Shoot Initiation Research Articles

Фенотипическая и генетико-селекционная оценка березы повислой (Betu-la pendula ROTH) и березы пушистой (Betula pubescens EHRH.) ex situ и in vitro

The results of a study of birch test cultures obtained by different methods of ex situ pollination and introduced into culture in vitro are presented. To identify the phenotypic, cultural and cytological characteristics of two birch species naturally growing in the Central Black Earth Region - silver birch (diploid) and downy birch (tetraploid), test cultures of seed origin F1 and F2 were specially created. The article presents data on phenotypic (growth in height in the juvenile and reproductive periods of ontogeny), genetic selection (seed propagation system) variability and some characteristics of the introduction of F1 seed progeny of breeding forms of local birch species into in vitro culture. To predict the growth characteristics of seed progeny of breeding forms of local birch species obtained by different methods of pollination - self-pollination and open pollination at different stages of ontogeny (at the age of 2 and 10 years), a statistical processing of the feature of growth in height of families was carried out using the rank Spearman's correlation coefficient ρ. Species specificity was shown in response to different pollination methods, morphogenic activity of self-fertile and self-sterile forms of these birch species on a nutrient medium ½ MC + 6-benzylaminopurine 1 mg/l, primary shoot formation of explants and regenerative capacity in these species, the nature of morphogenesis (formation of the main escape). For the polyploid species (downy birch), a higher level of self-fertility was established, as well as a more intensive growth of shoots in height than for drooping birch; primary explants in the former had a different character of morphogenesis (there was a joint initiation of the main shoot with adventitious ones) than in silver birch (formation of the main shoot), which indicates a greater adaptive potential for polyploid species and their greater opportunities for inclusion in genetic breeding experiments and obtaining valuable breeding forms.

Influence of Thidiazuron on in vitro regeneration potential of Kaempferia parviflora Wall. Ex Baker from Eastern India

An in vitro regeneration system was standarized on a traditional medicinal plant of Kaempferia parviflora. The rhizome sprouted buds upon transfer to MS basal medium with kinetin (3mg/l) and naphthalene acetic acid (0.5mg/l) were found optimum exhibiting maximum percent of shoot initiation as 90.6 ± 0.2. Maximum number of shoots as 8.1 ± 0.3 and maximum number of roots as 22.0 ± 0.3 were found in benzyladenine (3mg/l), naphthalene acetic acid (1mg/l) and thidiazuron (0.5mg/l) combination. Then the micropropagated plants were transferred to field for successful establishment as 90%. Furthermore, the morphological characteristic analysis was done between the conventional and micropropagated plants which showed very less significant variation. Thus the present protocol reports the propagation of K. parviflora plantlets which could be used for commercial exploitation in future.

Standard Operating Procedure for Micropropagation of Melia dubia Cav.: An Important Fastgrowing Tree

Due to increasing demand to meet out industrial requirement as a raw material , soft wood forest species are under tremendous pressure across the globe. The demand of fast growing Melia dubia is one of them. The usual approach of regeneration for this plant is through seed is unable to produce large scale plants. The present investigation aimed to develop the Standard Operating Procedure through Tissue culture method for mass multiplication of M.dubia using nodal segment . Results showed that the highest shoot initiation response (86.6%) was recorded in Murashige and Skoog medium supplemented with additives, NAA (0.1 mg l-1) and Kinetin (0.5mg l-1). Maximum response of shoot multiplication with highest shoot length of 5.5 cm was obtained in MS medium supplemented with combinations of Ascorbic acid (50 mg l-1) and Kinetin 1 mgl-1. For rhizogenesis, MS + 3.0 mgl-1 IBA (93.3 %) demonstrated superior in terms of the percentage of cultures with root induction, the average number of roots, and the average length of roots per explant. In conclusion present study ensures the successful mass multiplication of M. dubia, demonstrating the importance of tissue culture in the expansion of this economically significant multipurpose tree.

Meta-Topolin-induced regeneration and ameliorated rebaudioside-A production in genetically uniform candy-leaf plantlets (Stevia rebaudiana Bert.)

