Protocol For Shoot Regeneration Research Articles

Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

In vitro Regeneration of Multiple Shoots in Abelmoschus esculentus (L.) Moench (Okra) via Apical Shoot Meristem Culture

Introduction: To develop the efficient protocol for multiple shoot regeneration of A.esculentus by apical root culture method. Abelmoschus esculentus L., commonly known as okra, is a member of the Malvaceae family, which is widely consumed due to its high nutrient content and potential therapeutic properties. Okra contains various bioactive compounds that ex-hibit antibacterial properties and may be useful in treating type-2 diabetes, digestive diseases, and liver detoxification. To select the plant for the present and prepare the efficient protocol for the development of multiple shoot regeneration culture method. Methods: In this study, we developed an efficient protocol for multiple shoot regeneration of A. esculentus using the apical shoot culture method. Mature shoot apex explants of the germinated A. esculentus genotype CoBhH1 were cultured on Murashige and Skoog (MS) medium supple-mented with different concentrations of cytokinins (BAP and TDZ) and auxins (IAA and NAA) to determine the optimal conditions for shoot induction. Results: The highest number of multiple shoots (27.04 shoots) was obtained with 0.8 mg/L TDZ. Excised shoots were cultured on MS medium supplemented with GA3, which induced elongation of the shoots to a maximum of 8-10 cm. Regenerated plantlets were successfully transferred to soil, with a 100% survival rate and no differences in morphology or growth characteristics com-pared to control plants. Rooting was achieved with 1 mg/L IBA. Conclusion: This study provides an efficient protocol for multiple shoot regeneration of A. esculentus through apical shoot culture, which has potential applications in plant breeding and genetic engineering.

Prediction and optimization of indirect shoot regeneration of Passiflora caerulea using machine learning and optimization algorithms

BackgroundOptimization of indirect shoot regeneration protocols is one of the key prerequisites for the development of Agrobacterium-mediated genetic transformation and/or genome editing in Passiflora caerulea. Comprehensive knowledge of indirect shoot regeneration and optimized protocol can be obtained by the application of a combination of machine learning (ML) and optimization algorithms.Materials and methodsIn the present investigation, the indirect shoot regeneration responses (i.e., de novo shoot regeneration rate, the number of de novo shoots, and length of de novo shoots) of P. caerulea were predicted based on different types and concentrations of PGRs (i.e., TDZ, BAP, PUT, KIN, and IBA) as well as callus types (i.e., callus derived from different explants including leaf, node, and internode) using generalized regression neural network (GRNN) and random forest (RF). Moreover, the developed models were integrated into the genetic algorithm (GA) to optimize the concentration of PGRs and callus types for maximizing indirect shoot regeneration responses. Moreover, sensitivity analysis was conducted to assess the importance of each input variable on the studied parameters.ResultsThe results showed that both algorithms (RF and GRNN) had high predictive accuracy (R2 > 0.86) in both training and testing sets for modeling all studied parameters. Based on the results of optimization process, the highest de novo shoot regeneration rate (100%) would be obtained from callus derived from nodal segments cultured in the medium supplemented with 0.77 mg/L BAP plus 2.41 mg/L PUT plus 0.06 mg/L IBA. The results of the sensitivity analysis showed the explant-dependent impact of exogenous application of PGRs on indirect de novo shoot regeneration.ConclusionsA combination of ML (GRNN and RF) and GA can display a forward-thinking aid to optimize and predict in vitro culture systems and consequentially cope with several challenges faced currently in Passiflora tissue culture.

