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Methods for hybridseed produciton



METHODS OF HYBRIDSEED PRODUCTION

Hybrid is produced by crossing between two genetically dissimilar parents. Pollen from male parent (Pollen parent) will pollinate, fertilize and set seeds in female (seed parent) to produce F1 hybrid seeds. For production of a hybrid CROSSING between two parents is important, the crossing process will results in heterosis. In self pollinated cross it is difficult to cross but in cross pollinated crops it is easier.

In nature to create genetic variability and for its wider adaptation in different environmental conditions, flowering plants has adopted many mechanisms for cross pollination. Cross-pollination results in genetic heterogeneity and show wider adaptations. Flowering plants have evolved a number of devises to encourage cross-pollination. Those mechanisms are;

  1. Dicliny:Flowers are unisexual. In monoecious plants male and female flowers are borne on the same plant eg., cucurbits, maize, castor and coconut. In dioecious plants male flowers are borne on different plants eg., papaya, cannabis, mulberry.

  2. Dichogamy:Time of anther dehiscence and stigma receptivity are different forcing them for cross-pollination. The time gap between the two may vary from one day to many days. In protoandry anthers dehisce earlier than the stigma receptivity eg; maize, sunflower. In protogyny stigma become recetive earlier than the anther dehisce eg., Pearl millet mirabilis.

  3. Self-incompatibility:self fertilization in avoided by recognizing the self pollen by the stigma. Eg., Brassica, Petunia, Lilium .

  4. Herkogamy:there is spatial separation of the anthers and stigma. Their relative position is such that self fertilization cannot occur. The stigma projects beyond the anthers and therefore pollen cannot land on stigma. Eg., Lucerne stigma is covered with a waxy film. The stigma does not become receptive until this waxy membrane is broken by visit of honeybees resulting in cross-pollination.

  5. Male sterility:Absence or atropy or mis or malformed of male sex organ (functional pollen) in normal bisexual flower. Male sterility is of three types: genetic male sterility, cytoplasm sterility and cytoplasmic- genetic male sterility.

  6. A combination of two or more of the above mechanisms may occur in some species. This improves the efficiency of the system in promoting cross-pollination


Requisites of hybridseed production:

  1. Breeders responsibilities:

    • Develop inbred lines

    • Identification of specific parental lines

    • Develop system for pollen control

  2. Major problems for breeders & producers

    • Maintenance of parental lines

    • Separation of male and female reproductive organs

    • Pollination


Characteristics ofparental lines.

Female Parent Male Parent

High seed yield Good pollen production

Good seed characteristics Long shedding period

Male sterility Plant height

Lodging resistant Fertility restoration

Basic procedures for hybrid seed production

  1. Development and identification for parental lines

  2. Multiplication of parental lines

  3. crossing between parental lines and production of F1

Commercial hybrid seed production demands crossing technique which is easy and also economic to maintain parental lines. Only few crossing mechanisms have been adopted for commercial hybrid seed production they are;

  1. Hand emasculation and pollination

  2. Self-incompatibility

  3. Dicliny : monoecious and dioecious

  4. Male sterility

These techniques are specific to crop floral biology and flowering behaviour. These techniques have their own advantages and disadvantages. Based on the crop behaviour and crossing technique have been adapted for production of hybrid seeds commercially.

Among different techniques self-incompatibility and sex expression have significance particularly in vegetable and flower hybrid seed production.

  1. Hand emasculation and pollination:

    Hybrid seeds are produced manually by modifying the plant structure by removal of male organ from female plant before anthesis. This system is possible only when the male and female parts of a single flower or plants are separate. This is being adopted in bisexual perfect flowers where the androecium is removal with case. By removing the anther column / or male part from female line, the sterility of female line is created and is dusted with the pollen of desired male parent.

  2. Self Incompatibility:

    Self-incompatibility is a mechanism which avoids self fertilization through recognition of self pollen in or on stigma on the female pistil. But when pollen from other plant carried by wind or insects are accepted and sets seeds.

  3. Self-incompatibility will prevents self pollination (inbreeding) and promotes crosspollination (out breeding) and creates genetic variability. SI are seen in hermaphrodite and homomorphic flowers. Self-incompatibility is a widespread mechanism in flowering plants that prevents inbreeding and promotes outcrossing. The self-incompatibility response is genetically controlled by one or more multi-allelic loci, and relies on a series of complex cellular interactions between the self-incompatible pollen and pistil.

