In vitro culture of lemon juice vesicles

This study explores the possibility of culturing Citrus limon (L.) Burm. f. cv. Eureka (lemon) juice vesicles as isolated intact tissues capable of retaining their unique growth structure in vitro. Isolated juice vesicles either attached to or detached from the endocarp/mesocarp (albedo) tissue of origin were grown on various nutrient media using several physical environments. Various growth responses achieved in vitro from cultured vesicles are described. Intact vesicles attached to endocarp/mesocarp tissue were found to grow up to 6 months in culture as a distinct tissue with a minimum of adverse callus proliferation. Callus formation from some cultured explants occurred on all media and physical environments tested. Callus production eventually obliterated and irreversibly altered the normal development of juice vesicles. Inherent vesicle physiology rather than the tissue culture environment was the major determining factor affecting culture growth. Reducing the concentration of carbohydrates (fructose, glucose or sucrose) added to media from 3 to 0.01% or 0.0% reduced, but still did not totally prevent, callus production. Treatment of vesicles with 1000 mg/l ascorbic acid or citric acid, or 0.1 mg/l indole-3-acetic acid or abscisic acid enhanced the occurrence of normal appearing vesicles.Mention of a trade name or proprietary product or vendor does not constitute approval or guarantee of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.     

GROWTH RESPONSES FROM WHOLE FRUIT AND FRUIT HALVES OF LEMON CULTURED IN VITRO

A comparative growth study was conducted on juice vesicles cultured in the form of various fruit explant types (equatorially bissected fruit halves, longitudinally bissected fruit halves, one-eighth sections of fruit, one-quarter sections of fruit, whole carpel segments, 2 or 3 mm thick equatorial slices of fruit, and 1 cm² fruit endocarp pieces) from 15 mm diam Citrus limon (L.) Burm. f. cv. Eureka lemons. Juice vesicles within equatorial fruit halves produced the least amount of callus. Furthermore, these juice vesicles grew similarly to juice vesicles occurring in the tree grown fruit. A study of cultured equatorial fruit halves using 10–45 mm diam lemons was then conducted. Fruit half cultures containing juice vesicles could be readily established from 15–45 mm diam lemons. Vesicles from 10 mm diam fruit halves, however, invariably produced callus. Vesicles cultured within fruit halves produced proportionately less callus as their fruit diam increased. Juice vesicles cultured in 15–30 mm diam fruits lost their original green color and turned opaque as they matured (i.e., after 3–6 months in culture). A method is also presented, whereby whole lemon fruits can be established and maintained in vitro. Lemons, 35–45 mm in diam, were the best explant sources for establishing whole fruit cultures. Juice vesicles in whole fruit cultures may remain viable for up to 8 months in culture.     

Computerized Optimization of the Relative Growth of Callus Cultures of Orange Fruit Tissues7

The induction and growth of callus cultures from different partsof the fruit of Coorg mandarin orange (Citrus reticulata, Blanco)and the hormonal combinations that can maximize the growth witha basal MS medium, have been explored using Response SurfaceMethodology. The relative growth, protein, peroxidase pattern,and GC fingerprinting of volatiles have been studied. Calluscultures from juice vesicles were found to be more similar totheir explant counterpart than those from albedo, for all propertiesstudied. Key words: Callus culture, Citrus, albedo, juice vesicles, response surface methodology, growth optimization     

Differential effects of sucrose, abscisic Acid, and benzyladenine on shoot growth and callus formation in the abscission zone of excised citrus buds

The omission of sucrose from the basal medium stimulated callus formation in bud explants of Citrus sinensis (L.) Osbeck. Moreover, it increased the abscisic acid-induced callus proliferation reported earlier in the presence of 5% sucrose (Altman and Goren, Physiol. Plant. 32: 55, 1974). The inhibition of callus formation by the addition of sucrose was not due to the high osmotic potential of the medium. Benzyladenine induced callus formation slightly, in all sucrose concentrations up to 5%. The high level of sucrose was required, however, for the growth of shoots from buds cultured on both basal and benzyladenine-containing media.     

