Seed Technology

Breeder Seed production

Seed plays a major role as a basic input to cotton productivity and sustained production. Studies indicated that use of good quality seeds alone could increase production by up to 20 per cent. Good seeds enable the farmers to maintain uniform and vigorous stand of crop, which serve as a catalyst to make other input, productive and cost effective.

The inherent variability in a variety gets exposed in seed multiplication chain from Breeder Seeds to Certified Seed production. Hence, it is absolutely essential to maintain genetic purity in nucleus seed by practicing proper progeny row evaluation and selection for morphological uniformity, yield and quality parameters.

Unless the nucleus / breeder seed is of highest purity and quality, the seed multiplied from it cannot be regarded as of satisfactory genetic purity. Different institutions follow different methods for producing nucleus and breeder seeds. This chapter intends to help the cotton breeders to adopt a uniform procedure for producing nucleus and breeder seeds and for maintenance breeding in cotton.

Flower structure and natural out crossing

Cotton flowers are extra-axillary, terminal and solitary and are borne on the sympodial branches. The flower is subtended by an involucre of usually three unequal leaf like bracts. Bracteoles, alternating with the bracts on the inside of the involucre or standing on either side of the small bract, may be present. The calyx, consisting of five undiverged sepals, is persistent and shaped as a shallow cup (Figure 3). The calyx adheres tightly to the base of the boll as it develops.

The corolla is tubular, consisting of five obcordate petals alternating with calyx lobes and overlapping the next one in the series in a convolute manner. In some varieties, a spot of purple, sometimes called ‘eye’, is found on the claw (base) of the petals. On the first day after anthesis, the corolla changes into pinkish hue and then into red during succeeding days (Figure 4). It withers and falls off on the third day, together with the staminal column and stigma leaving the ovary, calyx and involucre intact.

The stamens are numerous and united to form a tubular sheath which surrounds the pistils except for the exposed portion of style and stigma at the tip. Under normal conditions, the pollen grains are viable upto 24 h and thereafter lose potency and fail to effect fertilization. The time taken by the pollen tube to traverse the style varies according to the variety and environment. In general, 10-13 h duration is required to traverse the entire length of style. Generally, the interval between pollination and fertilization varies from 36-40 h. The pollen grains of cotton are heavy, sticky and warty leaving little chance for wind pollination. Insects are the natural agents for the pollen transfer. It is typically entomophilous, pollinated by insects; the rate of cross-pollination is generally 3-20 %.

The pistil consists of 3-5 undiverged carpels corresponding to the locular composition of a fully mature dehisced boll. The ovules are attached to parietal placenta of each locule. The style varies in length and splits near the apex into three, four or five parts depending on the number of carpels. The dehiscence of the boll is along the dorsal sutures

Pollen Biology

There are approximately 10,000 pollen grains in a flower and the mature pollen grains are trinucleate. Pollen grains are large, spheroid and spinate with a diameter of over 120 microns. If conditions are favourable, the pollen grains begin to germinate as soon as they reach the receptive stigma. When the atmospheric conditions are congenial, they even tend to germinate in the flower itself. Under normal conditions, the pollen grains are viable upto 24 h and thereafter lose potency and fail to effect fertilization. However, pollen grains from younger buds may remain viable for upto 44 h and even for a longer period. Frequently, more than one tube emerges from the pollen grains on germination. The wall of pollen tube shows characteristic lamellae and the tip shows only a slight swelling in normally growing pollen tubes. The tendency to branching is not so pronounced in rapidly growing tubes as in slow growing ones. The diameter of pollen tube varies with the ploidy level, the tetraploids having larger pollen grains and tubes.

The pollen tubes after passing between papillate cells of the stigma bend down sharply into the soft transmitting tissues of the style and ovary. The course of pollen tubes is determined since each stigmatic lobe is directly in line with its corresponding locule and the ovules therein, and a direct connection is established between them through the conducting strand and the nutritive placental ridge.

The time taken by the pollen tube to traverse the style varies according to the species, variety and environment. In general, 10-13 h duration was taken to traverse the entire length of style. The pollen tube grows through the placenta, travel up the funiculus, and finally reach the micropyle. Sometimes, the pollen tube may cross from one placenta to another. In the micropyle, the pollen tube follows either a straight or zigzag path depending upon the alignment of the exostome and endostome, sometimes growing for a short distance between the two integuments, or the inner integument and the nucellus and forking at the tip. Two to four layers of nucellar tissue are digested during its passage into the embryo sac. Often more than one pollen tube enters the micropyle, but only one effects fertilization. Metaxenia has also been observed in that staple length and boll characters may be altered. Generally, the interval between pollination and fertilization varies from 36-40 h.

