[Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA)].
Conventional breeding for dual resistance of disease and pest of Musa cultivars remains a difficult endeavor, as the plant is polyploidic and high in sterility. Biotechnological techniques, eg., genetic engineering, in vitro mutation breeding, or protoplast fusion, may overcome the difficulties and improve the germplasm. Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for any of the biotechnological breeding methods. In this study an embryogenic cell suspension was established from immature male flower of Musa acuminata cv. Mas (AA), a popular commercial variety of banana in the South-East Asian region. After culture for 5-6 months on callus induction media, which consisted of MS salts, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 4.1 micromol/L biotin, 5.7 micromol/L indoleacetic acid (IAA), 5.4 micromol/L naphthaleneacetic acid (NAA), other vitamins, 87 mmol/L sucrose, and solidified with 7 g/L agarose, meristematic globules and yellow, friable embryogenic cultures were induced from the explants of 1-15th row young floral hands of immature male flowers. Of the four treatments of 2,4-D, 9 micromol/L was the most effective on the callus induction, it transformed 40.96% and 7.45% of the cultivated male floral hands into callus and embryogenic callus respectively. The explants to produce highest frequency of the embryogenic calli were floral hands of 6 to 12th rows, which generated 5.79% of the embryogenic calli. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 micromol/L 2, 4-D. After sieving selection of the cultures using a stainless steel metallic strainer with pore sizes of 154 microm at 15 day intervals for 3 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Based upon the growth quantity and growth rate of ECS, it was determined that the appropriate inoculum was 2.0 mL PCV ECS/30 mL medium in 100 mL flask, and the appropriate subculture cycle was 15 days. Planting of 6 months old ECS on semi-solid medium of somatic embryo induction and development (MSD) resulted in approximately 280 x 10(3) somatic embryos/mL PCV ECS. MSD contained SH macronutrients, micro-nutrients, Fe-EDTA and MS vitamins supplemented with 4.5 micromol/L biotin, 680 micromol/L glutamine, 2 mmol/L proline, 100 mg/L malt extract, 1.1 micromol/L NAA, 0.2 micromol/L zeatin, 0.5 micromol/L kinetin, 0.7 micromol/L N6-(2-isopentenyl) adenine, 29 mmol/L lactose, 130 mmol/L sucrose and solidified with 2g/L gelrite. After 3 months of maturity on MSD, 17.28% of the somatic embryos were germinated on germination media (MG), consisted of MS salt, Morel and Wetmore vitamins, 0.2 micromol/L 6-BA, 1.1 micromol/L IAA, 87 micromol/L sucrose and solidified with 2 g/L gelrite; and 14.16% of the somatic embryos could develop into normal plantlets on rooting media contained the same composition as that of MG but without auxin and cytokinin.
Wei
Yue-Rong
YR
The Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, China.
Huang
Xue-Lin
XL
Li
Jia
J
Huang
Xia
X
Li
Zhe
Z
Li
Xiao-Ju
XJ
D015084:2,4-Dichlorophenoxyacetic Acid / Q000494:pharmacology; D003470:Culture Media; D035264:Flowers / Q000187:drug effects / Q000196:embryology* / Q000502:physiology*; D028521:Musa / Q000187:drug effects / Q000196:embryology* / Q000502:physiology*; D018521:Plant Physiological Phenomena; D012038:Regeneration; D046509:Tissue Culture Techniques / Q000379:methods*
D003470:Culture Media; D015084:2,4-Dichlorophenoxyacetic Acid