In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp]

A requirement for generating transgenic pigeonpea [Cajanuscajan (L.) Millsp] plants is the development of a highly efficientin vitro regeneration procedure. This goal was achieved byusing germinated seedlings grown on B5 medium supplemented with 10 mgl^–1 6-benzylaminopurine, which induced differentiatingcallus formation in the cotyledonary node region. The calli were transferred onB5 medium with 0.2 mg l^–1 6-benzylaminopurine toobtain shoot induction. Elongated shoots were then further cultured on a B5hormone-free medium for rooting. Using this regeneration system transgenicpigeonpea plants were obtained both by particle bombardment andAgrobacterium tumefaciens-mediated gene transfer. Thepresence of the transgenes in the pigeonpea genome was confirmed by GUS assays,PCR and Southern hybridisation. The transgenic rooted plants were successfullytransferred to soil in the greenhouse. GUS and PCR assays of T1 progeniesconfirmed that the transgenes were stably transmitted to the next generation.This is the first report of successful use ofAgrobacteriumas well as particle bombardment for production of transgenic pigeonpea plants.     

Alleviation of fungicide-induced phytotoxicity in greengram [Vigna radiata (L.) Wilczek] using fungicide-tolerant and plant growth promoting Pseudomonas strain

This study was designed to explore beneficial plant-associated rhizobacteria exhibiting substantial tolerance against fungicide tebuconazole vis-à-vis synthesizing plant growth regulators under fungicide stressed soils and to evaluate further these multifaceted rhizobacteria for protection and growth promotion of greengram [Vigna radiata (L.) Wilczek] plants against phytotoxicity of tebuconazole. Tebuconazole-tolerant and plant growth promoting bacterial strain PS1 was isolated from mustard (Brassica compestris) rhizosphere and identified as Pseudomonas aeruginosa following 16S rRNA gene sequencing. The P. aeruginosa strain PS1 solubilized phosphate significantly and produced indole acetic acid, siderophores, exo-polysaccharides, hydrogen cyanide and ammonia even under tebuconazole stress. Generally, tebuconazole at the recommended, two and three times the recommended field rate adversely affected the growth, symbiosis, grain yield and nutrient uptake in greengram in a concentration dependent manner. In contrast, the P. aeruginosa strain PS1 along with tebuconazole significantly, increased the growth parameters of the greengram plants. The inoculant strain PS1 increased appreciably root nitrogen, shoot nitrogen, root phosphorus, shoot phosphorus, and seed yield of greengram plants at all tested concentrations of tebuconazole when compared to the uninoculated plants treated with tebuconazole. The results suggested that the P. aeruginosa strain PS1, exhibiting novel plant growth regulating physiological features, can be applied as an eco-friendly and plant growth catalyzing bio-inoculant to ameliorate the performance of greengram in fungicide stressed soils.     

Carbon allocation and partitioning in Vigna radiata (L.) Wilczek as affected by additional carbon gain

Carbon allocation to the source leaf, export and partitioning to the sink were studied in mungbean supplied by additional carbon from the source leaves subjected to high CO2 concentrations (600 and 900 cm3 m-3) in three metabolic and functional source-sink combinations. The plants were pruned to a source-path-sink system. With CO2 enrichment there was an appreciable increase in net photosynthetic CO2 uptake in earlier formed and physiologically younger leaves. Most of the carbon fixed as a result of enrichment was translocated out of the source leaf within one diurnal cycle. The carbon remaining in the source leaf was unchanged. Partitioning of extra carbon into starch or sugar depended upon the amount of extra carbon synthesized. The unloading of the extra carbon into sinks depended on whether it was used for growth or stored. Under increased carbon content, the leaf as a sink was able to reorganize its metabolic reactions more rapidly to maintain the required gradient for unloading than the pod acting as the sink.     

