Hybridization –Types, Procedure, advantages, and limitations.

Hybridization is the process in which two organisms that belong to different varieties, subspecies, species, or even genera are crossed, and it results in the formation of a hybrid that carries genetic characters from both parents. It is the crossing that occurs naturally or by human interference where the genetic materials from two distinct parents are brought together without changing the actual genetic content of the species. It is the process that becomes a strong evolutionary force because the exchange of characters between two distant populations is responsible for creating new variations in nature. Natural hybridization is referred to as the contact between two populations which have evolved separately for long periods, and when they interbreed, the offspring show mixed characters of the two lineages.

Historically, hybridization was taken as a taxonomic difficulty but now it is considered an important evolutionary mechanism that affects plants and animals by modifying their adaptation, survival, and formation of new species. The results of hybridization are diverse because in some cases it promotes adaptation, increases genetic diversity and sometimes forms new species, especially in plants when polyploidy occurs. In other cases, it may cause harmful effects like extinction of rare species by genetic swamping or merging of two distinct populations into one.

In agriculture, hybridization is used widely because it helps in forming synthetic populations and in producing better crop varieties which have high yield, better resistance to diseases and good tolerance to environmental conditions. It is used in crop improvement since it allows new gene combinations to appear, and these combinations is responsible for characters like better growth, stress tolerance and increased production.

Objectives of Hybridization

• It is used to combine desirable characters from two different parents into a single hybrid.
• It is the process that helps in increasing genetic variation within a population.
• It is used for developing new varieties that show better yield and improved growth.
• It helps in producing hybrids with higher resistance against diseases and pests.
• It is used to introduce specific traits like drought tolerance, salinity tolerance or better adaptability.
• It is the method that supports genetic rescue by improving the fitness of weak or declining populations.
• It helps in the formation of new species in plants when polyploidy occurs.
• It is used in agriculture to obtain superior crop types suitable for different climates and soils.

Types of Hybridization

1. Intra-varietal Hybridization– It is the crossing done within the same variety. The hybrids formed maintain the general identity of the variety, and this process is used to increase variation inside the variety so that better individuals can be selected.

2. Inter-varietal or Intraspecific Hybridization– This type is the crossing between two different varieties of the same species. It is used to combine desirable characters present in separate varieties, and the offspring usually show mixed characters from both parents.

3. Interspecific or Intrageneric Hybridization– It is the process of crossing two different species that belong to the same genus. This type is used when certain characters like better resistance or adaptability is not available within a single species.

4. Introgressive Hybridization– In this process, particular genes from one species are transferred into another species by repeated backcrossing. It is used mainly for introducing a useful trait from a wild species into a cultivated one.

5. Distant or Wide Hybridization– This type occurs when crosses are made between different species of the same genus or even between different genera of the same family. It is used to introduce completely new traits when such characters are absent in the species being improved.

Procedure of Hybridization

The procedure of hybridization involves a series of steps, and each step is important because it ensures that the desired characters from both parents is properly combined in the hybrid.

1. Selection of Parents– This is the first step where suitable parents are selected based on the required characters. The parents are usually chosen from well-adapted lines that show the desired traits.
2. Selfing of Parents (Artificial Self-pollination)– It is done to make the parents more homozygous. Selfing helps in removing unwanted characters and the parent line becomes more stable for crossing.
3. Emasculation– It is the removal of anthers from the female parent before pollen is released. This is required to stop self-pollination so that only the selected male parent can pollinate the flower. Different methods used are hand emasculation (forceps or scissor method), hot water treatment, cold water treatment, alcohol treatment, suction method, male sterility and chemical gametocides.
4. Bagging– After emasculation, the flower is covered with a bag made of butter paper or cloth. It prevents unwanted pollen from reaching the stigma and protects the flower until crossing is done.
5. Tagging– A tag is attached to the emasculated flower. It contains information like the date of emasculation, date of crossing and the names of male and female parents. This helps in proper record keeping.
6. Crossing (Artificial Cross-pollination)– Pollen from the male parent is collected and placed on the stigma of the emasculated female flower. This is the main step where the hybrid is formed. Pollen is usually applied with the help of a brush or forceps.
7. Harvesting and Storing the F1 Seeds– After pollination, the crossed flowers mature and the F1 seeds are collected. These seeds are dried, threshed and stored with tags for proper identification.
8. Raising the F1 Generation– In the next season, the stored seeds are planted to raise the F1 hybrid plants. These plants show the combined characters of both parents.

