Monomorphic Banding Pattern Research Articles

Micropropagation, micromorphological evaluation and genetic homogeneity validation of Cucumis collosus (Rottl.) Cogn.: A wild progenitor of melon

Cucumis collosus (Rottl.) Cogn., a wild ancestor of melon cultivars, is an underutilized cucurbit of arid horticulture having nutraceutical and therapeutic significance. C. collosus has great potential for enhancing the genetic base of cultivated melon and other cucurbits. This investigation is the first report on a robust and reliable micropropagation system for C. collosus (var. AHK-119) using nodal explants, followed by micromorphological and genetic stability assessment. More than 95% explants showed axillary shoot-bud induction on MS (Murashige and Skoog, 1962) medium containing 0.5 mg L−1 BAP and developed a maximum of 3.2 ± 0.6 shoots measuring 3.2 ± 0.1 cm length. After evaluating the various parameters, the best shoot multiplication (Shoot Number 34.9 ± 1.7, Shoot Length 6.7 ± 0.9 cm) was achieved on MS medium having half-strength of nitrates (825 mg L−1 NH4NO3 and 950 mg L−1 KNO3) + 0.25 mg L−1 6-benzylaminopurine (BAP) + 0.1 mg L−1 Kinetin (Kin) + 0.1 mg L−1 Indole-3-acetic acid (IAA) + additives (50 mg L−1 ascorbic acid and 25 mg L−1 each of adenine sulfate, L-arginine and citric acid), after 4 weeks. Micro-shoots were rooted using three approaches: In Vitro Continuous Treatment (IVCT), In Vitro Pulse-Treatment (IVPT) and Ex Vitro Pulse Treatment (EVPT). Of these, EVPT approach proved to be most successful in terms of rooting response (80%), root number (6.4 ± 0.9) and root length (5.7 ± 0.7 cm). The foliar micro-morphology analysis of in vitro leaves (IVL), ex vitro leaves (EVL) and mother plant leaves (MPL) demonstrated notable adaptive alterations during the transitional stages and produced structurally stable plants similar to the mother plant. Furthermore, the validation of genetic homogeneity using Start Codon Targeted (SCoT) and Inter Simple Sequence Repeats (ISSR) markers revealed monomorphic banding patterns among the micropropagated and the mother plant. The discussed method can be applied for large-scale commercial production of C. collosus for arid horticulture, urban/vertical farming, breeding purposes and improvement of other cucurbits.

Efficient plant regeneration via indirect organogenesis and genetic stability assessment by molecular markers in an endangered medicinal plant Operculina turpethum (L.) Silva Manso

Operculina turpethum (L.) Silva Manso is reported to be effective against several diseases including tumor, jaundice, gastrointestinal disease etc. The plant is facing extinction due to overexploitation and inefficient conservation strategies. In this work, an effective approach for micropropagation of O. turpethum by indirect organogenesis from stem explant is devised. Induction of callus culture was carried out on Murashige and Skoog medium enriched with benzyl adenine (3mg/L). Subsequently, the callus cultured on MS medium with 1mg/L BA + 1mg/L kinetin generated a maximum of 3.20 ± 0.7 number of shoots/0.5gm callus within 35 days of incubation. On MS medium with Indole-3-acetic acid (IAA)(0.2mg/L), 98.4% of the shoots were successfully rooted. The in vitro regenerated plantlets were acclimatised and shifted to outdoor condition with a success rate of 95%. The genetic stability of micro propagated plantlets was assessed using PCR-based molecular markers, random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) which yielded a total of 27 and 31 scorable bands respectively. The monomorphic banding pattern of in vitro propagated plants confirms their genetic homogeneity. This work is report on in vitro plant regeneration through indirect organogenesis and evaluation of genetic fidelity of micropropagated plantlets of O. turpethum.

