A Comprehensive Genome-Wide Investigation of the Cytochrome 71 (OsCYP71) Gene Family: Revealing the Impact of Promoter and Gene Variants (Ser33Leu) of OsCYP71P6 on Yield-Related Traits in Indica Rice (Oryza sativa L.).

The cytochrome P450 (CYP450) gene family plays a critical role in plant growth and developmental processes, nutrition, and detoxification of xenobiotics in plants. In the present research, a comprehensive set of 105 OsCYP71 family genes was pinpointed within the genome of indica rice. These genes were categorized into twelve distinct subfamilies, where members within the same subgroup exhibited comparable gene structures and conserved motifs. In addition, 105 OsCYP71 genes were distributed across 11 chromosomes, and 36 pairs of OsCYP71 involved in gene duplication events. Within the promoter region of OsCYP71, there exists an extensive array of cis-elements that are associated with light responsiveness, hormonal regulation, and stress-related signaling. Further, transcriptome profiling revealed that a majority of the genes exhibited responsiveness to hormones and were activated across diverse tissues and developmental stages in rice. The OsCYP71P6 gene is involved in insect resistance, senescence, and yield-related traits in rice. Hence, understanding the association between OsCYP71P6 genetic variants and yield-related traits in rice varieties could provide novel insights for rice improvement. Through the utilization of linear regression models, a total of eight promoters were identified, and a specific gene variant (Ser33Leu) within OsCYP71P6 was found to be linked to spikelet fertility. Additionally, different alleles of the OsCYP71P6 gene identified through in/dels polymorphism in 131 rice varieties were validated for their allelic effects on yield-related traits. Furthermore, the single-plant yield, spikelet number, panicle length, panicle weight, and unfilled grain per panicle for the OsCYP71P6-1 promoter insertion variant were found to contribute 20.19%, 13.65%, 5.637%, 8.79%, and 36.86% more than the deletion variant, respectively. These findings establish a robust groundwork for delving deeper into the functions of OsCYP71-family genes across a range of biological processes. Moreover, these findings provide evidence that allelic variation in the promoter and amino acid substitution of Ser33Leu in the OsCYP71P6 gene could potentially impact traits related to rice yield. Therefore, the identified promoter variants in the OsCYP71P6 gene could be harnessed to amplify rice yields.

Mitochondrial markers differentiate two distinct phylogenetic groups in indigenous rice landraces of northeast India: an evolutionary insight.

The inheritance of the mitochondria genome and its diversity is unique for genetic and evolutionary studies relative to nuclear genomes. Northeast India and Himalayan regions are considered as one of the centres of indica rice origin. Also, rice diversity in northeast India is very distinct and highly suited for evolutionary studies. Although reports are available on the genetic diversity of indigenous northeast rice landraces, its relationship with the wild relatives is not yet properly explored and understood. In an attempt, mitochondrial markers were used to study the evolutionary relationship between the 68 landraces of northeast India and wild relatives (O. rufipogon and O. nivara) along with IR64 (indica) and Nipponbare (japonica) were taken as reference cultivars. Phylogenetically, the findings include two distinct clusters in the indigenous northeast India landraces representing indica and japonica groups. Further, the wild relatives and ~60% of northeast India landraces were identified to be closely related to the Nipponbare cluster. Besides, landraces of northeast India grouping with the indica group (IR64) are characterized by the absence of wild relatives. This indicates that there are two distinct evolutionary paths in the origin of northeast Indian rice landraces based on mitochondrial markers diversity and it is proposed that the inheritance of mitochondria, mitonuclear genome interactions, and bottleneck events could have genetically separated these two phylogenetically unique groups of northeast rice landraces.

Marker-Assisted Improvement for Durable Bacterial Blight Resistance in Aromatic Rice Cultivar HUR 917 Popular in Eastern Parts of India.