Comparative efficacy of pedigree selection and selective intermating in greengram (Vigna radiata (L.) Wilczek)

Summary Selective intermating and pedigree selection methods were applied simultaneously to highly heterogeneous and heterozygous base populations of greengram in order to compare their relative efficacy in terms of evolving the number of productive lines as well as their production potential. Selection after two cycles of selective intermating was found to be a better method than traditional pedigree selection. The demerits of pedigree selection and merits of selective intermating are discussed. It is suggested that selective intermating replace the widely adopted but less effective pedigree selection for generating promising new material in such autogamous crops as greengram.Part of Ph.D. Thesis submitted by the senior author to the Haryana Agricultural University, Hisar, India     

Diversity in greengram (Vigna radiata (L.) Wilczek) germplasm collection and its potential use in crop improvement

Greengram or mungbean (Vigna radiata (L.) Wilczek), which originated in the Indian sub-continent, is an important pulse crop in India. Genetic diversity and patterns of variation among 111 accessions representing the better agronomic types from the entire collection of the National Bureau of Plant Genetic Resources were studied. Wide variation in morphological and agronomic traits viz. number of pods per plant, yield per plant, 100-seed weight, fruit setting capacity, flowering period, maturity, number of pod bearing peduncles, plant height, number of primary branches, length of branch, nodulation and leafiness was observed. Grain yield was found to be significantly correlated with 100-seed weight and pod length. The data were subjected to cluster and principal components analysis. The accessions were grouped into six discrete and well-defined clusters. The study demonstrated the patterns of variation at the population level. The multivariate analysis was useful in identifying a group of accessions with yield enhancing traits within a highly diverse group of accessions and their potential value in greengram improvement is suggested.     

微波辅助提取木豆根中染料木素工艺

以木豆根为原料,利用微波辅助提取技术进行提取,在单因素实验的基础上对提取条件进行了考察,根据BBD(Box-Behnken design)实验设计原理,采用3因素3水平的响应面分析法,以木豆根中主要异黄酮染料木素(genistein)为指标,对提取过程进行优化,得到最佳工艺参数为：提取温度为68℃,固液比为32∶1 mL·g^-1,乙醇浓度为78%,提取功率700 W,提取时间15 min。在最佳提取条件下染料木素的提取率可达到0.465±0.032 mg·g^-1。本研究对于微波提取技术的应用及木豆根的开发和利用都具有显著的意义。     

Halopriming of seeds imparts tolerance to NaCl and PEG induced stress in Vigna radiata (L.) Wilczek varieties

The investigation was carried out to study the effect of halopriming on NaCl and polyethylene glycol-6000 (PEG-6000) induced stress tolerance potential of three Vigna radiata (L.) Wilczek varieties, with varied abiotic stress tolerance potential. Halopriming is a seed priming technique in which the seeds were soaked in various salt solutions (in this study NaCl was used). The results of the study indicated that the application of stresses (both NaCl and PEG) induced retardation of growth attributes (measured in terms of shoot length, fresh weight, dry weight) and decrease in physiological attributes like total chlorophyll content, metabolites, photosynthetic and mitochondrial activity of the seedlings in all three V. radiata (L.) varieties. However, halopriming of the seeds could reduce the extent of decrease in these biological attributes. NaCl and PEG stress also caused increase in MDA content (a product of membrane lipid peroxidation) in all the varieties studied and this increase was significantly minimized under halopriming. From the present investigation it was evident that among the green gram varieties studied, Pusa Vishal, a NaCl tolerant variety showed enhanced tolerance to NaCl and PEG induced stress, when the seeds were subjected to halopriming followed by Pusa Ratna (stress sensitive variety). Pusa 9531 (drought tolerant variety) also showed positive halopriming effects but it was less significant when compared to other two varieties. It could be concluded that halopriming improved the drought and salinity stress tolerance potential of all varieties and it was significantly higher in the Pusa Vishal as compared to Pusa 9531 and Pusa Ratna.     

Isolation and characterization of a green tissue-specific promoter from pigeonpea [Cajanus cajan (L.) Millsp.

