不同激素对青钱柳外植体和愈伤组织褐化的影响

以青钱柳(Cyclocarya paliurus)叶片为研究材料, 采用单因素完全随机试验法比较不同激素(6-BA、GA₃和NAA)浓度组合对叶片外植体褐化的影响; 在此基础上, 分别采用二因素、单因素完全随机试验法比较6-BA+NAA不同浓度组合、不同基本培养基对愈伤组织褐化的影响。结果表明, 改良MS+1.0 mg∙L ^-1 6-BA+1.0 mg∙L ^-1 GA₃+0.3 mg∙L ^-1 NAA是抑制青钱柳叶片外植体褐化的最佳培养基, 其褐化率为0, 愈伤组织诱导率100%; 改良MS+0.5 mg∙L ^-1 6-BA+0.2 mg∙L ^-1 NAA是抑制愈伤组织褐化的最佳培养基, 其褐化率为0, 愈伤组织增殖倍数达4.80倍, 愈伤组织呈黄绿色或黄色, 颗粒状, 颗粒小而紧密、质硬且表面干燥。该方法有效解决了青钱柳叶片外植体和愈伤组织褐化问题, 为青钱柳组织培养褐化控制提供了一条简单高效的离体培养途径, 也为青钱柳叶片离体再生体系的建立奠定基础。     

不同激素对青钱柳外植体和愈伤组织褐化的影响

以青钱柳(Cyclocarya paliurus)叶片为研究材料, 采用单因素完全随机试验法比较不同激素(6-BA、GA₃和NAA)浓度组合对叶片外植体褐化的影响; 在此基础上, 分别采用二因素、单因素完全随机试验法比较6-BA+NAA不同浓度组合、不同基本培养基对愈伤组织褐化的影响。结果表明, 改良MS+1.0 mg?L ^-1 6-BA+1.0 mg?L ^-1 GA₃+0.3 mg?L ^-1 NAA是抑制青钱柳叶片外植体褐化的最佳培养基, 其褐化率为0, 愈伤组织诱导率100%; 改良MS+0.5 mg?L ^-1 6-BA+0.2 mg?L ^-1 NAA是抑制愈伤组织褐化的最佳培养基, 其褐化率为0, 愈伤组织增殖倍数达4.80倍, 愈伤组织呈黄绿色或黄色, 颗粒状, 颗粒小而紧密、质硬且表面干燥。该方法有效解决了青钱柳叶片外植体和愈伤组织褐化问题, 为青钱柳组织培养褐化控制提供了一条简单高效的离体培养途径, 也为青钱柳叶片离体再生体系的建立奠定基础。     

多酚氧化酶抑制剂对蝴蝶兰叶外植体褐变的影响

将多酚氧化酶(PPO)抑制剂添加到酶反应液中,抗坏血酸和半胱氨酸在0.5mmol/L就完全抑制蝴蝶兰PPO活性。300mg/L柠檬酸和100~200mg/L亚硫酸氢钠分别添加到培养基中,可使蝴蝶兰外植体褐变程度降低;采用抑制剂浸泡处理外植体,对外植体褐变抑制效果最好的为50mg/L抗坏血酸,外植体在褐变发生前PPO活性低于对照。     

