| Nobile-type Dendrobium,with diverse colors,graceful posture and long lasting blooms,possesses great ornamental value and has occupied an important position in the potted flower market.However,at present,both the quality and quantity of nobile-type Dendrobium in the market of our country lag far behind the foreign countries.One of the reasons is that the elite varieties suitable for production are not sufficient.Currently,the screening cycle is too long,due to the variety selection for elite nobile-type Dendrobium is usually carried out at the flowering stage after three to four years cultivation.Plantlets obtained mainly by cuttings are far from meeting the demand of commercial production,since the low propagation coefficient and long cycle period.The majority of varieties introduced costly from foreign countries by enterprises have disappeared before large-scale production,on account of several unfavourable factors such as cultivation conditions,market demand and production technology on one hand and the lack of suitable conservation technologies on the other hand.The excellent germplasm resources are losing seriously.Therefore,comprehensive research on variety fast-screening,propagation and conservation of elite nobile-type Dendrobium is imperative,in order to promote the commercial production at home.In the present study,the early screening system for elite varieties of nobile-type Dendrobium was further discussed based on our previous study,tissue culture conditions were selected,the vitrification cryopreservation procedure and its related mechanism were also investigated.The main results are as follows:(1)Flowering quality was found to be the key indicator of cultivar quality of nobile-type Dendrobium.Plant height,internode length and putrescine(Put)content in shoot tips of young cuttings from single-type varieties(with only one flowering stalk)were significantly correlated to the flowering quality,which indicates that they could be used as the reference indexes for early screening of elite cultivars;The intemode length,leaf number,plant height,number of roots,root thickness and Put content in shoot tips of young cuttings from clustered-type plants(with multiple flowering stalks)were closely related to the flowering quality,which recommends that they could be used as the reference indicators of early screening for superior cultivars.The technology of early screening for superior cultivars of nobile-type Dendrobium in two types was established.(2)The rapid propagation system of elite cultivar ’Senhe 2006’ by tissue culture through both clustered shoots and protocorm-like bodies(PLBs)ways was established.The multiplication rate was up to 5.1 times and plantlets were easily acclimatized with a survival up to 90%in ex-vitro conditions.In the case of PLBs way,1/2MS+0.5 mg·L-1 TDZ+2.0 g·L-1 casein hydrolysate was suitable for PLBs induction,1/2MS+2.0 g L-1 casein hydrolysate was suitable for PLBs multiplication,1/2MS+1.0 mg·L-1 Kin+0.1 mg·L-1 NAA was efficient for PLBs differentiation;The suitable medium for induction and proliferation of adventitious buds was 1/2MS+0.1 mg·L-1 TDZ+1.0 mg·L-1 BA+0.5 mg·L-1 Kin+0.2 mg·L-1 NAA;The optimum medium for strengthening and rooting was MS+0.5 mg·L-1 IBA+0.1 mg·L-1 NAA+100.0 g·L-1 banana homogenate(BP).All cultures were incubated at 23 ± 2℃ under a 14-h photoperiod at a light intensity of 40 pmol·m-2·s1.In addition,the established system was also suitable for the propagation of ’Senhe 2002’.(3)The vitrification cryopreservation of shoot tips/PLBs from ’Senhe 2006’ was established:About 2-3 mm shoot tips(or 3 mm PLBs)were excised using a stereomicroscope and precultured on solid MS medium with 0.3 M sucrose at 4℃ for 24(or 48)h.After preculture,shoot tips(or PLBs)were transferred into 200 μL centrifuge tubes with loading solution and incubated at 25℃ for 60(or 40)min,with an exchange of fresh solution at half-time intervals.Then,loading solution was replaced by the precooled vitrification solution PVS2 and dehydrated at 0℃ for 40 min with an exchange.Tubes with fresh PVS2 were plunged into the liquid nitrogen.For propagation purpose,they were rinsed in unloading solution twice,for 10 min each time,after rewarmed in a 37℃ water bath for 60 s,and then cultured on recovery growth medium.The survival percent of cryopreserved shoot tips and PLBs detected by 2,3,5-triphenyltetrazolium chloride(TTC)staining was 79.2%and 85.0%respectively,but both of them were without regrowth.(4)The mechanism of the reduced survival in shoot tips and PLBs from ’Senhe 2006’ were investigated during vitrification.The results showed each treatment involved in the vitrification protocol reduced the survival in varying degrees;Survival decrease in shoot tips mainly occurred during preculture,loading and rewarming;While in PLBs,it decreased significantly after preculture,loading,PVS2 dehydration and rewarming.Studies on the changes of reactive oxygen species(ROS),antioxidants and oxidative products suggested that oxidative injury caused by ROS-induced oxidative stress was associated with the decreased survival of shoot tips and PLBs from nobile-type Dendrobium during vitrification cryopreservation,but the ROS involved varied among biological materials and cryopreservation stages(5)Four kinds of exogenous proteins(CAT,MDH,HSP70 and PDH)at a range of concentrations were added at the key steps,where the survival of shoots tips and PLBs from ’Senhe 2006’ decreased significantly,to detect their effects on the cryopreserved survival.The addition of exogenous CAT,MDH and HSP70,which are involved in alleviating oxidative stress,had a positive effect on the vitrification cryopreservation of shoot tips and PLBs,but their effects were species,adding stage and concentration dependent;The cryopreserved survival of shoot tips and PLBs increased by 15.3%and 18.9%at most,respectively.Wheras,PDH did not perform as expectedIn conclusion,the early screening system for elite varieties of nobile-type Dendrobium was roundly optimized,the time of screening was shortened by two years and the efficiency was enhanced The rapid propagation system by tissue culture was established,and it provided a technical surport for the large-scale propagation of elite cultivars.Studies on the damage mechanism during vitrification cryopreservation showed that ROS-induced oxidative stress was recommended to be associated with the cryoinjuries of shoot tips and PLBs from nobile-type Dendrobium;Addition of some exogenous proteins,with a capability of anti-ROS,could be used as an efficient path for the improvement of cryopreservation protocol. |
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