Hybridization Methods of Plant Breeding in Self-Pollinated Groups

There are several methods of improvement of self-fertilized crops by hybridization. These are:

1. Pedigree method or breeding
2. Bulk method or breeding
3. Single seed descent method
4. Back cross method.
5. Multiple cross method

1. Pedigree Method

The pedigree method is the selection technique used mainly for self-pollinated crops. It is the process in which individual plants and their progenies are selected generation after generation so that the desirable characters become fixed. It is the method where a complete record of the ancestry is maintained, and this record helps in studying how the characters are inherited in each generation.

1. First Year (F1 Generation)– The plants selected for hybridization are crossed, and F1 seeds are obtained. These seeds contain the genetic material from both parents.
2. Second Year (F1 Generation)– The F1 plants are grown with enough space so that good quantity of F2 seeds is produced. These seeds are important for forming a large segregating population.
3. Third Year (F2 Generation)– Around 2,000 to 10,000 F2 plants are grown. This generation shows wide variation, and from this population nearly 200 to 500 superior plants are selected.
4. Fourth Year (F3 Generation)– The selected F2 plants are grown in rows, and each row is evaluated separately. From each row, the best 3 to 5 plants are selected and harvested.
5. Fifth and Sixth Year (F4, F5 Generations)– Selection is continued in the same way as in the F3 generation. By the end of the F5 generation, nearly 20 to 50 families remain for the next evaluation.
6. Seventh Year (F6 Generation)– By continuous self-pollination, most lines become homozygous and uniform. The plants that show uniform desirable characters are harvested, and their seeds are bulked to form the base of a new variety.
7. Eighth Year (F7 Generation)– Preliminary yield trials are carried out to examine the performance of the lines. This step is important to check the yield and quality.
8. Ninth to Eleventh Year (F8–F10 Generations)– The superior lines are tested again to confirm their performance. Several characters like height, maturity, lodging, disease resistance and quality are examined.
9. Twelfth to Thirteenth Year (F10, F11 Generations)– The selected variety is multiplied and seeds are prepared for release. These seeds are then given to farmers for cultivation.

Merits of the Pedigree Method

• It allows continuous selection of desirable plants in each generation.
• It helps in studying the inheritance of characters because complete ancestry records is maintained.
• It is suitable for improving self-pollinated crops where homozygosity is required.
• It helps in removing unwanted segregants at early generations.
• It allows the breeder to observe individual plant performance in different generations.
• It produces stable and uniform lines by successive selfing and selection.
• It helps in selecting for traits that show clear expression in segregating generations.

Demerits of the Pedigree Method

• It is a time-consuming method because selection continues for many generations.
• It requires large field area for growing segregating populations.
• It needs careful record keeping of each plant and its progeny.
• It becomes difficult when characters do not express clearly in early generations.
• It demands skilled workers because slight mistakes in tagging or records can affect the whole program.
• It is costly as continuous evaluation and maintenance of families is required.

2. Bulk Method or Breeding

The bulk method is the selection procedure used mainly for self-pollinated crops. It is the process where the segregating population from F2 to F5 is grown in bulk without much individual selection, and the next generation is formed from the bulked seeds. Natural selection acts during the early generations, and proper selection is started only when the lines become more stable. This method was introduced by Nilsson-Eule of Sweden.