Bacterial blight (BB) is a devastating disease of rice in the tropics of Indian sub-continent, where the presence of Xoo races with varying levels of genetic diversity and virulence renders disease management extremely challenging. In this context, marker-assisted improvement of plant resistance has been proven as one of the most promising approaches for the development of sustainable rice cultivars. The present study demonstrates the marker-assisted introgression of the three BB resistant genes (Xa21 + xa13 + xa5) into the background of HUR 917, a popular aromatic short grain (ASG) rice cultivar in India. The performance of the resulting improved products (near isogenic lines (NILs), HR 23-5-37-83-5, HR 23-5-37-121-10, HR 23-5-37-121-14, HR 23-65-6-191-13, HR 23-65-6-237-2, HR 23-65-6-258-10 and HR 23-65-6-258-21) establishes the utility of marker-assisted selection (MAS) approach for accelerated trait introgression in rice. The MAS-bred lines carrying three introgressed genes showed broad spectrum BB resistance (lesion length, LL of 1.06 ± 1.35 cm to 4.61 ± 0.87 cm). Besides, these improved lines showed the complete product profile of recurrent parent HUR 917 along with the enhanced level of durable BB resistance. The improved introgression lines with durable BB resistance would contribute to sustainable rice production in India, particularly in the Indo-Gangetic plane that has substantial acreage under HUR 917.

Identification of microRNAs That Provide a Low Light Stress Tolerance-Mediated Signaling Pathway during Vegetative Growth in Rice.

Low light intensity affects several physiological parameters during the different growth stages in rice. Plants have various regulatory mechanisms to cope with stresses. One of them is the differential and temporal expression of genes, which is governed by post-transcriptional gene expression regulation through endogenous miRNAs. To decipher low light stress-responsive miRNAs in rice, miRNA expression profiling was carried out using next-generation sequencing of low-light-tolerant (Swarnaprabha) and -sensitive (IR8) rice genotypes through Illumina sequencing. Swarnaprabha and IR8 were subjected to 25% low light treatment for one day, three days, and five days at the active tillering stage. More than 43 million raw reads and 9 million clean reads were identified in Swarnaprabha, while more than 41 million raw reads and 8.5 million clean reads were identified in IR8 after NGS. Importantly, 513 new miRNAs in rice were identified, whose targets were mostly regulated by the genes involved in photosynthesis and metabolic pathways. Additionally, 114 known miRNAs were also identified. Five novel (osa-novmiR1, osa-novmiR2, osa-novmiR3, osa-novmiR4, and osa-novmiR5) and three known (osa-miR166c-3p, osa-miR2102-3p, and osa-miR530-3p) miRNAs were selected for their expression validation through miRNA-specific qRT-PCR. The expression analyses of most of the predicted targets of corresponding miRNAs show negative regulation. Hence, miRNAs modulated the expression of genes providing tolerance/susceptibility to low light stress. This information might be useful in the improvement of crop productivity under low light stress.

Androgenesis in indica rice: A comparative competency in development of doubled haploids.

Rice is critical to global food security which demands immediate attention to meet the ever-growing population. Development of improved variety is the major focus area of research, in which doubled haploid (DH) technology plays a vital role. Since, androgenesis shows its potential in DH production, this method was not capitalized specially in indica rice due to due to its recalcitrant nature to tissue culture. Success of androgenesis is governed by many important factors such as stage of anther, pre-treatment conditions, accurate concentrations of media, and plant growth regulators. Though reports of androgenesis are abundant in rice, most of them either used japonica or a specific cultivar of indica rice ecotypes. In this study, a media combination was established which is successful in producing doubled haploids from F1s of Savitri x Pokkali, IR20 x Mahulata along with the popular indica hybrids of Arize 8433DT, Arize 6453, Arize Bold, and Swift Gold. Out of 12 different media combinations tested, and 5 different durations of cold-treatments studied, N6 media with 2,4-D (2.0 mg/l) and BAP (0.5 mg/l) with 7th day cold pre-treatment was found to be most effective in all of the F1s for callus induction. Among all the F1s, rice hybrid, Arize 8433DT showed highest of 52% callus induction. In case of green shoot regeneration, MS media with NAA (0.5 mg/l), BAP (2.0 mg/l) and Kn (1.0 mg/l) (MS+C4) was found to be the most efficient of six treatments studied with highest of 58.25% regeneration in Arize 8433DT. Further, MS+C4 in combination with proline (5.0 mg/l) increased the regeneration rate to 85.99%. Besides, MS media with NAA (1.0 mg/l), Kn (0.1 mg/l) and 50 g/l sucrose was found to be most efficient for supporting root induction in all F1s. This study claims the establishment of genotype independent androgenic protocol for indica rice which could be capitalized in indica rice improvement.

Role of transporters in plant disease resistance.