Direct and indirect shoot regeneration from leaf explants of Centratherum punctatum Cass., A wild ornamental plant

Centratherum punctatum Cass. is an important wild ornamental medicinal plant belonging to Asteraceae family. It has anti-inflammatory, anti-cancerous, anti-hypersensitive, anti-bacterial and anti-fungal properties and is commonly used in hair care preparations. In this study we describe a protocol for high frequency direct and indirect shoot regeneration from leaf explants. In vitro derived leaves were cultured on MS medium supplemented with different concentrations of TDZ, ZN or BAP (0.1–2.0 mg/L) alone or in combination with NAA (0.1–0.7 mg/L) for shoot regeneration and varying concentrations of 2, 4-D or NAA (0.2–2.0 mg/L) for callus induction. Optimum direct shoot regeneration response was obtained from in vitro derived leaves in MS medium supplemented with 0.5 mg/L TDZ (61.4 ± 3.90% response; 27.4 ± 1.99 shoots per explant; mean shoot length of 4.8 ± 0.81 cm). Out of the 7, 14, 21 and 28-day-old leaves, 14-day-old leaves resulted in maximum shoot induction indicating the influence of explant age. Lamina with petiole exhibited a significant increase in shoot regeneration compared to that without petiole. Pretreating the explants with 1.0 mg/L TDZ for 24 h prior to culture produced the highest response of 92 ± 1.84% with a mean number of 38.7 ± 1.24 shoots per explant. 2, 4-D was found favourable for callus induction than NAA for callus induction from leaf explants of C. punctatum. The 21-day-old leaf showed the optimum callus induction. The optimum shoot regeneration from callus was obtained in 1.5 mg/L TDZ (84.2 ± 4.09% response with 58.2 ± 2.46 shoots per explant). Well-developed shoots with 3.0–4.0 cm length were rooted on half strength MS medium containing 0.5 mg/L IBA. The rooted shoots were transplanted to soil and acclimatized with 96.8 ± 3.26% success. Analysis of clonal fidelity of the micropropagated plants through SCoT marker analysis revealed the genetic similarity of mother plant and in vitro derived plants.

Knockout of phytoene desaturase gene using CRISPR/Cas9 in highbush blueberry.

Among the New Plant Breeding Techniques (NPBTs), the CRISPR/Cas9 system represents a useful tool for target gene editing, improving the traits of the plants rapidly. This technology allows targeting one or more sequences simultaneously, as well as introducing new genetic variations by homology-directed recombination. However, the technology of CRISPR/Cas9 remains a challenge for some polyploid woody species, since all the different alleles for which the mutation is required must be simultaneously targeted. In this work we describe improved protocols adapting the CRISPR/Cas9 system to highbush blueberry (Vaccinium corymbosum L.), using Agrobacterium-mediated transformation. As a proof of concept, we targeted the gene encoding for phytoene desaturase, whose mutation disrupts chlorophyll biosynthesis allowing for the visual assessment of knockout efficiency. Leaf explants of in vitro-cultured blueberry cv. Berkeley has been transformed with a CRISPR/Cas9 construct containing two guide RNAs (gRNA1 and gRNA2) targeting two conserved gene regions of pds and subsequently maintained on a selection medium enriched with kanamycin. After 4 weeks in culture on the selection medium, the kanamycin-resistant lines were isolated, and the genotyping of these lines through Sanger sequencing revealed successful gene editing. Some of mutant shoot lines included albino phenotypes, even if the editing efficiencies were quite low for both gRNAs, ranging between 2.1 and 9.6% for gRNA1 and 3.0 and 23.8 for gRNA2. Here we showed a very effective adventitious shoot regeneration protocol for the commercial cultivar of highbush blueberry "Berkeley", and a further improvement in the use of CRISPR/Cas9 system in Vaccinium corymbosum L., opening the way to the breeding mediated by biotechnological approaches.

Protoplast-Based Transient Expression and Gene Editing in Shrub Willow (Salix purpurea L.).

Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

Silver nitrate reduces hyperhydricity in shoots regenerated from the hypocotyl of snapdragon cv. Maryland Apple Blossom

