【作者】 张国裕；

【导师】 程智慧；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2003， 硕士

【摘要】 以“二细一粗”银条作为研究试材，对银条茎尖分生组织的培养条件、不同处理对提高茎尖再生植株脱毒率的效果进行了研究，并利用不同外植体探讨了银条再生快繁的适宜途径。目的是获得脱毒种株并进性大量、快速的繁殖，以解决生产中银条由于结籽率低，且难以培养成苗，长期以根状茎进行无性繁殖所造成的病毒累积的问题，并为进一步进行银条的营养利用、生理代谢、生物育种研究奠定基础。试验结果表明： 银条的离体培养以MS作为基本培养基最为适宜，再生苗生长快速，植株健壮。茎尖分生组织培养在MS+6-BA 0.5 mg/L+NAA 0.1mg/L+蔗糖4％+琼脂6.5g/L上，成苗率可达74.1％，最适宜茎尖的培养；经指示植物法、电镜观察检测，再生植株的脱毒率随茎尖切割长度的减小而提高，但过小的茎尖操作、培养困难，培养周期长，成苗率低。0.5mm大小的茎尖成苗率与脱毒率相对较高，分别为71.4％、30.0％，较为适合培养；茎尖的重复切割培养与接种植株进行预热处理均有显著提高脱毒率的作用，但同时也降低了成苗率。植株在42℃高温下，经15d热处理后，0.5mm大小茎尖切割培养成苗率与脱毒率分别达到50.0％、57.1％，重复切割培养3次后则分别为28.6％、87.5％。为减小操作难度，缩短周期，银条植株经42℃，15d热处理后，切取0.5mm大小茎尖进行一次培养较为适宜。 不同生育时期的外植体愈伤组织的诱导能力不同，刚展开的幼嫩叶片、粗壮的幼嫩茎段及花蕾长度在0.5 cm左右时的花药最适宜愈伤的诱导，诱导率最高。幼嫩茎段与花药在MS+6-BA 2.0mg/L+NAA 1.5mg/L+IAA 0.5mg/L培养基上，幼嫩叶片在MS+6-BA 2.5mg/L+NAA 1.5mg/L+IAA 1.0mg/L上，愈伤组织诱导效果最好，愈伤生长快速，颜色鲜艳，继代培养不褐化。愈伤组织不定芽的分化中，花药诱导形成的愈伤难以形成不定芽；叶片与茎段诱导的愈伤可以在MS+6-BA 1.0 mg/L+NAA 0.8mg/L+GA₃ 2.0mg/L上分化形成不定芽，诱导分化率分别为71.4％、78.6％。 对幼嫩叶片、幼嫩茎段与茎尖进行不定芽的直接诱导与增殖，发现银条叶片组织未能再生；茎尖、茎段组织直接诱导较大不定芽的再生率较低，平均每个外植体最多可诱导不定芽5～7个。而通过茎段先诱导出不定芽芽团，再进行芽的伸长这一途径，每个外植体诱导的不定芽数平均可提高到14.3个，且芽团经5次继代增殖再生能力没有衰退表现，再生植株生产潜力非常巨大，是一条较优的再生途径。茎段不定芽芽团诱导培养基为添加6一BA 5.Om留L的MS培养基;增殖培养基为MS+6一BA 4.5m叭+GA3 1 .Om留L;不定芽伸长培养基以MS+6一BA 1.5 mg几+N AA 0.4m叭+GA30.5m叭较好。 对不同温度、光照条件、以及蔗糖、6一BA、CCC浓度对离体培养条件下银条根状茎诱导的影响进行研究表明:全黑暗的培养条件是银条根状茎诱导的必需因子，短日照（8扮d）与长日照（14扮d）均促使甸旬茎发育成侧枝;高浓度蔗糖有利于根状茎的诱导，单独使用低浓度的蔗糖（3%）不能诱导根状茎产生而形成黄化的侧枝，再添加6一BA 5.Om留L十CCC 50Om留L后虽然可以形成根状茎，但单株产量很低。6一BA与CCC对根状茎形成具有明显的促进作用，能增加根状茎数，提高单株产量。银条根状茎的诱导温度以20℃为宜，提高温度会降低根状茎数。 再生苗在1/2 Ms+N AA o.sm留L十活性炭0.3%上生根效果最好，将生根的完整植株移栽到消过毒的1蛙石:1珍珠岩的基质上，成活率高达95.8%。更多还原