Plants being sessile have evolved numerous mechanisms to meet the changing environmental and growth conditions. Plant pathogens are responsible for devastating disease epidemics in many species. Transporter proteins are an integral part of plant growth and development, and several studies have documented their role in pathogen disease resistance. In this review, we analyze the studies on genome-wide identifications of plant transporters like sugars will eventually be exported transporters (SWEET), multidrug and toxic compound extrusion (MATE) transporters, ATP-binding cassette (ABC) transporters, natural resistance-associated macrophage proteins (NRAMP), and sugar transport proteins (STPs), all having a significant role in plant disease resistance. The mechanism of action of these transporters, their solute specificity, and the potential application of recent molecular biology approaches deploying these transporters for the development of disease-resistant plants are also discussed. The applications of genome editing tools, such as CRIPSR/Cas9, are also presented. Altogether the information included in this article gives a better understanding of the role of transporter proteins during plant-pathogen interaction.

Anther Culture Efficiency in Quality Hybrid Rice: A Comparison between Hybrid Rice and Its Ratooned Plants.

An immense increase in human population along with diminished lands necessitates the increase of rice production since, it serves the human population as a staple food. Though rice hybrids (RH) are showing considerable yield enhancement over inbreds in terms of both quality and quantity, farmers' adoption of hybrid rice technology has been much slower than expected because of several constraints such as seed cost and quality. Doubled haploid (DH) technology was considered useful for the development of inbred lines from rice hybrids in a single generation. Androgenesis shows its significance in development of DHs in rice which requires an efficient method to establish the production of large population. To start the anther culture, anthers are the main component of androgenesis to be isolated from unopened spikes. However, the duration of spikes availability for anther culture coupled with the segregation of rice hybrids in the next generation requires the main crop be ratooned to reduce the cost of cultivation. Therefore, the efficiency of the androgenic method was tested in main crop using a quality indica rice hybrid, 27P63 and its ratooned ones. The effects of various factors such as cold temperature pre-treatment of boots, treatment duration, and different combination of plant growth regulators (PGR) on callus response along with shoot regeneration were tested for development of DHs from both ratooned and non-ratooned plants. The N6 medium supplemented with 2.0 mg/L 2,4-D (2,4-dichlrophenoxy acetic acid), 0.5 mg/L BAP (6-benzylamino purine), and 30 g/L maltose was found to be most effective for callusing as compared to MS (Murashige and Skoog) medium. The N6 media inducted calli showed maximum response rate for green shoot regeneration in MS media supplemented with 0.5 mg/L NAA (1-napthaleneacetic acid), 0.5 mg/L Kn (Kinetin; 6-furfurylaminopurine), 1.5 mg/L BAP and 30 g/L sucrose after 2 weeks of culture. The pre-treatment of spikes at 10 °C for 2 d followed by a 7th and 8th d were found to be most effective for callusing as well as for regeneration, producing a total of 343 green plants from ratooned and main rice hybrid, 27P63. Morpho-agronomic trait-based assessment of ploidy status revealed 94.46% diploids, 3.49% polyploids, 0.58% mixploids, and 1.45% haploids. Microsatellite markers could authenticate all 324 fertile diploids as true DHs. Though this study shows a reduction in generation of DHs from ratooned plants as compared to the main crop, manipulation of chemical factors could optimize the method to enhance the production of considerable number of DHs. Utilization of ratooned of hybrid rice in androgenesis would save time and cost of cultivation.

Differential Expression of Genes at Panicle Initiation and Grain Filling Stages Implied in Heterosis of Rice Hybrids.

RNA-Seq technology was used to analyze the transcriptome of two rice hybrids, Ajay (based on wild-abortive (WA)-cytoplasm) and Rajalaxmi (based on Kalinga-cytoplasm), and their respective parents at the panicle initiation (PI) and grain filling (GF) stages. Around 293 and 302 million high quality paired-end reads of Ajay and Rajalaxmi, respectively, were generated and aligned against the Nipponbare reference genome. Transcriptome profiling of Ajay revealed 2814 and 4819 differentially expressed genes (DEGs) at the PI and GF stages, respectively, as compared to its parents. In the case of Rajalaxmi, 660 and 5264 DEGs were identified at PI and GF stages, respectively. Functionally relevant DEGs were selected for validation through qRT-PCR, which were found to be co-related with the expression patterns to RNA-seq. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated significant DEGs enriched for energy metabolism pathways, such as photosynthesis, oxidative phosphorylation, and carbon fixation, at the PI stage, while carbohydrate metabolism-related pathways, such as glycolysis and starch and sucrose metabolism, were significantly involved at the GF stage. Many genes involved in energy metabolism exhibited upregulation at the PI stage, whereas the genes involved in carbohydrate biosynthesis had higher expression at the GF stage. The majority of the DEGs were successfully mapped to know yield related rice quantitative trait loci (QTLs). A set of important transcription factors (TFs) was found to be encoded by the identified DEGs. Our results indicated that a complex interplay of several genes in different pathways contributes to higher yield and vigor in rice hybrids.