Present report ascertains the efficacy of meta-Topolin (mT) for improved in vitro mass propagation of stevia (Stevia rebaudiana Bert.) with high-frequency shoot initiation, multiplication, and proliferation over other cytokinins. Shoot tips were inoculated on Murashige and Skoog (MS) medium fortified with 6-benzyladenine, kinetin, mT, thidiazuron, and zeatin individually, at different concentrations ranged from 0.5—1.5 mg L−1. Among various combinations, MS medium + 1.5 mg L−1 mT developed the maximum number (15.33) and length (57.33 mm) of shoots with a greater number of leaves (38.67) after 4 weeks of culture. Elongated shoots were then transferred for root induction on MS medium fortified with a indole-3-butyric acid (IBA), indole-3-acetic acid, and α-naphthalene acetic acid individually, at concentrations ranged from 0.5—1.5 mg L−1. MS medium + 1 mg L−1 IBA was found as the superior rooting medium with the maximum number (10.67) and length (19.00 mm) of roots after next 4 weeks of culture. Rooted plantlets were acclimatized with 95% survival rate in sand followed by sand-soil (1:1; w w−1) mixture in next 8 weeks. Proper growth and development of acclimatized plantlets without any abnormalities was confirmed by micromorphological assessment (at cellular level), plant pigment analyses as well as leaf gas exchange measurement. Stability of ploidy level of the acclimatized plantlets was confirmed by flow cytometry. Furthermore, genetic fidelity of the acclimatized plantlets with their mother plant was confirmed by various molecular markers such as primers from directed amplification of minisatellite-region DNA, conserved DNA-derived polymorphism, inter simple sequence repeats, and start codon targeted polymorphism markers. High-performance liquid chromatography analysis indicated that acclimatized plantlets produced significantly higher (2.4-folds) rebaudioside-A (96.50 mg g−1 dry weight) in comparison to mother plant (40.20 mg g−1 dry weight). The current report, for first time, accounts the mT-mediated large-scale in vitro propagation of stevia ensuring genetic fidelity and simultaneously ameliorating the rebaudioside-A production.

Effect of Drought Stress on Leaf Productivity and Liquor Quality of Tea: A Review

Tea (Camellia sinensis (L.) Kuntze) is a highly valued plant known for its refreshing taste, medicinal properties, and health benefits. It is an evergreen shrub or small tree belonging to the Theaceae family. The productivity and quality of tea leaves and liquor are strongly influenced by edaphic and environmental factors. However, the cultivation of tea faces significant challenges due to the increasing occurrence of drought associated with global warming and climate change. In this review, we have summarized the potential effects of drought on the growth, productivity, and liquor quality of tea. Drought exerts substantial impacts on the physiological, biochemical, and morphological features of tea plants. For instance, under drought stress, there is a reduction in leaf activity, including chlorophyll production, photosynthetic rate, and CO2 uptake. Drought periods also lead to decreased shoot initiation and extension rates. However, it is important to note that responses to drought can vary depending on factors such as tea variety, cultivar diversity, agricultural management practices, and study techniques. While drought-induced biochemical damage may occur, it is often reversible, and the plant can recover upon rehydration. Irrigation strategies employed during dry periods have been shown to have a significant positive effect on tea yields, making it a promising option for enhancing productivity in drought-prone regions. Furthermore, the cultivation of drought-tolerant tea cultivars, along with the application of micro and macro-nutrients, as well as hormone treatments, can contribute to improving the post-drought recovery process. Studying the influence of drought on tea productivity offers an opportunity for frontier research, aiming to understand the intricate relationship between tea leaf productivity and liquor quality. Additionally, investigating the factors that contribute to stress recovery in tea plants holds promise for enhancing cultivation practices and ensuring the sustainability of tea production in the face of changing climatic conditions.