牛蒡组织培养和高频植株再生的研究

通过对牛蒡(A rctium lapp a L.)不同外植体、不同激素配比的比较研究,建立了牛蒡离体培养高效植株再生体系.牛蒡子叶与下胚轴切段在含2.0 m g/L 2,4-D和0.5~2.0 m g/L BA的M S培养基中愈伤组织诱导率可以达到87%~100%;在1.0~3.0 m g/L NAA和0.5~2.0 m g/L BA的M S培养基上通过愈伤组织间接分化或外植体直接分化形成不定芽,其中愈伤组织分化率可达100%;下胚轴的分化率明显高于子叶,在1.0 m g/L NAA和1.0 m g/L BA的M S培养基上下胚轴直接分化率达77.3%.组织学观察发现牛蒡再生有器官发生和体细胞胚发生两种途径.将生长状态良好的不定芽转至含1.0 m g/L IBA和1.0 m g/L NAA的1/2 M S培养基上生根,移栽,成活率达到93.3%.从诱导愈伤组织到组培苗在珍珠岩中过渡成活,大约需要13周.组培苗次年开花并结实,生长形态特征正常.     

Repression of asexual embryogenesis in vitro by some plant growth regulators.

A factor that represses asexual embryogenesis has been observed in the Rutaceae, with particularly high concentrations in the naturally monoembryonic cultivars. This investigation was an initial step towards identifying the factor. Citrus reticulata Blanco Ponkan mandarin nucellus explants and Daucus carota L. 'Queen Anne's Lace' callus were employed to examine effects of known plant growth regulators and to determine possible identity of one or more of them with the repressive factor. The chalazal halves of ovules of C. media L. 'Citron of Commerce' were used as control repressor source. Embryo initiation and growth of both test tissues were depressed markedly by 2,4-D, abscisic acid and ethephon. Slight inhibitions were obtained with IAA, kinetin and gibberellic acid. Recovery from the repressor did not occur readily in Citrus nucellus following recultures in citron-ovule-free medium; carrot callus resumed normal embryogenesis immediately upon transfer to suppressor-free medium. The repression by natural sources apparently involved the combined action of some or all natural hormones that are generically related to the above.     

Production of Virus-tree Nucellar Plantlets from Unfertilized Ovules of Citrus in vitro

On a series of Murashige and Tucker (MT) media supplemented with different growth regulators, the 8-week-old unfertilized ovules of Washington navel orange (Citrus sinensis) were able to regenerate perfect plantlets via somatic embryogenesis or organogenesis. The sorts and combinations of exogenous hormones had remarkable effects on the induction, growth and differentiation of its callus. It was found that the most suitable induction medium was MT medium supplemented with 1.0 mg/l BA and 0.5mg/l 1AA. The most suitable differentiation medium was MT medium supplemented with 1.0 mg/l IBA. It was proved by indicator plant examination that the nucellar plantlets free of citrus exocortis virus (CEV) and citrus tristeza virus (CTV) had been obtained from infected trees.     

扁桃愈伤组织诱导及分化的研究

以扁桃优良品种'Naporeil'的茎段、叶片和花药作为外殖体,分别对其进行愈伤组织诱导和分化研究,以筛选愈伤组织的最佳诱导增殖培养基、分化培养基和生根培养基.结果表明,该品种以茎段、花药作为外殖体最易诱导获得愈伤组织,叶片不适宜作为外殖体诱导愈伤组织;愈伤组织的最佳诱导增殖培养基均为B5+0.5 mg/L 2,4-D+0.2 mg/L 6-BA+1.0 mg/L NAA,愈伤组织诱导率为100%,增殖倍数最高可达7倍;茎段愈伤组织的分化培养基为MS+0.2 mg/L NAA+0.8 mg/L 6-BA+0.5 mg/L ZT,分化率为71%;花药愈伤组织未见分化.由茎段愈伤组织再分化获得的不定芽在1/2MS+0.5 mg/L IBA培养基上诱导生根,并给以黑暗预处理可使生根率达80%以上.     