The potentiality of rice microsatellite markers in assessment of cross-species transferability and genetic diversity of rice and its wild relatives.

The main aim of this study is to assess the potentiality of SSR markers for the identification of the cross-species transferability frequency in a large set of the diverse genome types of wild relative rice along with cultivated rice. Here, we used 18 different rice genotypes representing nine different genome types with 70 SSR markers to investigate the potentiality of cross-species transferability rate. The overall cross-species transferability of SSR markers across the 18 rice genotypes ranged from 38.9% (RM280 and RM447) to 100% (RM490, RM318, RM279, RM18877 and RM20033, RM19303) with an average of 76.58%. Also, cross-species transferability across chromosome ranged from 54.4% (chromosome 4) to 86.5% (chromosome 2) with an average of 74.35%. The polymorphism information content of the markers varied from 0.198 (RM263) to 0.868 (RM510) with a mean of 0.549 ± 0.153, showing high discriminatory power. The highest rate of cross-transferability was observed in O. rufipogon (97%), The highest rate of cross-species transferability was in O. rufipogon (97.00%), followed by O. glaberrima (94.20%), O. nivara (92.80%), Swarna (92.80%), O. longistaminata (91.40%), O. eichingeri (90%), O. barthii (88.50%), O. alta (82.80%), O. australiensis (77.10%), O. grandiglumis (74.20%), O. officinalis (74.20%), Zizania latifolia (70.00%), O. latifolia (68.50%), O. brachyantha (62.80%), Leersia perrieri (57.10%) and O. ridleyi (41.40%) with least in O. coarctata (28.50%). A total of 341 alleles from 70 loci were detected with the number of alleles per locus ranged from 2 to 12. Based on dendrogram analysis, the AA genome groups was separated as distinct group from the rest of the genome types. Similarly, principal coordinate analysis and structure analysis clearly separated the AA genome type from the rest of the genome types. Through the analysis of molecular variance, more variance (51%) was observed among the individual, whereas less (14%) was observed among the population. Thus, our findings may offer a valuable resource for studying the genetic diversity and relationship to facilitate the understanding of the complex mechanism of the origin and evolutionary processes of different Oryza species and wild relative rice.

Correction: Blast resistance in Indian rice landraces: Genetic dissection by gene specific markers.

[This corrects the article DOI: 10.1371/journal.pone.0211061.].

Blast resistance in Indian rice landraces: Genetic dissection by gene specific markers.

Understanding of genetic diversity is important to explore existing gene in any crop breeding program. Most of the diversity preserved in the landraces which are well-known reservoirs of important traits for biotic and abiotic stresses. In the present study, the genetic diversity at twenty-four most significant blast resistance gene loci using twenty-eight gene specific markers were investigated in landraces originated from nine diverse rice ecologies of India. Based on phenotypic evaluation, landraces were classified into three distinct groups: highly resistant (21), moderately resistant (70) and susceptible (70). The landraces harbour a range of five to nineteen genes representing blast resistance allele with the frequency varied from 4.96% to 100%. The cluster analysis grouped entire 161 landraces into two major groups. Population structure along with other parameters was also analyzed to understand the evolution of blast resistance gene in rice. The population structure analysis and principal coordinate analysis classified the landraces into two sub-populations. Analysis of molecular variance showed maximum (93%) diversity within the population and least (7%) between populations. Five markers viz; K3957, Pikh, Pi2-i, RM212and RM302 were strongly associated with blast disease with the phenotypic variance of 1.4% to 7.6%. These resistant landraces will serve as a valuable genetic resource for future genomic studies, host-pathogen interaction, identification of novel R genes and rice improvement strategies.