Effect of Explants Type and Plant Growth Regulators on the Initiation and Multiplication of Vegetative Shoots of Stevia rebaudiana Bertoni in vitro

This study was carried out in the Plant Tissue Culture Laboratory of the College of Agriculture - Karbala Unaiversity for the period from 2022 to 2023. The study included the use of explants and different combinations in the sterilization, initiation and multiplication of the vegetative shoots of the Stevia plantiv vitro, the study was carried out in three phases: the first included sterilization of explants (shoot tips and lateral buds) using sodium hypochlorite at concentrations of (0, 0.5, 1, 1.5, 2)% for periods of (5, 10, 15) minutes. The second included the initiation of shoots cultures by cultivating the shoot tips and lateral buds on the MS medium with different concentrations (0, 0.5, 1, 1.5) mg l-1 BA and (0, 0.1, 0.2, 0.3) mg l -1 NAA, the third stage was carried out by cultivating the growths resulting from the shoot tips cultivated in the previous stage on the MS medium prepared with different concentrations (0, 1, 2, 3, 4) mg l-1 BA and (0, 0.1, 0.2, 0.3, 0.4) mg l-1NAA. The results of the study showed the superiority of the developing apical in achieving the lowest contamination rate of 38% compared to the side buds that achieved a contamination rate of 40.67%, and the concentration of 2% of NaOCl achieved the lowest pollution rate of 8.33%, and the duration of 15 minutes achieved the lowest pollution rate of 24%. The results of the initiation experiment showed the superiority of the shoot apical as a explants that achieved the highest response rate to emergence of 70% compared to the lateral shoots that recorded a response rate of 54.69%, and the concentration of 1 mg l-1 BA was significantly superior and achieved a response rate of 82.5% compared to the comparison treatment that gave a rate of 82.5%. A response of 32.5%. As for the results of the vegetative multiplication experiment, the results showed the superiority of BA at a concentration of 3 mg l-1 in achieving the highest rate in the number and length of branches was 33.35 branches Plant-1, 8.20 cm respectively. compared to the comparison treatment that achieved the lowest rate, and the concentration of 0.3 mg l-1 NAA was superior in achieving the highest average of the number and length of the vegetative total reached 30.84 plant branch-1, 7.65 cm respectively, while the comparison treatment achieved the lowest mean for the studied traits.

THE INFLUENCE OF MINIMAL NPK FERTILISATION ON THE QUANTITY AND QUALITY OF APPLE YIELDS

During an annual cycle, fruit trees go through developmental stages, characterised by a higher demand for some mineral elements, called critical periods, which coincide with the phenophases of bud opening and shoot initiation. Research on mineral nutrition in fruit growing has highlighted that the maximum consumption of nutrients is during the period of shoot growth, fruit development and differentiation of fruit buds. In apple, flowering is influenced more by the time of fertiliser application and the form of nitrogen than by the amount applied, as apple has high requirements for the element phosphorus in the phenophases of intense shoot growth and wood maturation. Fertilisation with NPK increased apple yield for both fertilisation treatments compared to the unfertilised control treatments. After applying 270 kg·ha⁻¹ NPK in the first year of the experiment, very significant production increases were obtained, 4.46t·ha⁻¹, with a content of 16% dry matter, 13.13°Brix soluble matter and a titratable acidity of 0.465 mg malic acid/100 g.

Sanitation of Apple Cultivars from AP Phytoplasma and ApMV and ACLSV Viruses Using In Vitro Culture and Cryo-Knife Therapy in Liquid Nitrogen

Systemic infections with phytoplasmas and viruses threaten the production of healthy plant material under the fruit species certification system. We tested the possibility of sanitation using in vitro culture and cryotherapy. The starting material of the cultivars Golden Delicious (clones A and B), Virginia Crab, and Panenské zlepšené was taken from in vivo plants that tested positive for apple proliferation phytoplasma. The Táborita cultivar was obtained from already established in vitro cultures that had tested positive for apple proliferation phytoplasma, apple mosaic virus, and apple chlorotic leaf spot virus. Cultivars Golden Delicious A, Virginia Crab, and Panenské zlepšené were sanitated from the phytoplasma in the first step, i.e., by sterilization and a subsequent transfer to in vitro conditions. Golden Delicious B remained infected with the phytoplasma, and both viruses, after the in vitro culture phase and together with Táborita, were subjected to cryotherapy by vitrification. In Golden Delicious B, three out of thirteen initial shoot tips regenerated after a liquid nitrogen treatment. Four mericlones were regenerated from 10 initial cryopreserved shoot tips of Táborita. None of the three pathogens were detected by PCR in the regenerated Golden Delicious B mericlones. On the contrary, in the case of Táborita, infection with all the pathogens was detected after regeneration. The results obtained indicate the potential applicability of in vitro cultivation techniques or, if necessary, subsequent cryopreservation as a method for sanitizing against systemic microbial contamination. However, further research on the relationship between pathogens and specific genotypes is needed.