We developed an efficient protocol for shoot regeneration from hypocotyl explants in snapdragon (Antirrhinum majus L.) cv. Maryand Apple Blossom by plant growth regulators (PGRs), followed by ethylene synthesis inhibitor Aminoethoxyvinylglycine (AVG), ethylene precursor 1-Aminocyclopropane-1-carboxylate (ACC), and ethylene action inhibitor silver nitrate (AgNO3). The combination of benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) did not induce shoots, whereas naphthaleneacetic acid (NAA) and Zeatin (Zt) induced shoots. The combination of 0.05 mg/L NAA and 2.5 mg/L Zt resulted in the maximum shoot regeneration percentage and a reasonable number of shoots per explant. However, the regenerated shoots exhibited hyperhydricity and failed to develop into normal plants when introduced into plant growth media. Addition of ACC and AVG to the media containing 0.05 mg/L NAA and 2.5 mg/L Zt inhibited shoot regeneration, while AgNO3 (2.0 mg/L) marginally improved shoot regeneration and successfully reduced hyperhydricity. Shoots grown in AgNO3-containing media showed a reduction in ethylene related gene expression and reactive oxygen species (ROS) accumulation; the leaves showed reduced intercellular space as well as an increase in the number of open stomata under AgNO3 treatment. Moreover, shoots regenerated from AgNO3-containing media easily developed into normal plants in plant growth medium. This study highlights the importance of specific PGRs in shoot regeneration, as well as the role of AgNO3 in reducing the hyperhydricity of shoots regenerated in vitro. These findings will contribute to a better understanding of the hyperhydricity mechanism. We expect this protocol to be useful for in vitro shoot regeneration and genetic transformation of the snapdragon cultivar used in this study.

An Optimized Protocol for Seed Sterilization and Shoot Regeneration from Mature Embryo in Wheat (Triticum Aestivum L.) var. HD2967

In the present study, an efficient protocol for seed sterilization was developed. This protocol used a 1.0 % bavistin solution, which provides a protective response against fungal contamination. It was observed that there was no significant difference among the effective sterilization treatments in comparison to seed germination and seedling length under in vitro conditions. Furthermore, the embryo excised from bavistin-treated seeds showed improvement in callus induction and shot regeneration in var. HD2967. 0.1% HgCl2-SDS solution used for seed sterilization was also optimized with 2.5 to 3.0 minutes as the best-suited duration, providing a completely sterilized environment for the seeds for healthy and fast growth. In addition, plant growth regulators (PGRs) were further used to screen the most effective concentration for callus to shoot formation using mature embryos. Auxin concentration of 2.0 mg/l in MS agar media was quite effective for callus induction from embryo explant. For shoot regeneration (SI-7) combination is suitable for a maximum number of shoots per callus. This combination of 2, 4-D, Zeatin, CuSO4, and TDZ showed good regeneration in the wheat variety HD2967. The optimized protocol displayed 100% callus induction and up to 80-85% regeneration in HD2967, with a high reproducibility rate paving the way for regenerating plantlets from mature wheat embryos.

Callus via Shoot Organogenesis and Plant Regeneration of Stevia rebaudiana Bertoni

Stevia rebaudiana Bertoni is important medicinal plant which is native between southern Brazil and northern Paraguay. A reliable protocol for shoot organogenesis and regeneration was developed for an antidiabetic medicinal plant species. Regeneration via indirect shoot organogenesis was established from two types of explant (leaf and stem) on MS medium [1] with different concentrations and combinations of plant growth regulators (PGR) such as 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and gibberellic acid (GA3) for inducing organogenic callus of S. rebaudiana. In the present investigation the highest percentage of response was shown by 90% and 80% callogenesis from leaf and stem explants on MS medium supplemented with 2.0 mg/l 2,4-D + 0.5 mg/l BAP + 0.5 mg/l GA3 (MSC3) was observed. The high frequency of regenerants was observed from S. rebaudiana leaves at 0.5 mg/l 2,4-D + 2.0 mg/l BAP + 0.5 mg/l GA3 (MSR3). Optimal shoot multiplication and rooting were obtained on 2.0 mg/l BAP and 0.1 mg/l indole- 3-butyric acid (IBA), respectively, followed by successful acclimatization of regenerants (90% survival) to greenhouse condition. The purpose of this study was to establish a reliable S. rebaudiana regeneration protocol that can be used to facilitate the cultivation, propagation and planting of this plant species.