Effect of multiple allelic combinations of genes on regulating grain size in rice.

The grain size is one of the complex trait of rice yield controlled by a plethora of interaction of several genes in different pathways. The present study was undertaken to investigate the influence of seven known grain size regulating genes: DEP1, GS7, GS3, GW8, GL7, GS5 and GW2. A wide phenotypic variation for grain length, grain width and grain length-width ratio were observed in 89 germplasm. The correlation analysis showed a strong association among these three grain traits viz. GL, GW, GLWR and TGW which play important roles in determining the final rice grain size. Except for GW2, all six genes showed strong association with grain size traits. A total of 21 alleles were identified with an average of 2.1 allele/locus in 89 germplasm of which seven alleles were found to be favourable alleles for improving the grain size with the frequency range of 24 (26.97%) to 82 (92.13%); the largest was found in GS5 followed by GW8, GL7, DEP1, GS3 and GS7 genes. Through ANOVA, four markers (GS3-PstI, S9, GID76 and GID711) of three genes (GS3, DEP1 and GL7) were found significantly associated with all the three traits (GL, GLWR and TGW). Concurrent results of significant associations of grain size traits with other markers were observed in both analysis of variance and genetic association through the general linear model. Besides, the population structure analysis, cluster analysis and PCoA divided the entire germplasm into three sub-groups with the clear-cut demarcation of long and medium grain types. The present results would help in formulating strategies by selecting suitable candidate markers/genes for obtaining preferred grain shape/size and improving grain yield through marker-assisted breeding.

Correction: Use of molecular markers in identification and characterization of resistance to rice blast in India.

[This corrects the article DOI: 10.1371/journal.pone.0176236.].

Use of molecular markers in identification and characterization of resistance to rice blast in India.

Rice blast disease caused by Magnaporthe oryzae is one of the most destructive disease causing huge losses to rice yield in different parts of the world. Therefore, an attempt has been made to find out the resistance by screening and studying the genetic diversity of eighty released rice varieties by National Rice Research Institute, Cuttack (NRVs) using molecular markers linked to twelve major blast resistance (R) genes viz Pib, Piz, Piz-t, Pik, Pik-p, Pikm Pik-h, Pita/Pita-2, Pi2, Pi9, Pi1 and Pi5. Out of which, nineteen varieties (23.75%) showed resistance, twenty one were moderately resistant (26.25%) while remaining forty varieties (50%) showed susceptible in uniform blast nursery. Rice varieties possessing blast resistance genes varied from four to twelve and the frequencies of the resistance genes ranged from 0 to 100%. The cluster analysis grouped the eighty NRVs into two major clusters at 63% level of genetic similarity coefficient. The PIC value for seventeen markers varied from 0 to 0.37 at an average of 0.20. Out of seventeen markers, only five markers, 195R-1, Pi9-i, Pita3, YL155/YL87 and 40N23r corresponded to three broad spectrum R genes viz. Pi9, Pita/Pita2 and Pi5 were found to be significantly associated with the blast disease with explaining phenotypic variance from 3.5% to 7.7%. The population structure analysis and PCoA divided the entire 80 NRVs into two sub-groups. The outcome of this study would help to formulate strategies for improving rice blast resistance through genetic studies, plant-pathogen interaction, identification of novel R genes, development of new resistant varieties through marker-assisted breeding for improving rice blast resistance in India and worldwide.

Development and validation of cross-transferable and polymorphic DNA markers for detecting alien genome introgression in Oryza sativa from Oryza brachyantha.

African wild rice Oryza brachyantha (FF), a distant relative of cultivated rice Oryza sativa (AA), carries genes for pests and disease resistance. Molecular marker assisted alien gene introgression from this wild species to its domesticated counterpart is largely impeded due to the scarce availability of cross-transferable and polymorphic molecular markers that can clearly distinguish these two species. Availability of the whole genome sequence (WGS) of both the species provides a unique opportunity to develop markers, which are cross-transferable. We observed poor cross-transferability (~0.75 %) of O. sativa specific sequence tagged microsatellite (STMS) markers to O. brachyantha. By utilizing the genome sequence information, we developed a set of 45 low cost PCR based co-dominant polymorphic markers (STS and CAPS). These markers were found cross-transferrable (84.78 %) between the two species and could distinguish them from each other and thus allowed tracing alien genome introgression. Finally, we validated a Monosomic Alien Addition Line (MAAL) carrying chromosome 1 of O. brachyantha in O. sativa background using these markers, as a proof of concept. Hence, in this study, we have identified a set molecular marker (comprising of STMS, STS and CAPS) that are capable of detecting alien genome introgression from O. brachyantha to O. sativa.