An Efficient Protocol for In vitro Regeneration and Acclimatization of Banana (Musa spp.) cv. Grand Naine

Aim: The study was undertaken with a view to standardize a protocol for in vitro regeneration of Banana cv. Grand naine using shoot tip of sucker as an explant
 Methodology: In present study, explants sterilized with different sterilizing agents such as Tween-20 (1%), Bavistin (0.5-1%), Streptomycin sulphate (250 mg/L), Ascorbic acid (150 mg/L) + Citric acid (100 mg/L), HgCl2 (0.1%) and 70% ethanol.Sterilized explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth hormones for shoot initiation BAP alone (1.5, 2.0, 4.0, 6.0 mg/L) and BAP (3.0 mg/L) in combination with IAA and IBA (2.0 mg/L), elongation BAP (3.0 mg/L) and NAA (1.0, 1.5, 2.0 mg/L) and rooting IAA (1.0, 1.5 mg/L) and IBA (1.0, 1.5 mg/L). Primary and secondaryhardening was done in potting mixture containing autoclaved black soil: vermicompost: cocopeat (1:1:1) and garden black soil, cocopeat and red soil (1:1:1) respectively.
 Results: In present investigation 1% Bavistin (fungicide) showed maximum respond to prevent fungal contamination. Highest shoot initiation (100%) was observed on a MS medium fortified with BAP (1.5 mg/L). Maximum shoot length (10.7 cm) was recorded on a MS medium supplemented with BAP (3.0 mg/L) + NAA (2.0 mg/L) + Activated charcoal. Maximum root initiation was observed on half strength MS medium supplemented with IAA (1.5 mg/L). In vitro regenerated plantlets hardened on the mixture of autoclaved black soil: vermicompost: cocopeat (1:1:1). After 14 weeks In vitro plantlets transferred in green house for acclimatization where, 80% survival rate was recorded.
 Conclusion: Regeneration protocol was successfully standardized. Therefore, itcan be used for large scale propagation of healthy, disease and virus free planting material and In vitro propagation helps to meet higher demand of healthy planting material within shorter period.

Tissue culture of gerunggang (Cratoxylum arborescens (Vahl) Blume): multipurpose native species of Indonesian peatland

Gerunggang (Cratoxylum arborescens (Vahl) Blume) is a tree native to Indonesian peatlands that has great potential as an alternative fiber-producing species for the pulp and paper industry because it is adaptable and does not have the potential to disrupt local ecosystems. The species also contains important anti-cancer compounds for the pharmaceutical industry. These industries require large quantities of raw materials to meet their needs, which cannot be met by the supply of conventional plant seedlings. Tissue culture is one of the propagation biotechnologies that can be used as an alternative to provide more efficient germplasm. In this study, we developed a protocol for gerunggang propagation using tissue culture technique. The results showed that the combination of 1 mg/l 6-benzylamino purine (BAP) and 0.5 mg/l naphthalene acetic acid (NAA) in Murashige & Skoog media provided the fastest bud break time (2 weeks before the first subculture), with the highest shoot initiation (3.0 ± 0.2 cm), and after 4 weeks of incubation, the highest auxiliary shoot elongation (7.4 ± 0.1 cm). The significant effect of this hormone concentration also increases the nodule multiplication coefficient (NMC), which is 7.5. In vitro root response on the same propagation medium showed good growth with an average root length of 4.8 ± 0.2 cm. Acclimatization of plantlets was successful under ex vitro conditions and resulted in good growth after 2 and 3 months in the greenhouse. The advantage of applying the tissue culture method to gerunggang is that it can produce about seven times more seedlings from one explant in about 4 weeks. These results will be very useful for further research in the field and require a more intensive and comprehensive study.