    Types of Self-Incompatibility (SI)

    1. Heteromorphic self-incompatibility:

      In this system flowers are of different morphology of the reproductive parts. The morphological differences can be seen visibly in flowers this will coincide with crossibility. The characters affecting this type of SI are style length, filament length, pollen size, exine sculpturing. The presence or absence of other SI mechanisms will not affect cross pollination in heteromorphic SI.

      • Heteromorphic distyle: eg., Primula

        The flowers of primula have style at two different heights.Has two types of flowers

        1. Thrum flower: short style and long anther

        2. Pin flower: has short anthers and long style

        But these two are cross compatible.
        Pin x Pin (ss x ss) : Incompatible mating
        Pin x Thrum (ss x Ss) : Compatible mating
        Thrum x Pin (Ss x ss) : Compatible mating
        Thrum x Thrum (Ss x Ss) : Incompatible mating

      • Heteromorphic Tristyle: eg., Lythrum

        Three types of flowers;

        1. Short style and stamens are mid and long

        2. Mid style and stamen are short and long

        3. Long style and stamen are short and mid

        When homomorphic flowers are crossed it results in incompatible mating and when hetromorphic flowers are crossed it results in compatible mating.

    2. Homomorphic flowers

      Flowers morphological are same, so mating types cannot be recognized by morphological features. This types of self-incompatibility is controlled by ‘S’ alleles. For crossing parental ‘S’ alleles should be different, then only fertilization takes places and seed sets.

      Two types of self-incompatibility;

      1. Sporophytic self-incompatibility

      2. Gametophytic self-incompatibility

      Sporophytic self-incompatibility (SSI)

      SSI is less common or rare when compared to GSI. The rejection of pollen is controlled by S loci which is dominant. The dominance relationship are like S1> S2>S3>……. This type of self-incompatibility is controlled by diploid genotypes of the sporophyte (pollen). Pollen will not germinate on the stigma of the flower that contains either of the two alleles in the pollen so, rejected.

      Gametophytic self-incompatibility (GSI)

      GSSI is more common when compared to SSI. The rejection and acceptance of pollen is controlled by ‘S’ loci. Unlike SSI, incompatibility is controlled by haploid genotype of pollen itself.

      S1 pollen can germinate on pistil of S1S2 but due to common S1 allele the pollen tube growth seizes. Similarly in S2 the pollen tube growth seizes. When S3 pollen come in contact with pistil of S1S2 there will be normal growth of pollen tube and fertilization takes place, this is called as partial compatibility. Where as, S3 and S4 pollen can pierce in to the style (S1 S2) and cause fertilization, called as complete compatibility.

    3. In single gene system there are three types of mating systems they are;

      S1S2 X S1S2 : 0% compatibility
      S1S2 X S2S3 : 50% compatibility
      S1S2 X S3S4 : 100% compatibility

      Pollen rejection and acceptance of cross pollen is a mechanism of complex interactions.

      There are three main interactions;

      1. Pollen –stigma interaction seen in SSI

      2. Pollen- style interaction seen in GSI

      3. Pollen tube- ovule interaction seen in GSI

        Pollen tube- ovule interaction is very rare and seen in some cases of cocoa, pollen tube reaches ovule and effect fertilization but incompatible combination will degenerate the embryo at early stage development only resulting in no seed set.

      1. Pollen-stigma interaction

        This type of interaction is seen in SSI. It includes dry stigma. The stigma has a hydrated layer of proteins know as pellicle. This pellicle is involved in incompatible reaction. Within a few minutes of pollen reaching stigmatic surface, the pollen releases an exine exudates which is glycoprotein. Due to ‘S’ allele proteins interactions induces immediate callose formation in the papillae, this is in direct contact with pollen. Also this callose is deposited on the protruding pollen tube preventing further germination of the pollen. Here stigma is the site of incompatibility reaction. If once pollen cross this stigmatic barrier, there is no further rejection of pollen.

        In case of wet stigma seen in GSI, when pollen sits on the stigma, pollen coat stimulate an unusual form of stigmatic reaction focused beneath the areas of coating. This results in the deposition of fibrogranular electron opaque layer by the extracellular vesicles in the outer layer of the papillae wall. This results in blockage of water passage or loss of osmotic competence by pollen resulting in drying of pollen.

        Pollen tube - style interaction:This is common in gametophytic self-incompatibility. The pollen can germinate and pollen tube growth will pierce the stigma. But the rate of the pollen tube growth is very slow when compared to compatible pollen. This slow growing pollen tube will stops due to exhaustion of reserve materials and deposition of ‘S’ allele polysaccharide at the tip of the pollen tube which blocks the growth of tube. This deposition is limited to the tip and thus will not affect the other compatible pollen tube growth.