The pollen grains of cotton are heavy, sticky and warty leaving little chance for wind pollination. Insects are the natural agents for the pollen transfer. Honeybee (Apis mellifera L.) is the main vector for pollination apart from other honeybees, bumble bees, leaf cutting bees and a few dipterans. The main pollinating insects differ due to distribution of insects and ecological condition and their capability also differ on account of visiting behaviour, body size and fully pubescent insects such as the yellow breast wood bee, heavy flower wasp and black spinytibial bee are highly efficient for pollination.

Pollination and fertilization

Cotton is an often-cross-pollinated crop with 3 to 20% cross-pollination, depending upon the insect activity and environment in which the parents are grown. Hybrid seed production is not easy unlike in other crops like maize, sunflower, bajra etc., which are highly cross-pollinated and possess additional favourable contrivances and high crossed seed output per pollination. The flowers are solitary in to axils of leaves. Hence, emasculation and pollination are essential in cotton, which make hybrid seed production a tedious, costly and difficult process. Even if genetic or chemical method of emasculation are practiced in specific crosses, physical transfer and dusting of pollen grains on stigma of each emasculated flower is essential and has not been circumvented by pollination employing honey bees or other effective techniques. The problem of nicking is not there in cotton since the flowering extends over a period of time.

In commercial seed production, emasculation and hand pollination is a daily activity, extending for a period of 30 to 60 or even 90 days. For obtaining good quality seeds, it is advisable to restrict the pollination to 30-40 days of effective flowering.

Being an indeterminate crop, cotton has a continuous flowering habit and flowering starts 50 to 60 days after sowing. The effective flowering interval in a growing season range from 45-90 days, depending upon the variety/species. The flowers are produced in regular intervals of 3 and 5 days, respectively, on horizontal and vertical axes of sympodial branches of the main stem. The opening of flowers follows a spiral course in acropetal and centrifugal succession. Among the various systems to produce hybrid cotton, the traditional hand emasculation (Doak’s method) of female parent in the evening hours followed by pollination in the next morning has been widely adopted for commercial seed production. Before starting emasculation in commercial hybrid seed production plots, all the opened and uncrossed flowers are removed completely from the female parent plots to avoid mixing of crossed boll with naturally set bolls. In the process of emasculation first the bracts are removed from the selected fully grown flower bud. Then, the petals together with androceum, which along with anther sac are removed gently and smoothly by cutting them with thumb nail without damaging the stigma, style, ovary or even the white covering of the ovary. After emasculation, the flower buds are covered with red tissue paper or straw tube so as to avoid the contamination of the emasculated flower by exotic pollen. The emasculation in the female parent bud is done during the evening hours from 3.00 to 6.00 PM. The following care must be taken during emasculation

• Cutting from the external layer of the calyx into the internal perianth layer should not be too deep in order to prevent damaging the ovary, and the white membrane covering the outer wall of the ovary.
• Do not strip off the bracts. To avoid breaking the stigma, the calyx and perianth should not be pulled off too violently.
• To emasculate completely, be sure that no anthers are left on the pistil.

The stigma of the emasculated female flower is highly receptive from 8 to 10 AM on the subsequent day of emasculation. At this time, pollination has to be carried out using pollen from the male flower, for which the flower about to open are to be collected, the bracts and petals removed and kept open under sun in a plastic tray in the morning hours (8 to 10.0 AM.) of the day of pollination. Normally, one flower from male parent can be used to pollinate 5-8 emasculated buds depending upon the parental species, genotypes, season of crop growth etc. During pollination, the flower is held in right hand, with exposed androecium shaken gently or rubbed on the receptive stigmatic surface of the emasculated bud. Dusting of sufficient pollen must be ensured on all the stigmatic lobes for the proper development of all the locule. The emasculated flower should be handled with care to avoid snapping.

Soon after pollination, the red bag or tube in the female flower is replaced with white bag or tube, to get protection from foreign pollen. The uncrossed flowers left in female parental plots are removed and destroyed on each day after completion of pollination. A trained labour can emasculate nearly 300-400 flowers in the evening and pollinate the same number of flowers in the morning hours of the following day, depending upon the age group, skill and supervision. In this method, successful boll setting ranges between 30 and 80 % depending on the hybrid, stages of crop, crossing efficiency and crop husbandry. Inspite of high investment and process being tedious and time consuming, hybrid seed production is a profitable venture.

