Previously we have about of plant tissue culture (PTC) for the rapid multiplication. In PTC we can use various parts such shoot tip for shoot tip culture, leaf, etc. as explant.
Imagine producing thousands of identical, healthy plants from just a small piece of tissue using PTC technique.
Now let think about the plant and environment interaction! You will find abundant of organism affecting the plant such as bacterial, virus, fungi. among them Virus is one the factor that reduced crop yield and there is very limited treatment for virus.
It is difficult to identify whether plant is infected with virus at time we are taking it as culture as some time it may present asymptomatic. It is also fact that virus distribution in different parts of the plant have difference in infection (present high/low concentration/ or absent in some part).
This difference in distribution of infection of virus led the hypothesis that it could be used to produce the virus free plant. The meristem is one of such part in the part that is free from infection of virus (see example below).
Here, we’ll explore how this powerful tissue known as the meristem, can help us rapidly propagate plants and protect them from viruses, benefiting agriculture on a large scale.
In the core of the shoot tip, we find a very special type of tissue called the meristem. The meristem consists of undifferentiated cells capable of dividing continuously, which is why it serves as the perfect starting material for shoot tip culture. These cells not only divide but also give rise to other cells that grow, stretch, and differentiate, allowing the plant to develop.
Meristematic tissue is present in various parts of a plant. For example, at the very tip of the shoot, we have the shoot apical meristem, responsible for the plant’s vertical growth. Similarly, in the roots, we have the root apical meristem. Depending on where they are located, meristematic tissues are categorized into three types:
- Apical Meristem: Found at the tips of roots and shoots, facilitating vertical growth.
- Intercalary Meristem: Located between the nodes of plants, like grasses, and responsible for increasing plant height.
- Lateral Meristem: Found along the sides of roots and stems, contributing to the plant’s thickness and girth.
The shoot apical meristem is especially important in shoot tip culture because it contains totipotent cells, meaning these cells have the ability to regenerate into a whole new plant.
As we dive deeper into the topic, it’s important to differentiate between meristem culture and meristem tip culture. Meristem culture involves only the true meristematic dome, while meristem tip culture includes the meristematic dome plus a few surrounding leaf primordia. Although the terms are often used interchangeably, in practice, meristem tip culture is more common for producing virus-free plants
Shoot tip culture is a vital technique in plant biotechnology, widely used for rapid clonal propagation and the production of virus-free plants.
This method utilizes the meristematic tissues at the shoot tips—regions where active growth occurs—to produce identical copies of a plant quickly.
Plants produced from shoot tip culture are not only true to type but can also be free from viruses and other pathogens, making them highly valuable in agriculture and horticulture.
Table of Contents
Key Concepts:
Shoot Tip Culture:
- This technique involves the in vitro (in a lab) culture of shoot tips or apical meristems—tiny growing points at the tips of stems or branches.
- The explant (small piece of tissue) is placed on a nutrient medium under sterile conditions to grow and develop into a whole plant.
Clonal Propagation:
- Clonal propagation refers to the asexual reproduction of plants to produce exact genetic copies (clones) of the parent plant.
- Shoot tip culture allows for the mass production of genetically identical plants, ensuring uniformity in crops like fruits, flowers, and ornamentals.
- It is faster than traditional methods of vegetative propagation, like cuttings or grafting, and can produce thousands of plants from a single shoot tip in a short time.
Brief history of Shoot tip culture
- 1946: Ball successfully cultures the meristem tips of Nasturtium (Tropaeolium majus), creating rooted plants.
- 1949: Li~maset and Cornuet discover that viruses are often absent or undetectable in the apical meristem, highlighting its potential for virus-free plant production.
- 1952: Morel and Martin experiment with Dahlias, proposing that isolating and culturing apical meristems can yield virus-free plants.
- 1955: Morel and Martin confirm their hypothesis by producing virus-free plants from six potato cultivars using meristem tip culture.
- 1960: Morel induces multiple protocorm formations in Cymbidium orchids, enhancing the technique for rapid clonal multiplication.
- 1962: The discovery of cytokinin and improved tissue culture media by Murashige and Skoog further enhances meristem tip culture, making it commercially viable for mass propagation of various crops.
- 1985: Advances in cryobiology extend the use of meristem tip culture to germplasm storage.
Recent developments: Meristem tip culture is adopted as a tool for gene transfer in higher plants, reflecting its versatility in modern plant biotechnology.
Process of Shoot Tip Culture:
- Selection of Explant– A small piece of the shoot tip or apical meristem (around 0.1 to 1 mm) is carefully excised from the donor plant. The explant is usually free from vascular tissues, as viruses are often found in these areas.
- Sterilization: To prevent contamination, the shoot tip is treated with sterilizing agents like sodium hypochlorite or alcohol.
- Inoculation: The sterilized shoot tip is placed on a nutrient medium containing essential nutrients, growth regulators (like auxins and cytokinins), vitamins, and sometimes antibiotics to support growth.
- Growth & Multiplication: In the sterile, controlled environment of the culture medium, the shoot tip starts to grow and produce new shoots.
- Over time, these shoots can be divided and subcultured to produce more plants.
- Rooting: Once the shoots have developed, they are transferred to a medium that promotes root formation, leading to the development of a complete plant.
- Acclimatization: The plants are then gradually introduced to external conditions (temperature, humidity, light) before being planted in soil.
Applications of Shoot Tip Culture:
- Rapid Clonal Propagation:
- Allows mass production of plants in a relatively short time.
