Decoding leaf reddening
Onset of leaf reddening occurs when the night temperatures start to decline below 20 °C followed by bright sunshine. Due to low temperature and high light intensity photo-inhibition occurs in the plants. Photo-inhibition succeeded by free radical formation was proved in the study. Free radicals in turn resulted in chlorophyll degradation to a tune of 50%. This caused the cotton plants to accumulate more anthocyanins in leaves and non-foliar green organs to protect chlorophyll pigments from further oxidation.
Abiotic stress management
“Alarm photosynthesis pathway explored in cotton New metabolic pathway “Alarm photosynthesis was explored in cotton. Expression analysis (qPCR) of the major gene of this pathway, i.e. GLP1/oxalate oxidase (OxO) gene in leaves as well as in different tissues confirmed its presence at gene level. Proteins extracted showed the bands of OxO in the gel. Among the four cotton spp., desi cotton had more oxalate oxidase expression, whereas water stressed leaf samples were observed to possess more OxO activity than control. Overall, expression of OxO in cotton leaves at both gene and protein level confirms the existence of this “Alarm photosynthesis pathway in cotton.
In vitro fibre development
Three-day old fertilised ovules of LRA 5166 were cultured in vitro to study the factors associated with fibre elongation. The best result could be obtained when ovules were cultured on TM medium with IAA (1.0 mg.L-1) + GA (1.0 mg.L-1) + BA (1.0 mgL-1) for 15 days and later sub-cultured on TM medium with IAA (2.0mgL-1) + BA (0.5 mgL-1) with a fibre length of 15- 17mm in 25-30 days of culture.
Pruning of Cotton for second fruiting cycle
Cotton crop raised during the previous summer season were subjected to pruning by clipping the main stem at 45 cm above the soil and pruning the redundant branches soon after harvest. In general, sprouting of new sympodial branches started within 8-10 days of pruning and flowering initiated within 25-30 days while it took about 45-50 days in normal crop. Hybrids grown under elevated CO2 atmosphere produced more bolls than ambient grown plants. Pruned plants had more bolls compared to the normal seeded crop. Pruning cotton crop after harvest leads to second fruiting cycle resulting in additional yield of 50 – 60% and save around Rs 8000/-. By extending the crop, additional biomass production of 10 tonnes/ha means additional 3.0 tonnes of C fixed for every hectare of cotton cultivation.
Source-Sink alteration
A tool to improve cotton productivity Bud and boll shedding is a major problem in cotton. This can be overcome by foliar application of ethylene (5.7 to 8.6 mM) at onset of square formation. This leads to synchronous flowering and boll opening enhancing yield by 25%. The technology has been approved and part of the Package of Practices in Gujarat, Andhra Pradesh and Karnataka.
Root architecture in cotton
Roots are very important organ for plant growth and development. In cotton, morphological adaptive response to excess water is the formation of adventitious roots and hypertrophied lenticels. Accessions Nagpur 9, DTS- 108-09, CNH-09-7, 1430 performed better under drought stress with efficient root traits such as tap root length (60 cm), lateral root length (40 cm) and root dry weight (1.23 g) under drought condition in PVC pipes harvested at 90 days after planting.
Evaluation of PGPR and microbial inoculants to alleviate drought stress in cotton (G.hirsutumL.)
Microbial inoculants play an important role in imparting drought tolerance in several crop plants. The study investigates the potential of PGPR and microbial inoculants for alleviating drought stress in cotton. The output could provide another sustainable strategy to mitigate the effects of drought in cotton.
A significant difference was observed between the control and bacterial inoculation treatments in enhancing root and shoot traits in cotton at 45 DAI (days after inoculation). Among the six isolates evaluated, Pseudomonas sp. (5R) showed better shoot and root traits under drought stress.
Out of these, Sphingomonas sp. treated cotton plants showed better drought stress tolerance than control in terms of plant height, leaf chlorophyll content (37.4 SPAD) and relative water content (85 %).