English

The efficient plantlets regeneration of peanut is the most important and a primary step to successfully transform gene and apply recently developed genome editing techniques for crop yield improvement. The purpose of this research is to develop protocol for peanut cultivars applying different concentration of hormones for selected peanut cultivars and develop plantlets regeneration protocol. There is no previously reported protocol for the Chinese peanut cultivar N3 and Yu-hua-14. We optimized shoot and root regeneration protocol for two peanut cultivars. Both cultivars showed positive response for the cytokinin plant growth hormone 6-benzylaminopurine (BAP) and thidiazuron (TDZ). The highest shooting rate (97%) was found in a medium supplemented with 4 mg/L BAP and (94.33%) for 1 mg/L TDZ. Hence, more shoot initiation was observed at higher concentration of BAP as compared to TDZ. However, the maximum root regeneration (81%) was found on medium containing 0.3 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D) and the highest rooting rate (96.33%) was found in a medium containing 1 mg/L α-naphthalene acetic acid (NAA), indicating lower concentration of NAA induce more rooting rate than 2,4-D treatment. In this study, cotyledonary node was used, and this method was found to be efficient and rapid for in vitro peanut regeneration. © 2022 Friends Science Publishers

Standardization of protocol for shoot regeneration in Bacopa monnieri (L.) Pennell using leaf explants

Bacopa monnieri (L.) Pennell commonly called ‘Brahmi’ is used as a medicinal herb since ages. It is known for its revitalizing and nootropic actions in Ayurvedic medicine as it strengthens memory as well as intellect. This activity of the plant has been accredited to a mixture of triterpenoid saponins, sterols and alkaloids. Of these saponins, Bacoside-A and Bacoside-B have been found to be the most important. Present work aimed to establish direct and indirect shoot regeneration protocol in Bacopa monnieri (L.) Pennell using leaf explants. It was found that MS medium supplemented with 1.0 mg/l 2, 4-D and 0.5 mg/l BAP was best for callus formation (53.33%). Leaf explants showed both direct and indirect regeneration and MS medium supplemented with 6.0 μ mol/ was found to be best for shoot regeneration.

High-frequency adventitious shoot organogenesis from in vitro stem explants of Scutellaria araxensis Grossh.

The present study introduced an in vitro shoot organogenesis protocol for the medicinal plant Scutellaria araxensis (Lamiaceae). Stem, leaf, and petiole explants were cultured in half-strength Murashige and Skoog (MS) medium containing different concentrations of 6-benzylaminopurine (BAP) alone or in combination with thidiazuron (TDZ), indole-3-butyric acid (IBA), or α-naphthalene acetic acid. Callus formation occurred from stem and petiole explants in most cultures; however, in leaf explants, it was observed only in cultures containing 0.5 mg/l BAP supplemented with TDZ at all concentrations. The highest frequency of indirect shoot induction (100 and 90%) with an average of 20.33 and 12 shoots per explant was observed in stem-derived calli cultured on half-strength MS medium containing 2.0 mg/l BAP plus 0.5 and 1.5 mg/l TDZ, respectively. The best direct shoot organogenesis (40%) was observed in stem explants cultured on half-strength MS medium containing 0.5 mg/l BAP and 0.5 mg/l IBA with a mean of 18 shoots per stem explant. The regenerated micro-shoots were elongated on a medium fortified with 0.5 mg/l gibberellic acid and then successfully rooted in half-strength MS medium supplemented with 0.5 mg/l IBA. The obtained plantlets were acclimatized in a growth chamber with a survival rate of 100%. This study is the first report of a simple and efficient in vitro shoot organogenesis and regeneration protocol for S. araxensis by using stem explants, which could be useful for the conservation, genetic manipulation, and exploitation of biological molecules of this valuable genetic source.