Morphological and biochemical characterization of Passiflora quadrangularis L. - A source of vegetable from East Siang district, Arunachal Pradesh, India

Present research investigation was aimed at morphological and biochemical assessment of Passiflora quadrangularis L. commonly known as giant granadilla and locally called as vegetable squash grown as vegetable crop by the Adi tribe of Arunachal Pradesh. Seven genotypes collected during survey were characterized for different morphological and biochemical traits. Results showed that average fruit weight was 432.57g/fruit, with juice content 100.11 mL/fruit, vitamin C content 25.79 mg, vitamin A content 1.65 mg, Mean total flavonoids content was 16.75 mg/100 g of fruit juice, total soluble solids 12.040 Brix, antioxidant activity (DPPH) 6.07 %, titratable acidity 1.69 %, total carbohydrates 9.95 %, phenol content 338.38 mg/100 g of leaf was noted among the genotypes tested. The mean anthocyanin content in leaf was 1.20 mg/100 g, tendril 0.90 mg/100 g and petiole 1.69 mg/ 100 g among the genotypes. Seed protein profiling of Passiflora quadrangularis L. with SDS- PAGE showed diverse molecular weights ranging from 11 KD to 163.53 KD. However, monomorphic banding pattern among the protein profiling of giant granadilla was recorded among the selected genotypes. The results of the study show that the collected genotypes are belonged to Passiflora quadrangularis L. and are good source of nutritive value which can be used as source of vegetable.

In vitro Propagation of Curcuma caesia Roxb. via Bud Culture Technique and ISSR Profiling of the Plantlets for Genetic Homogeneity

An in vitro propagation protocol has been developed for Curcuma caesia Roxb., an endangered medicinal plant by Foundation for Revitalisation of Local Health Traditions and Central Forest Department of India. The plant bears poorly germinated seeds and produces two-storage organs: rhizomes and multiple root tubers. Only rhizomes have medicinal-economic values. They serve as propagules too, which results in a shortage of planting material. Therefore, a complete one-year production cycle of C. caesia has been standardized through in vitro propagation including explants establishment (one month), subculture cycles (seven months), rooting (one month) followed by primary hardening (one month) and secondary hardening (two months). Dormant shoot buds on rhizome served as explants for culture initiation on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA) and kinetin (KIN) in combination with citric acid (CA), adenine sulfate (AdS) and indole- 3-acetic acid (IAA). Maximum bud break (70%) was obtained on MS with 8 mg L-1 BA, 8 mg L-1 KIN, 100 mg L-1 CA, 200 mg L-1 AdS and 2 mg L-1 IAA (standard medium). Shoot production potential continued on this medium during the subsequent seven-month-long subculture cycle. The in vitro raised shoots rooted best on ½-strength MS containing 1 mg L-1 indole-3-butyric acid. Plantlet survival rate was >95% after acclimatization. The genetic homogeneity of plantlets with the mother plant was analyzed using Inter Simple Sequence Repeats which generated a monomorphic banding pattern to confirm the uniformity of in vitro raised plantlets of C. caesia.

Genetic Evaluation of In Vitro Micropropagated and Regenerated Plants of Cannabis sativa L. Using SSR Molecular Markers.

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.

Molecular genetic diversity analysis in short duration pigeonpea [Cajanus cajan (L.) Millsp.

In the present study, 48 short duration pigeonpea genotypes were used to analyse molecular diversity employing 20 pigeonpea specific microsatellite markers. Among the 20 microsatellite markers, 11 markers produced polymorphism and nine markers showed monomorphic banding patterns. A total of 35 alleles were generated and the number of alleles produced by different markers ranged from one to three with an average of 1.75 alleles per marker. The polymorphic markers viz., PGM 82, CcM 1615, CcM 2857 and CcM 1277 produced a maximum of three alleles. The allele size ranged from 150 bp (CcLG08_RFQI4) to 290 bp (CcM 1277). The average PIC value obtained was 0.269. The data analysis for microsatellite markers showed high dissimilarity among 48 genotypes. A dendrogram was constructed using Unweighted Pair Group Method with Arithmetic Average (UPGMA) analysis, 48 genotypes were grouped into seven clusters with the similarity coefficient ranged from 0.43 to 1.00. Among the seven clusters, subcluster 1 of cluster I was the largest with fifteen genotypes followed by nine genotypes in the subcluster 2 of cluster II. From this study, it is concluded that the diverse pigeonpea genotypes viz., ICPL 19009, ICPL 19019, ICPL 19030 (cluster I), ICPL 19002 (cluster VI) and CO 6R (cluster VII) could be utilized in the breeding program based on microsatellite marker and cluster analysis. Keywords: Pigeonpea, microsatellite markers, molecular genetic diversity, Polymorphic Information Content, UPGMA clustering.

