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Tissue Culture

Tissue Culture facility at LAES Keravat came into operation in 1992 and was funded by German Development Service (GDS). Its main operations was receiving, maintaining and also producing materials for research and distribution to farmers. The Tissue Culture laboratory in Keravat has a medium, kitchen, office and the growth/culture room. The First tissue culture trials were carried out on sweet potato (ipomea batatas) and Galip (Canarium Indicum). Success rate of Galip tissue culture using the embryo and apical buds was good, however a quarter of all established explants were contaminated by mould agents like Penicillium and Aspergillus spp. On the other hand, sweet potato varieties were received from overseas and multiplied and about 300 plants were produced and transferred to pots.

Project/Research

Activities carried out by the laboratory are routine maintenance of the crop germplasm in the laboratory and multiplication of disease free sweet potato materials for the Immediate Quarantine Facilities Screen house and multiplication of potato plantlets for Mid Altitude areas in the New Guinea Islands Region.

The tissue culture laboratory maintained a germplasm collection of disease free sweet potato, potato, yam cassava, taro and other crop varieties such as banana. The project is hoping to introduce other crops such as pitpit, noni and Galip in to tissue culture in the future.

Objectives
The project has two main objectives as follows;

  1. To establish and conserve germplasm collection of sweet potato, potato and other selected crop species in tissues culture
  2. To multiply genetic materials for Immediate Quarantine Facility Screen House and research projects
  3. Activities
    The main activities involved in tissue culture work at Keravat are as follows;

    1. COLLECTION OF MATERIALS

  • Explants must be taken from the most pathogen-free parts of the host plant. The plant parts may include buds, suckers, and seeds; shoot tips or meristem tip and/ or nodal parts of the plant.
  • The meristem tip and/or shoot tip are free from virus as it is the most isolated and clean part of the plant. That is why one main use of meristem tip culture is to eliminate pathogens.

2. CLEANING AND SURFACE STERILISING OF EXPLANT

  1. Clean explants under running tap water to get rid of any dead tissues or dirt/contaminants on surface of explant.
  2. Immerse the material in a chemical solution called sterilant for alternative period of timing. This process is called surface
    sterilization.
  3. The explants are rinsed consecutively in 3 to 4 rinses of sterilized distilled water.
  4. 3. MEDIA PREPARTION

    Nutrients are essential for the growth and development of the plant; without water and mineral nutrients a plant cannot live inside the cultures vials. Carbohydrate such as sucrose is an essential constituent to the culture media in which plants invitro grow on. Plants that grow invitro have tissues under this condition not fully autotrophic, therefore sugars must be added to the culture medium. Other organic substances such as plant growth regulators also play an important role in the growth and development of invitro plants. A gelling agent is added to make the culture media semi-solid. Below are the steps involved in preparation of a crop medium.

    1. Chemicals are weighed on an electronic balance and mix in an amount of distilled water
    2. PH is calibrated
    3. The medium is mixed together on magnetic stirrer and the crop medium is measured
    4. It is poured into culture vials and autoclaved at a temperature of 1210C for 15 -20 minutes.

    4. INITIATING PLANTS (Culturing explants into tissue culture)

  • Culturing is the actual action taken by inoculating the desired crop parts or explants into culture vessels containing suitable prepared nutrient media.
  • The process is carried out in a laminar airflow cabinet which is preferred to be clean because there is continuous inflow of air which sucks and filters out any existing dust particles or contaminants that maybe available on the surface of the working station.
  • The cultures are monitored regularly by removing contaminated cultures, treating cultures with antibiotic where necessary, checking lights and air conditions, removing and discarding high contaminated cultures and making subcultures for maintenance and cleaning purposes.

5. SUB– CULTURING

Sub- culturing is another process done when the following situation occurs;

  1. The plantlet previously cultured produces multiples of plantlets or shoots.
  2. The culture is contaminated and is cleaned.
  3. It is very important for continuous cleaning because this action reduces the occurrence of contamination and eventually result in the production of clean planting material. It is also important for maintenance and multiplication of the material.

    6. How are the plants “hardened off” from tissue culture to nursery?

    The plantlets have the following characteristics when they are transferred from the lab to the screen house:
  • Fewer Palisade cells – not able to produce its food.
  • Have poor vascular connection between roots and shoots and thus reduce water conduction.
  • The cuticle or waxy layer is poorly developed and so this result in great water loss through evaporation when the plantlet is transferred to a less humid in vivo environment.
    Therefore, when transferring from culture vessels to nursery to hardened off, the plantlets need to be placed into clean, moist, free draining potting mix, in high humidity under shade in a screen house.

7. Deflasking and Potting mix

Deflasking is the process by which the plantlet is removed from the vessel and agar in the roots are thoroughly washed off under running tap water.

At LAES, Keravat, hardening off is carried out in two main ways.

  1. Potting Mix
  2. A mixture of topsoil, sawdust, and sand is prepared.

  3. Jiffy pots and potting mix

Ready mix compost, available from Farmset

8. Potting mix
Potting Mix. A mixture of topsoil, sawdust, and sand.

The potting mix is prepared by sterilizing using heat
The plantlet is transferred to the potting mix ensuring that all agar or gelling agent is washed off and roots are not broken off. It is placed in the nursery under controlled humidity in the screen house and monitored daily

9. WATERING
The plantlets are monitored daily by watering twice every day and any appearance of weed or insects is taken care of by pulling out and spraying with insecticide etc.

As time goes by and the plant starts to grow, the humidity is gradually reduced and irradiance of the sunlight is increased until when the plant is hardened off fully and ready to be transplanted to the field.

PROJECT OUT PUT

The current outputs of the project are;
1. Supply of disease – free planting materials especially sweet potato,
2. Recovering some lost materials through tissue culture
3. Production of other crops such as taro, noni, galip, pitpit through tissue culture.

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