ANALISIS SESAR-SESAR MAJOR SEMENANJUNG MALAYSIA DENGAN TEKNIK GRAVITI DEKONVOLUSI EULER 3D

The application of biotechnology in upland rice improvement programs depends on the availability of efficient regeneration protocols. Although protocols for shoot regeneration of upland rice are available, none has been reported for pigmented cultivars. This study reports on a protocol for callus induction and regeneration of Tadong, a pigmented upland rice cultivar from Sabah. For callus induction, immature embryos were cultured on media containing 2,4-Dichlorophenoxyacetic (2,4-D) at various concentrations (0 – 2.5 mg/L) and on different types of media (MS; MSB5; N6B5; N6). To induce shoot regeneration, callus explants were cultured on MS medium supplemented with combinations of 6-Benzylaminopurine (BAP) at various concentrations (0 – 3.0 mg/L) and 1-Naphthaleneacetic acid (NAA) at 1.0 mg/L. To induce shoot development, callus explants were pre-treated with Thidiazuron (TDZ) at various concentrations (0-1.0 mg/l) and exposed to different desiccation periods (0 – 72 hours). 2,4-Dichlorophenoxyacetic at 2.5 mg/L and N6B5 medium resulted in the highest percentages of explant forming callus which were 60.3 ± 17.0 % and 58.7 ± 9.8 % respectively. The regeneration media failed to induce shoot on callus explants, instead, green spots were formed on the surface of the callus. The green spots were stimulated to develop into shoots when the callus explants were pre-treated with 0.5 mg/L TDZ or exposed to partial desiccation for 24 h, the percentages of explant forming shoot were 35.7 ± 4.8 % and 47.7 ± 6.8 % respectively. Shoots developed into complete plants on hormone-free MS medium and acclimatized.

CALLUS FORMATION AND PLANT REGENERATION FROM IMMATURE EMBRYOS OF PIGMENTED UPLAND RICE (Oryza sativa cv. Tadong)

The application of biotechnology in upland rice improvement programs depends on the availability of efficient regeneration protocols. Although protocols for shoot regeneration of upland rice are available, none has been reported for pigmented cultivars. This study reports on a protocol for callus induction and regeneration of Tadong, a pigmented upland rice cultivar from Sabah. For callus induction, immature embryos were cultured on media containing 2,4-Dichlorophenoxyacetic (2,4-D) at various concentrations (0 – 2.5 mg/L) and on different types of media (MS; MSB5; N6B5; N6). To induce shoot regeneration, callus explants were cultured on MS medium supplemented with combinations of 6-Benzylaminopurine (BAP) at various concentrations (0 – 3.0 mg/L) and 1-Naphthaleneacetic acid (NAA) at 1.0 mg/L. To induce shoot development, callus explants were pre-treated with Thidiazuron (TDZ) at various concentrations (0-1.0 mg/l) and exposed to different desiccation periods (0 – 72 hours). 2,4-Dichlorophenoxyacetic at 2.5 mg/L and N6B5 medium resulted in the highest percentages of explant forming callus which were 60.3 ± 17.0 % and 58.7 ± 9.8 % respectively. The regeneration media failed to induce shoot on callus explants, instead, green spots were formed on the surface of the callus. The green spots were stimulated to develop into shoots when the callus explants were pre-treated with 0.5 mg/L TDZ or exposed to partial desiccation for 24 h, the percentages of explant forming shoot were 35.7 ± 4.8 % and 47.7 ± 6.8 % respectively. Shoots developed into complete plants on hormone-free MS medium and acclimatized.

Enhanced in vitro Shoot Regeneration and Biochemical Properties of Stevia rebaudiana using Chitosan

Stevia rebaudiana is a herbaceous perennial plant with great global demand due to its beneficial steviol glycosides(SGs) content. Current conventional breeding technique is unable to cater the need for more S. rebaudiana planting materials. Therefore, an improved in vitro shoot regeneration protocol was developed for S. rebaudiana by using chitosan. The highest fresh weight of plant (0.586 ± 0.176 g/explant), dry weight of plant (0.056 ± 0.02 g/explant) and plant height (4.94 ± 1.17 cm/explant) with maximum number of leaves (25.33 ± 6.95 /explant) were observed on explants grown in optimun treatment of MS basal medium supplemented with 1.0 mg/L of 6-benzyaminopurine (BAP) and 60 mg/L of low molecular weight (MW) chitosan after 4 weeks of culture. Scanning electron microscopy (SEM) analysis showed that new shoot primordia and shoot bud formation can be seen as early as day 3 and 5 of cultures on optimun treatment. Further biochemical assays showed that total phenolic acid content, total protein content and total hydrolyzed sugar content were recorded higher in explants cultured in optimun treatment as compared to control media. In contrast, total chlorophyll content and total flavonoids content were reduced in optimum treatment. Meanwhile, no significant difference in antioxidant activity was observed. All cultures from the optimal treatment were successfully regenerated, acclimatized and grew well with 100% survival rate. Taken together, an enhanced and efficient shoot regeneration protocol of S. rebaudiana was successfully developed which will be useful for rapid and large-scale micropropagation in future.