Selfing

To maintain parental genotype purity, selfing is a general practice. The unopened flower buds are selected for selfing and are usually done in the afternoon. For selfing the flower buds, different techniques are practiced at different places. Generally, in the low wind areas, selfing can be achieved by rolling the cotton lint over the tip of the bud and a paste of mud is applied over the rolled cotton lint to ensure the intactness of the lint. To identify the bolls at the time of bursting of selfed flowers, the lint is also rolled and pasted with mud in the pedicle of the bud. In other places, selfing is practiced by tying a thread at the tip of the bud to prevent from opening and thereby ensuring 100% self pollination. Figure 6 presented below indicates the process of selfing. Figure7 shows the appearance of sterile and fertile anther column in cotton flower. Figure 8 to 11 indicates the sequence of hand emasculation and pollination

Roughing

Adequate and timely rouging is extremely important in seed production. They should be removed at the earliest possible before flowering. It is wise to remove the whole plant and not just the flower by hand. Sometimes there may be differences in crop growth, variation in height, flowering duration and maturation etc., some plants flower very early and some plants flower very late. If the variety used is genetically pure there won’t be variation in the field. If variations are observed, it is mainly due to genetic contamination (or) admixtures of seeds. Because of these, such field will be rejected for seed production. In order to avoid all these defects, plants which are deviating from the concerned variety / hybrid on morphological characters should be rouged out. Remove all the diseased and infested plants from seed production plot.

Land requirement

Land to be used for production of cotton seed must be free of volunteer plants of cotton. There should be no hard pan in the sub-surface layer and also it should be free from the hard layer of carbonates that interferes in the proper development of plants, and therefore the seed.

Isolation

Cotton is mainly a self-pollinated crop but natural cross-pollination to the extent of 10 to 50% in G. hirsutum and 1 to 2% in G. barbadensehas been recorded in various countries.Actual isolation requirement for cotton varies according to the extent of natural cross-pollination. In India, a minimum isolation distance of fifty meters for foundation seed class and thirty meters for certified seed class production from fields of other varieties of the same species, other species and fields of the same variety not confirming to variety purity requirements for certification is mandatory.

If male-sterile lines are used for producing hybrid seed, following eligibility requirements are to be considered;

• An inbred line to be eligible for certification shall be from a source such that its identity may be assured and approved by the certification agency.
• Hybrid seed to be eligible for certification shall be the progeny of two approved inbred lines, one of which shall be male-sterile.
• An inbred line shall be a relatively true breeding strain resulting from self-pollination with selection.
• The foundation class seed shall consist of an approved male-sterile line to be used as a female parent and an approved inbred line to be used as a male parent for the purpose of producing hybrid seed.
• A male-sterile line shall be a strain carrying cytoplasmic-genetic male sterility, which sheds no viable pollen and is maintained by the normal sister strain, which is used as a pollinator.
• The certified-class seed shall be the hybrid seed to be planted for any use except seed production.

In cotton, the following stages of seed production are taken up in seed production chain.

Nucleus and Breeder seed production in varieties The initial handful of seeds obtained from selected individual plants of a particular variety, for the purpose of purifying and maintaining that variety, by the originating plant breeder and its further multiplication under his own supervision or the supervision of a qualified plant breeder, to provide Breeder seed constitutes the basis for all further seed production.

Nucleus seed refers to the seed produced by the breeder, who developed the particular variety or by any other breeder of the institution, where the variety was developed, which is directly used for the multiplication as breeder seed. The Association of Official Seed Certifying Agencies (AOSCA) defines breeder seed as the seed material directly controlled by the originating or the sponsoring breeder or institution, providing for the initial and recurring increase of foundation seed.

Nucleus Seed Production

To start a nucleus seed production programme, a base source is a pre-requisite. For released varieties, it may be breeder seed or nucleus seed. Sufficient single plants (minimum 200 plants) may be selected from the base source. The selected plants should confirm to the basic morphological (phenotypic) characters given at the time of release of variety. The selected plants are individually observed for various distinguishing morphological characters (Table 1), and are selfed. Mean and standard deviation for various agronomic characters are to be worked out and the plants that lie within the mean + SD for all the characters are selected. Individual plant selection is made on morphological characters identical or typical to released variety in field. The selected plants are subjected to fibre quality evaluation to determine 2.5% span length, micronaire, uniformity ratio and bundle strength. Plants which conform to the basic fibre characters given in the release proposal or which lie within mean are selected (Flow diagram 1).

The selfed seeds from the selected plants are grown in a randomized replicated design. The row length of plants may be determined based on the availability of seeds. Normally, two rows of two replications per progeny are grown. Appropriate spacing should be adopted to enable full expression of characters. It will be better to take up nucleus seed production in areas where the variety is most adopted and during the best growing season.

Normally, the progeny rows should be grown in compact fields with proper isolation. The nucleus seed production plot should be critically observed by the breeder for all the morphological character during different growth periods. If any progeny of any plant is found to be deviant at any stage for any character, the whole progeny should be rejected. The lines unusually susceptible to pests and diseases may also be rejected. Selfing is to be done to maintain the purity. The progenies are studied for various morphological characters and the data are analysed statistically. The progenies that fall within the mean + CD @ 5 % are selected. The fibre test and micro spinning test are conducted and only progenies that confirm to the original fibre characters are selected. Equal quantity of selfed seeds of selected progenies is bulked to constitute the nucleus seed. If large quantity of breeder seed is required, next stage of progeny bulk seed may be considered as nucleus seed. If it is required, the cycle of selection could be repeated again, to further ensure uniformity.

