Studies On The Key Techniques Of Industrial-scale Micropropagation In2Local Characteristic Cultivars In Fujian

Fujian province is one of the major banana production areas in China, and the localcharacteristic cultivars play an important role in production. The local characteristic cultivars of‘Fuzhoubajiao’(Musa spp., ABB groups) and ‘Tianbaoaijiao’(Musa spp., AAA groups) in Fujianwere selected as the materials to discuss some key technologies, such as explants induction,proliferation, rooting, transplanting and acclimatization. Furthermore, the genetic stability wasdetected by ISSR. Finally, the industrial-scale micropropagation system was established and theeconomic benefit was analysed. The main results were as follows:1The establishment and optimization of in vitro regeneration systemin Fujian local characteristic banana cultivars1.1In vitro culture of Musa spp. cv. Fuzhoubajiao (ABB group)Fuzhoubajiao sucers were used as the explants to study the effects of different disinfectiontime on contamination rates and germinating rates. The clustering buds were used to study theeffects of different factors on proliferation and rooting. The influence of different cultureconditions, such as temperature, light quality and inoculation way on plantlet growth werecompared.①The establishment of sterile-plantlets system from the explants of banana. The explantswas sterilized by0.1%HgCl2, the result showed that the contamination rate was lower for20min(39.3%) than that for15min(46.7%), and the germinating rate from the explants was higherfor15min(59.1%) than that for20min(52.9%). The explants primary germination time was35days.②The optimization of the proliferation culture in Fuzhoubajiao. The orthogonal design wasused for comparing the effects of different salt concentrations and the different concentrations of6-BA and NAA on the proliferation. The results showed that the best medium for Fuzhoubajiaoproliferation was MS+3.0mg/L6-BA+0.2mg/L NAA, with proliferation coefficient of3.59. Meanwhile, the effects of different temperature conditions and the different light conditionsand inoculation ways on proliferation were compared. The results showed that the most suitabletemperature for proliferation was28°C; adding some amount of red light was beneficial to plantletdifferentiation; the shallow liquid medium was superior to solid medium for grow of plantlets.③The optimization of rooting in Fuzhoubajiao. On the basis of the1/2MS+1.0g/L ACrooting medium,, the effect of different concentrations of NAA, IBA and sugar on rooting werecompared and the statistical analysis was conducted by the method of assignment score combiningorthogonal design, The results showed that the best medium for Fuzhoubajiao rooting was1/2MS+0.3mg/L NAA+0.3mg/L IBA+20g/L sugar+1.0g/L AC.Meanwhile, the effect of the two inoculation methods of the removal of the top of the bud ornot on rooting was compared. The results showed that the no removal of the top of the bud wasbeneficial to growth.1.2Optimization of in vitro culture of Musa spp. cv. Tianbaoaijiao (AAA group)On the basis of the in vitro culture of Fuzhoubajiao, the effects of different factors onproliferation and rooting were compared in Tianbaoaijiao.①The optimization of the proliferation culture in Tianbaoaijiao. The orthogonal design wasused for comparing the effects of different salt concentrations and the different concentrations of6-BA and NAA on the proliferation. The results showed that the best medium for Fuzhoubajiaoproliferation was MS+5.0mg/L6-BA+0.3mg/L NAA, with proliferation coefficient of3.61.②The optimization of rooting in Tianbaoaijiao. On the basis of the1/2MS+1.0g/L ACrooting medium, the effect of different concentrations of NAA, IBA and sugar on rooting werecompared and the statistical analysis was conducted by the method of assignment score combiningorthogonal design, The results showed that the best medium for Tianbaoaijiao rooting was1/2MS+0.1mg/L NAA+0.3mg/L IBA+40g/L sugar+1.0g/L AC.2Acclimatization and transplanting of the local characteristicbanana plantlets in FujianThe rooted plantlets were used as the transplanting materials, and the effects of different timeof acclimatization, different matrixes and different seasons on the transplanting were compared.The comparative experiment of different acclimatization time indicated that the survival ratewas the lowest