3.8 Explant
Regeneration
1. Count the survival of the explants 2–3 weeks after rewarming
and calculate the percentage of survival (see Note 12).
2. Transfer the garlic and shallot explants into culture vessels
containing regeneration medium.
3. Count the regrowth of the explants 6–10 weeks (mint, garlic)
and 8–12 weeks (potato, shallot) after rewarming (see
Fig. 3eÀh).
4 Notes
1. Murashige and Skoog medium can be prepared from single
micro- and macronutrients and vitamins. For convenience
and standardization, powdered medium sourced from a commercial supplier is recommended (Duchefa Biochemie BV,
Haarlem, the Netherlands).
2. To support plant growth and propagation, a combination of
0.1 mg/L NAA (1-naphthaleneacetic acid) and 0.5 mg/L 2iP
(6-(y-y-Dimethylallylamino)purine) can be added to the
media. Also other hormone combinations were published and
proved to be suitable under our conditions, such as 0.1 mg/L
kinetin and 0.1 mg/L IAA (indole-3-acetic acid) or 2 mg/L
BAP (6-Benzylaminopurine) and 0.2 mg/L NAA
(1-naphthaleneacetic acid).
3. For garlic inflorescences, several regeneration media have been
tested successfully. Alternatively, a medium containing MS
medium with vitamin, 0.3 mg/L IAA, 2.0 mg/L 2iP, 3%
sucrose, and 1% agar at pH 5.8 or a medium containing MS
medium with vitamin, 0.3 mg/L IAA, 2.0 mg/L BAP, 3%
sucrose, and 1% agar at pH 5.8 can be used.
4. Check bulbs for insects and sclerotic parts. The storability of
bulbs improves when insects or sclerosis are avoided.
5. Mites are vectors for virus diseases. Therefore, large mite populations promote the spread of infections by directly transporting fungal spores and, hence, weaken also the bulbils and bulbs.
Make sure that mites do not colonize bulbils and bulbs below
the outer scales.
6. Plants can be colonized by microbial endophytes, mainly bacteria, which cannot be removed by surface sterilization. The
outbreak of these microorganisms is visible by white or yellowish exudates or as a more or less permanent tiny veil surrounding the basis of the explant. It can be either tolerated by the
plants or contribute to its death. During sub-cultivation,
microbial endophytes can accumulate and restrict the number
of propagation cycles. Culture vessels showing high endophytic
contaminations need to be removed.
658
Angelika Senula and Manuela Nagel
Regeneration
1. Count the survival of the explants 2–3 weeks after rewarming
and calculate the percentage of survival (see Note 12).
2. Transfer the garlic and shallot explants into culture vessels
containing regeneration medium.
3. Count the regrowth of the explants 6–10 weeks (mint, garlic)
and 8–12 weeks (potato, shallot) after rewarming (see
Fig. 3eÀh).
4 Notes
1. Murashige and Skoog medium can be prepared from single
micro- and macronutrients and vitamins. For convenience
and standardization, powdered medium sourced from a commercial supplier is recommended (Duchefa Biochemie BV,
Haarlem, the Netherlands).
2. To support plant growth and propagation, a combination of
0.1 mg/L NAA (1-naphthaleneacetic acid) and 0.5 mg/L 2iP
(6-(y-y-Dimethylallylamino)purine) can be added to the
media. Also other hormone combinations were published and
proved to be suitable under our conditions, such as 0.1 mg/L
kinetin and 0.1 mg/L IAA (indole-3-acetic acid) or 2 mg/L
BAP (6-Benzylaminopurine) and 0.2 mg/L NAA
(1-naphthaleneacetic acid).
3. For garlic inflorescences, several regeneration media have been
tested successfully. Alternatively, a medium containing MS
medium with vitamin, 0.3 mg/L IAA, 2.0 mg/L 2iP, 3%
sucrose, and 1% agar at pH 5.8 or a medium containing MS
medium with vitamin, 0.3 mg/L IAA, 2.0 mg/L BAP, 3%
sucrose, and 1% agar at pH 5.8 can be used.
4. Check bulbs for insects and sclerotic parts. The storability of
bulbs improves when insects or sclerosis are avoided.
5. Mites are vectors for virus diseases. Therefore, large mite populations promote the spread of infections by directly transporting fungal spores and, hence, weaken also the bulbils and bulbs.
Make sure that mites do not colonize bulbils and bulbs below
the outer scales.
6. Plants can be colonized by microbial endophytes, mainly bacteria, which cannot be removed by surface sterilization. The
outbreak of these microorganisms is visible by white or yellowish exudates or as a more or less permanent tiny veil surrounding the basis of the explant. It can be either tolerated by the
plants or contribute to its death. During sub-cultivation,
microbial endophytes can accumulate and restrict the number
of propagation cycles. Culture vessels showing high endophytic
contaminations need to be removed.
658
Angelika Senula and Manuela Nagel