Expression of rbcS genes encoding small subunit of rubisco, most abundant protein in green tissue, is regulated by at least three parameters--tissue type, light conditions and stage of development. One of the green tissue-specific promoters of rbcS gene family was isolated from pigeonpea by PCR. Expression of uidA gene encoding beta-glucuronidase in the transgenic tobacco plants under the control of pigeonpea rbcS promoter, clearly showed that this promoter was as strong as pea rbcS3A promoter characterized earlier. Study of the sequence similarity with pea rbcS3A promoter, especially the region (boxes I and III) that is required for rbcS3A expression, showed more than 50% divergence. In contrast, pigeonpea promoter sequence isolated in the present study was more similar to that of spinach and rice rbcS promoters.     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062     

Comparative efficiency of four selection methods for deriving high-yielding lines in mungbean [Vigna radiata (L.) Wilczek]

The comparative efficiency of four selection methods, viz., honeycomb (HC), pedigree selection (PS), single-seed descent (SSD) and the bulk method (BM), was assessed in three crosses of mungbean. The lines derived by each method, along with check varieties, were yield-tested in a compact family block design in F₅ and F₆ generations during summer and kharif of 1990. On the basis of the mean of the lines, the range, the number of superior lines over the best check, and the proportion of the top 10% lines in all the crosses and generations, the honeycomb method exhibited superiority over PS, SSD and BM for yield per plant and its component traits. PS, SSD and BM did not differ from each other. The honeycomb and SSD methods were found suitable for deriving superior lines for seed yield and pods per plant in mungbean.     

Growth of Foliage of Pigeon pea (Cajanus cajan [L.] Millsp.) and Disease Progress of Alternaria Blight

Vanderplank developed a model based on the logistic growth system to calculate the apparent infection rate (r) and to estimate plant disease progress. By estimating his r and p^∧ (infection rate corrected for growth) the results were frequently negative. Kushalappa and Ludwig later developed another model for adequately correcting the host growth. The present model is a modification of Kushalappa and Ludwig's model in which the results are positive in case of Alternaria blight of pigeon pea (Cajanus cajan [L.] Millsp.). The disease intensity increased despite host growth resulting into an epidemic. A factor accounting for leaf fall due to blighting has been taken into consideration.     

Comparison of single seed descent,selective intermating and mass selection for seed size in greengram (Vigna radiata (L.) Wilczek)

Summary Three selection methods (single seed descent (SSD), mass selection and selective intermating) were applied simultaneously to a highly heterogeneous and broadly based population of greengram. Progeny developing after two cycles of selection were evaluated for yield and seven other economic characters. The relative efficacy of each selection method was judged on the basis of the number of high yielding progeny, mean yield of top 10% progeny, and mean of the highest yielding progeny. Selection after two cycles of selective intermating was found to be the best method for generating productive progeny although mass selection favouring smaller seeds was an equally efficient method. Both of these were found superior to SSD selection.     

Selection and characterization of mannitol-tolerant callus lines of Vigna radiata (L.) Wilczek

An osmotically (mannitol) tolerant callus line of Vigna radiata (L.) Wilczek has been isolated from callus cultures grown on modified PC-L2 medium supplemented with increasing concentrations of mannitol. The tolerance was stable and retained after growth in the absence of mannitol selection for 2 months. The growth of the tolerant line, in the presence of mannitol (540 mol m^-3) was comparable to that of a sensitive callus line growing in the absence of mannitol. This line not only grew well on media containing up to 720 mol m^-3 mannitol, but also required 450 mol m^-3 mannitol for its optimal growth. Osmotically tolerant callus also showed increased tolerance to NaCl (0–250 mol m^-3) stress as compared to sensitive callus. Accumulation of Na⁺ was lower, and the level of K⁺ was more stable in osmotically tolerant than in sensitive calli, when both were exposed to salt. The free proline content of both tolerant and sensitive calli increased on media supplemented with mannitol or NaCl. However, the proline content of sensitive callus was higher than in tolerant callus in the presence of same concentrations of mannitol or NaCl.Abbreviations NAA -naphthaleneacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Yield, growth and physiological responses of mung bean [Vigna radiata (L.) Wilczek] genotypes to waterlogging at vegetative stage