Breeder Seed Production

The nucleus seed is utilised to produce the breeder seeds. It is important to take up the breeder seed production in the areas where the variety is most adopted and during the best growing season. The crop is sown with proper isolation (at least 50 metres) and the plot is critically observed by the breeder for all the morphological characters. If any plant is a deviant at any stage for any of the characters, the plant should be rouged out immediately.

Important distinguishing morphological characters in cotton (As per DUS Testing Procedure)

Schematic diagram of Nucleus and Breeder Seed Production

Quality improvement

The following technologies were identified for seed quality improvement

Effect of polymer and pesticide coating on viability, vigour and longevity of cotton seeds

Seed treatment with imidaclorpid @ 5 g kg-1 alone or coated with polymer @ 5 ml kg-1 + carbendazim @ 2 g kg-1 is effective in control of sucking pests up to 45 days after sowing. Coating of seeds with polymer @ 5 ml kg-1 or polymer + carbendazim or polymer + cabendazim + imidacloprid or Chlothianidine or Thiamethoxam either alone or in combination prolonged the seed viability and quality for a period of sixteen months.

Effect of relative humidity levels on physiological and biochemical changes in cotton seed during storage

With an increase of storage period and relative humidity there was a progressive decline in viability, dry matter of seedling, vigour, seed oil content, coupled with rapid increase of seed moisture content, free fatty acid accumulation, electrical conductivity of seed leachate, free sugars and storage fungi in all the genotypes Anjali, Surabhi, LRA5166 and AKH4 stored at 40%, 60%, 80% relative humidity and along with silica gel (Control) for eight months under ambient condition.

Effect of seed treatments on the maintenance of viability and vigour in cotton seeds under ambient storage

Freshly harvested and delinted seeds of cotton varieties LRA 5166 (treated with neem leaf powder @ 10g kg-1) and Surabhi (treated with neem kernel powder @ 10g kg-1) packed in paper bag and stored under ambient condition for 32 months, excelled other treatments by registering higher seed quality with extended the storage life.

Influence of spacing and micronutrient spray on yield and quality of seed

Sequential foliar application with diammonium phosphate 2 %, MgSO4 1 %, boron 0.1 %, ZnSO4 2 % at 45, 50, 60, 75 days after sowing enhanced the seed cotton and quality seed yield at 120 x 60 cm spacing in Cv.Anjali

Detopping with defoliation treatment maximizes seed yield in cotton

The beneficial effects of detopping at 120 days and spraying of ethrel @ 450 g a.i. acre-1 at 160 days after sowing exhibited in terms of reduced plant height with increased number of sympodia, bolls, boll weight, seed cotton yield and seed yield in LRA 5166

Seed soaking and foliar application of hormones and nutrients for enhancement of productivity and quality of cotton seed

Seed soaking in succinic acid @ 0.2% and spraying of NAA @10 ppm on 60 and 75 days enhanced the field emergence (4%), plant stand establishment (98%), Seed cotton yield and quality seed yield in cv.Sumangala.

Enhancement of hybrid cotton seed yield and quality through parental crop management

In hybrid Savitha, the boll setting efficiency was high due to pollination in the early phases of flowering (up to 5th week) and there on declined significantly. Seeds formed during that period were highly vigorous and viable. The parental seed crop management with seed soaking treatment and foliar application of growth hormones and nutrients indicated that soaking of female parent seeds in succinic acid @ 0.2 % for six hours before sowing and pollination up to five weeks from flower initiation and simultaneously foliar application of boron @0.1 % at 60 DAS, MgSO4 1 % at 75 DAS or foliar application of boron @ 0.1 % at 60, 75 & 90 DAS alone found good for production of high quality hybrid seed.

Control of seed deterioration in cotton genotypes

Cotton seeds stored under laboratory conditions for one, two, three and four years, dry dressed with a mixture of calcium oxychloride and calcium carbonate (3:1) @ 5 g kg-1 and iodine vapours impregnated in the calcium carbonate @ 3g kg-1 and kept in airtight container under ambient condition for seven days had shown increased seed vigour and viability due to halogenation, irrespective of age of seeds and reduced the pathogenicity tested in cv. 70 E, Bikaneri Narma, AC.738, PIL.43,PIL.8, .7 and M.12 stored seeds.

Performance and productivity of F1 and F2 seeds of Intra hirsutum hybrids

The morphological and reproductive traits of ten F2 generations of pre released intra hirsutum hybrids were compared along with F1. A decrease in plant height, sympodia plant-1, seed index and lint index in F2 over F1 was observed in all the cultures. However an increase in the number of bolls plant-1 and seed cotton yield plot-1 was observed in H1, H2, H4, H7, H9, H10, Savita and NHH 44. The fiber property of F2 plants such as 2.5%span length of H3, H6, was on par with savita. Uniformity ratio of H1, H2, H4, H7, H9, and H10 found superior to either of Savita and NHH 44. The micronaire of all the cultures was on par with NHH 44 and inferior to Savita. The fiber elongation of H7 and H9 were superior to Savita and NHH44.