Molecular genetic diversity in dual purpose and land races of pigeonpea (Cajanus cajan (L.) Millsp.)

Molecular genetic diversity was assessed among 32 dual purpose and land races of pigeonpea using 20 pigeonpea specific SSR markers. Among 20 markers, 15 markers revealed polymorphism, while five markers exhibited a monomorphic banding pattern. The polymorphic markers produced a total of 43 alleles with an average of 2.15 alleles per marker. The markers CcM 0008 and CcM 1026 produced the highest number of alleles (4). The Polymorphic Information Content (PIC) value of the markers ranged from 0.06 (PGM 16) to 0.64 (CcM 0008) with an average of 0.19. A dendrogram constructed using UPGMA distinguished 32 genotypes into 10 clusters. Cluster I was the largest with 15 genotypes followed by cluster V with five genotypes. The Neighbour- joining tree produced based on the weighted average for dissimilarity matrix grouped the 32 genotypes into eight groups. Among them group II was the largest comprising of 10 genotypes. Based on the molecular genetic diversity study using SSR markers, the genotypes Kunnathur local, CRG 13-01, BSR 1 and Pillayakothur local were found diverse among the genotypes and had good grain and vegetable pigeonpea traits could be used in the breeding programme. The results indicated that SSR markers provide a more definitive separation of clusters indicating a higher level of efficiency for determining the relationship among pigeonpea genotypes. Keywords:  Pigeonpea, Dual purpose, Land races, Genetic diversity   &nbsp

Thidiazuron Induced In Vitro Clonal Propagation of Lagerstroemia speciosa (L.) Pers.—An Important Avenue Tree

A high throughput regeneration protocol has been developed for Lagerstroemia speciosa through node explants under the regime of various plant growth regulators (PGRs). This protocol can provide an alternative mode to seed-grown plants and minimize the cost–time of regeneration, significantly. Murashige and Skoog (MS) medium containing various combinations of PGRs exhibited a marked stimulatory effect on morphogenesis. Of the various combinations tried, node explant pretreated with thidiazuron (TDZ; 5.0 µM) for 4 weeks and followed with transfer into MS medium containing 1.0 μM 6-benzyladenine (BA) and 0.25 μM α-naphthalene acetic acid (NAA) was reported to be the best treatment as it resulted in a maximum number of 24.5 shoots with an average shoot length of 7.1 cm per explant in 90% of cultures after 12 weeks of incubation. The in vitro-generated shoots rooted satisfactorily in the adopted ex vitro method of rooting, which saves time and cost. Among the different treatments, the greatest rooting percentage (85%) was observed in the 200 μM IBA-treated shoots, with the highest root number (8.7) and length (3.4 cm) occurring after 4 weeks. Four months after being transferred to ex vitro, some of the physiological attributes of the in vitro-propagated plants were examined and compared to the ex vitro plants. Further, analysis of the genetic integrity in tissue culture-raised plantlets along with the parental tree was accomplished through DNA-based RAPD technique. The monomorphic banding pattern obtained by the RAPD primers resulted in a high level of genetic uniformity in regenerated plants.

M-brigde- and elicitor-assisted enhanced post-storage germination of Rauvolfia serpentina synthetic seeds, their genetic fidelity assessment and reserpine estimation