Establishment of an efficient shoot regeneration system in vitro in Brassica rapa

To improve the genetic transformation system for Brassica rapa L., we established a high-efficiency shoot regeneration protocol. A double haploid line ‘CX001’ with strong regeneration ability was identified from among 25 genotypes. Cotyledons from 4-d-old ‘CX001’ exhibited high-frequency (77.14%) shoot regeneration when explants were cultured on Murashige and Skoog (MS) basal medium supplemented with 1.0 mg L−1 6-benzylaminopurine (BA) and 0.5 mg L−1 α-naphthaleneacetic acid (NAA). The highest regeneration frequency (85.40%) and the maximum shoots number per explant (1.79) were achieved on medium containing 2.0 mg L−1 BA and 0.5 mg L−1 NAA. The addition of 2.5 mg L−1 AgNO3 was critical to shoot regeneration of the cotyledonal explants. The highest rooting rate was obtained on MS medium supplemented with 0.1 mg L−1 NAA. Flow cytometric analysis indicated that there was no ploidy variation between the mother plant and the regenerated plants. Regenerated plantlets with healthy roots were domesticated and transplanted into pots under greenhouse conditions. A reliable and efficient shoot regeneration protocol was established, which lays the foundation for genetic transformation in Brassica rapa.

Propagation Studies in Sapodilla [Manilkara zapota (L.) P. Royen]: A Review

Sapota [Manilkara zapota (L) P. Royen] is an important commercial fruit crop of Sapotaceae family grown for fresh fruits and chicle production. Sapota when propagated by vegetative means, produce true to type plant material and reduces juvenile period and result in early commercial production. Various propagation techniques on different rootstocks were tried for the purpose. Among different rootstocks, khirni (Manilkara hexandra) was found most successful and is used for commercial plant propagation as it possesses high graft compatibility with sapota and plants produce fruits of excellent quality with high yield. Various vegetative propagation techniques have been standardized and used on large scale depending upon season and region. The general techniques used for sapota propagation are softwood grafting, veneer grafting, side grafting, inarching and air layering. Softwood grafting is employed in regions with moderate temperature and high relative humidity which prevent desiccation of scion stick. Veneer and side grafting using mature scion stick is also found effective in regions with variable climates. Inarching technique possess high graft survival rate and is practiced by private nurseries. Propagation through cuttings is not successful in sapota even when treated with rooting hormones. Micro-propagation in sapota by tissue culture techniques have been tried but application remains limited to academics only. This may be due to high phenolic contents and sap exudation in explant. Special protocols for successful root and shoot regeneration are required to be standardized for commercial scale plant propagation.

In vitro tissue culture protocol of ancient einkorn (Triticum monococcum ssp. monococum) wheat via indirect shoot regeneration