Comparison of performance of model bulk, superior bulk and ordinary bulk of plants selected from progenies

In LRA5166 and Sumangla pooled seeds of single plant progenies of superior bulk (A), model bulk (B) and ordinary bulk (C) were raised along with breeder seed (BS). The plants from superior bulk of both the varieties came to flowering earlier than the ordinary bulk. In case of LRA 5166, the model bulk performed equally with superior bulk and breeder seed. The number of sympodia recorded was maximum especially in superior bulk of sumangala (15.6) when compared with ordinary bulk (13.7). Superior bulk of LRA 5166 and model bulk of sumangala showed better performance for morphometric character such as plant height. Maximum yield/ha was recorded in model bulk of Sumangala followed by breeder seed of LRA 5166. Better performance for fiber quality parameters like span length and bundle strength was registere in superior bulk followed by model bulk and breeder seed.

Cataloguing (a) morphological and (b) genetic marker using molecular technique available in the currently cultivated cotton varieties and hybrids

Morphological markers of cotton varieties LRA5166 and Sumangala (G. hirsutum) were studied for easy identification during winter 2003, 2004, 2005, seasons using breeder seeds. The visible morphological characters such Seedling pigmentation, Growth habit, Density of foliage, Plant: Stem hairiness, Pubescence on stem Bract: number of serration No. of teeth in the bracts Boll surface Fibre: strength Fibre: fineness (micronaire value) were found useful in variety and hybrid identification. Genetic marker-The genomic DNA from single seed of hybrid Savita and its parents T7 (female) and M12 (male) was isolated and random primers were used for RAPD analysis. The primer OPA 01, 02 and 03 showed the presence of DNA sequence in female, hybrid and male parent of hybrid Savita. The primer OPC 06 very clearly indicated the presence of similar DNA sequence in hybrid and parents. The primer OPA 07 and 08 indicated the presence of male and female specific DNA sequences which are present in hybrids. These DNA segments help to identify the presence of male or female seeds in the hybrid seed lot.

Effect of supplemental foliar nutrients on seed yield and quality of cotton seed

In Anjali seed crop, foliar application of DAP @ 2 % + Boron @ 0.6 Kg/ha + Zinc @ 0.5% on 70, 90 and 110 days after sowing significantly improved the seed quality and recorded higher seed yield. Application of DAP @ 2 % (T1), Boron @ 0.6 Kg/ha (T2), Zinc @ 0.5% (T3) individually exhibited positive effect on seed quality improvement, and the beneficial effect is high when it is applied simultaneously.

Enhancement of planting quality of seed and stand establishment of cotton through sequential pelleting

The delinted seeds of cotton cv. Anjali were coated using Polymer @ 3g kg-1 with following ingredients for pelleting. Seed protectant: Bavistin @ 2g kg-1, Thiram @ 2g kg-1, Cruiser @ 3g kg-1, Imidacloprid @ 7g kg-1; Bio inoculants: Trichoderma viridie @ 4g kg-1, Pseudomonas florescence @ 10g kg-1; Nutrients: DAP @ 20g kg-1, Micro nutrient mixture@ 10g kg-1 and Gypsum @ 20g kg-1; Botanicals: Arappu leaf powder @ 50g kg-1 (Albizia amara), Neem leaf powder (Azadirachta indica) @ 20g kg-1 and Turmeric (Cucumin sp) rhizome powder @ 20g kg-1 . Following are the best five combination and sequence of coating for seed quality improvement. 1.Thiram + Gypsum + Micronutrient + Gau chao + DAP, 2. Thiram + DAP + Gau chao+ Micronutrient + Gypsum, 3. Arappu leaf powder + Turmeric rhizome powder + Neem leaf powder, 4. Neem leaf powder + Turmeric rhizome powder + Arappu leaf powder, 5. Bavistin+ Neem leaf powder + Arappu leaf powder + Turmeric rhizome powder

Standardization of laboratory techniques for variety identification

Seed physiological laboratory techniques such as Phenol test, Modified phenol colour reaction, test, Ferrous sulphate test, Potassium hydroxide test, Sodium hydroxide test, GA3 soak test, 2,4-D soak test, Hydrogen peroxide soak test were conducted to assess the reaction and physiological expression of the cotton hybrid and their parental seeds. The hybrids used are HB 224 (LRA 5166 x P 4), Surya (T 13 x M 12), Savita (T 7 x M 12), Varieties: Surabhi, Anjali, Supriya, and SVPR 2. The observations revealed the following Phenol test: no response to phenol test; hence no grouping could be possible; Modified phenol test: no response to modified phenol test, hence no grouping could be possible; Ferrous sulphate test: all the genotypes turned into black colour; Potassium hydroxide test: The cotton genotypes could be classified based on colour development; Sodium hydroxide soak test: The cotton genotypes could be classified based on colour development; GA3 soak test: Based on hypocotyl length the cotton genotypes were grouped into three categories; 2, 4-D soak test: no genotypes germinated at 0.5 ppm, 2, 4-D solution; Hydrogen peroxide soak test: Based on the response of cotton genotypes to hydrogen peroxide, the genotypes studied were classified.