The present study reports an amelioration of synthetic seed germination efficiency with a simple intervention of filter paper (M-bridge) drenched in ½Murashige and Skoog liquid (½MSL) medium in Rauvolfia serpentina. Synthetic seeds were developed from in vitro shoot tips using 3% (w/v) sodium alginate and 75 mM calcium chloride. Media for synthetic seed gemination were tested in cotton bed drenched with sterile water (SW), cotton bed drenched with ½MSL medium, M-bridge drenched with ½MSL medium, and ½MS semisolid (½MSS) medium alone. Synthetic seeds inoculated on ½MSL or SW or ½MSS media, exhibited slower germination and inferior performance; however, with the involvement of M-bridge, fastest germination (5.6 ± 0.4 days) and maximum proliferation (4.8 ± 0.7 shoots with 42.8 ± 2.4 mm length, and 19.8 ± 2.7 leaves/synthetic seed after 42 days of inoculation) were recorded. Among the tested elicitors (silver nitrate, salicylic acid and sodium nitroprusside) at their different concentrations supplemented in ½MSL medium (with M-bridge), 50 µM silver nitrate resulted in 100% germination frequency and resulted in germination within 5 days. Further, the synthetic seeds were stored for 42 days at − 20 °C, 8 °C, and 25 °C. Maximum germination efficiency was obtained in case of seeds stored at 8 °C with respect to earliness in germination (6.9 ± 0.3 days), germination percentage (71.8 ± 3.4%) and subsequent proliferation (4.9 ± 0.6 shoots with 18.9 ± 0.9 mm length, and 8.9 ± 0.7 leaves/synthetic seed). Clonal fidelity analysis of the in vitro regenerants performed using RAPD markers exhibited monomorphic banding pattern. The reserpine content of the regenerants was found to be maximum following 42 days-storage at 8 °C (126.9 µg/g), followed by a substantial decline in the synthetic seed-derived regenerants following 42-days-storage at 25 °C (120.7 µg/g) or − 20 °C (121.8 µg/g).

Accelerated mono-phasic in vitro mass production of banana propagules and their morpho-cyto-genetic stability assessment

A unique one-step in vitro propagation protocol via concurrent shoot-root development was established in banana cv. Grand Nain. 6-benzyladenine, kinetin, meta-Topolin, thidiazuron (TDZ), and zeatin (0.5—1.5 mg L − 1) individually supplemented in Murashige and Skoog medium were tested for multiple shoot-root regeneration from shoot tip explants. The earliest fresh shoot (∼7 days) and root (∼5 days) initiation, highest number of shoots (∼21) and roots (∼16) with maximum root elongation (16.5 cm) were recorded in 0.5 mg L − 1 TDZ. For all intents and purposes, TDZ (0.5 mg L − 1) was recorded to be the optimum formulation in terms of the highest simultaneous shoot and root formation that was corroborated further by matrix plot analysis. On the other hand, network plot analysis and principal component analysis illustrated the association between plant growth regulators based on their effect on growth attributes under study. Histological studies on shoots, roots, and leaves revealed a consistent growth and development of plantlets under in vitro condition. Cent percent plantlets were survived during acclimatization, and performed best in cocopeat in comparison to other substrata (vermiculite, perlite, vermicompost-soil, soil-sand, and sand only). The cytology-, flow cytometry- and ISSR primer-assisted fidelity assessments of the in vitro-regenerants exhibited numerical chromosomal as well as ploidy stability and monomorphic banding patterns without any polymorphism within the in vitro-regeneranted plantlets and with their mother plant. Based on the comprehensive information developed in this study, this protocol should be of immense importance to facilitate single-step large-scale production of true-to-type planting materials and to explore the possible comparative physico-chemical roles of the plant growth regulators used in this study.

​Genetic Diversity Analysis in Extra Early Pigeonpea [Cajanus cajan (L.)] Genotypes using SSR Markers

Background: The present study aims at assessment of genetic diversity in 43 extra early pigeonpea genotypes with 41 simple sequence repeat markers. Methods: In the present investigation, 41 HASSR markers were used to assess the genetic diversity. Similarity matrices were utilized to construct the unweighted pair group method with Arithmetic average dendrogram using NTSYs PC software. Result: Out of 41 HASSR markers, 21 markers were showed distinct banding pattern, while 11 markers were showed monomorphic banding pattern and remaining 9 markers showed no banding pattern or not amplified. Cluster analysis done using HASSR markers revealed that ICPL87119 was found to be the most diverse. This genotype also found to be resistant to wilt. Based on similarity coefficients and cluster analysis genotypes ICPL87119, ICPL84031, ICPL88014, ICPL161, ICPL87091 and CORG9701 were genetically more distant from other genotypes studied and these varieties can be used for their desirable characteristics in breeding programs for pigeonpea improvement.