1. First Year (F1 Generation)– Hybridization is carried out and F1 seeds are produced. These seeds contain characters from both parents.
2. Second Year (F1 Generation)– Nearly 50 to 100 F1 plants are grown, and all the seeds from these plants are harvested together to form the F2 population.
3. Third Year (F2 Generation)– The F2 plants are grown and their seeds are harvested in bulk again. This keeps the genetic variation intact.
4. Fourth Year (F3 Generation)– The F3 plants are cultivated and F4 seeds are harvested in bulk. No selection is usually done in this stage.
5. Fifth Year (F4 Generation)– The same procedure is followed. F4 plants are grown and all seeds are collected in bulk.
6. Sixth Year (F5 Generation)– Bulk propagation continues till most lines reach the required level of homozygosity. Usually this stage marks the end of the bulk phase.
7. Seventh Year (F6 Generation)– The seeds are now space-planted and individual plant selection begins. This is the start of active selection.
8. Eighth Year (F7 Generation)– The progeny of each selected plant is grown separately. The best progenies are identified and kept for further improvement.
9. Ninth Year (F8 Generation)– Preliminary yield trials are carried out to check the performance of selected lines.
10. Tenth to Twelfth Year (F9–F12 Generations)– Multi-location trials are conducted to examine stability and adaptability. The best line is then multiplied for seed distribution.

Merits of the Bulk Method

• It is simple because detailed pedigree records are not required.
• It needs less labour as early generations do not require much attention.
• Natural selection acts during the bulk phase and may increase the number of superior genotypes.
• It is useful for studying the survival of genes and genotypes under different environmental conditions.

Demerits of the Bulk Method

• It takes a long time to develop a new variety since early selection is delayed.
• The breeder has limited control because natural selection mainly acts in early generations.
• Information on inheritance of characters is not available due to the bulk nature of the population.
• Natural selection may favor plants that survive, but these may not be the highest-yielding or most desirable types.

3. Single Seed Descent Method

The Single Seed Descent (SSD) method is the breeding technique used mainly for self-pollinated crops. It was first suggested by Dr. Goulden in 1939. In this method, one seed from each plant is taken to form the next generation. It allows the segregating generations to advance quickly without much selection in the early stages, and the population becomes homozygous in the later generations. After the lines become stable, proper selection is carried out.

• Hybridization (F1 Generation)
  The method begins with hybridization between two selected varieties. The F1 seeds formed contain the characters of both parents.
• F2 Generation (Bulk Plot Planting)
  The F1 seeds are planted in bulk. No selection is done at this stage. A single seed from each F2 plant is collected to maintain the population.
• F3 to F6 Generations (Single Seed Descent)
  In each of these generations, plants are grown from a single seed taken from every plant of the previous generation. The population is grown in bulk plots, and no selection is carried out. This step helps in maintaining the genetic variability.
• F7 Generation (Single Plant Selection)
  By this time, most plants become homozygous. Now individual plant selection is started, and the best plants showing desirable characters are identified.
• F8 Generation (Plant or Head Rows)
  The progeny of each selected plant is grown in separate rows. These rows help in evaluating the lines because each row comes from one selected plant of the previous generation.
• F9 to F10 Generations (Preliminary Yield Trials)
  Preliminary yield trials are carried out to study the performance of selected lines. Characters like yield and disease resistance are observed.
• F10 and Beyond (Yield Trials)
  Advanced yield trials are conducted at different locations to check stability and adaptability. The superior lines are then taken forward for release as new varieties.

Merits of the Single Seed Descent Method

• It helps in reaching homozygosity quickly because generations advance fast.
• It preserves the genetic diversity since one seed from every plant is taken, keeping the population broad.
• It needs less space and fewer resources in the early generations.
• It reduces labour in the beginning as no selection is done during the early segregating generations.

Demerits of the Single Seed Descent Method

• Selection is delayed to later generations, so early chances of selecting superior plants are missed.
• There is a risk of losing good plants because only one seed from each plant is carried forward.
• Even after fast advancement to homozygosity, the total time needed for yield trials and testing remains long.

4. Back Cross Method

The back cross method is the breeding technique used mainly to improve a good variety by adding one specific character from another parent. It was proposed by Harlan and Pope in 1922. This method is used in both self-pollinated and cross-pollinated crops. The purpose of this method is to transfer a single desirable character like disease resistance, drought tolerance or early maturity into a well-performing variety without disturbing its other characters.

The recurrent parent is the desirable variety used again and again in crossing, while the non-recurrent or donor parent provides only the needed trait.