Variety Identification of cotton genotypes through electrophoresis

Variety Identification of cotton genotypes through electrophoresis were conducted for the development of predictable banding pattern due to single seed protein globulin in cotton seed. Initially total soluble protein from hybrids Savita (T7 x M12) and Kirti (Suman x BN) were extracted and loaded in an SDS-PAGE and run in a gel unit. The gel indicates proper formation of bands due to differential movement of protein molecules. Cotton varieties like Anjali, LRA5166, Sumangala, L 604, Sahana, PKV Rajat, Suvin, K9, K10, K11, AKH-4, G27, LD327, Arogya, Surabhi, MCU5 and MCU5VT were also used for the extraction of single seed globulin. The development of seed protein profile showed variability among the genotypes. In genotypes LRA 5166 and Anjali single seed proteins were extracted and separated through electrophoresis for the identification of genetic purity among the certified seed lots. The protein profile pattern obtained out of seed storage protein is used for the identification of genotypes.

Genetic purity testing of hybrid seeds using electrophoresis technique in Cotton

The genetic purity testing of cotton hybrids and parental lines viz., Surya (HLS 329 x M 12), DHH 11 (CPD 423 x CPD 420) was conducted through profiling of seed protein globulin. The methods adopted for extraction of seed protein were 1.SDS – PAGE of salt soluble proteins, 2.SDS – PAGE of salt soluble globulins 3. SDS – PAGE of methanol ppt fraction. The method. SDS – PAGE of salt soluble proteins was found suitable. The Electrophoresis analysis for two hybrids (Suyra and DHH 11), using Salt Soluble Globulins expressed polymorphic bands of proteins. The presence of protein bands, position and intensity of bands recorded for all the samples showed greater differences and were utilized for variety characterization and identification. The protein band observed for surya (hybrid), M12 (Male parent) and HLS 329 (Female parent) are different in expression of bands. The expression of bands is nine in hybrid and eight in parents. The additional band expressed at Rm value 0.439 will facilitate for the identification of presence of female parental seeds in the hybrid (Surya) seed lot. The band expressed at Rm value 0.465 in Surya and HLS 329 confirm that the particular F1 seed would have been derived from using HLS 329 as female parent. This will facilitate the identification of other seeds particularly the male in hybrid seed lot or other cotton variety seed in hybrid seed lot. Similarly, the presence of band at Rm values 0.325 and 0.465 in CPD 423 (female) will facilitate the identification of admixtures in hybrid DHH 11.

Effect of sowing time on production and quality of cotton hybrid seed in different zones

In seven cotton hybrids Savita (T7 X M12), Surya (T13 X M12), Sruti (70E X RSP 4), Kirti (Suman X BN), HB 224 (LRA 5166 X P4), LHH 144 (PIL 48 X PIL 8), NHH 44 (BN 1 X AC 738) it was found that sowing of parents from twentieth August to fifth September resulted in higher hybrid seed yield with enhanced boll setting %.

18.Film coating of cotton seeds with polymers

Film coating cotton seeds having initial viability of 90 %, with polymer polykote @ 3ml/kg of seed diluted with 5 ml water combined with carbendazim (Bavistin) @ 2 g /kg, stored in cloth bag under ambient condition prevented seed deterioration up to 18 months of storage and can retain the viability of 77%. By coating the seeds with polykote @ 3ml/kg of seed diluted with 5 ml water combined with carbendazim (Bavistin) @ 2 g /kg and Imida cloprid @ 7g /kg when seeds are stored in polythene bags, viability can be maintained to 76%

