Optimized Recovery of Cryostored Dormant Buds of Mulberry Germplasm.

A two-step freezing cryoprotocol preceded by desiccation to 15 to 25% moisture content was developed and successfully applied to winter dormant buds of mulberry (different Morus spp.) of a core set comprising 238 accessions studies in our laboratory. The survival and recovery percentage of diverse accessions cryobanked for various periods were tested under in vitro conditions, and several factors were analyzed to determine their role in optimizing the recovery of low-viability accessions. The effect of rates of freezing and thawing (both fast and slow), were tested and recovery compared. Recovery conditions such as dark incubation and rehydration in sterile moist moss grass for different durations after cryopreservation led to a higher survival percentage compared to controls. Two different recovery culture media were compared for their efficiency in survival. On average, the survival under in vitro culture conditions using optimized conditions was high: above 60% in majority of the accessions. Dormant buds showed viability in the range of 25 to 100% with an average of 50.4%. The recovery percentage of winter dormant buds after cryopreservation via slow freezing and slow thawing with rehydration by moist moss grass for 2 h was recorded in the range from 63.3 to 90.9% with an average of 81.05%. Without rehydration, it ranged from 50 to 75% with an average of 60.4%. Regeneration of cryopreserved mulberry germplasm after 6 years of storage indicated no survival loss over different years of storage, and 33-40% of the accessions showed viability above 40%, up to a maximum of 100%. Maximum shoot formation (100%) was obtained from Morus alba. The majority of the accessions were rooted in vitro within 20-25 days of subculture in the auxin rich rooting media, except in wild species M. latifolia and M. laevigata, which took longer (45 to 60 days) for root development. All the rooted plantlets were then transferred to the field and successfully established in a glasshouse.

Retraction Note to: Inferring Phylogenetic Relationships of Indian Citron (Citrus medica L.) Based on rbcL and matK Sequences of Chloroplast DNA.

The Editor-in-Chief and the publisher have retracted this article [1] because of significant overlap with previously published articles [2-5]. Ajit Uchoi, Surendra Kumar Malik, Ravish Chaudhary, Susheel Kumar, M.R. Rohini, Digvender Pal, and Sezai Ercisli disagree with the retraction. The publisher was not able to get in contact with Rekha Chaudhury, she did not respond to any correspondence about this retraction.

Inferring Phylogenetic Relationships of Indian Citron (Citrus medica L.) based on rbcL and matK Sequences of Chloroplast DNA.

Phylogenetic relationships of Indian Citron (Citrus medica L.) with other important Citrus species have been inferred through sequence analyses of rbcL and matK gene region of chloroplast DNA. The study was based on 23 accessions of Citrus genotypes representing 15 taxa of Indian Citrus, collected from wild, semi-wild, and domesticated stocks. The phylogeny was inferred using the maximum parsimony (MP) and neighbor-joining (NJ) methods. Both MP and NJ trees separated all the 23 accessions of Citrus into five distinct clusters. The chloroplast DNA (cpDNA) analysis based on rbcL and matK sequence data carried out in Indian taxa of Citrus was useful in differentiating all the true species and species/varieties of probable hybrid origin in distinct clusters or groups. Sequence analysis based on rbcL and matK gene provided unambiguous identification and disposition of true species like C. maxima, C. medica, C. reticulata, and related hybrids/cultivars. The separation of C. maxima, C. medica, and C. reticulata in distinct clusters or sub-clusters supports their distinctiveness as the basic species of edible Citrus. However, the cpDNA sequence analysis of rbcL and matK gene could not find any clear cut differentiation between subgenera Citrus and Papeda as proposed in Swingle's system of classification.

An efficient regeneration and rapid micropropagation protocol for Almond using dormant axillary buds as explants.

An efficient in vitro protocol was standardized for Almond (Prunus dulcis) propagation using dormant axillary buds as explants. Explants were cultured on Murashige and Skoog (MS) and woody plant medium (WPM) supplemented with different concentration/combination(s) of phytohormones. MS basal medium showed lowest shoot induction and took longest duration for shoot initiation. Multiple shoots were induced in MS medium supplemented with the combination of BAP (0.5 mgL(-1)). Cultures showed poor response for rooting in all combinations of plant growth regulators (PGRs) and took 90 days for initiation. Rooting was higher in half strength of MS than in full-strength. The highest root induction (33.33%) was recorded in half MS medium supplemented with 0.1 mgL(-1) IBA (indole-3-butyric acid) followed by full strength of MS medium (20%) supplemented with IBA (0.1 mgL(-1)). α-Naphthalene acetic acid (NAA) was less effective for rooting than IBA. The highest root induction (25%) was found in half strength of MS medium supplemented with 0.1 mgL(-1) NAA followed by full strength of MS medium (20%). The protocol developed would be of use in mass propagation of almond and also support in vitro conservation.

Genetic diversity and phylogenetic analysis of Citrus (L) from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA.

The north-eastern region of India is reported to be the center of origin and rich in diversity of Citrus (L.) species, where some wild and endangered species namely Citrus indica, Citrus macroptera, Citrus latipes, Citrus ichagensis and Citrus assamensis exist in their natural and undisturbed habitat. In order to have comprehensive information about the extent of genetic variability and the occurrence of cryptic genomic hybridity between and within various Citrus species, a combined approach involving morphological, cytogenetical and molecular approaches were adopted in the present study. Cytogenetic approaches are known to resolve taxonomic riddles in a more efficient manner, by clearly delineating taxa at species and sub species levels. Male meiotic studies revealed a gametic chromosome number of n = 9, without any evidence of numerical variations. Bivalents outnumbered all other types of associations in pollen mother cells (PMCs) analyzed at diplotene, diakinesis and metaphase I. Univalents were frequently encountered in nine species presently studied, though their presence appropriately did not influence the distributional pattern of the chromosomes at anaphases I and II. The molecular approaches for phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 of rDNA using maximum parsimony method and Bayesian inference have thrown light on species inter-relationship and evolution of Citrus species confirming our cytogenetical interpretations. The three true basic species i.e. Citrus medica, Citrus maxima and Citrus reticulata with their unique status have been resolved into distinct clades with molecular approaches as well. C. indica which occupies a unique position in the phylogenetic ladder of the genus Citrus has been resolved as a distinct clade and almost behaving as an out-group. The presences of quadrivalents in C. indica also echo and support its unique position. From our study it is amply clear that C. reticulata also has close relation to C. ichagensis, as these species have clustered together, denoting their close genetic relationship. On the other hand, our studies did not demonstrate a clear differentiation between subgenera Citrus and Papeda at the rDNA level. The combined approach of cytogenetical and molecular analysis did complement our early karyological findings and helped in resolving many a taxonomic riddles.

Karyological studies in ten species of Citrus(Linnaeus, 1753) (Rutaceae) of North-East India.

Ten Citrus (Linnaeus, 1753) species of North-East India have been karyo-morphologically analysed. All studied species had 2n=18 chromosomes without any evidence of numerical variation. All the chromosomes were found to be of metacentric and sub-metacentric in all the species; the morphology of the chromosomes showing size difference only. Symmetrical karyotype which does not have much difference in the ratio of longest to shortest chromosome in all the species was observed. Three species, Citrus grandis (Osbeck, 1757), Citrus reticulata (Blanco, 1837) and Citrus medica (Linnaeus, 1753) are identified as true basic species from asymmetry studies of karyotypes as they reflect on the primitive nature of their genomes. Citrus indica (Tanaka, 1937)occupies a special taxonomic position within the genus Citrus as a progenitor for other cultivated species.