Micropropagation of strawberry (Fragaria ananassa “pajaro”)by meristem culture method

Experiments were carried out to examine the effects of different combinations of plant growth regulators in vitro micropropagation of strawberry plants. Specimens were sterilised by a different concentration of sodium hypochlorite - NaOCl (1, 2, and 3%) for 20 mins. A meristem was separated and cultured on Murashige and Skoog (MS) containing 0.3 mg/l BAP for 8 weeks. Initial shoots were transferred to MS with various concentrations of BAP (0, 0.1, 0.3, and 0.5 mg/l) and kinetin (0, 0.1, 0.2, and 0.3 mg/l) in either single or combination. After 6 weeks of culture, the regenerated shoots were transferred on 1/2 MS with different concentrations of NAA (0, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/l) and BAP (0, 0.1, 0.3, and 0.5 mg/l) in either single or combination. The results showed that the lowest percentage of contamination (10%) and the highest percentage of regeneration (80%) response were achieved on MS media by sterilisation with 3% NaOCl for 20 mins. The highest shoot proliferation per explant (16.2 shoots/explant) was reached on MS with 0.3 mg/l BAP plus 0.1 mg/l kinetin. 1/2 MS media with 0.1 mg/l NAA plus 0.1 mg/l BAP induced 14.4 roots per shoot and produced the longest roots (9.1 cm). However, the higher concentration of NAA (0.3-1.0 mg/l) or in combination with BAP resulted in callus formation. The plantlets, thus developed, were hardened and successfully established in soil containing organic fertiliser: Trichoderma: coconut peat (1:1:1 v/v/v).

In vitro clonal propagation of Tanacetum cinerariifolium and establishment of an ex situ collection of selected clones

Dalmatian pyrethrum Tanacetum cinerariifolium (Trevir.) Sch. Bip. (Asteraceae) is a perennial herb endemic to the eastern coast of the Adriatic Sea. The species is widely cultivated in many countries for its bioactive compounds pyrethrins, which are used as natural insecticides. Plants derived from seeds vary greatly in pyrethrin content; therefore, the vegetative propagation of high-quality individuals is very important for the establishment of agricultural pyrethrum crops. The present study deals with rapid in vitro multiplication of pyrethrum, ex vitro adaptation of selected clones and creation of an ex situ collection, as a first step towards introducing the species into agriculture in Bulgaria. Seeds from a private ex situ collection in Bulgaria and from a natural Croatian population were used as initial material for in vitro cultures initiation. Basal MS medium (Murashige and Skoog 1962) or MS supplemented with different concentrations of kinetin and indole-3-butyric acid were used for seed germination and multiplication of one-seed derived clones by consecutive subcultivations. The propagation effectiveness was evaluated as a number of new plants obtained per initial shoot. Considerable losses were noticed due to both endophytic contaminations and necrosis, especially on media supplemented with plant growth regulators. These problems were overcome by medium optimization: adding an antibiotic and modifying the medium to increase the calcium concentration using CaCO3. In the best medium variant (basal MS + 200 mg/L Medaxone + 75 mg/L Ca) no more infected plants were observed, and the percentage of necrotic plants decreased threefold, which resulted in formation of 38.06±10.11 new plants per initial shoot for a period of 7 months. Three hundred and sixty plants were ex vitro adapted in a phytotron (88% surviving rate), then 16 plants from 4 selected clones were transferred to the ex situ collection and bloomed twice from the very first growing season (June and September). The number of the flower heads increased in the second year of field cultivation and an average of 328±138 capitula per plant were counted for the best clone. The first trials to establish a pilot plantation of pyrethrum are promising.

Regeneration of Raja (Musa AAB Group) and Kepok (Musa ABB Group) bananas on various stages of in vitro culture

Banana group can be divided into two groups based on their method of its utilization. Banana group that can be eaten immediately after ripening is called "dessert banana", e.g., Raja, while the group that needs particular processing before consumption is called "plantain banana", e.g., Kepok. This study aimed to compare the growth of Raja banana with Kepok plantain through experiments at three stages of the in vitro culture media: the shoot initiation, shoot multiplication, and plantlet acclimatization, also to compare the growth of Raja bananas with Kepok plantains in response to cytokinins BAP and TDZ at shoot multiplication stage. Planting material was in the form of rhizomes that was prepared with the same size. The experiment was performed using a completely randomized design. Results showed that Kepok plantain could adapt faster to the media condition than Raja banana; the phenomenon was evident in the growing speed of Kepok during shoot initiation stage. The proportion of the B genome did not show a direct effect on shoot induction at the multiplication stage or enlargement and organ formation at the acclimatization stage. Shoot induction at the multiplication stage depended more on the composition of the media used. The combination of BAP 3 mg L-1 and TDZ 0.01 mg L-1 in MS media produced the best shoot induction rate, and TDZ 0.01 mg L-1 in MS media had the highest shoot elongation rate.
 Keywords: banana, BAP, genome, PGR, plantain, TDZ