Plant regeneration from callus cultures of Vitex trifolia (Lamiales: Lamiaceae): a potential medicinal plant.

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supplemented Murashige & Skoog medium, and incubated at 25 +/-2 degrees C under a light intensity of 61 micromol/m2s from cool white fluorescent lamps and a 16 h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10 microM BAP in combination with 0.54 microMNAA. Addition of 135.74-271.50 microM adenine sulphate (Ads) and 0.72-1.44 microM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10 microM BAP in combination with 0.54 microM NAA, 271.50 microM Ads and 1.44 microM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23 microM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Plant regeneration from callus cultures of Vitex trifolia (Lamiales: Lamiaceae): a potential medicinal plant / Regeneración de plantas a partir de cultivos de callos de Vitex trifolia (Lamiales: Lamiaceae): una planta medicinal potencial

Vitex trifolia is a shrub species with popular use as a medicinal plant, for which leaves, roots and flowers have been reported to heal different distresses. The increasing exploitation of these plants has endangered its conservation, and has importantly justified the use of biotechnological tools for their propagation. Our aim was to present an efficient protocol for plant regeneration through organogenesis; and simultaneously, to analyze the genetic homogeneity of the established clonal lines by Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Plantlet regeneration was achieved in callus cultures derived from stem, leaf and petiole explants of V. trifolia on a differently supple mented Murashige & Skoog medium, and incubated at 25±2ºC under a light intensity of 61µmol/m2s from cool white fluorescent lamps and a 16h photoperiod. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more rapidly from stem and petiole explants as compared to leaf explants on medium containing 11.10µM BAP in combination with 0.54µMNAA. Addition of 135.74-271.50µM adenine sulphate (Ads) and 0.72-1.44µM gibberellic acid (GA3) to the culture medium increased the growth of shoot buds. The highest rate of shoot bud regeneration responses was obtained in stem explants using 11.10µM BAP in combination with 0.54µM NAA, 271.50µM Ads and 1.44µM GA3. In vitro rooting of the differentiated shoots was achieved in media containing 1.23µM indole butyric acid (IBA) with 2% (w/v) sucrose. Regenerated plantlets were successfully established in soil with 86% survival under field condition. Randomly Amplified Polymorphic DNA and Inter Simple Sequence Repeat markers analyses have confirmed the genetic uniformity of the regenerated plantlets derived from the second up to fifth subcultures. This protocol may help in mass propagation and conservation of this important medicinal plant of great therapeutic potential.

Vitex trifolia es una especie arbustiva de uso popular como planta medicinal, sus hojas, raíces y flores se han reportado para la cura de diferentes aflicciones. El aumento de la explotación de estas plantas ha puesto en peligro su conservación y ha justificado el uso de herramientas biotecnológicas para su propagación. El objetivo de esta investigación fue presentar un protocolo eficiente para la regeneración de estas plantas a través de la organogénesis, y analizar la homogeneidad genética de las líneas clonales establecidas por ADN polimórfico amplificado aleatoriamente (RAPD) mediante la repetición de marcadores de inter secuencia simple (ISSR). La regeneración de plántulas se logró en cultivos de callos derivados de explantes de tallo, hoja y pecíolo de V. trifolia en un medio diferenciado Murashige & Skoog, que se incubaron a 25±2ºC bajo una intensidad de luz de 61μmol/m2s con lámparas fluorescentes blancas y un fotoperíodo de 16h. La tasa de regeneración de brotes se correlacionó positivamente con la concentración de las hormonas en el medio nutritivo. Los brotes se regeneraron más rápidamente a partir de explantes de tallo y pecíolos en comparación con explantes de hoja. La mayor tasa de regeneración de brotes se obtuvo en los explantes de tallo utilizando 11.10μM BAP en combinación con 0.54μM NAA, 271.50μM Ads y 1.44μM GA3. Este protocolo puede ayudar a la propagación masiva y conservación de esta importante planta medicinal de gran potencial terapéutico.

Factors influencing rapid clonal propagation of Chlorophytum arundinaceum (Liliales: Liliaceae), an endangered medicinal plant.

