Here is a detailed protocol for the in vitro propagation of papaya, focusing on organogenesis and multiplication. It provides guidance on addressing challenges such as callusing and loss of green colouration.

Papaya (Carica papaya L.) can be efficiently propagated in vitro using shoot tips or axillary buds as explants. Begin by culturing these explants on a Murashige and Skoog (MS) medium, which is a reliable basal medium for papaya micropropagation. To achieve the best results for shoot induction and multiplication, it is recommended to add 0.5 mg/L 6-benzylaminopurine (BAP), 0.1 mg/L naphthaleneacetic acid (NAA), 0.5 mg/L gibberellic acid (GA3), and 40 mg/L adenine sulfate to the MS medium. This combination, known as the proliferation medium (PPRM), supports continuous shoot proliferation and maintains healthy green explants with minimal callus formation. Shoots can be maintained on this medium for extended periods, with multiplication rates exceeding 20 shoots per explant. The shoots typically retain the morphological characteristics of the parent plant (González et al., 2021).

If poor shoot growth or excessive callusing is observed, transfer the shoots to an elongation medium (PELM), which is composed of MS basal salts, 0.1 mg/L NAA, 0.5 mg/L kinetin and 1 mg/L GA₃. This step helps to recover shoot vigour and prepares them for rooting (González et al., 2021). To induce roots, use a half-strength MS medium supplemented with 1 mg/L indole-3-butyric acid (IBA). Agar can be replaced with expanded vermiculite to improve root quality. Once the roots are well developed, acclimatise the plantlets gradually under high humidity, then transfer them to soil. It is recommended that humidity is reduced progressively over four weeks, and survival rates of up to 90% have been reported using this approach (González et al., 2021).

Studies have shown that a combination of approximately 0.54 mg/L BAP and 0.1 mg/L NAA is optimal for shoot multiplication. Media lacking NAA result in fewer shoots and leaves, so maintaining a low level of auxin is important for robust organogenesis and minimising unwanted callus formation (Efendi & Putra, 2017; Wardani et al., 2019). For rooting, it is best to use a medium without BAP, since the presence of auxins can inhibit root development (Wardani et al., 2019; Nguyen et al., 2018).

In summary, successful in vitro propagation of papaya requires careful adjustment of plant growth regulators: moderate BAP and low NAA concentrations for shoot induction and a shift to auxin-rich, cytokinin-free media for rooting. Regular subculturing and prompt transfer to the appropriate medium at each stage will help to maintain explant health and reduce callusing, thereby facilitating efficient organogenesis and multiplication (González et al., 2021; Efendi & Putra, 2017; Wardani et al., 2019; Nguyen et al., 2018).

References:

• Efendi, D., & Putra, M. (2017). Optimisation of in vitro lateral shoots multiplication of papaya (Carica papaya L.) “Callina” with BAP and NAA. Journal of Tropical Crop Science, 4(3), 102–107. https://doi.org/10.29244/JTCS.4.3.102-107
• González, J., Granero, M., & Encina, C. (2021). In vitro long-term cultures of papaya (Carica papaya L.). Horticulturae. https://doi.org/10.21203/rs.3.rs-262638/v1
• Nguyen, V., Yen, C., & Hsieh, C. (2018). Effect of nutritional and growth hormonal factors on in vitro regeneration of papaya (Carica papaya L. cv. Red Lady). Journal of the National Science Foundation of Sri Lanka, 46(4), 499–507. https://doi.org/10.4038/JNSFSR.V46I4.8631
• Wardani, F., Efendi, D., Dinarti, D., & Witono, D. (2019). Perbanyakan Pepaya (Carica papaya L.) ‘Sukma’ In Vitro dari Eksplan Tunas Pucuk sebagai Respon terhadap BA dan NAA. Jurnal Agronomi Indonesia (Indonesian Journal of Agronomy), 47(2), 132–138. https://doi.org/10.24831/jai.v47i2.25115

In vitro propagation of papaya (Carica papaya L.), particularly the Tainung and Aliança cultivars, can be challenging due to issues like chlorosis and excessive callus formation. However, optimizing the culture medium and hormonal balance can enhance shoot induction and multiplication.