Effects of Light Intensity on in Vitro Regeneration Of Stevia Rebaudiana Bertoni, Bacopa Monnieri L. And Solanum Tuberosum L.

Light is the most essential factor controlling plant growth, morphogenesis, metabolism and chlorophyll content of plant cells, tissues and organ culture. The present investigations were carried out to find out effects of different light intensities (210, 500, 1000, 1500, 2000, 2500 and 3000 lx) on the regeneration of three plants, namely Stevia rebaudiana Bertoni., Solanum tuberosum L. and Bacopa monnieri L. Results showed that light intensity has a direct effect on plant growth in vitro. There was less time for shoot initiation and the highest mean number of shoots/explants (9.75 ± 3.862) of S. rebaudiana was observed from the nodal explants when the culture was maintained at 1000 lx light conditions. At maximum light intensity (3000 lx) best result was recorded for S. tuberosum. A distinct intensity of light, 1500 lx showed the best outcome both for the number and length of shoots of B. monnieri (p = 0.00). The present study also demonstrated the relationship between root growth and light condition. Bangladesh J. Bot. 52(1): 53-60, 2023 (March)

In vitro Propagation and Assessment of Genetic Homogeneity using RAPD and ISSR Markers in Tinospora cordifolia (Wild.) Hook. F. & Thoms, An Important Medicinal Plant of Nepal

The Menispermaceae family includes the widely glabrous, succulent, climbing shrub Tinospora cordifolia (Gurjo), which has been found to have a variety of pharmacological and ethnomedicinal characteristics. T. cordifolia is also one of the most commercially exploited plants in pharmaceuticals. The nodal segment explants were initially cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyl amino purine (BAP), and/or Kinetin (KIN) among which MS +BAP (2.0 mg/l) induced shoot initiation after 7 days of post-inoculation. The nodal segments were then excised and treated with various concentrations of BAP, BAP with KIN, and coconut water for the proliferation among which 5 mg/l induced significant nodal segment proliferation (9.0 nodal segments/per explant) and shoot length (8.0 cm). The efficacy of coconut water in increasing the nodal segment proliferation of T. cordifolia was tested and the shoot proliferation increased significantly at 5% and 10% of coconut water, however, the maximum response of shoot number (23.0), shoot length (12 cm) was in the MS medium supplementation with BAP (5mg/l) and 10% coconut water. The genetic fidelity of these plants was also confirmed by random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers in wild and in vitro cultures. This protocol is an efficient way for the in vitro mass propagation of true-to-type plantlets of T. cordifolia which provides a basis for germplasm conservation and sustainable utilization.

Transcriptome Analysis Reveals the Heat Stress Response Genes by Fire Stimulation in Michelia macclurei Dandy