【Abstract】 The culturation of the tip meristem of many flower Betony which was about 0.3-0.5 mm in length with 1 or 2 primordia leaves had been investigated on different culture medium. At the same time, the effect of different techniques on the virus-free rate of regeneration planlets by tip meristem cultured in vitro, the regeneration and rapid propagation system were studied, too. The results showed:As the basic medium for many flower Betony culturation in vitro, MS was the best in all media include White, MA, B5 and Ne. The optimal medium for the tip meristem culture was MS+6-BA 0.5 mg/L+NAA 0.1 mg/L+sucrose 4%, on which 74.1% plantlet ratio was produced. The different treatment to produce virus-free plantlet by the tip meristem culture was studied. The results showed that the size of excised shoot tip was negative correlated with the virus-free rate of plantlet. Too smaller shoot tip make operation and regeneration difficult. The attempt of using 0.5 mm length shoot tip was successed with 71.4% plantlet rate and 30.0% virus-free rate produced. The virus-free rate could be increased markedly by using the tip meristem culturation technique together with the techniques such as cut of stem tip more than one times, higher temperature pre-treatment of the planr material. However the survival rate might be degraded. The suitable method to obtain virus-free plantlet was to pre-treat the plants at 42℃ for 15 days firstly, and then to cut the shoot tip(0.5 mm long) one times. 50.0% survival rate and 57.1% virus-free rate were gained with this method.Explants in different development period showed differences in callus induction.The younger and tender the explants were, the better the callus induction would be. Leaf discs, stem segments and anther could all be induced to produce callus, and the induction rate could reach 100%. However, only 61.2% induction rate was got from anther. The callus from variant explants showed differences in buds differentiation. Adventitious buds can be obtained from leaf and stem callus ,but not from anther callus.The best medium for leaf discs to induce callus was MS+6-BA 2.5 mg/L+NAA 1.5 mg/L+IAA 1.0 mg/L. It wassuitable to induce callus from stem segments and anther on MS+6-BA 2.0 mg/L+NAA 1.5 mg/L+IAA 0.5 mg/L. The best adventitious buds induction medium for leaf and stem callus was MS+6-BA 1.0 mg/L+NAA 0.8 mg/L+GA3 2.0mg/L, with 71.4%, 78.6% induction rate, respectively.The stem apex, leaf disc and stem segments as explants were used to study the effects of different hormones treatments on the adventitious bud induction and propagation.The results showed that stem apex and stem segments had low reproducing rate with only got 5-7 buds per explant in longer buds induction, while leaf disc had not showed ability to reproduce.The best way to get more reproducing plantlet (14.3 per explant)was to lengthen the bud from the bud lump which was gotten from the induced stem segments on MS medium supplemented with 6-BA 5.0 mg/L. The bud lump can be propagated for a long time and the reproducing ability would not decreased on MS medium with 6-BA 4.5 mg/L and GA3 1.0 mg/L. The better medium for bud elongation is MS+6-BA 1.5 mg/L +NAA 0.4 mg/L +GA3 0.5 mg/L.The effects of the photoperiod, temperature, sucrose, 6-BA and CCC on in vitro stolon induction were studied. The results showed that dark cultural condition is essential to stolon induction. Short photoperiod (8h/d) and long photoperiod (14h/d) would make stolons grow into branches. Lower sucrose concentration (3%) inhibited stolon induction and only got feeble branches were produced. If 6-BA 5mg/l and CCC 500mg/L be supplemented to the medium, smaller stolon could be formed. This procession was apparently promoted by higher sucrose concentration. 6-BA and CCC were not essential to stolon induction, but they could significantly accelerate the development of stolon and enhance the average output per plantlet. The situable temperature was 20℃. Higher temperature may degrade the stolon number.1/2MS medium with 0.5 mg/L NAA and 3% activit更多还原