when the plantlets were transplanted after acclimatization for1d, and the yellowleaves and wilting symptoms in plants occurred; the survival rate was the highest when theplantlets were transplanted after acclimatization for7d.The comparative experiment of different matrixes indicated that the best matrix was gardensoil: peat soil: river sand (3:2:1) or peat soil: coconut chaff: river sand (1:1:1), with the survivals rates of97.5%and95%.Besides, the transplanting experiment was carried out for comparing the effects of spring andautumn on the transplanting, respectively. The result showed that the plant grew faster in autumnthan in spring, and the survival the rate was more than95%.3The analyses of the genetic stability of the plantlets by ISSR in thelocal characteristic banana plantlets in FujianThe plantlets from the generation0,11,12,13,14,15were selected for detecting the geneticstability by ISSR in Fuzhoubajiao and Tianbaoaijiao.The selected12random primers were used for PCR amplification from genomic DNA’s of6specimens in Fuzhoubajiao, and the total of155bands was amplified. With the first generation asthe reference, the variant rates of the generation11,12,13,14,15were1.50%,3.76%,20.30%,9.02%and14.25%, respectively. The genetic similarity coefficients of the11~12th generationwere closer to that of the first generation, between0.877and0.927, which of the13~15thgeneration were farer, and genetic similarity coefficient between0.748and0.832. The aboveresult showed that the genetic traits were relatively stable within12generations. Therefore, it wasproposed that the subculture time should not exceed12generations in the production.The selected12random primers were used for PCR amplification from genomic DNA’s of6specimens in Tianbaoaijiao, and the total of153bands was amplified. With the first generation asthe reference, the variant rates of the generation11,12,13,14,15were2.98%,5.22%,10.44%,8.21%and9.70%, respectively. The genetic similarity coefficients of the11~15th generation werebetween0.823and0.930, and the genetic distance was the furthest between the generation0and13. The above result showed that the genetic traits in Tianbaoaijiao were relatively stable within15generations. Therefore, it was proposed that the subculture time should be between12and15generations in the production.4Establishment of the industrial-scale micropropagation mode andanalyses of economic benefits in local characteristic banana cultivarsin FujianThe main industrial-scale micropropagation technical chain of local characteristic bananacultivars in Fujian was: the establishment of sterile-plantlets system from the explants (using theauxiliary buds as explants)→the bud proliferation culture (proliferation by bud clusters)→plantletrooting→acclimatization→transplanting→sales. The main technical parameters were as follows:The sterile-plantlets system from the explants was under the conditions of0.1%HgCl2sterilization for15min; the proliferation coefficient was3.59on the medium of MS+3.0mg/L6-BA+0.2mg/L NAA; the rooting rate was95%on the medium of1/2MS+0.3mg/L NAA+0.3 mg/L IBA+20g/L sugar+1.0g/L AC; and the survival rate of transplanting was97.5%(thetransplanting matrix was garden soil: peat soil: river sand=3:2:1).The establishment of the sterile-plantlets system in Tianbaoaijiao was referenced to that inFuzhoubajiao; the proliferation coefficient was3.61on the medium of MS+5.0mg/L6-BA+0.3mg/L NAA; the rooting rate was98.33%on the medium of1/2MS+0.1mg/L NAA+0.3mg/LIBA+40g/L sugar+1.0g/L AC; and the survival rate of transplanting was95%(the transplantingmatrix was garden soil: peat soil: river sand=3:2:1).The economic benefit was analyzed in case of the annual output of1million tube plantsaccording to the established main technical system, which would cost288474.73Yuan and pertube plant costed0.21Yuan. According to the market price of0.5Yuan per20-30cm height oftube plant, the total profit was211525.27Yuan, which the profit was0.29Yuan per plant. It wasfound that the charge for the labor costs was the highest with the cost of153088.70Yuan, whichaccounted for53.07%of the total costs. The electricity costs were the second with the cost of58849.85Yuan, accounting for20.40%of the total costs. The fixed assets were the third with thecost of41173.50Yuan, accounting for13.69%of the total costs. The proportion of the three partsreached87.74%.