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Picloram-induced enhanced callus-mediated regeneration, acclimatization, and genetic clonality assessment of gerbera

BackgroundGerbera jamesonii Bolus ex Hooker f. (African daisy) is listed among the top five most important ornamental plants in the global floricultural industry. To satisfy its demand, the floriculture industry relies on reproducible and effective propagation protocol while retaining the genetic uniformity of G. jamesonii. The present study, for the first time, reports the potential of picloram for enhanced induction of organogenic calli from leaves of G. jamesonii and its high-frequency indirect regeneration. ResultsThe fastest induction of calli with maximum fresh and dry weight was recorded in the Murashige and Skoog (MS) semisolid medium supplemented with 1 mg/l picloram. In addition, callus induction was observed in 2,4-dichlorophenoxy acetic acid- and α-napthaleneaceticacid-supplemented media but with delayed response and reduced fresh and dry weight. The proliferated calli were transferred to shoot induction media containing MS salt and 0.5–1 mg/l N6-benzylaminopurine, kinetin, or thidiazuron. A mean number of ~6 shoots per callus were developed after 5 days of culture in the MS medium supplemented with 1 mg/l kinetin, with a mean length of 5.2 cm. Successful rooting of shoots was achieved in the MS medium fortified with 1.5 mg/l indole-3-acetic acid, wherein the earliest root initiation (~5 days), as well as the maximum number (~9) and length (~4.8 cm) of roots, were recorded. Complete plantlets were primarily acclimatized in sand before being transferred to a mixed substrate (of soil, sand, tea leaf waste, and cow urine) that secured >90% survival and further growth of the plantlets. Eventually, clonal fidelity of the in vitro regenerants assessed via inter-simple sequence repeats (ISSR) primers exhibited a monomorphic banding patterns that suggested genetic integrity within the plantlets as well as with their mother plant. ConclusionsThe results of the present study should be of interest for commercial propagation and mutagenesis- as well as genetic transformation-related research.

Identification of Point Mutation in Exon 3 of Leptin Gene in Munjal Sheep

Background: Leptin is a varied hormone which plays vital role in body development by regulating the balance between food intake and energy expenditure by signaling to the brain. Leptin has diverse effect on controlling appetite, energy metabolism, growth, reproduction, body composition and immunity. The present study was aimed to screen candidate point mutation (g.332G greater than A) in the targeted genomic region of leptin gene in Munjal sheep. Methods: A total of 50 Munjal sheep were selected and genomic DNA was isolated in Automated Maxell RSC DNA/ RNA purification system by using Maxwell RSC whole blood DNA kit. Reported set of primers was used to amplify 463bp fragment encompassing targeted region (exon 3) of leptin gene. PCR-RFLP was performed to genotype targeted point mutation in our resource population. PCR products were digested by Cail 1 restriction enzyme to genotype g.332G greater than A (at 332th nucleotide of exon 3 leptin gene) non-synonymous mutation (Arg to Gln). Result: All studied samples resolved into monomorphic banding pattern, revealed only AA (463bp single band bp) genotype. The absence of candidate mutation in our resource population might be due to small sample size.

English

Eurycoma longifolia is a medicinally potent plant found in the tropical forest of South-East Asia. Every part of the plant, especially the root is traditionally used as an aphrodisiac, anticancer and anti-inflammatory. E. longifolia is conventionally propagated by seeds but with low germination rate and efficiency. This has made an in vitro propagation of E. longifolia a desirable alternative. Hence, this study reports an effective method of direct organogenesis of shoot. In vitro seedling’s leaves were cultured on Murashige and Skoog (MS) medium containing 1.0 mg L-1 6-benzylaminopurine (BAP), producing 1.8 ± 0.5 shoots per leaf with a regeneration frequency of 68.2%. The shoot buds were directly formed from leaves without intermediate callus formation. To obtain complete plantlets, the shoots were in vitro rooted with an average number of 4.2 ± 0.4 roots per shoot in half-strength MS (½MS) medium supplemented with 0.5 mg L-1 indole-3-butyric acid (IBA). Regenerated plantlets were successfully acclimatized to field conditions with an 85% survival rate. Genetic fidelity of the micropropagated plantlets was evaluated using Simple Sequence Repeat (SSR) and Inter Simple Sequence Repeat (ISSR) analysis. The results showed that the monomorphic banding patterns of in vitro raised plantlets and their mother plant were similar, confirming its homogeneity and the reliability of the multiplication system. © 2021 Friends Science Publishers

