doi: 10.15389/agrobiology.2019.1.140eng

UDC 635.6:576.3/.7.086.83:58

Funded by Da Nang University Science and Technology Development Foundation by project No. B2017-DN03-13



M.L. Nguyen1, T.H.T. Ta1, T.N.B.T. Huyen1, A.V. Voronina2

1University of Education The University of Da Nang, Vietnam, Danang city, 459 — Ton Duc Thang st., e-mail (✉ corresponding author),,;
2Timiryazev Russian State Agrarian University—Moscow Agrarian Academy, 49, ul. Timiryazevskaya, Moscow, 127550 Russia, e-mail

Nguyen M.L.
Huyen T.N.B.T.
Ta T.H.T.
Voronina A.V.

Received November 4, 2018


Anther culture is one of the powerful techniques to pure line production for the short time. This method can be applied to accelerate the breeding process of F1 hybrids of bitter melon (Momordica charantia L., family Cucurbitaceae), which is an important commercial crop in tropical and subtropical countries of South America, Asia and Africa. Although, there are several factors affecting the success of this method, the effective protocol of bitter melon anther culture has not been developed at present. The results of this study showed that the microspore developmental stage has a significant (p ≤ 0.05) effect on speed and rate of callus formation. It was revealed that the frequency and the rate of callus formation and the morphology of callus substantially depend on the composition and concentration of growth regulators in the nutrient medium. In addition, the dynamics of callus formation in bitter melon anther culture was first studied. The main objective of the work was to study the influence of the microspore developmental stage and the composition of nutrient medium on the ability of callus formation in bitter melon anther culture in vitro. Plants of the F1 hybrid bitter melon Diago 26 were grown in field in Dailoc district, Quangnam province (Vietnam) in 2018 according to the standard technique for obtaining commercial fruits. The buds harvested at 5-7 am were stored in plastic bags in dark condition at 4 °С for 1 day. Before culturing of anther, flower buds were surfaced sterilized using 70 % ethanol and 5 % sodium hypochlorite (NaOCl). Anther removed from flower buds were inoculated in the induction medium in the horizontal laminar air flow; then incubated at 25±2 °С and a photoperiod of 16 h light/8 h dark for 4 weeks. Three variants of the Murashige-Skoog (MS) nutrient mediums were used, differing in composition and concentration of growth regulators: MK1 with the addition of 1.0 mg/l 2.4-D and 1.5 mg/l BAP; MK2 — 1.5 mg/l 2,4-D and 1.0 mg/l BAP; MK3 — 1.5 mg/l NAA, 1.0 mg/l BAP and 0.5 mg/l kinetin. The completely randomized design with statistical analysis using the software IBM SPSS Statistics Base 22 and Microsoft Excel 2013 were carried out. The cytological analysis result showed that each anther contained microspores with different development stages. Therefore, it is impossible to separate microspores at the unique developmental stage being suitable for anther culture; however, there is always a predominant stage. In this study, buds were divided into two main groups. The first group consisted of buds with the size of 4.0-5.0 mm with early and mid uninucleate microspores. The second group included buds with the size of 5.1-6.5 mm with late uninucleate and binucleate microspore. After inoculation, the beginning of calli formation was observed within the first week for anther of the buds with the size of 4.0-5.0 mm and within the second week for the buds with the size of 5.1-6.5 mm. The highest frequency of callus formation (93.75±2.55 %) was observed on MS medium supplemented with 1.0 mg/l of 2,4-D and 1.5 mg/l of BAP (MK1). Most calli were formed during the second and third week after cultivation. Also, there was the significant difference about the morphology of the calli obtained on three nutrient medium. Calli on medium MK1 were yellow, strongly dense and calli on MS medium with the addition of 1.5 mg/l of 2,4-D and 1,0 mg/l of BAP (MK2) being green, strongly dense. Green-yellow, dense calli were obtained on medium supplemented with 1.5 mg/l NAA and 1.0 mg/l BAP and 0.5 mg/l Kinetin (MK3). However, the effect of the developmental stage of microspores on the morphology of calli was not revealed. Despite receiving a large number of calli, the formation of embryoids was not observed.

Keywords: Momordica charantia L., bitter melon, callus, doubled haploid, anther culture,microspore stage.




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