Heat stress due to external heat sources such as fire is an ecological problem for plants. When forest plants suffer from fire, high temperatures cause an array of morphological, physiological, and biochemical changes, which affect growth and development. Michelia macclurei Dandy is an evergreen broad-leaved tree species with the characteristics of fast growth, strong adaptability, and good fire-resistance. Some studies have improved the understanding of how fire behavior affects physiology, function and mortality, but the extreme heat response genes and mechanisms need improved understanding. In this study, we conducted a fire experiment (slight and severe) and RNA-Seq in M. macclure. The de novo assembly obtained 104,052 unigenes, and 48.46% were annotated in at least one public database. Specifically, 4458 and 4810 differentially expressed genes (DEGs) were identified in slight and severe fire treatment groups, respectively. In two treatment groups, 612 unigenes were differentially expressed, which were enriched in ‘oxidoreductase activity’ in the molecular function (MF) category of Gene Ontology (GO) enrichment analysis, suggesting the core role of oxidoreductase activity in response to extremely high temperatures in M. macclurei. In KEGG enrichment analysis of DEGs, the ‘plant hormone signal transduction’ is overrepresented, suggesting that this process plays an important role during heat response in M. macclurei. In the pathways of cytokinine and salicylic acid, some vital DEGs were enriched, which were related to cell division, shoot initiation, and disease resistance, and the potential interactions during heat stress were discussed. Moreover, the DEGs linked to heat stress response were identified, including heat shock factors, stress enhanced protein, signal transduction, photosystem, and major transcription factors. The qRT-PCR examination of various tissues, expression dynamics, and treatments revealed that the genes coding for the heat shock protein HSF30, stress enhanced protein, and photosystem I reaction center subunit II exhibited particularities in leaf tissue. Genes coding for heat shock proteins displayed a distinct expression pattern between fire treatment and conventional heat stress, which could signify the distinctive function of HSPs and the mechanism of heat responses. Altogether, these may interact to respond to fire stress through alterations in cellular processes, signaling transduction, and the synthesis and degradation of response proteins in M. macclurei. The results of this study provide a crucial transcriptional profile influenced by heat stress in M. macclurei, and could be of great use to explore the fire prevention mechanisms of fire-resistant tree species.

Colchicine-mediated in vitro polyploidization in gerbera hybrid.

An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in vitro-developed shoots (tips) were treated individually with 0.1%, 0.25% and 0.5% (w/v) colchicine solutions for 4, 6, 8, and 12h. The colchicine-treated shoot tips were then inoculated on Murashige and Skoog (MS) medium fortified with 1.5mg/l meta-Topolin for multiple shoot proliferation and later transferred into 1.5mg/l indole-3-acetic acid-fortified MS medium for rooting of shoots. The ploidy levels of the colchicine-treated and regenerated plantlets along with the non-treated oneswere confirmed via flow cytometry analysis and metaphasic chromosome count. The highest frequency oftetraploid plantlets (50%) were obtained when shoot tips were treated with 0.1% colchicine for 4h. Morphological observations revealed that inducedtetraploidplantlets exhibited delayed fresh shoot initiation, fewer but longer shoots, as well as fewer but broader leaves. Likewise, the studyof stomata revealed that in comparison to their diploid counterparts, the tetraploidplantlets exhibited less frequent yet significantly larger stomata, and higher number of chloroplasts. The tetraploids were recorded with significantly higher chlorophyll, carotenoid, and anthocyanin content during the photosynthetic pigment analyses. During ex vitro acclimatization and field growth, the tetraploid plants exhibited delayed proliferation but with higher vigor and thickened broad leaves. The genetic uniformity among the diploid and the tetraploidplants was confirmed using conserved DNA-derived polymorphism(CDDP), directed amplification of minisatellite-region DNA(DAMD), inter simple sequence repeats(ISSR), and start codon targeted(SCoT) polymorphism marker systems. The tetraploids developed in the present study would be of immense importance for the genetic improvement of gerbera as far as its ornamental values are concerned.

In vitro callus and shoot initiation of Black cohosh (Actaea racemosa. L) pre-incubated in darkness treatments

