qatar. It is not easy to be propagated by classical horticultural methods. Seed dormancy was broken by scratching via sand paper. Several plant growth regulator were used to initiate callus including 2, 4, D, NAA and BAP and their combinations. The results showed that the best plant growth regulators to induce callus were both 2.0 mg/l 2, 4, D and 1.5 mg/l IBA. The obtained callus will be treated to regenerate new plantlets. Adventitious shoots and roots formation will be induced and a large number of in vitro plants will be produced. The in-vitro grown clones will be hardened (acclimatized) for greenhouse and later field conditions. The in-vitro plants will be removed from the cultures; medium will be removed by washing with running water and sown in pots. The pots will be covered with plastic sheets to keep high humidity and gradual removal of the plastic will harden the plants for greenhouse.Conclusions Recently, in-vitro culture technique of desert plants has received importance because it can be used for the fast propagation and ex situ conservation of endangered plants. The success of micro-propagation and in vitro conservation of the selected endangered plants depends on the best choice of the explants, the efficiency of the sterilization method and correct plant growth regulator. The best in-vitro conservation of the selected plant species is in MS media with the following plant hormones 2, 4, D, NAA. IBA and BAP.Acknowledgements «This study was made possible by UREP grant # UREP19-209-1-037 from the Qatar national research fund (a member of Qatar foundation). The statements made herein are solely the responsibility of the author(s).»