Assessment of Genetic Stability of Micropropagated Bambusa balcooa Roxb. using RAPD Marker

Bambusa balcooa Roxb. was in vitro propagated by optimizing protocol using nodal segment from secondary branches with 100% success in MS liquid media containing 100 mg/l Myo-inositol, 3% sucrose supplement with 4.4 - 26.64 μM BAP for shoot multiplication, and 2.69 - 32.26 μM NAA for root induction. The highest shoot multiplication (14.53 ± 0.33 folds), shoot length (5.9 ± 0.6 cm), shoot number per explants (4.0 ± 0.24), and rooting (89.3 ± 0.33%) was obtained in MS liquid media supplement with 13.32 μM BAP (shooting) and 26.88 μM NAA (rooting) and 1% aqueous leaf extract of Artemisia vulgaris L. (EAV). Twenty RAPD (Random amplified Polymorphic DNA) primers were used individually to amplify DNA of tissue culture-raised plants and the mother plant where 8 primers yielded monomorphic banding patterns with reproducible, clear, scorable bands (2.8 per primer) ranging from 250 to 1800 bp respectively which revealed the micropropagated plants of B. balcooa retained their genetic stability.
 Plant Tissue Cult. & Biotech. 31(1): 81-95, 2021 (June)

Micropropagation and clonal fidelity assessment of acclimatized plantlets of Crotalaria longipes Wight & Arn. using ISSR markers

An efficient protocol for micropropagation of Crotalaria longipes Wight & Arn., an endemic and endangered Indian legume, was developed. Nodal explants cultured on Murashige and Skoog (MS) supplemented with Thidiazuron (TDZ) 1.0 mg L−1 and NAA 0.5 mg L−1 showed maximum shoot induction. The multiplied shoots were subsequently sub-cultured every 3 weeks on the same medium. The separated microshoots cultured on half-strength MS basal salts supplemented with 0.5 mg L−1 indole-3-butyric acid (IBA) showed maximum root induction. After 4 weeks of culture, the rooted shoots (> 6 cm) were planted in pots containing coco peat and garden soil (1:1) covered with plastic domes and maintained at 25 oC for 2 weeks. After 2 weeks, the potted plants were transferred to a glasshouse, maintained at 25 °C with a relative humidity of 80%. The acclimatized plants were then successfully established in soil with an 80% survival rate. Clonal fidelity assessment of acclimatized plantlets was confirmed using inter simple sequence repeat (ISSR). All the in vitro developed plants showed monomorphic banding pattern similar to the mother plant, thus ascertaining the true-to-type nature of the in vitro raised plants.

New varieties of tomato - morphological aspects and molecular characterisation with RAPD and SSR markers

This study presents the main morphological features and the first molecular investigations of four new tomato varieties (Solanum lycopersicum), aiming to obtain their complete characterisation. Evaluation with the standard descriptors for tomato revealed specific and distinct traits for each analysed variety. The molecular analyses for variety identification started with testing three methods for DNA extraction. With an optimized method, which doesn’t need liquid nitrogen for plant tissue disruption, good quality DNA was obtained, in adequate quantities, and well preserved when stored at -20 °C. To highlight the genetic differences among the analysed tomato varieties, nine RAPD primers and ten SSR primers were tested. Of these, the optimal amplification protocols for five RAPD primers and five SSR primers were established. The amplified products obtained with RAPD primers revealed an average number of bands per primer of 8.8 and a total rate of polymorphism of 59.1%; with OPB10 primer was seen the highest number of DNA bands (11), and with OPA07 primer was registered the highest degree of genetic variability among the studied varieties (77.7%). Two SSR markers (SSR 20 and SSR T107) amplified monomorphic banding patterns corresponding to 170 base pairs and 250 base pairs, respectively, for all varieties; with SSR T7, SSR T62, and SSR T70 primers were generated multiple amplification bands, with a different distribution of the bands into the agarose gel for each analysed tomato variety.