Exploiting the epigenetic transgenerational inheritance of stress responsive traits for imparting abiotic stress tolerance in cotton
If the epigenetic change in response to environmental conditions is inherited, the epigenetic inheritance may allow the plants to continually adjust its gene expression to fit its environment without changing its DNA code. The potential value of epigenetic inheritance is reflected directly by the influence of variations in DNA methylation on important agricultural traits such as flowering time, plant height, drought tolerance and yield. The use of Epigenetic Regulating Chemicals (ERCs) for improving stress tolerance in plants is gaining importance. The following study was devised to explore the possibility of using epigenetic inheritance for imparting abiotic stress tolerance in cotton.
The drought tolerance imparted by epigenetic regulated chemicals (ERCs) through seed treatment, in varieties like Suraj and LRA 5166 was inherited upto fourth generation. This is evident from the fact that the ERCs like 5 azacytidine, sulfamethazine, epigallocatechin gallate and nicotinamide improved the relative water content, SPAD values, proline content, nitrate reductase activity, chlorophyll stability index and total soluble sugars and reduced the excised leaf water loss when compared to untreated control. Among the ERCs, 5 azacytidine improved the key drought tolerant traits in both Suraj and LRA 5166.
The epigenetic regulating chemicals (ERCs) like Sulfamethazine 10 pM (68.3%) and 5 Azacytidine IQpM improved the relative water content over control in Suraj and 5 Azacytidine 40 pM recorded higher RWC in LRA 5166. Higher total soluble sugars (TSS) content was recorded by 5 Azacytidine 40 pM treated plants in case of Suraj and Epigallocatechin gallate 100pM treated plants of LRA5166.
Evaluation of PGPR and microbial inoculants to alleviate drought stress in cotton (G .hirsutum L.)
The bio stimulants like PGPR play significant role in alleviation of drought stress. This project aims toevaluate the PGPRs collected from cotton rhizosphere for their potential to improve the drought tolerance in cotton.
Out of seven best native PGRs selected. Sphingomonas sp. treated cotton plants showed better drought stress tolerance than control. While evaluating their In-vitro growth promotion activity, three bacterial isolates; Micrococcus luteus, Agrococcus sp and Solibacillus isronesis exhibited higher activity in terms of IAA (1, 3 Indole Acetic Acid). Also, maximum ACC deaminase activity was found in drought tolerant bacterial isolate Micrococcus luteus.
Leaf reddening in Cotton Causes
The major causes for leaf reddening in cotton are mineral nutrient stress, water logging, drought, low temperature stress, high light stress, incidence of sucking pests, strong winds, microbes, choice of genotype, certain insecticides, herbicides like 2,4-D and spraying of cocktail of incompatible combination of chemicals. Abiotic stresses in general create a transient nutrient deficiency in plants, as the uptake of mineral nutrients is hampered due to various reasons. Reduction in uptake of mobile nutrients like, N, P, K and Mg under abiotic stress conditions, leads to translocation of nutrients from older leaves to newer leaves at the top. These stresses thus accelerate senescence of older leaves by degradation of chlorophyll. Accumulation of anthocyanin pigment occurs to protect the leaves from further damage.
