(c) What is the importance of pollen storage ? Explain the methods adopted for storage of pollen grains. Add a note on test tube fertilization.

Importance of Pollen Storage

Pollen storage is a cornerstone in modern plant breeding, genetic conservation, and fundamental research. Its significance stems from the ability to extend the functional lifespan of pollen grains, which are naturally ephemeral.

• Overcoming Flowering Asynchrony: Many plant species or varieties flower at different times, making cross-pollination difficult. Storing pollen allows breeders to synchronize pollination events, enabling hybridization between parents that would otherwise not flower concurrently.
• Facilitating Hybridization and Breeding Programs: Stored pollen provides a ready supply of male gametes, improving the efficiency of breeding programs. It eliminates the need to continuously grow pollen-producing parent plants, saving resources and time. This is particularly useful for fruit trees with long juvenile phases where producing pollen for hybridization can be time-consuming.
• Germplasm Conservation: Pollen banks act as vital reservoirs of genetic diversity. They preserve desirable traits and valuable genotypes, including those of endangered species or unique cultivars, against genetic erosion. This complements seed banks and other germplasm conservation strategies, especially for species that do not produce viable seeds or are clonally propagated.
• International Exchange of Genetic Material: Stored pollen can be easily transported across geographical boundaries, facilitating the exchange of exotic nuclear genetic diversity between research institutions and breeding centers globally, without the logistical complexities of transporting whole plants or seeds.
• Research in Pollen Biology and Allergies: A continuous supply of viable pollen is essential for studying pollen physiology, germination mechanisms, pollen-pistil interactions, and for research into pollen allergies.
• Commercial Crop Production: In some commercial fruit productions, stored pollen can be used for supplementary pollination to achieve desired fruit set and yield, especially when natural pollination is insufficient.

Methods Adopted for Storage of Pollen Grains

The primary goal of pollen storage methods is to reduce metabolic activity and prevent degradation, thereby extending viability. Methods are broadly categorized into short-term and long-term storage.

1. Short-Term Pollen Storage

These methods aim to extend pollen viability from days to a few weeks or months by controlling environmental conditions.

• Temperature and Humidity Control:
  • Refrigeration (0-10°C): Storing pollen in refrigerators significantly slows down metabolic processes. Optimal relative humidity (typically 0-30% RH) is often maintained using desiccating agents like silica gel to prevent moisture absorption, which can reduce viability. For instance, pollen of certain apple cultivars stored at 4°C can prolong viability 1-5 fold.
  • Desiccation: Reducing the moisture content of pollen grains is crucial for extending viability. Pollen is dried under controlled conditions, often with desiccants such as silica gel or saturated salt solutions to achieve specific low humidity levels (e.g., 7-20% moisture content for cryopreservation).
• Storage in Organic Solvents: Some studies have explored storing pollen in non-polar organic solvents like diethyl ether, benzene, or petroleum. This method aims to induce a state of "absolute dormancy" where metabolic activity is almost halted. However, its efficacy varies greatly among species.
• Controlled Atmosphere Storage: Manipulating the gas composition in the storage environment, such as reducing oxygen levels or increasing CO2, can sometimes extend pollen viability by further lowering metabolic rates. However, excessive CO2 can also suffocate pollen.

2. Long-Term Pollen Storage (Cryopreservation)

Cryopreservation is the most effective method for long-term preservation, extending pollen viability for years, or even decades.