黑莓外植体褐化影响因素分析及适宜培养条件筛选

以黑莓(Rubus spp.)品种'Kiowa'为实验对象,研究了基本培养基类型、外植体类型、枝条暗处理时间、防褐化剂以及光照度对组织培养中外植体褐化及腋芽诱导的影响.结果表明,在供试的6种基本培养基(MS、1/2 MS、B5、1/2 B5、N6和1/2 N6)中,在MS培养基上外植体的褐化率较低(37.78%)、腋芽诱导率最高(67.89%)且生长状况良好;褐化程度与外植体的木质化程度及取材部位均有一定的关系,以1年生半木质化枝条的上部茎段为外植体,褐化率最低(19.67%)、腋芽诱导率较高(91.89%)且生长状况良好;接种前进行一定时间的暗处理对外植体褐化有一定的抑制作用,对枝条暗处理3 d,防褐化效果最佳;在培养基中添加不同类型和浓度的防褐化剂对外植体褐化和腋芽诱导也有一定的影响,以添加3 000 mg·L-1活性炭或200 mg·L-1 VC的防褐化效果较好;培养过程中的光照度与外植体褐化也有一定的关系,在1 500 lx光照度下外植体的褐化率较低(15.66%)且腋芽诱导率和生长状况均较好.根据实验结果,筛选出防止黑莓外植体褐化的适宜培养条件为:以1年生半木质化枝条的上部茎段为外植体,15 ℃暗处理3 d后,消毒并接种在添加了3 000 mg·L-1活性炭或200 mg·L-1 VC的MS培养基(含20 g·L-1蔗糖、5.6 g·L-1琼脂及0.2 mg·L-1 6-BA,pH 5.8)上,置于光照度1 500 lx的条件下培养.     

降低卷丹百合组培褐变技术研究

以卷丹百合Lilium lancifolium鳞片为外植体,采用1/2MS+0.8 mg·L~(-1) 6-BA+0.2 mg·L~(-1) NAA为基本培养基,研究不同消毒时间的外植体消毒效果,探讨聚乙烯吡咯烷酮(PVP)、抗坏血酸(V?)、活性炭(AC)以及光、暗两种培养条件对卷丹百合组织培养过程中褐变的影响。结果表明,用75%酒精消毒30 s、0.1%HgCl_2消毒10 min、2%NaClO消毒6 min的消毒效果最好,污染率为33.33%,成活率和诱导率均为66.67%。在培养基中加入PVP、V?、AC和暗培养均能减轻褐变的发生,其中以4.5 mg·L~(-1) PVP处理对卷丹百合褐变的抑制效果最好,外植体褐变率最低为30%,愈伤组织诱导率为70%。     

二乔杜鹃无菌株系建立的初步研究

以二乔杜鹃顶芽为外植体,探讨不同花色类型、不同消毒时间、培养基中不同生长调节剂组合对其无菌株系建立的影响。结果表明,以0.1%升汞作表面消毒剂,顶芽外植体的最适消毒时间为10 min;在相同消毒条件下,白花类型的污染率低于红色类型,红花类型外植体的褐变率低于白花类型;含较高浓度ZT的初代培养基中外植体的褐变率较低。     

花生幼叶不定芽诱导与快速繁殖

以花生品种‘粤油7号’6~8 d龄幼叶为外植体进行植株再生研究。结果表明,外植体在MS+0.6 mg/L NAA+8 mg/L 6-BA+1 mg/L AgNO₃+3 mg/L Gln培养基上,诱导不定芽效果好,经两次继代培养出芽率达81.03%,每个外植体平均出芽数达5.79个。经伸长培养基MS+2 mg/L 6-BA+4 mg/L GA₃培养,不定芽伸长长度达1.0~2.0 cm。试管苗在培养基1/2 MS+0.5 mg/L NAA+3 mg/L IBA上生根率达86.15%,不定根粗而长,有侧根,移栽成活率达90%,结实率100%。     

柑桔实生苗大量增殖研究

将枳和酸橙的实生试管苗上、下胚轴和子叶切割成若干段分别进行离体培养,结果表明,枳的各个切段在添加10^-4或5×10^-5摩尔BA的MS培养基中,不定芽形成率最高,平均每个外植体不定芽发生总量分别达41个和33.5个,其中以上胚轴发生的不定芽最多,占全部不定芽总量的66.6%。在上、下胚轴中,则以第一切段发生的不定芽最多。添加KT的所有处理,不定芽均较少,形成不定芽最多的处理也仅及N₀B₁处理的四分之一。发根量则以添加10^-5摩尔NAA的培养基最多。将枳的不定芽扦插于添加5×10^-6或10^-6摩尔NAA的MS培养基中,15-20天即可发根成苗。在完全相同的各个处理中,酸橙的不芽定形成量明显少于枳。     