🧪 Optimal Culture Medium for Shoot Induction

A study by González et al. (2021) recommends the following Murashige and Skoog (MS)-based medium for shoot induction and multiplication:

6-Benzylaminopurine (BAP): 0.5 mg/L

Naphthaleneacetic Acid (NAA): 0.1 mg/L

Gibberellic Acid (GA₃): 0.5 mg/L

Adenine Sulfate: 40 mg/L

This combination, known as the Proliferation Medium (PPRM), supports continuous shoot proliferation with minimal callus formation and maintains healthy green explants. Shoots can be maintained on this medium for extended periods, with multiplication rates exceeding 20 shoots per explant.

🌱 Addressing Chlorosis and Callus Formation

If chlorosis or excessive callus formation occurs, it may indicate an imbalance in the hormonal composition. Reducing the concentration of BAP and NAA can help mitigate these issues. For instance, a study on the 'Horana Papaya Hybrid 01' variety found that 1.0 mg/L BAP combined with 0.1 mg/L NAA resulted in high shoot induction without callus formation.

🌿 Elongation and Rooting

Once shoots are sufficiently developed, transfer them to an Elongation Medium (PELM) composed of:

MS Basal Salts: Full strength

NAA: 0.1 mg/L

Kinetin: 0.5 mg/L

GA₃: 1.0 mg/L

This medium promotes shoot elongation and prepares the plantlets for rooting.

For rooting, use a half-strength MS medium supplemented with 1 mg/L Indole-3-Butyric Acid (IBA). Agar can be replaced with expanded vermiculite to improve root quality.

🌤 Acclimatization

After rooting, acclimatize the plantlets gradually under high humidity, then transfer them to soil. It is recommended that humidity is reduced progressively over four weeks, and survival rates of up to 90% have been reported using this approach.

🔬 Genotypic Variability

It's important to note that in vitro propagation protocols can vary based on the papaya cultivar. For example, the 'Horana Papaya Hybrid 01' variety showed optimal shoot induction with 1.0 mg/L BAP and 0.1 mg/L NAA. Therefore, it may be beneficial to tailor the hormonal concentrations to the specific cultivar being propagated.

By adjusting the hormonal balance and culture conditions, successful in vitro propagation of papaya cultivars like Tainung and Aliança can be achieved.

In vitro propagation of papaya (Carica papaya L.), particularly the Tainung and Aliança cultivars, can be challenging due to issues like chlorosis and excessive callus formation. However, optimizing the culture medium and hormonal balance can enhance shoot induction and multiplication.

🧪 Optimal Culture Medium for Shoot Induction

A study by González et al. (2021) recommends the following Murashige and Skoog (MS)-based medium for shoot induction and multiplication:

6-Benzylaminopurine (BAP): 0.5 mg/L

Naphthaleneacetic Acid (NAA): 0.1 mg/L

Gibberellic Acid (GA₃): 0.5 mg/L

Adenine Sulfate: 40 mg/L

This combination, known as the Proliferation Medium (PPRM), supports continuous shoot proliferation with minimal callus formation and maintains healthy green explants. Shoots can be maintained on this medium for extended periods, with multiplication rates exceeding 20 shoots per explant.

🌱 Addressing Chlorosis and Callus Formation

If chlorosis or excessive callus formation occurs, it may indicate an imbalance in the hormonal composition. Reducing the concentration of BAP and NAA can help mitigate these issues. For instance, a study on the 'Horana Papaya Hybrid 01' variety found that 1.0 mg/L BAP combined with 0.1 mg/L NAA resulted in high shoot induction without callus formation.

🌿 Elongation and Rooting

Once shoots are sufficiently developed, transfer them to an Elongation Medium (PELM) composed of:

MS Basal Salts: Full strength

NAA: 0.1 mg/L

Kinetin: 0.5 mg/L

GA₃: 1.0 mg/L

This medium promotes shoot elongation and prepares the plantlets for rooting.

For rooting, use a half-strength MS medium supplemented with 1 mg/L Indole-3-Butyric Acid (IBA). Agar can be replaced with expanded vermiculite to improve root quality.

🌤 Acclimatization

After rooting, acclimatize the plantlets gradually under high humidity, then transfer them to soil. It is recommended that humidity is reduced progressively over four weeks, and survival rates of up to 90% have been reported using this approach.

🔬 Genotypic Variability

It's important to note that in vitro propagation protocols can vary based on the papaya cultivar. For example, the 'Horana Papaya Hybrid 01' variety showed optimal shoot induction with 1.0 mg/L BAP and 0.1 mg/L NAA. Therefore, it may be beneficial to tailor the hormonal concentrations to the specific cultivar being propagated.

By adjusting the hormonal balance and culture conditions, successful in vitro propagation of papaya cultivars like Tainung and Aliança can be achieved.