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In vitro Germination,Callus Induction and Primary Haustorium Organogenesis in the Parasitic Plant Cynomorium songaricum

Xin YUE, Gui-Lin CHEN

Bulletin of Botanical Research 2020, 40 (6): 846-854. DOI: 10.7525/j.issn.1673-5102.2020.06.007

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Cynomorium songaricum Rupr. is an obligate parasitic plant widely used as traditional Chinese medicine and Mongolian medicine. Here， we firstly describe protocols for in vitro germination， callus induction and haustorium organogenesis in C.songaricum Rupr. In this study， adequate concentrations of gibberellic acid（GA₃），_{in combination with other plant growth regulators in the medium， promoted embryo development and germination of C.songaricum seeds. The highest callus induction rate from seeds（13.7%） after a 40 d incubation was obtained with B₅ medium adding 2，4-dichlorophenoxyacetic acid（2，4-D； 1.0 mg·L^-1）， kinetin（KT；0.5 mg·L^-1） and GA₃（1.0 mg·L^-1. This resulted in callus_{formation in 13.7%±3.1% of seeds. Addition of 2，4-D（0.5 mg·L^-1） and KT（0.25 mg·L^-1） yielded highest haustorium organogenesis from calluses. Some primary haustorium branched to form adventitious roots of 3-4 cm in length. Subsequently， the tip of each adventitious root formed a nascent primary haustorium， which was then branched out into adventitious roots. The role of auxins（2，4-D in this study） in the formation of primary haustorium and adventitious roots from seed callus in C.songaricum was also discussed.}}

Treatment	Plant growth regulators(mg·mL^-1)		Frequency of haustorium formation(%)	Number ofhaustorium
Treatment	2.4-D	KT	Frequency of haustorium formation(%)	Number ofhaustorium
1	0.00	0.25	0.0±0.0^b	0.0±0.0^c
2	0.25	0.25	33.3±23.1^b	3.7±0.6^b
3	0.50	0.25	66.7±30.6^a	5.3±2.5^ab
4	0.75	0.25	13.3±11.5^b	6.7±2.1^ab
5	1.00	0.25	16.7±5.8^b	8.3±1.5^a

Table 2 Effect of plant growth regulators on haustorium induction in the callus of C.songaricum seed

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B5 medium was added 0.25 mg·L-1 KT and various 2，4-D amounts（Table 2） to induce haustorium organogenesis from the callus. For all media， pH was adjusted to 6.0 before agar（3%） addition. Each medium was then submitted to autoclave（25 min， 120oC）. Sterile triangular flasks were filled with 60 mL haustorium differentiation medium， and five calluses were added to each flask. Incubation was carried out away from light at 25±1oC. Three independent assays consisting of five triangular flasks were performed.

No haustoriuml hair was observed on the surface of the primary haustorium. Furthermore， the structure was similar to that of the primary haustorium from seed germination. The number of haustorium per callus was determined. The best results were obtained with 1.0 mg·mL-1 2，4-D，_{which yielded_{six haustorium per callus by Day 60. The callus broke through the radicle and formed root-like organs of 3 to 4 mm in length（Fig.3A）. The tops of these structures were enlarged to form globular shaped organs（primary haustorium； Fig.3A-B）. Scanning electron microscopy indicated that the haustoriuml top was composed of a uniform fossa surrounded by a thick ribbon like structure（Fig.3D-E） with protrusions（Fig.3F）， increasing to approximately eight on Day 60 and remaining constant thereafter（Table 2）. Interestingly， some of the primary haustorium branched to form adventitious roots of 3 to 4 cm in length（Fig.3C）. The tip of each adventitious root formed nascent primary haustorium， which then branched out into adventitious roots.}}

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