• Initial Hybridization (F1 Generation)
  The process begins by crossing the recurrent parent with the donor parent. The recurrent parent is generally used as the female. The F1 hybrids carry characters from both parents.
• Backcrossing (BC1 Generation)
  Instead of selfing the F1 plants, they are crossed back with the recurrent parent. This produces the BC1 generation. Plants are selected showing major characters of the recurrent parent along with the required trait from the donor.
• Subsequent Backcrossing (BC2, BC3 Generations)
  The selected BC1 plants are again crossed with the recurrent parent. This is repeated for several backcross generations. In each generation, plants that carry the desired trait and resemble the recurrent parent is selected. Usually 5 to 6 backcrosses are needed.
• Final Selection and Selfing
  After the desired combination of characters is obtained, the selected plants are self-pollinated to make them homozygous. This stabilizes the transferred character.
• Field Testing and Release
  The improved line is tested in field trials to compare its performance with the original recurrent parent. After confirmation, the seeds are multiplied and released for cultivation.
• Special Considerations
  This method works easily when the character is controlled by a dominant gene. If the trait is recessive, extra steps are needed for identifying the right plants. It is also useful in interspecific crosses where characters like male sterility are transferred from one species to another.

Merits of the Back Cross Method

• It is effective for transferring single important traits like disease resistance without changing other characters of the recurrent parent.
• Environmental factors influence the method very little, and several generations can be raised in one year using off-season nurseries.
• It helps in preserving almost all characters of the recurrent parent except the trait added from the donor.
• It reduces the need for long yield trials because the recurrent parent is already a good performer.
• It does not require very large populations, which makes it suitable when resources are limited.

Demerits of the Back Cross Method

• Improvement is restricted to only the specific trait being transferred, and the new variety is not better in other aspects.
• Several backcross generations are required, making the process long and costly.
• There is a chance of transferring unwanted linked traits along with the desired one, a problem known as linkage drag.

5. Multiple Cross Method

The multiple cross method is the breeding procedure used when several desirable characters from different inbred lines are to be combined into one genotype. It is also called the composite cross method. This method is mainly used when the characters are controlled by single genes and each parent contributes one useful trait.

• Initial Crosses
  Several pure lines are selected based on their desirable characters. These lines are crossed in pairs such as A × B, C × D, E × F and G × H. Each pairwise cross produces F1 hybrids carrying characters from both parents.
• Double Crosses
  The F1 hybrids obtained from the initial crosses are then crossed again. The hybrid formed from (A × B) × (C × D) is crossed with the hybrid formed from (E × F) × (G × H). This results in a complex hybrid that contains the characters of all the original lines.
• Further Breeding
  The hybrids formed after the double crosses are grown and further improved by using the pedigree method or the bulk method. The breeder selects the method depending on the characters to be fixed and the objectives of the breeding program.

Merits of the Multiple Cross Method

• It is useful for combining several monogenic traits from different parents into one genotype.
• The hybrids obtained usually show wider adaptation under different environmental conditions.

Demerits of the Multiple Cross Method

• The productivity of multiple cross hybrids may be lower than that of single-cross hybrids.
• Its usefulness becomes limited in areas where disease pressure or risk is not very high.

Hybridization Methods of Plant Breeding in Cross-Pollinated Crops

Hybridization in cross-pollinated crops is the process where different inbred lines are crossed to obtain improved hybrids showing desirable characters. Several methods are used depending on the purpose of the breeding program and the nature of the crop. The common methods include single cross, double cross, three-way cross, top cross and synthetic cross.