Einkorn indirect shoot regeneration was obtained from leaf, coleoptile, and root explants cultured on full, ½, and ¼ strength Murashige and Skoog (MS) medium containing 0.5 to 10 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D). Calluses derived from coleoptile and root explants were cultured on full-strength MS medium supplemented with 0.5 to 5 mg L−1 thidiazuron (TDZ) for 6 wk to induce plant regeneration. Only callus cultures derived from coleoptile explants regenerated shoots. For root induction, 1.0 to 10 mg L−1 indole-3-acetic acid (IAA) was used for 4 wk. The highest rate of callus formation (93%) was from root explants cultured on full-strength MS medium with 4 mg L−1 2,4-D. Coleoptile explants had the highest callus formation (100%) after culture on full-strength MS medium with 3 to 6 mg L−1 of 2,4-D. The highest indirect shoot regeneration (7.0 ± 1.1 shoots produced per callus with a 67% shoot formation frequency) was from calluses derived from coleoptile explant cultured on full MS medium supplemented with 5 mg L−1 of TDZ. Of the different IAA concentrations investigated for rooting, the greatest number of roots per explant (7.7 ± 0.088 roots produced per regenerated shoot with a 100% root formation frequency) was observed on MS medium supplemented with 6 mg L−1 IAA. This indirect shoot regeneration protocol for coleoptile-derived einkorn wheat callus will be useful for future wheat genetic and improvement studies.

Regeneration of Genetically Stable Plants from in Vitro Vitrified Leaves of Different Carnation Cultivars.

This study was conducted to investigate the efficacy of shoot regeneration from different leaf types (normal leaves and vitrified leaves) from three different carnation cultivars ‘Kumbuyl’, ‘Denev’, and ‘Jinju’ using different combinations of 3-indole butyric acid (IBA) and thidiazuron (TDZ) concentrations. The shoot tips cultured on Murashige and Skoog (MS) basal media (Type 1 media) produced normal leaves, while those cultured-on media supplemented with plant growth regulators and/or vitamin (Type 2 media and Type 3 media) produced vitrified leaves for all cultivars. Culture of normal leaf segments on MS medium containing different combinations of IBA and TDZ concentrations induced callus in all treatments; however, the callus was unable to induce shoots and finally became necrotic. In contrast, no callus induction was observed in the control (hormone-free treatment). When vitrified leaf segments underwent the same treatments, shoots were induced from the vitrified leaves (derived from Type 2 media) but were unhealthy and gradually died, whereas those induced from Type 3 media were vitrified and healthy. The optimal combination for the best shoot regeneration and number of shoots per explants varied depending on the genotypes used. The vitrified shoots induced from the leaves of Type 3 media transformed into normal shoots and survived well under greenhouse conditions. According to the results of random amplified polymorphic DNA (RAPD) analysis, the banding patterns of twelve primers that were detected in vitrified leaf-induced normalized shoots were identical to those of normal in vitro grown plants, indicating that no genetic variation had occurred during the procedure. Taken together, this study indicates that vitrified leaves can be used for shoot regeneration of recalcitrant carnation cultivars, regardless of the genotypes and types of vitrified leaves. However, as the number of shoots per explants was still low, further investigation is warranted to obtain a more efficient shoot regeneration protocol for genetic transformation of the cultivars.

Meta-topolin and β-cyclodextrin enhance multiple shoot and root production in black gram Vigna mungo (L.) Hepper

The recalcitrant nature of black gram is the major constraint of in vitro regeneration and agrobacterium- mediated genetic transformation, to overcome this, a productive shoot regeneration protocol has been achieved in black gram cultivar T9 using 7-day old cotyledonary node explants excised from in vitro- raised black gram seedlings using meta-topolin. An aromatic cytokinin,meta-topolin along with BA (1.5+0.5 mg/L) in 0.50 strength MS medium with 1.5% (w/v) sucrose exhibited a maximum number of multiple shoots (32.0±0.37 shoots/explant) at the end of 6 weeks of culture. The shoots were elongated in (6.40±0.50 cm/shoot) in MS medium supplemented with GA3 (2.0 mg/L). A maximum number of roots (9.60±0.50/shoot) and root length (11.20±0.73 cm/shoot) were obtained in combination with β-cyclodextrin(a cyclic oligosaccharide;1.5 mg/L) and IBA (1.5 mg/L). The rooted plantlets were hardened and acclimatized with least mortality rate of 2% in pot mixture consisting red soil:sand:farm yard manure (FYM) (2:1:1) and grown in green house with 85% relative humidity. Ploidy levels were analyzed using flow cytometry which confessed the chromosomal stability in invitro raised plants similar to parent plants. This protocol may be useful for producing transgenic black gram with desirable agro-traits in Indian cultivars.