A study was conducted to examine the physiological response of contrasting mung bean (Vigna radiata) genotypes viz., T 44 & MH–96–1 (tolerant) and Pusa Baisakhi & MH–1K–24 (sensitive) under waterlogging conditions. Plants were waterlogged at vegetative stage (30 days after sowing) for 3, 6 and 9 days. Waterlogging resulted in decreased leaf area, crop growth rate, root growth and nodules number, membrane stability index, photosynthesis rate, chlorophyll and carotenoid contents, flowering rate, pod setting, yield and altered dry matter partitioning. Sensitive genotypes showed large reductions in aforementioned physiological traits and slow recovery in photosynthesis rate. On the other hand, tolerant genotypes maintained higher photosynthetic rate, chlorophylls and carotenoids, growth rate, membrane stability and fast photosynthetic recovery under waterlogging. After 9 days of exposure to waterlogging, photosynthetic rate and yield losses in most sensitive genotype (MH-1K-24) were 83 and 85 %, respectively. On an average, photosynthetic loss at 3, 6 and 9 days of waterlogging was 43, 51, and 63 %, respectively, while grain yield loss was 20, 34 and 52 % respectively.     

Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer

Two experiments were carried out from 1981 to 1983 in Vertisol field at ICRISAT Center, Patancheru, India to measure N₂-fixation of pigeonpea [Cajanus cajan (L.) Millsp.] using the¹⁵N isotope dilution technique. One experiment examined the effect of control of a nodule-eating insect on fixation while another in vestigated the effect of intercroping with cereals on fixation and the residual effect of pigeonpea on a succeeding cereal crop. Although both experiments indicated that at least 88% of the N in pigeonpea was fixed from the atmosphere, one result is considered fortuitous in view of the differential rates of growth of the legume and the control, sorghum [Sorghum bicolor (L.) Moench]. The difference method of calculation in dieated negative fixation and the results emphasized the problem of finding a suitable nonfixing control. In a second experiment, when all plants were confined to a known volume of soil to which¹⁵N fertilizer was added in the field, these problems were overcome, and isotope dilution and difference methods gave similar results of N₂-fixation of about 90%. In intercropped pigeonpea 96% of the total N was derived from the atmosphere. This estimate might be an artifact. There was no evidence of benefit from N fixed by pigeonpea to intercropped sorghum plants. Plant tissue¹⁵N enrichments of cereal crops grown after pigeonpea indicated that the cereal derived some N fixed by the previous pigeonpea. Thus residual benefits to cereals are not only an effect of ‘sparing’ of soil N.     

Influence of cadmium stress and arbuscular mycorrhizal fungi on nodule senescence in Cajanus cajan (L.) Millsp

Cadmium (Cd) causes oxidative damage and affects nodulation and nitrogen fixation process of legumes. Arbuscular mycorrhizal (AM) fungi have been demonstrated to alleviate heavy metal stress of plants. The present study was conducted to assess role of AM in alleviating negative effects of Cd on nodule senescence in Cajanus cajan genotypes differing in their metal tolerance. Fifteen day-old plants were subjected to Cd treatments--25 mg and 50 mg Cd per kg dry soil and were grown with and without Glomus mosseae. Cd treatments led to a decline in mycorrhizal infection (MI), nodule number and dry weights which was accompanied by reductions in leghemoglobin content, nitrogenase activity, organic acid contents. Cd supply caused a marked decrease in nitrogen (N), phosphorus (P), and iron (Fe) contents. Conversely, Cd increased membrane permeability, thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2), and Cd contents in nodules. AM inoculations were beneficial in reducing the above mentioned harmful effects of Cd and significantly improved nodule functioning. Activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) increased markedly in nodules of mycorrhizal-stressed plants. The negative effects of Cd were genotype and concentration dependent.     