Possible measures which can be taken to prevent leaf reddening in cotton
- Foliar spray of 2 % DAP along with 1% Potash or Potassium Nitrate at about 90 DASFoliar spray of 2 % Urea, 0.5 % Zinc Sulphate and 0.2 % Boron twice at 15 days intervalFoliar spray of 19:19:19 (1%) or 2 % DAP with 1 % Magnesium Sulphate at peak boll formation stage.Avoid planting susceptible genotypes like RCH-2 Bt and Mallika BtAvoid spraying insecticides like Methomyl and ThiodicarbPlanting improved desi cotton varieties which are highly tolerant to abiotic and biotic stressesPlanting short duration varieties which can escape biotic and abiotic stressesPracticing early sowing so as to enable the plants to avoid stress during the critical stagesPlanting desi varieties/hybrids with less nutrient demand under rainfed conditionsFollowing moisture conservation measures like mulching and nutrient conservation measures like intercropping and crop rotation
Causes of leaf reddening and yield loss in cotton plants with red foliage in parts of Central India
Elucidated that in parts of central india, onset of leaf reddening occurs when the night temperatures start declining below 200 C followed by brighter sunshine during the day time. This was supported by biochemical analysis and gene expression studies. Due to low temperature and high light intensity photoinhibition occurred in plants. Photoinhibition was succeeded by the formation of free radicals as proved in the study. The free radicals in turn resulted in chlorophyll degradation to a tune of 50 %. This caused the cotton plants to accumulate more anthocyanins in leaves and nonfoliar green organs to protect the chlorophyll pigments from further oxidation.
As the non foliar organs (bracts and capsule wall of the bolls) were reported to make 24% of photosynthetic contribution during boll development stage, the yield loss occurred in cotton plants due to 50 % loss of chlorophyll pigments in them (proved in the study).
Spraying of CICR Nutrient Consortia at 15 days intervals was found to delay cold induced leaf reddening by around 10 days
Germplasm lines –A treasure mine for water logging tolerance
Phenotyping of 2700 Gossypium hirsutum lines was done at CICR Nagpur during 2015-16 and screening was performed based on plant height, Height to Node Ratio, timing of lenticel formation, formation of adventious roots, number of red leaves and yellow leaves, stem girth and yield and yield components. Out of 2700 lines, 211 tolerant lines and 44 susceptible lines were shortlisted. During the year 2016-17, the shortlisted lines were further phenotyped at CICR, RS, Coimbatore, based on general morphological characters reflecting growth, SPAD values, timing of formation of Lenticels, formation of adventitious roots, detection of iron deficiency symptoms and Nitrate reductase activity. Ten highly tolerant and four highly susceptible lines were identified for Marker Assisted Breeding. Key enzyme assays which contribute to tolerance to water logging were done in susceptible and tolerant lines used for marker assisted breeding. The Sucrose synthase activity in roots was found to be higher in susceptible lines IC356708 and IC357607 both under control and water logged conditions. However when compared to susceptible ones, the tolerant genotypes LRA5166 and IC63998 recorded almost 2 fold increase in sucrose synthase activity under water logged conditions when compared to other genotypes. The lactate dehydrogenase activity in roots was higher in tolerant varieties both under control and water logged conditions. Lactate formation is an important indicator of the ability of a plant to survive hypoxia without extensive cell damage.
Among the lines, LRA 5166 recorded higher number of sympodia, higher root lactate dehydrogenase activity under stress conditions. Higher leaf alcohol dehydrogenase activity was recorded by both LRA 5166 (4.8 D340 nm/min) and IC63998 (5.8 D 340 nm/min) under stress conditions compared to susceptible ones. There is no notable difference in root alcohol dehydrogenase activity and root pyruvate decarboxylase activity among tolerant and susceptible ones under stress conditions. Leaf area reduction of LRA 5166 under stress (1783.5 cm2) conditions compared to control (4215.8 cm2) may be one of the adaptations to water logging. The Nitrate reductase activity in roots was higher in tolerant varieties both under control and water logged conditions. Hence the plants can cope up with nitrogen assimilation under hypoxic conditions.
Phenotyping of germplasm lines for drought tolerance
Among the three hundred and fifty Gossypium hirsutum germplasm accessions screened, 5 accessions namely Nagpur 9, SGNR -27, F-1226, DTS-108-09 and IC 357406 were identified as drought tolerant based on mid day wilting, leaf water potential, epicuticular wax content and relative water content. Shortlisted 104 germplasm lines were phenotyped during summer by the traits such as mid-day wilting, relative water content and epicuticular wax contents. Fourteen drought tolerant lines and 7 susceptible lines were identified. IC 357406 and Nagpur 9 were found to be highly tolerant to drought. Four hundred and eighty one germplasm lines were analysed for epicuticular wax content to correlate with drought tolerance. Four lines were identified to have high epicuticular wax content of above 200µg/cm2.