Identification of suitable treatments for cotton seeds

A field experiment was conducted the following seed treatment to assess the seed cotton productivity. T1- No treatment (control), T2- Hydration (10 -12h) and drying at room temperature, T3-2% CaCl2 hydration at room temperature and surface drying, T4- Hydration with 50 ppm GA3 and surface drying at room temperature, T5 – Hydration (10 -12h) and drying at room temperature and dry dressing with Thiram 0.25%, T6-0.5% KNO3 hydration and drying at room temperature, T7- Seed soaking in H2O2 @ 40 mM for 6 hrs and drying at room temperature, T8- Seed soaking in KH2PO4 @ 100 ppm and drying at room temperature, T9- Seed soaking in Succinin Acid @ 0.2% before sowing and drying at room temperature, T10-2% KCl hydration at room temperature and surface drying. During the crop growth all reproductive traits were recorded. It was observed that all seed treatments have enhanced the productivity measured in terms of Plant Height, Number of Sympodia, Number of Bolls/plant and per boll Weight, however, treatments such as Hydration (10 -12h) and drying at room temperature and dry dressing with Thiram 0.25%, Seed soaking in H2O2 @ 40 mM for 6 hrs and drying at room temperature, Seed soaking in Succinin Acid @ 0.2% before sowing and drying at room temperature were more efficacious.

Influence of pulsed magnetic seed treatment on seed cotton productivity

Seeds of twenty two cotton genotypes were subjected to pulsed magnetic seed treatment @ 5 hours for fifteen days continuously with different field strength as T 1 – Control; T 2- Field intensity(nT) 750nT, 1500nT, 500nT; T 3- Field intensity(nT) 1500nT, 500nT, 750nT; T 4- Field intensity(nT) 500nT, 750nT; T 5- Field intensity(nT) 750nT. Seed quality assessment showed significant enhancement in viability in all the genotypes especially with treatment of seed in magnetic field intensity of 750nT followed by 1500nTand 500nT. However, when the seeds are sown in the field for the estimation of the effectiveness of seed treatment in terms of seed cotton productivity, the treatment effect was non-significant, though there were numerical differences. Significant difference among genotypes was observed, irrespective of magnetic field strength for seed treatment.

Relative performance of seed treatments on cotton seed quality enhancement and productivity

Ten halo priming seed treatment, CaCl2 hydration @2%, KH2PO4 priming @450 ppm, KCl priming @1.0% and MnSO4 seed soaking @0.1% enhanced, significantly the seed viability by 17 to 19 % and seedling vigour. Among the acids such as Boric acid @ 0.001%, ascorbic acid @300mg/lit, salicyclic acid@300mg/lit, succinic acid@0.2% used for priming cotton seeds under ambient condition, succinic acid@0.2% primed seeds recorded significantly higher viability (84%). Cotton seeds coated with eleven coating materials and it was found that among those Arappu leaf powder @ 100 g/kg and Turmeric rhizome powder @ 20g/kg increased the seed viability by 10%, seedling length and vigour. The five growth hormones used for priming the seeds such as GA3 @100 ppm, IAA@100 ppm, IBA@100 ppm, BAP@10 ppm and Zeatin@20 ppm, seed priming with GA3 @100 ppm and Zeatin@20ppm enhanced seed viability significantly by 7% and seedling length. Among the 12 botanicals and organics used for priming cotton seeds Prosopis leaf extract @1.5%, Neem leaf extract @ 1%, Dasakavya @ 2.5% and Panchakavya@10% enhanced the seed viability significantly by 10 to 13% with significant increase in seedling length. Dry seed treatments with protectants showed five to nine percent seed viability enhancement, among the eight treatments applied Thiram @ 2g/kg (5%), Bavistin @ 2g/kg (6%), Trichoderma @ 10g/kg (6%) and Turmeric rhizome powder @ 20g/kg (9%) enhanced seed viability and seedling vigour. Seeds treated with Imida chlorpid (7g/kg) alone has shown numerical increase in seed viability with significant increase on seedling vigour. The productivity of treated seeds observed in terms of field emergence, seedling growth, dry matter accumulation, number of sympodia, bolls/plant, boll weight and seed cotton yield/plant showed that priming seed treatment with CaCl2 @2%, H2O2 @ 60mM, KH2PO4@450ppm, Succinic Acid @0.2%, KCl@1.0%, Prosopis leaf extrac t@1.5%, MnSO4 @ 0.1% and Seed coating with trichoderma @10g/kg and Turmeric rhizome powder@ 20g/kg, Neem leaf extract @ 1.0%, Dasakavya @2.5%, Panchakavya @10%, Thiram @ 2g/kg, Bavistin @ 2g/kg, Trichoderma @10g/kg, MnSO4 @ 0.1%, Arappu leaf powder @ 100 g/kg, Turmeric rhizome powder @ 20g/kg, significantly increased the field emergence, seedling growth, dry matter production, boll number/plant, boll weight, seed index and seed cotton yield/plant. The fiber properties such as 2.5 span length, uniformity ratio, fiber strength, and micronaire has not been influenced due to seed treatments.