The effect of 24-epibrassinolide and clotrimazole on the adaptation of Cajanus cajan (L.) Millsp. to salinity

The objective of this study was to evaluate the effects of one of brassinosteroids (24-epibrassinolide) and clotrimazole, (an inhibitor of brassinosteroid synthesis) on plant growth parameters, parameters related to leaf gas exchange (photosynthetic and transpiration rates; stomatal conductance; water use efficiency), photosynthetic pigment content and osmolyte (sugars and proline) content in Cajanus cajan exposed to salinity. Salt stress??caused by NaCl treatment??affected values of all parameters analyzed. The effects were ameliorated by 24-epibrassinolide and intensified by clotrimazole. The hormone increased fresh mass of the plant, shoot dry mass, leaf area, water content of leaves and roots, photosynthetic pigments, sugar concentration, photosynthetic rate, and water use efficiency. The effects of hormone were less evident in the absence of salt. However, under this condition the application of clotrimazole affected the values of parameters studied, indicating the importance of brassinosteroid synthesis for the normal development of the plant.     

Genetic variation of trypsin and chymotrypsin inhibitors in pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives

Variation in the trypsin inhibitors (TIs) and the chymotrypsin inhibitors (CIs) among 69 pigeonpea [Cajanus cajan (L.) Millsp.] strains from a wide geographical distribution and among 17 accessions representing seven wild Cajanus species was studied by electrophoretic banding pattern comparisons and by spectrophotometric activity assays. The TI and CI electrophoretic migration patterns among the pigeonpea strains were highly uniform but varied in the inhibitor band intensities. The migration patterns of the inhibitors in the wild Cajanus species were highly species specific. The mean TI activity of pigeonpea strains (2279 units) was significantly higher than that of the wild Cajanus species (1407 units). However, the mean CI activity in the pigeonpea strains (62 units) was much lower than that in the wild species (162 units). Kenya 2 and ICP 9151 were the lowest and the highest, respectively, in both the TI and CI activities among all the pigeonpea strains used in this study. A highly-significant positive correlation was observed between the TI and CI activities. The Bowman-Birk type inhibitors with both TI and CI activities were identified in all the pigeonpea strains and also in the accessions of all the wild species except C. volubilis (Blanco) Blanco. The C. volubilis accession ICPW 169 was found to be null for both CI bands and CI activity. Environment, strain, and environment x strain interaction showed highly-significant effects on both the TI and CI activities. Growing the pigeonpea strains at a different environment from their area of adaptation increased TI and CI activities and also altered the maturity period.     

Identification and confirmation of quantitative trait loci controlling resistance to mungbean yellow mosaic disease in mungbean [Vigna radiata (L.) Wilczek]

Mungbean yellow mosaic India virus (MYMIV) is a major constraint on mungbean production in South and Southeast Asia. The virus belongs to the genus Begomovirus, causing yellow mosaic disease and subsequently yield loss up to 75–100 %. The present study employed F₂ and BC₁F₁ populations derived from a cross between susceptible (BARImung 1; BM1) and resistant (BARImung 6; BM6) mungbeans to identify quantitative trait loci (QTLs) associated with resistance to MYMIV. Resistance to the virus was evaluated using F_2:3 and BC₁F_1:2 populations under field conditions in two locations in Bangladesh in 2012. A total of 1,165 simple sequence repeat markers from different legumes were used to detect the polymorphism between BM1 and BM6. Sixty-one polymorphic markers were used to construct a linkage map comprising 11 linkage groups. Composite interval mapping consistently identified two major QTLs, qMYMIV2 on linkage group 2 and qMYMIV7 on linkage group 7, conferring the resistance in both F₂ and BC₁F₁ populations. qMYMIV2 and qMYMIV7 accounted for 31.42–37.60 and 29.07–47.36 %, respectively, of the disease score variation, depending on populations and locations. At both loci, the resistant alleles were contributed by the parent BM6. qMYMIV2 appeared to be common to a major QTL for MYMIV resistance in mungbean reported previously, while qMYMIV7 is a new QTL for the resistance. The markers linked to the QTLs in this study are useful in marker-assisted breeding for development of mungbean varieties resistant to MYMIV.     

In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [Cajanus cajan (L.) Millsp.] cv. JKR105

In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L⁻¹ 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L⁻¹ 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.