DOF1 transcription factor for improving Nitrogen Use Efficiency
Nitrogen assimilation in plants requires carbon skeleton (2-oxoglutalate, 2-OG), produced from photosynthetic metabolic intermediates in addition to inorganic nitrogen present in soil. Increased production of carbon skeleton might stimulate nitrogen assimilation in plants. DOF1 (DNA-binding with one finger) transcription factor regulates the key enzymes linking C and N metabolism and was found to improve Nitrogen Use Efficiency (NUE) under nitrogen limiting conditions. An attempt was made to study the expression of DOF1 transcription factor and downstream genes such as Pyruvate Phosphate Dikinase (PPDK), Pyruvate Kinase (PK) involved in carbon metabolism and Nitrate Reductase (NR), Nitrite Reductase (NiR), Glutamate Synthase (GT), Glutamine Synthetase (GS) involved in nitrogen metabolism in plants grown under varying levels of Nitrogen in sand culture. The NR and NiR expression levels were higher in cotton irrespective of the expression levels of DOF1. The expression levels of chl ab binding protein were high in N limiting conditions when compared to 100 % N indicating N deficiency induced senescence of cotton leaves. Dof1 transcription factor expression was low under 0% N when compared to 100 % N. However at 25 % N the expression level of DOF1 transcription factor and other downstream genes were high, indicating the possibility of increasing nitrogen Use Efficiency (NUE) under nitrogen limiting conditions by over expressing DOF1 in cotton.
Gene expression of six transcription factor in response to drought was studied in six genotypes against well-watered control. Basic helix–loop helix (GhbHLH), GhVATpase (Vacuolar H+ ATPase) and GhNCED (9-cisepoxy carotenoid dioxygenase) showed significant up regulation only in Nagpur 9 and LRA5166 and significant down regulation in 2389. Ghdreb1A (dehydration responsive element binding protein) and Ghnac9 (NAC transcription factor) showed significant up regulation in all the studied genotypes. Gh1433f gene, Suraj and Nagpur 9 showed significant up regulation of the gene. Significant up regulation of GhALDH7 was noticed in Nagpur 9, LRA 5166, 891 and Suraj genotypes.
Host Plant Resistance Pink Bollworm
Normal flowers and pink boll worm (PBW) infested flowers were collected from Gossypium hirsutum variety Suraj. Methanolic floral extract were collected from individual flowers. Average terpene content in PBW infested flowers was higher (9.7 mg/g linalool equivalents) when compared to 6 mg/g linalool equivalents in normal flowers. Organic compounds in the methanolic extract of PBW infested flowers and normal flowers were identified using GC-MSMS. Relative abundance of β Caryophyllene is twofold higher in PBW infested flowers than normal flowers. In PBW infested flowers slightly higher relative abundance of methyl ester of pentadecanoic acid and linolenic acid, the precursor of jasmonic acid, is seen than in normal flowers. The terpenes, 2,thujene, copaene, α guaiene, epizonarene which are found in normal flowers are not detected in PBW infested flowers. The terpenes, cubebene and the fatty acid esters, 2-methyl octadecane and 11-butyl docosane which are found in PBW infested flowers are not detected in uninfested flowers. β Caryophyllene was found to attract the natural enemies of crop pests.
WhiteFlies
Epicuticular wax content was quantified from Gossypium hirsutum germplasm lines categorized as (i) high whitefly and high CLCuD (ii) low whitefly and high CLCuD and (iii) low whitefly and low CLCuD. The epicuticular wax content ranged between 38 to 66 µg/cm2 in the first category, 23 to101 µg/cm2 in the second category where as in the third category, quantity of wax extracted was 291.5 to 368 µg/cm2. This indicates that germplasm lines with higher epicuticular wax content had lower incidence of whiteflies and hence lower CLCuD incidence. However temporal and spatial variations in wax contents were observed. Line Nos. 53 (IC357886), F2383, CISA 310, CISA 614, 276, 279, 198, 85 and 5 possessed high epicuticular wax content. Line No. 126 (IC 358823) had very low epicuticular wax content.