Effect of pulsed magnetic seed treatment on aged cotton seeds

Cotton seed lots of genotypes namely Sahana, Supriya, Surabhi, JLH-168 and JCC-1 aged 12, 24 and 36 months, treated with pulsed magnetic field strength of 500 nT, 750 nT and 1500 nT for five hours a day for 15 days continuously and in combination of 500 nT (15 days), 750nT+1500nT (30 days), 500nT+750nT+1500nT (45 days) revealed that germination in 36 months stored Sahana seeds increased significantly 22%, 22% and 29% respectively, when seeds were exposed to 1500 nT, 750nT+1500nT and 500nT+750nT+1500nT pulsed magnetic field strength, over untreated seeds. In 24 months, old seeds the improvement was 17, 20 and 24%, respectively. In 12 months, old seeds this was 14, 11 and 11 % respectively. This indicates that more aged seed lots are more responsive to seed treatments that less aged seed lots.

Performance of PPFM seed treatment on germination enhancement under differential soil moisture

Delinted cotton seeds were primed with one percent PPFM culture formulation sown in pots filled with sand medium and soil moisture regulated from 10 to 50% for 12 days, showed an improvement in germination (71.5%) as against (57.5 %) untreated seeds at 10% soil moisture. The arithmetic increase of seed germination of primed seeds was noticed up to 35% soil moisture. Sowing primed seeds on soil moisture above 35% did not encourage seed germination. Sowing primed seeds in 15 to 25% soil moisture followed by watering once in two days also favoured seed germination. Therefore, cotton seed priming with PPFM would be useful for taking up seed sowing even when soil moisture is sub optimal provided that the same soil moisture would be maintained till the end of germination period.

Studies to improve the seed setting efficiency in cotton

Seed setting efficiency in cotton flower differ significantly due to pollination attractants and the duration of application. Among the pollination attractants Rose extracts (10%) Dasakavya (10%) recorded higher seed setting efficiency. The interaction of Pollination attractants and varieties showed no significance; however, the duration of spray and varieties have shown significant difference. The pollination attractant had also shown significant difference for seed index and seed cotton yield. Among the seed setting treatments tried foliar application of CICR-Consortia (S) (0.1%) and NAA @40 ppm have enhanced the seed setting efficiency and seed index over control. DUS testing for the establishment of Distinctiveness, Uniformity and Stability was carried out for 471 new candidate varieties, 328 Variety of common Knowledge, 2 Farmers variety, 184 Essentially derived variety and 183 Initial variety.

Implementation of PVP legislation and Distinctiveness Uniformity and Stability (DUS) Testing in Cotton

Objectives: Establishment and maintenance of database on extant cotton varieties Conduct of DUS test of New, VCK and Farmers varieties Maintenance breeding of reference cotton varieties Morphological characterization of cotton varieties Registration of extant cotton varieties

DUS Test centers

Maintenance of Reference collection at CICR, CBE

DUS testing at participating centers

Status of applications for protection of new/extant/essentially derived/common knowledge varieties under PPV&FRAct,2001

ICAR-CICR has been maintaining and multiplying CICR released Non Bt varieties (Suraj, Surabhi, LRK 516, LRA 5166, CNA 1003, 1028,1032,1054 & five notified Bt varieties (Suraj Bt, Rajat Bt, PKV 081 Bt, GJHV 374 Bt, RS 2013 Bt 6) developed and released by ICAR-CICR for demonstrations and commercialization during the years 2019-21. The quality TFL seeds of Bt varieties produced to the tune of 45.87q during 2019, 2020 and 2021 & were distributed among the State Seed Corporations and State Agriculture Universities. Efforts for commercialization of the above Bt varieties were also undertaken after entering an MOU with Private Seed Companies, SAUs & State Seed Corporations. The quality seeds of 994.5 Kg were distributed under various field demonstrations and 1021 Kg were supplied for commercialization of Bt varieties through MOUs signed with different private seed companies as well as State Seed Corporations for Maharashtra and Telangana.

There is lot of demand for Non Bt varietal seed also from Organic farmers/ NGOs, hence Breeder as well as TFL seed production of these were taken up during these years. Thus a total Breeder Seed of 38.40q; TFL seed of 2.318q were produced. TFL seed is being produced at Farmers’ fields also in Participatory mode to the tune of 12.56q. Regular visits were made to educate & train them on Isolation distance & rogueing to maintain varietal genetic purity. Later the Seed Cotton was purchased on buy back system after signing MoU at 10% more than the market value/rates.

Besides Cotton production, to maintain Isolation Distance & add revenue to the existing Revolving Fund, other crops like Red Gram, Gram, Linseed, Lakodidal, Wheat etc were taken up. Thus under this ICAR Seed Project, various classes of Seed were produced for MSSC, Akola & NSC, Haryana viz., 308q Certified Seed (CS) of Wheat cv. PBW 550 & HD 2967, 107.4q CS of Red Gram cv. BSMR-736, 52.4q CS of Gram cv.Jaki 9218, 3.23q Foundation Seed (FS) of Linseed cv. PKV NL 260 & 6.0q CS of Lakodidal cv.Mahavitra.