甘蔗茎尖培养中减轻酚害

甘蔗茎尖组织预先在无菌水中浸泡30 min后,再接种到附加6-BA 2.0 mg·L-1、NAA 0.1 mg·L-1的MS培养基中,可减轻外植体的褐变;茎尖诱导培养基中直接附加适量的活性炭(AC,以0.02%为宜,最高不超过0.05%),减轻茎尖组织酚害的效果最理想.另外,在培养基中附加抗氧化剂(如:聚乙烯吡咯烷酮,PVP),或根据外植体的褐变情况及时转移到新鲜的培养基中,均能减轻甘蔗茎尖组织的酚害.     

柠檬酸和抗坏血酸对蝴蝶兰叶外植体褐变发生的影响

目的:探究柠檬酸和抗坏血酸对蝴蝶兰叶片外植体褐变发生的影响以及对PPO活性变化影响的作用机理.方法:以褐变率和褐变指数为参考数据,分析柠檬酸和抗坏血酸对外植体PPO活性和PPO反应产物积累的影响以及与外植体褐变发生的关系.结果:分别用100mg/L柠檬酸共培养和50mg/L抗坏血酸浸泡处理叶片外植体,经离体培养3d,褐变率分别比对照降低94.9%和54.9%,离体培养6d,褐变指数低于对照的0.53,分别为0.46和0.36,同时PPO活性降低.结论:推测柠檬酸抑制褐变的原因是直接与酶蛋白作用,抗坏血酸则与新生醌类物质结合.     

蝴蝶兰叶片外植体褐变过程中PAL基因的表达变化

研究了蝴蝶兰(Phalaenopsis sp.)叶片外植体褐变过程中PAL基因表达的变化。结果表明,在整个褐变过程中,外植体的PAL基因表达出现差异,离体培养第3天的表达明显提高,一直到第8天还维持较高表达水平,以后随着外植体褐变的加重,PAL基因表达水平逐渐降低。与对照相比,在Fe盐浓度加倍为55.6 mg L-1培养基中培养的外植体PAL基因表达水平提高发生的时间比对照早,培养第2天就明显增强,随培养天数的延长,一直维持较高的表达水平;其PAL活性也高于对照,两种培养条件下,外植体总酚含量都随着其褐变加重而增加,说明PAL基因表达与蝴蝶兰外植体褐变过程相关。     

亳芍茎尖组织培养的研究

选取亳芍茎尖为试验材料,探究不同培养条件对亳芍组织培养的影响,结果表明:亳芍茎尖在1/2MS+6-BA 1.0 mg/L培养基上培养39 d后,茎尖分化出芽的同时也形成较多的丛生芽;丛生芽在1/2MS+6-BA1.0mg/L培养基上增殖速度最快;来自不同启动培养基上的丛生芽在相同培养基上,接种15 d后观察,不同来源的丛生芽长势不同,30 d后仍存在一定的差异;幼苗在1/2MS+IBA 0.1生根效果最好。     

植物组织培养外植体褐变的研究进展

外植体的褐变是组织培养过程中的主要障碍。目前一般认为影响褐变的因素主要有外植体材料、培养基和培养条件。为了防止褐变的发生,主要可以从外植体和培养条件、进行细胞筛选和材料的预处理、抑制剂或吸附剂的加入等方面入手。酚、酚酶的区域性分布假说、自由基伤害假说、保护酶系统假说是褐变机理方面较为成熟的3种假说。     