      2. Three-way hybrid

        Two SI lines are crossed to get an heterozygote and again crossed with self-compatible parent which has suppressors locus and give F1 hybrid which is self-compatible

      3. Double cross hybrid

      4. Triline hybrid

        Hybrid seed production is done by crossing of two inbreed lines. Have to maintenance these inbreed lines which are self-incompatible. For success of self pollination need to eliminate SI. There are many methods to over come SI

        • Bud pollination (Tatebe, 1939)

        • Washing stigma surface/ pollen grains with organic solvents (Roggen 1974)

        • Mechanical and electric methods (Roggen & Van Dijk, 1972; 1973)

        • CO2 Treatment (Nakanishi et al., 1969)

        • High temperature treatment (Visser, 1977)

  4. Modification of sex

    Hermophrodite flowers has both male and female reproductive organ in a single flower.

    1. Complete flower; flowers containing all the four whorls viz., sepals, petals, androecium and gyneocium.

    2. Incomplete flower: flowers missing any of the one whorl.

    3. Imperfect Flowers- Sexual distinctness: Monoecious (Maize) vs. Dioecious (Hollies, Poplars)

    4. Perfect Flowers- Gamete Maturation Time (Dicogamy):

    5. Production of this unisexual/ imperfect flowers will naturally leads;

      • Outcrossing avoids the deleterious effects of inbreeding depression

      • Promotes heterozygosity, genetic variability, and genetic exchange,

      • Advantageous to the long-term survival and adaptation of a species.

    Monoecious:

    Flowers are unisexual and are present at different position on the same plant. Eg. cucumber. Terminal flowers are male flower. In the middle of the plant is female favouring crosspollination.

    Dioecious: male flowers and female flowers are in different plant. So called as male plant and female plant.

    Sex modification through hormones and chemicals

    Sex expression in dioecious and monoecious plants is genetically determined and can be modified to a considerable extent by environmental and introduced factors such as mineral nutrition, photoperiod, temperature, phytohormones . Amongst these, phytohormones have been found to be most effective agents for sex modification and their role in regulation of sex expression in flowering plants has been documented. The morphological differences in various sex types and their specific metabolic characteristics result from the possession of specific patterns of proteins, enzymes and other molecules. Modification of sex expression in cucurbits has been induced both by changing the environmental conditions and by applying treatments with growth regulators. Auxin treatments increase the female sex tendency while gibberellins cause a shift towards maleness.

    Hormones & Chemicals inducing Femaleness: Auxins- NAA, Etherl, Ethephon, Cytokinis- BA, Brassinosteriods etc.

    Hormones & Chemicals inducing Maleness: GA3, AgNO3, ABA Thio porpinic acid, Pthalimide, Paclobutrazol etc.

    In cucumber AgNO3 found to be potent inhibitors of ethylene action leading to femaleness.It should be sprayed when first true leaf is fully expanded. Gibberlic acid spray will leads to excessive elongation and weakening of plants and there will be increased number of mall formed male flowers with less pollen. In gynoecious cucumber there will be increased number of male nodes when sprayed with silvernitrate and gibberlic acid, which made possible for multiplication of gynoecious in hybrid seed production.

    Environmental sex modification

    Environment has greater influence on the sex modification. But due to introduction of photosensitive varieties or hybrids in modern era of agriculture it has gained less importance. However in seed production it has its influence on sex expression. In cucumber, high temperature and long day length (> 14 hours) favours male flowers. High temperature will extends the flowering of female flowers. As the temperature increases form 19 0C to 230 C the node for first female flowers has also increased from 9.6 to 16.5 number. This clearly indicates that high temperature favours male and delays female flowering.

    Male sex expression of several plant species is favoured by high temperatures and female sex expression by low temperatures. Male sterile mutant of tomato developing male sterile flowers at a minimum temperature of 30°C and normal flowers at lower temperatures. In Brussels sprouts of low temperature effect on the development of the androecium. In onions a slight production of viable pollen by normally male sterile plants above 20 °C.


MaleSterility

Hybrid production requires a female plant in which no viable male gametes are borne. Emasculation is done to make a plant devoid of pollen so that it is made female. Another simple way to establish a female line for hybrid seed production is to identify or create a line that is unable to produce viable pollen. This male sterile line is therefore unable to self-pollinate and seed formation is dependent upon pollen from the male line.