- Plants such as banana, sugarcane, and orchids benefit greatly from this technique, as large quantities of high-quality plants can be produced year-round.
- Production of Virus-Free Plants:
- One of the most significant applications is the production of virus-free plants.
- Viruses are often absent in the meristematic regions (shoot tips) of infected plants, so culturing this tissue can produce plants that are completely free of the virus.
- This is especially crucial for crops like potatoes, strawberries, and citrus, which are prone to viral infections that reduce yield and quality.
- Preservation of Germplasm:
- Shoot tip culture can be used for the long-term storage of plant germplasm (genetic material), allowing for the conservation of rare or endangered plant species.
- Improvement of Crop Quality:
- By ensuring the plants are genetically identical, shoot tip culture maintains the desirable traits of the parent plant, such as disease resistance, high yield, or superior flower color.
Advantages of Shoot Tip Culture:
- Virus-Free Plants: Produces plants that are free of viruses and other pathogens, improving crop health and yield.
- Rapid Multiplication: Large numbers of plants can be produced in a short time from a single shoot tip, making it highly efficient.
- Genetic Uniformity: Ensures all propagated plants are genetically identical to the parent, maintaining the quality and traits of the original plant.
- Space Efficiency: Requires minimal space compared to traditional plant propagation methods.
Challenges in Shoot Tip Culture:
- Contamination: Maintaining sterile conditions is crucial; any contamination can ruin the culture.
- Cost: The initial setup, including lab equipment and skilled personnel, can be expensive.
- Acclimatization Stress: Plants cultured in controlled environments may face stress when transferred to natural soil conditions.
Example:
Experimental study 1
Use of Tissue Culture Techniques for Producing Virus-Free Plant in Garlic and Their Identification through Real-Time PCR
Study conducted by – Taşkın H, Baktemur G, Kurul M, Büyükalaca S.
Problem:
The main problem identified in garlic production in Turkey is the prevalence of pests and diseases, particularly those transmitted through contaminated seedlings. Key issues include:
- Significant yield losses (30% to 100%) due to nematodes, white rot disease, and viruses like onion yellow dwarf virus and leek yellow stripe virus.
- Ineffective chemical control methods for viral diseases, necessitating the need for virus-free plant propagation.
- Insufficient effectiveness of the shoot tip culture technique for producing virus-free plants.
Objective:
The study aims to address these issues through the following objectives:
- Compare the effectiveness of the meristem culture technique against the shoot tip culture technique for obtaining virus-free garlic plants.
- Evaluate the micropropagation success of two different nutrient media and two garlic species (A. tuncelianum and A. sativum).
- Assess the effectiveness of real-time PCR for the detection of viruses in garlic plants
Media and growth condition
- Garlic Species: A. sativum and A. tuncelianum propagated via meristem and shoot tip cultures.
- Surface Sterilization: Cloves treated with 25% sodium hypochlorite for 20 minutes, followed by 4-5 washes with sterile water.
- Extraction: Meristem and shoot tips extracted using sterile forceps and scalpels under a stereobinocular microscope in laminar flow.
- Culture Conditions: Placed in glass tubes with MS nutrient medium and incubated at 25°C, under 3000 lux light with an 8-hour dark and 16-hour light photoperiod.
- Micropropagation: Germinated meristem and shoot tips transferred to two nutrient media:
- Medium 1: MS with 0.5 mg/L 2-IP, 0.2 mg/L NAA, and 30 g/L sucrose.
- Medium 2: MS with 2 mg/L BA, 0.5 mg/L IBA, and 30 g/L sucrose.
- Observation Period: After 6 weeks, the number of shoots per plant recorded, and shoots were subcultured.
- Experimental Design: Completely randomized design with three replications and thirty explants per replication. Real-Time PCR Studies was done to detect virus.
Result
In vitro plants from meristem and shoot tip cultures tested for onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV) using real-time PCR assay.
Virus Detection:
- Meristem Culture: No viruses detected in garlic plants.
- Shoot Tip Culture:
- OYDV detected in 80% of A. tuncelianum and 73% of A. sativum plants.
- LYSV detected in 67% of A. tuncelianum and 87% of A. sativum plants.
Conclusion:
Shoot tip culture is a powerful tool in plant biotechnology, offering immense potential for rapid clonal propagation and virus elimination. It plays a vital role in ensuring the large-scale production of high-quality, disease-free plants, contributing to agricultural advancement and biodiversity conservation. As the demand for efficient crop production grows, shoot tip culture remains a key technique for sustainable plant propagation.
References
Taşkın H, Baktemur G, Kurul M, Büyükalaca S. Use of tissue culture techniques for producing virus-free plant in garlic and their identification through real-time PCR. ScientificWorldJournal. 2013 Jul 7;2013:781282. doi: 10.1155/2013/781282. PMID: 23935432; PMCID: PMC3725790.
In vitro plant regeneration using shoot tip culture in commercial cultivar of sugarcane*
SANDEEP BIRADAR, D. P. BIRADAR, V. C. PATIL, S. S. PATIL AND N. S. KAMBAR
Karnataka J. Agric. Sci., 22(1) :(21-24) 2009
Pant, B., & Thapa, D. (2012). In vitro mass propagation of an epiphytic orchid, Dendrobium primulinum Lindl. through shoot tip culture. African Journal of Biotechnology, 11(42), 9970-9974.
Saeed, N. A., Zafar, Y., & Malik, K. A. (1997). A simple procedure of Gossypium meristem shoot tip culture. Plant Cell, Tissue and Organ Culture, 51, 201-207.