Gibberellic acid and thidiazuron promote micropropagation of an endangered woody tree (Pterocarpus marsupium Roxb.) using in vitro seedlings

Pterocarpus marsupium Roxb., which belongs to the Fabaceae family, is a promising herbal drug producing forest tree widely known as the ‘Indian Kino’. An effective regeneration protocol using in vitro seedlings of P. marsupium under the gibberellic acid and thidiazuron regimen is described in the present study. Gibberellic acid was also tested to have an effect on in vitro rhizogenesis from microshoots. Combination of gibberellic acid and thidiazuron in culture media improved the germination percentage, multiplication and subsequent elongation of shoots. Murashige and Skoog medium containing 0.50 µM gibberellic acid with 0.50 µM thidiazuron was found to be the most effective treatment for producing the highest number of shoots per seedling (23.0) with an average shoot length 5.14 cm in 85% cultures, after 8 weeks. For in vitro rhizogenesis, 100 μM of indole 3-butyric acid was treated for 5 days in basal ends of isolated microshoots. Thereafter, pretreated microshoot was transferred to gibberellic acid (0.50 µM) supplemented MS medium. This treatment was found to be the most effective for in vitro root formation, where 10.54 roots with an average root length of 4.60 cm per microshoot in 80% cultures were recorded, after 4 weeks. During acclimatization photosynthetic traits and their attributes such as net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and Chlorophyll content were analyzed and these attributes were found to be fluctuating initially for 35 days, thereafter, an increasing trend was recorded up to 84 days of acclimatization. The well acclimatized plantlets were shifted to fields where they grew normally without any morphological changes with an 86.7% of survival. Genetic integrity in micropropagated plants was validated by DNA-based ISSR primers. These primers were amplified in monomorphic banding pattern suggesting a high-level of genetic uniformity in micropropagated plants. Overall, an effective and high throughput plant regeneration method has been developed for obtaining true-to-type regenerants of P. marsupium. The present study revealed the high throughput regeneration of P. marsupium through in vitro seedlings method which may be an alternative tool for mass propagation and germplasm conservation of woody trees directly from seed, thus reducing an explant preparation and recovery of seedlings capable of multiple shoot formation under the regimes of gibberellic acid and thidiazuron. Both the growth regulators are involved in the improvement of plantlet regeneration processes as well as maintenance of physiological stresses during in vitro cultures.

Plant regeneration through direct and indirect organogenesis, phyto-molecular profiles, antioxidant properties and swertiamarin production in elicitated cell suspension cultures of Swertia minor (Griseb.) Knobl

The present study reports an optimized protocol for high frequency in vitro plant propagation through direct and indirect organogenesis, phytochemical accumulation, molecular profiling and antioxidant evaluation for micropropagated Swertia minor, a promising alternative to industrially important Swertia chirayita. Moreover, the study also aimed at enhancing the production of antidiabetic and anti-obesity drug swertiamarin using an alternative technology of elicitated cell suspension cultures. Different types, concentrations and combination of cytokinins and auxins showed their effects during various in vitro growth stages. A combination of BAP (3.0 mg/l) and TDZ (1.0 mg/l) had a dominant role in promoting multiple shoot proliferation with production of an average of 19.1 ± 0.95 shoots/node in 85% response. MS medium added with IBA (2.0 mg/l) showed optimal response for in vitro rooting (9.2 ± 0.56 roots/shoot). In order to establish genetic stability, molecular marker-based profiling of micropropagated plants were done and 'monomorphic banding pattern were identical to the mother plants. 2,4-D (2.0 mg/l) supported the maximum callus induction and proliferation rate (95%). The wild-grown plants showed higher polyphenols content and antioxidant activities as compared to callus and in vitro derived plantlets. However, chitosan-treated (25 ppm) methanolic extract of cell biomass accumulated in cell suspension cultures produced higher contents of swertiamarin (1.45 mg/g DW) than salicylic acid and methyl jasmonate. The described protocol can be effectively used for the large-scale propagation, exploitation of active compounds and will serve as potential alternative to S. chirayita for fulfillment of over-growing industrial requirements. The present investigation addressed in vitro regeneration, callus culture, somatic embryogenesis, molecular profiling, secondary metabolite production, cell suspension culture studies for first time in Swertia minor.