Stem Weevil
Plant phytochemicals contributing to host plant resistance/susceptibility to stem weevil such as phenols, sugars, terpenoids and tannins were estimated from leaves and collar regions of nine cotton varieties chosen for the study. MCU3 recorded higher phenolic content in 16th to 19th day old seedlings in the collar region and in 15 th and 16 th day old seedlings in Baghubali. RCHB625 recorded higher phenolic content on 23rd and 24th day after sowing. LRa 5166 recorded higher phenolic content in leaves throughout the growth period. Total soluble sugars are higher in Mallika and RCHB625 at 15-16th DAS in collar region.LRA 5166, MCU 5 and Bahubali recorded higher terpenoid content from 15th to 16th days after sowing. MCU3 recorded higher tannin content on 15th, 16th, 18-21 st DAS. Hence MCU 3 showed resistance to stem weevil compared to other 8 varieties under study.
Express Nitrogen Guru
The gadget works based on the principle of transmittance of light by leaves. N deficiency causes changes in pigmental composition in cotton leaves. As the intensity of N deficiency increases, there is an increase in red pigment, anthocyanin and increase in green chlorophyll pigments in cotton leaves. The gadget detects N deficiency based on red, green, blue, hue, saturation and luminosity values of the leaf colour.
When the user presses the button, the program takes a picture of the leaf inserted into the leaf chamber. The image is then processed by the program to determine the average color parameters of the leaf. The gadget processes the average leaf colour and compares it to the standard nitrogen plot in its memory to determine the approximate N content of the plant. Based on pre-set thresholds, the device recommends corrective measures in the form of voice advisory both in English and vernacular language to the farmers.
Exploiting the transgenerational inheritance of epigenetic marks for drought tolerance
The epigenome (chemical “tags present in DNA) changes rapidly in response to signals from the environment. The epigenome remains flexible as environmental conditions continue to change. However if an epigenetic change in response to environmental conditions is inherited, the epigenetic inheritance may allow the plants to continually adjust its gene expression to fits its environment without changing its DNA code. Epigenetic inheritance is important for the understanding of phenotypic variations in nature. The potential value is reflected directly by the influence of variations in DNA methylation on important agricultural traits such as flowering time, plant height and pathogen resistance and yield. Recent evidences in plants indicate that few epigenetic traits are stably inherited across generations. Use of epigenetic regulating chemicals for improving stress tolerance in plants is gaining importance in recent days. Also studies have offered proof that it is possible to increase stress tolerance in the immediate progeny by exposing ancestral plants to mild/short-term stress signals due to transgenerational transmission of stress memory and associated patterns of gene expression.
An experiment was conducted to explore the possibility of using epigenetic inheritance for imparting abiotic stress tolerance in cotton. Suraj and LRA5166 seeds treated with Epigenetic Regulating Chemicals (ERCs) like 5-Azacytidine, Sulfamethazine, Epigallocatechin gallate and Nicotinamide along with Control (Water) in the first generation and generation advancement was done upto five generations and screened for drought tolerance. The results revealed that seed treatment with 5-Azacytidine contributes to drought tolerance atleast up to three generations in cotton varieties Suraj and LRA 5166 by enhancing most of the important drought tolerant traits involved in water conservation and osmolyte production.
Whole genome bisulphite sequencing revealed that the higher proportion of differentially hypo methylated regions was observed in Chromosome 9 and 12 in third generation plants of LRA 5166. In case of Suraj, higher proportion of differentially hypo methylated regions were observed in Chromosome 5 of third generation plants. This indicates increase in gene expression under drought stress conditions, thus imparting drought tolerance to cotton.