MSc

The study entitled “Anther culture of Capsicum annuum L. for doubled haploid
production” was carried out at the Department of Plant Biotechnology, College of
Agriculture, Vellayani, Thiruvananthapuram during 2019 - 2021. The objective of the study
was to develop haploids of Capsicum annuum var. Arka Meghana by anther culture.
Standardisation of the suitable stage of bud of Capsicum annuum var. Arka Meghana
for anther culture was carried out by staining the anthers of different bud stages with 1%
aceto-orcein and calculating the percentage of microspores at late uninucleate and early
binucleate stages. Buds collected at six days after bud initiation had 57% of microspores in
the late uninucleate stage and 34% in the early binucleate stage. Buds collected at nine days
after bud initiation had 21% of microspores in the late uninucleate stage and 49% in the early
binucleate stage. Hence the buds collected at six and nine days after bud initiation were found
to be most suitable for anther culture.
The optimum surface sterilization condition for buds was standardised by treating the
buds with 70% of ethanol and 4% of sodium hypochlorite for different time intervals. Surface
sterilization with 70% ethanol for 30 seconds followed by 4% sodium hypochlorite for 15
minutes was found to be most effective.
Anther culture was carried out in nine media compositions for optimization. Anthers
were inoculated onto full strength Murashige and Skoog (1962) medium (MS) and Dumas de
Vaulx (1981) medium (CP) with different concentrations of plant growth regulators Kinetin,
BA, NAA, IAA and 2,4-D with AgNO3 and activated charcoal as additives. The anthers were
incubated at 25°C for two and eight days in darkness. The treatment T5 - MS + 4.00 mg/L
NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10 mg/L AgNO3 incubated at 25°C for
two days in darkness showed callogenesis (3.17%) at six weeks after inoculation. All the
treatments incubated at 25°C for eight days in darkness showed response at sixth week of
inoculation with callogenesis varying from 3.75% to 15.38%. Among the nine treatments,
highest callogenesis of 15.38% was observed in the treatment T5 at sixth week of inoculation.
Globular embryo initiation (1.25%) was observed in the treatment T4 - MS + 4.00 mg/L NAA
+ 1.00 mg/L BA at sixth week of inoculation.
The six treatments that showed good response at 25°C incubation temperature and
eight days darkness were incubated at 35°C in two days darkness. All the treatments showed
response varying from 4% to 15.54% of callogenesis at second week of inoculation.
Callogenesis varying from 10.66% to 34.48% and embryonic calli induction varying from
3.17% to 17.24% was observed in the fourth week of inoculation. At sixth week of
inoculation callogenesis varied from 11.11% to 37.93% and embryonic callus induction
varied from 3.17% to 19.54%. Among the six treatments, the maximum callogenesis of
37.93% and embryonic calli induction of 19.54% was observed in the treatment T5 at fourth
week of inoculation.
Embryogenesis was observed in the treatment T7 – MS + 4.00 mg/L NAA + 0.50
mg/L BA+ 0.25% activated charcoal + 15 mg/L AgNO3 (1.82%) and treatment T6 - MS +
4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 15mg/L AgNO3 (1.33%) at
fourth week of inoculation.
At sixth week of inoculation, embryo induction (1.15%) was observed in the
treatment T5 – MS + 4.00 mg/L NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10mg/L
AgNO3. Highest embryogenesis of 2.72% was observed in the treatment T7 – MS + 4.00
mg/L NAA + 0.50 mg/L BA+ 0.25% activated charcoal + 15 mg/L AgNO3.
To conclude, among the treatments tested in Capsicum annuum var. Arka Meghana
the best treatment for indirect embryogenesis and direct embryogenesis was MS + 4.00 mg/L
NAA + 0.10 mg/L BA + 0.25% activated charcoal + 10 mg/L AgNO3 and MS + 4.00 mg/L
NAA + 0.50 mg/L BA + 0.25% activated charcoal + 15 mg/L AgNO3 respectively at culture
conditions of 35°C initial incubation temperature and two days darkness.