Chlorophytum arundinaceum is an important medicinal plant and its tuberous roots are used for various health ailment treatments. It has become an endangered species in the Eastern Ghats, and a rare medicinal herb in India, due to its excessive collection from its natural habitat and its destructive harvesting techniques, coupled with poor seed germination and low vegetative multiplication ratio. In order to contribute to its production systems, an efficient protocol was developed for in vitro clonal propagation through shoot bud culture. For this, multiple shoots were induced from shoot bud explants on Murashige and Skoog's medium supplemented with 2.5-3.0 mg/L BAP, 0.01-0.1 mg/LNAA and 3% (w/v) sucrose. Inclusion of Adenine Sulphate (25mg/L) in the culture medium improved the frequency of multiple shoot production and recovered the chlorotic symptoms of the leaves. Media having pH 5.9 and 4% sucrose showed significant improvement on shoot bud multiplication and growth. In vitro flowering was observed when the subcultures were carried out for over four months in the same multiplication media. Rooting was readily achieved upon transferring the shoots on to half- strength MS medium supplemented with 0.1 mg/L IBA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house, successfully established, and flowered in the field. This method could effectively be applied for the conservation and clonal propagation to meet the demand of planting materials.

Factors influencing rapid clonal propagation of Chlorophytum arundinaceum (Liliales: Liliaceae), an endangered medicinal plant

Chlorophytum arundinaceum is an important medicinal plant and its tuberous roots are used for various health ailment treatments. It has become an endangered species in the Eastern Ghats, and a rare medicinal herb in India, due to its excessive collection from its natural habitat and its destructive harvesting techniques, coupled with poor seed germination and low vegetative multiplication ratio. In order to contribute to its production systems, an efficient protocol was developed for in vitro clonal propagation through shoot bud culture. For this, multiple shoots were induced from shoot bud explants on Murashige and Skoog’s medium supplemented with 2.5-3.0mg/L BAP, 0.01-0.1mg/L NAA and 3% (w/v) sucrose. Inclusion of Adenine Sulphate (25mg/L) in the culture medium improved the frequency of multiple shoot production and recovered the chlorotic symptoms of the leaves. Media having pH 5.9 and 4% sucrose showed significant improvement on shoot bud multiplication and growth. In vitro flowering was observed when the subcultures were carried out for over four months in the same multiplication media. Rooting was readily achieved upon transferring the shoots on to half- strength MS medium supplemented with 0.1mg/L IBA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house, successfully established, and flowered in the field. This method could effectively be applied for the conservation and clonal propagation to meet the demand of planting materials. Rev. Biol. Trop. 59 (1): 435-445. Epub 2011 March 01.

Chlorophytum arundinaceum es una planta medicinal importante y sus raíces se utilizan en diversos tratamientos contra enfermedades. Se ha convertido en una especie en peligro de extinción en el Ghats Oriental y una hierba medicinal rara en la India, debido a la recolecta excesiva en su hábitat natural y la manera destructiva de cosecharla, asociado con una mala germinación y pobre multiplicación vegetativa. Para contribuir con sus sistemas de producción, se desarrolló un protocolo eficiente para la propagación clonal in vitro a través del cultivo de brotes. Para ello, los retoños múltiples fueron inducidos a partir de sus brotes en un medio Murashige y Skoog enriquecido con 2.5-3.0mg/L de BAP, 0.01-0.1mg/L de NAA y el 3% (w/v) sucrosa. La inclusión de sulfato de adenina (25mg/L) en el medio de cultivo mejoró la frecuencia de producción de brotes múltiples y se recuperaron los síntomas de clorosis de las hojas. Los medios con un pH de 5.9 y 4% de sucrosa mostraron una mejoría significativa en la multiplicación y crecimiento de las yemas. En la floración in vitro se observó cuando los subcultivos se llevaron a cabo durante más de cuatro meses para los mismos medios de multiplicación. El enraizamiento se logró fácilmente al transferir los brotes a un medio MS de intensidad media enriquecido con 0.1 mg/l de IBA y 2% (w/v) de sucrosa. Las plántulas micropropagadas maduraron en el invernadero, se establecieron exitosamente y florearon en el campo. Este método se podría aplicar para la conservación y propagación clonal con el fin de satisfacer la demanda de material de siembra.