Types of Hybridization Methods

• Single Cross– A single cross is the mating between two inbred lines. For example, A × B or C × D. The number of possible single crosses can be calculated by the formula n(n–1)/2. These hybrids usually show the highest hybrid vigour, but seed yield is sometimes low because inbreds used as parents may be weak.
• Double Cross– A double cross is obtained by crossing two single crosses. This involves four inbred lines. For example, (A × B) × (C × D). The number of double crosses is given by (n(n–1)(n–2)(n–3))/4. Double cross hybrids are widely used in crops like maize because they can give high yields without increasing the production cost.
• Three-Way Cross– A three-way cross is produced when a single hybrid is crossed with another inbred line, like (A × B) × C. This method uses the hybrid vigour of the single cross as the female parent and helps in getting good hybrid seed yield with normal grain size.
• Top Cross (Inbred × Variety Cross)– A top cross is the crossing of an open-pollinated variety with an inbred line. The variety is usually used as the female parent. It is used for making new hybrids and is also helpful in testing the combining ability of inbred lines.
• Synthetic Cross– A synthetic cross, also called a poly-cross, is made by crossing many inbreds or sibbed lines without controlled pollination. Seeds from pretested parents are mixed and grown in an isolated area, and natural cross-pollination takes place. This method is useful in forage crops where artificial pollination becomes difficult.

Hybridization Methods of Plant Breeding in Vegetatively Propagated Crops

In vegetatively propagated crops the hybridization method is different because these crops reproduce by asexual means. Crops like sugarcane and potato are multiplied through cloning, so the breeding work depends on selecting improved clones and crossing them to obtain better hybrids. The hybrids formed are again multiplied vegetatively so that the desirable characters remain unchanged.

• Selection of Improved Clones– Clones showing desirable characters like high yield, resistance and good quality are selected. These clones are grown under conditions that help in flowering and seed formation even though their normal reproduction is vegetative. This selection ensures that the chosen clones have the capacity to produce improved offspring.
• Crossing of Desirable Clones– The selected clones are then crossed. Hybridization is done by cross-pollinating the clones to combine their useful characters. The aim of this crossing is to bring the good traits of different clones into one hybrid.
• Multiplication by Cloning– After hybridization, the F1 hybrids are multiplied by vegetative means. Each hybrid plant acts as a new source of clones. These clones are produced through tissue culture, cuttings or other vegetative methods. This helps in preserving the characters of the hybrid without genetic change.
• Development of New Varieties– This method has been used in crops like sugarcane and potato. The new clones developed show better yield, improved quality and resistance when compared with the parent clones.

Uses of Hybridization

• It is used to combine desirable characters from two different parents into one hybrid.
• It helps in producing new varieties showing better yield and quality.
• It is used for introducing disease resistance, drought tolerance or other useful traits.
• It increases genetic variation in a population which helps in selection.
• It supports development of hybrids that show hybrid vigour.
• It helps in forming new species in plants when polyploidy occurs.
• It is used in agriculture to obtain crop types suitable for different climates and soils.

Advantages of Hybridization

• It creates new combinations of genes that may produce superior plants.
• It helps in improving yield, quality and resistance in crops.
• It brings desirable traits from two parents into one hybrid.
• It increases heterozygosity which often results in hybrid vigour.
• It provides a wider genetic base for future breeding work.
• It helps in developing varieties that can adapt to different environments.

Limitations of Hybridization

• It is a time-consuming process because several generations are needed.
• It requires large populations to recover desirable recombinants.
• There is a chance of transferring unwanted characters along with desirable ones.
• Hybridization needs skilled handling for crossing and emasculation.
• The hybrids may show sterility or poor performance in some crosses.
• It becomes difficult when parents differ widely in flowering time or compatibility.

Comparison between Pedigree and Bulk Methods

Pedigree Method

• Selection is done on individual plants from the F2 generation onwards.
• Artificial selection plays the major role, and natural selection has very little effect.
• Detailed pedigree records are maintained for each selected plant and its progeny.
• It usually takes about 14–15 years to develop and release a new variety.
• It is widely used because it allows precise selection of desirable characters.
• The breeder must give close attention from the beginning of segregation.
• Segregating generations are space-planted for easy observation.
• The population size is generally smaller.

Bulk Method

• Segregating generations like F2 onwards are maintained as bulk populations without individual selection.
• Natural selection is the main force acting on the population, with artificial selection applied only when needed.
• No pedigree records are maintained, which simplifies the procedure.
• It takes longer than 10 years for developing a new variety because early selection is delayed.
• It is used less frequently but useful when handling very large populations.
• Requires less attention from the breeder during the bulking period.
• Bulk populations are planted at commercial planting rates.
• Much larger populations are maintained, and natural selection helps in obtaining plants with extreme characters.