In hermaphrodite flowers pollens are non-functional or inactive or sterile while, female gametes functions normally. It is the inability of plant to produce or to release functional pollen as a result of failure of formation or development of functional stamens, microspores or gametes. Male sterility can be either genetic or cytoplasmic or cytoplasmic-genetic. This prevents autogamy and permits crosspollination. Promotes heterozygosity. Sterility is due to nuclear genes or Cytoplasmic gene or both.

In hybrid seed production process female is a male sterile line crossed with male fertility restorer line to get F¬1 hybrid.

Cytoplasmic male sterility

Cytoplasmic male sterility, as the name indicates, is under extra nuclear genetic control mainly mitochondrial genome. They show non-Mendelian inheritance and are under the regulation of cytoplasmic factors. In this type, male sterility is inherited maternally. In general there are two types of cytoplasm: N (normal) and the aberrant S (sterile) cytoplasms. These types exhibit reciprocal differences. Cytoplasmic male sterility (CMS) is caused by the extra nuclear genome (mitochondria or chloroplast) and shows maternal inheritance. Manifestation of male sterility in CMS may be either entirely controlled by cytoplasmic factors or by the interaction between cytoplasmic and nuclear factors.

  • Stamen (anther and filament) and pollen grains are affected

  • It is divided into:

    • Autoplasmic

      CMS has arisen within a species as a result of spontaneous mutational changes in the cytoplasm, most likely in the mitochondrial genome

    • Alloplasmic

      CMS has arisen from intergeneric, interpecific or occasionally intraspecific crosses and where the male sterility can be interpreted as being due to incompatibility or poor co-operation between nuclear genome of one species and the organellar genome another CMS can be a result of interspecific protoplast fusion. Cytoplasmic male sterility is used in hybrid seed production. In this case, the sterility is transmitted only through the female and all progeny will be sterile. This is not a problem for crops such as onions or carrots where the commodity harvested from the F1 generation is produced during vegetative growth. These CMS lines must be maintained by repeated crossing to a sister line (known as the maintainer line) that is genetically identical except that it possesses normal cytoplasm and is therefore male fertile.

      Disadvantages

      1. insufficient or unstable male sterile

      2. Difficulties in restoration system

      3. Difficulties with seed production

    Cytoplasmic-genetic male sterility

    Male sterility is controlled by an extranuclear genome and often nuclear genes can have the capability to restore fertility. When nuclear restorations of fertility genes (“Rf”) are available for CMS system in any crop, it is cytoplasmic-genetic male sterility; the sterility is manifested by the influence of both nuclear (Mendelian inheritance) and cytoplasmic (maternally inherited) genes. There are also restorers of fertility (Rf) genes, which are distinct from genetic male sterility genes. The Rf genes do not have any expression of their own unless the sterile cytoplasm is present. Rf genes are required to restore fertility in S cytoplasm which causes sterility. Thus N cytoplasm is always fertile and S cytoplasm with genotype Rf- produces fertile; while S cytoplasm with rfrf produces only male steriles. Another feature of these systems is that Rf mutations (i.e., mutations to rf or no fertility restoration) are frequent, so N cytoplasm with Rfrf is best for stable fertility.

    Cytoplasmic-genetic male sterility systems are widely exploited in crop plants for hybrid breeding due to the convenience to control the sterility expression by manipulating the gene–cytoplasm combinations in any selected genotype. Incorporation of these systems for male sterility evades the need for emasculation in cross-pollinated species, thus encouraging cross breeding producing only hybrid seeds under natural conditions.

    In cytoplasmic-genetic male sterility restoration of fertility is done using restorer lines carrying nuclear restorer genes in crops. The male sterile line is maintained by crossing with a maintainer line which has the same genome as that of the MS line but carrying normal fertile cytoplasm.

    Genetic Male sterility:

    Male sterility is controlled by mutations in nuclear genes in the single recessive genes affect stamen and pollen development, but it can be regulated also by dominant genes. MS alleles are generally recessive. A male sterile line is maintained by crossing with heterozygous male fertile line.

    Male sterile plants of monoecious or hermaprodite crops are potentially useful in hybrid program because they eliminate the labor intensive process of flower emasculation Constraint of the use of genetic male sterility

    • The maintenance of the male sterile line. Normally, a GMS line (A-line) is maintained by backcrossing with the heterozygote B-lines (Maintainer lines), but the progeny produced are 50% fertile and 50% male sterile


Solution:

  1. Identify marker genes that are closely linked to ms genes and affect some vegetative characters

  2. Use of environmental and chemical methods that can lead to production of 100% male-sterile seed



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