| In this study,the terrestrial medicinal orchids?Cremastra appendiculata,Dendrobium nobile and Bletilla striata?were used to study the factors and inductive pathways of multiple shoots occurring.On the basis of the successful establishment of chemical induction for multiple shoots of the terrestrial medicinal orchids,the transgenic induction of multiple shoots was also explored.The main results were as follows.1.In the current work,the protocorms from the germination of Cremastra appendiculata seeds were taken materials,which were used to investigate the effects of different mediums,different plant growth regulators kind or concentration,different active carbon concentration,different temperature and light intensity to protocorm proliferation of C.appendiculata.The results showed that the condition that media 1/2MS + 6-BA 1.0 mg·L-1 + IBA 1.0 mg·L-1 + AC 0.5 g·L-1,temperature of 15?,light intensity of 2000 lx was the best for protocorm proliferation of C.appendiculata.The protocorms grew into clump round bodies with better growth vigour.And the proliferation rate reached to 177.11% after 40 days.Effects of “Shoot cluster” proliferation of C.appendiculata by different major element content,different plant growth regulators kind or concentration,different active carbon concentration and the method of orthogonal design?TDZ,IAA,sugar and major element?,the protocorms of the best develop condition were taken materials.The results showed that the greatest multiplication coefficient of shoot?3.16?was observed on 1/4MS + TDZ 1.5 mg·L-1 + IAA 0.4 mg·L-1 + sugar 30 g·L-1.This culture conditions was the best for “Shoot cluster” induction and proliferation.2.In this study,the seeds of Dendrobium nobile were taken materials,which were used to investigate the effects of different 75% alcohol process-time and different 0.1% mercuric chloride process-time and the plant growth regulators which 6-BA and NAA on seed germination by using the method of orthogonal design.The results showed that the seed germination method after respectively putting them in 75% alcohol?time of 15 s?,0.1% mercuric chloride?time of 10 min?was the better way.When the treated seeds inoculated on the MS medium did not contains 6-BA and NAA,the higher rate of seed germination was obtained.The dark-green protocorms were taken materials,which were used to investigate the effects of the plant growth regulators which 6-BA and 2,4-D and AC on callus induction by using the method of orthogonal design.The basic medium was MS + sugar 30 g·L-1.The results showed that callus was green and grew well by MS + 6-BA 1.0 mg·L-1 + 2,4-D 1.0 mg·L-1 + AC 0.2 g·L-1 induced culture.To induce multiple shoots,the green callus was taken materials and MS + sugar 30 g·L-1 was taken basic media.Research the affect of the plant growth regulators which 6-BA and NAA and AC on multiple shoot induction by using the method of orthogonal design.The results showed that the condition that medium MS + 6-BA 1.0 mg·L-1 + NAA 0.1 mg·L-1 + AC 0.2 g·L-1 was conducive to multiple shoots induction,the induction rate reached to 80.0%.3.In this work,the seeds of Bletilla striata were taken materials,which were used to investigate the effects of different NAA concentration to seed germination of B.striata.The results showed that when seeds inoculated on MS + NAA 0.5 mg·L-1 + AC 0.5 g·L-1 + PP 75 g·L-1,the higher rate of seed germination was obtained.Effects of multiple shoots induction of B.striata by different 6-BA concentration,the seedlings of the similar develop condition were taken materials.The results showed that the condition that medium 3/4MS + 6-BA 0.1 mg·L-1 + NAA 1.0 mg·L-1 + IBA 0.15 mg·L-1 + AC 0.3 g·L-1 + BP 50 g·L-1 was conducive to multiple shoots induction,the induction rate reached to 87.5%.4.In this paper,the protocorms of Cremastra appendiculata,Dendrobium nobile and Bletilla striata were taken transformation materials.The optimal parameters?such as physiological state of material,approach,bacterium concertration,pre-culture time,co-culture time,infection time?for A.tumefaciens-mediated transformation of these three orchids were researched.The results showed that few protocorms of C.appendiculata showed blue when they were dyed and few showed negative with PCR checking.C.appendiculata transformation tests performed poorly.The genetic transformation suitable condition of Dendrobium nobile: 5 mm in diameter protocorm pre-cultured for 1 d which treated 3 min with sonication of 40 KHz,incubated for 10 min by Agrobacterium tumefaciens which OD600 is about 0.50.7,and then were placed on the co-culture medium with 2 d co-culture periods in darkness.The results showed that many transformants showed blue when they were dyed and many also showed positive with PCR checking.All of these results prove that ipt gene has been transformed into D.nobile.The genetic transformation suitable condition of Bletilla striata: 10 mm in length seedlings pre-cultured for 2 d which treated 3 min with sonication of 40 KHz,incubated for 10 min by Agrobacterium tumefaciens which OD600 is about 1.01.2,and then were placed on the co-culture medium with 2 d co-culture periods in darkness.The results showed that many transformants showed blue when they were dyed and many also showed positive with PCR checking.All of these results prove that ipt gene has been transformed into B.striata. |
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