A medicinal plant that is commonly overharvested in its native habitat, Black cohosh (Actaea racemosa L.) rhizomes are used to mitigate symptoms of menopause and are a common alternative to hormone replacement therapy. Developing an accurate protocol for initiation of Black cohosh in tissue culture is becoming a necessity. Wild harvesting practices are the main source of Black cohosh rhizomes processed by pharmaceutical companies and are causing a significant decrease of these wild plants, leading to an At-Risk status. Growing these plants in a plant tissue culture laboratory is more sustainable than current practices, and having an accurate protocol for initiation of these plants is crucial to this. Our lab has previously developed a protocol for initiation of Black cohosh and this research was meant to optimize this protocol. Leaf explants of Black cohosh were cultured in vitro to determine the optimal duration of time for initiation of callus and/or shoot under darkness, and appropriate plant growth regulators (PGR) for shoot growth. Leaf explants incubated under darkness for increasing amount of time (0 to 6 weeks of darkness) were analyzed at two stages of tissue development. Stage 1 involved initiation of leaf explant material under darkness (treatment) and measured the callus development in leaf explants; Stage 2 was induction of adventitious shoot growth and cell division and compared the effect of two PGRs, KT and BA, on shoot growth for each darkness treatment. Leaf explants were given a score using a culture chart that assigns a numerical score to leaf explants based on their physiological characteristics. These numerical scores took into account color of leaf explant, callus development, shoot growth, and presence of dead leaf tissue. Stage 2 measured the number of shoots produced per explant within culture containers between PGRs and the number of weeks in darkness. Overall trends indicated that mortality of leaf explants decreased as the duration of incubation under darkness increased with mortality of explants decreasing from 63.9% (T0 darkness treatment) to 2.8% (T6 darkness treatment). Culture scores of explants (including dead explants) was highest for T6 weeks of darkness (culture score 2.96 on average), and for culture scores without dead explants (culture score of 3.05). During the adventitious shoot growth stage, however, T5 weeks of darkness performed significantly higher (KT: 28 shoots/ BA: 17.3 shoots) than T6 weeks of darkness (KT: 12.9 shoots/ BA: 9.4) in Replication 2 for both plant growth regulators KT PGR performed better, on average, than BA PGR

PERFORMANCE OF DIFFERENT GROWING CONDITIONS AND LENGTH OF CUTTINGS ON BIOCHEMICAL PARAMETERS OF DRAGON FRUIT (HYLOCEREUS UNDATUS) SAPLINGS

Dragon fruit is getting wider popularity among the farmers and researchers as well. Due to high value benefits many farmers are cultivating this crop. This increasing demand of these fruit necessitates more advanced research on its better establishment. By keeping this in consideration, an investigation was taken to evaluate planted saplings under abiotic stresses for success and standardizing the growing conditions and length of cuttings. The results indicated that, the data on biochemical parameters at 50 days after first shoot initiation, the maximum shoot moisture content (79.33%) and total chlorophyll content (6.90 mg g-1) was recorded in shade net condition, whereas highest protein content (0.25 mg g-1), phenol content (44.47 mg g-1), proline content (49.92  g-1) and total sugars (5.86%) were recorded in the open field condition. However, the 35 cm length of cuttings exhibited highest total chlorophyll content (6.13 mg g-1) and total sugar content (5.88%), whereas maximum protein content (0.25 mg g-1), total phenol content (39.70 mg g-1) and proline content (46.51 g-1) was recorded in 45 cm length of cuttings. Therefore, treatment combination shade net condition with 35cm length of cuttings has reduced the abiotic stress as compared to other treatments at 50 days intervals after first shoot initiation so, shade net condition with 35 cm length of cuttings performed better under abiotic stresses.

Callus Induction and Regeneration Response of Mustard (Brassica spp.)

Mustard (Brassica spp.) is the third most important edible oil crop belonging to the family Brassicaceae (Cruciferae) and the genus Brassica. Due to the growing world population and increasing industrialization, the demand for edible oil and biofuels is increasing worldwide. Stress-tolerant mustard variety creation by transformation or genome editing procedures, as well as varietal in vitro regeneration responses, are critical challenges. The objective of the study was to design a tissue culture protocol for better use the explant by using hypocotyls and cotyledon explants to observe the in vitro response to callus induction and regeneration of three mustard types, namely BARI Sarisha-14, Binasarisha-4, and Binasarisha-9.Explants were cultured on Murashige and Skoog medium (MS) supplemented with different concentrations and combinations of NAA(10 mg mL-1), BAP (10 mg mL-1 for callus induction and shoot initiation medium , 2.5 mg mL-1 for shoot outgrowth medium), AgNO3(50 mg mL-1), GA3(2 mg mL-1), and IBA (10 mg mL-1)for callus induction, shoot initiation, shoot outgrowth, and root initiation. Binasarisha-4 showed the best performance with hypocotyls76.67% callus induction, 70.03% shoot initiation, 92.23% shoot outgrowth, and 40% root initiation followed by BARI Sarisha-14 (71.67%, 58.23%, 73.33% and 23.33% respectively) and Binasarisha-9 (54.17%, 38.87%, 45.83% and 11.11% respectively). It is recommended that in vitro hypocotyls culture of Binasarisha-4 can be used for frequent and successful genetic transformation in Mustard.