• Cryopreservation in Liquid Nitrogen (-196°C):
  • Principle: At ultra-low temperatures, all metabolic and enzymatic activities are virtually ceased, preventing cellular damage and maintaining pollen viability for very long durations.
  • Process:
    1. Collection and Dehydration: High-quality, viable pollen is collected. The moisture content is then carefully reduced (e.g., to 5-20% depending on the species) using desiccating agents (like silica gel or dry air) or saturated salt solutions. This step is critical to prevent ice crystal formation, which can damage cell structures during freezing.
    2. Packaging: Dried pollen is placed into sterile cryovials, which are labeled and sealed.
    3. Freezing: The cryovials are then rapidly cooled and immersed directly into liquid nitrogen (-196°C) or stored in its vapor phase (-150°C). Some protocols involve controlled freezing rates.
    4. Storage: The vials are stored in specialized cryotanks.
    5. Thawing and Rehydration: For use, pollen is rapidly thawed and often rehydrated under controlled conditions to restore its physiological activity before pollination.
  • Advantages: Offers the longest preservation period, high genetic stability, and is suitable for creating pollen cryobanks for a wide range of species, including many fruit and nut crops like pecan, walnut, pistachio, and date palm.
• Freeze Drying (Lyophilization):
  • Principle: This method involves freezing the pollen and then removing water by sublimation under vacuum. This gentle process preserves cellular structure.
  • Process: Pollen is rapidly frozen to sub-zero temperatures (e.g., -60°C or -80°C) and then subjected to a vacuum, causing ice to sublimate directly into vapor.
  • Application: Successfully preserves desiccation-tolerant pollen for extended periods, maintaining nutritional quality for several years.

Test Tube Fertilization (In Vitro Fertilization in Plants)

Test tube fertilization, or in vitro fertilization (IVF) in plants, refers to the process of achieving fertilization outside the plant body under controlled laboratory conditions. This technique is invaluable for overcoming various reproductive barriers encountered in conventional plant breeding.

Mechanism and Process:

1. Isolation of Gametes/Reproductive Structures: Male gametes (from pollen grains) and female gametes (egg cells within ovules) are isolated. Pollen is typically germinated in a suitable nutrient medium in vitro to produce pollen tubes. Ovules, or even isolated egg cells, are also cultured separately.
2. In Vitro Pollination: Instead of relying on natural pollination, pollen grains or pollen tubes are directly applied to cultured ovules, placentae, or even stigmas under sterile conditions.
   • In Vitro Stigmatic Pollination: Emasculated flowers are sterilized and cultured. Pollen from ripe anthers is then placed on the stigma in vitro. This method is used when premature ovary abscission prevents seed set in vivo.
   • In Vitro Placental Pollination: Unfertilized ovules attached to placental explants are dissected, cultured on a nutrient medium, and then pollinated with viable pollen. This is particularly useful for overcoming incompatibility barriers in the stigma or style.
   • In Vitro Ovular Pollination: Isolated ovules are cultured and directly pollinated with germinated pollen or pollen tubes.
3. Gamete Fusion: The pollen tube grows through the cultured female reproductive tissue and discharges male gametes, leading to fusion with the egg cell (syngamy) and the central cell (triple fusion), forming a zygote and primary endosperm nucleus, respectively. In some advanced cases, direct fusion of isolated sperm and egg cells has been achieved (e.g., in maize).
4. Embryo and Plantlet Development: The resulting zygote is then cultured to develop into an embryo, which subsequently grows into a plantlet in vitro. These plantlets can then be transferred to soil and grown to maturity.

Applications of Test Tube Fertilization:

• Overcoming Incompatibility Barriers: It bypasses pre-fertilization barriers such as pollen-stigma incompatibility, slow pollen tube growth, or bursting of pollen tubes in the style, which often hinder wide crosses (intergeneric or interspecific hybridization).
• Producing Novel Hybrids: Enables the creation of new hybrid plants that cannot be obtained through conventional crossing techniques, by fusing gametes from distantly related species.
• Haploid Production: Can be used for producing haploid plants through parthenogenesis.
• Studying Fertilization Physiology: Provides a controlled environment to observe and manipulate the cellular and molecular events of fertilization in higher plants.
• Genetic Transformation: Offers potential advantages in genetic transformation protocols, potentially avoiding the need for antibiotic or herbicide resistance genes as selection markers.