Micropropagation of Terminalia arjuna Roxb. from cotyledonary nodes

Cotyledonary node explants excised from 21 day old seedlings of T. arjuna produced multiple shoots when cultured on full strength MS or modified MS (1/2 strength major salts and Fe-EDTA) medium supplemented with different concentrations (0.1-1.0 mg/l) of BAP. Maximum 8.9 shoots/explant could be recorded after 30 days of inoculation on modified MS medium supplemented with BAP (0.5 mg/l). A proliferating shoot culture was established by reculturing the original cotyledonary nodes (2-3 times) on shoot multiplication medium after each harvest of the newly formed shoots. Shoots (each having 2-3 nodes/shoot) thus obtained were also used as a source of nodal explant that gave rise to 1-2 shoots when cultured on modified MS+BAP (0.5 mg/l) medium. Thus, 45-55 shoots could be obtained after 60 days of culture initiation from a single cotyledonary node. About 88% shoots rooted well after 15 hr pulse treatment with IBA (1 mg/l) in liquid MS medium followed by transfer to modified MS medium without IBA. About 80% of these plantlets were successfully acclimatized in plastic pots containing sand and soil mixture and 70% plantlets transferred in the field those survived even after 6 months of transplantation.     

魔芋茎尖组织培养和植株再生的研究

以魔芋茎尖、幼芽为外植本,接种于１／２ＭＳ＋１．０ｍｇ／ＬＢＡ＋０．０１ｍｇ／ＬＮＡＡ的培养基中,茎尖、幼芽逐渐生长直接形成幼芽或幼苗;或从茎尖、幼芽由来的膨大的块茎组织表面诱导出幼芽。膨大的块茎组织分割后接种于ＭＳ＋０．０１ｍｇ／Ｌ＋０．０１ｍｇ／ＬＮＡＡ的培养基中进行增殖培养,同时从增殖的块茎组织表面不断地诱导出幼芽。幼芽切块转入不含激素的ＭＳ培养基中,形成幼苗。幼苗切块转入ＭＳ＋０．１ｍｇ／ＬＮＡＡ的生根培养茎中,幼苗生根,形成完整的植株。试管苗移栽６个月后获得干块茎。     

Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant

Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.     

三七胚培养中的胚胎发生

三七成熟胚培养子MS+1 mg／L 1AA或NAA或2,4 — D的培养基上。二月后,在MS+1 mg／L 1AA或NAA的培养基上由外植体可诱导产生胚状体,但在含2,4 — D的培养基上只产生愈伤组织而无器官分化。胚状体转入MS+CA3 1 mg／L+IAAo.5 mg／L培养基上可发育成具胚根、根芽的小植株。     

In vitro cormlet production of saffron (Crocus sativus L. Kashmirianus) and their flowering response under greenhouse

A complete protocol for the saffron cormlet production under in vitro conditions and subsequent flowering under greenhouse conditions is described. Highest number of cormlets (70.0 ± 0.30) per corm slice (explant) could be regenerated on Murashige and Skoog (MS) half strength medium supplemented with thidiazuron (TDZ) (20 μM), Indole acetic acid (IAA) (10 μM), and sucrose (40 g/l). Maximum germination (90%) of these cormlets could be achieved on MS medium containing 6-benzyl amino purine (BAP) (20 μM) and α-naphthalene acetic acid (NAA) (15 μM). In order to increase the size of the in vitro raised cormlets, these were cultured on MS medium containing TDZ (15 μM) and IAA in the range of 1.5-30 μM. Maximum increase in cormlet size could be attained on TDZ (15 μM) + IAA (12.5 μM) + sucrose (30 g/l), and the average size of cormlets was 2.5g. In another experiment, apical vegetative buds of actively growing corms were cultured for cormlet development, and corms of size 2.5g could be developed on MS medium with NAA (15 μM), BAP (20 μM), and sucrose (30 g/l). The in vitro developed cormlets were dried under shade at 25 ± 2°C for 7 d. These were then planted in small cups containing clay loam soil and kept in green house at 20 ± 2°C. In vitro developed cormlets with mean weight 2.5 g showed maximum flowering (25%) as well as vegetative growth (55%), while only 19% cormlets of 2.0 g flowered. To our knowledge this is the first report on successful flowering from in vitro raised cormlets under greenhouse.