罗布麻愈伤组织诱导及植株再生

以罗布麻(Apocynum venetum L.)当年的成熟种子和5周龄的幼苗叶片为外植体,研究了不同激素组合、暗培养对愈伤组织及植株再生的影响.结果表明,幼苗作外植体诱导愈伤的最佳培养基为添加1.0 mg/L 6-BA 0.2 mg/L IBA的MS培养基;继代培养中1.0 mg/L 6-BA与0.2 mg/L IBA组合愈伤致密而生长迅速,长时间培养硬化的愈伤组织可用添加0.5 mg/L 6-BA和0.1 mg/L IBA培养基和初期暗培养获得大量质地疏松、增殖迅速的愈伤组织;再生苗诱导以0.5 mg/L 6-BA 0.2 mg/L IBA组合为佳;1/2MS附加NAA 0.6 mg/L为适宜的生根培养基,初步建立了罗布麻离体再生体系.     

Repression of asexual embryogenesis in vitro by some plant growth regulators

Summary A factor that represses asexual embryogenesis has been observed in the Rutaceae, with particularly high concentrations in the naturally monoembryonic cultivars. This investigation was an initial step towards identifying the factor.Citrus reticulata Blanco Ponkan mandarin nucellus explants andDaucus carota L. ‘Queen Anne's Lace’ callus were employed to examine effects of known plant growth regulators and to determine possible identity of one or more of them with the repressive factor. The chalazal halves of ovules ofC. media L. ‘Citron of Commerce’ were used as control repressor source. Embryo initiation and growth of both test tissues were depressed markedly by 2,4-D, abscisic acid and ethephon. Slight inhibitions were obtained with IAA, kinetin and gibberellic acid. Recovery from the repressor did not occur readily inCitrus nucellus following recultures in citron-ovule-free medium; carrot callus resumed normal embryogenesis immediately upon transfer to suppressor-free medium. The repression by natural sources apparently involved the combined action of some or all natural hormones that are generically related to the above. This paper is part of B. Tisserat's Ph.D. dissertation in Botany at the University of California, Riverside. The research was supported in part by the Elvenia J. Slosson Fellowship in Ornamental Horticulture awarded to T. Murashige.     

In Vitro Induction of Regeneration of Fruit-Like Structure in Lycopersicon esculentum Mill.

It is possible to induce in vitro regeneration of fruit-like structures from tissue pieces excised from young pistil and young fruit of Lycopersicon esculenturn Mill. These fruit-like structures could be cultured in vitro to maturation, meanwhile they became red coloured. Under dissection it was observed that the fruit-like structure consists of fruit flesh and fruit coats without seeds and plancentae. Tests of exogenous hormones and explant ages revealed: 1. Supplement with exogenous cytokinins alone could induce the explants excised from the pistils to regenerate the fruit-like structures, and the highest induced frequency (50%) was obtained when 0. 5 mg/L zeatin was supplemented. Exogenous auxin seems unnecessary. 2. When tissues of young fruit were used as explants, all explants excised from 4—12 mm (diameter) young fruits could be induced to differentiate the fruit-like structures on medium supplemented with exogenous hormones. However, the hightest frequency of induction (62.5%) was obtained only in tissues of explants excised from 8 mm (diameter) sized fruit and cultured on medium supplemented with 2 mg/L of 6-BAP and 1 mg/L of NAA where regermination of the fruit-like structures took place. In order to investigate the expression of cell totipotency arisen in the regeneration of the fruit-like structure a concept of partial expression of plant cell totipotency was proposed and discussed.     

Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration

The effects of cocultivation with Agrobacterium tumefaciens, regeneration and selection conditions on the transformation efficiency of citrange (Citrus sinensis L. Osbeck×Poncirus trifoliata L. Raf.) have been investigated. Factors such as cocultivation period, preculture of explants, use of acetosyringone or feeder plates during cocultivation, cocultivation on a medium rich in auxins, postcultivation in darkness, and different kanamycin concentrations for selection were assessed. A 3-day cocultivation on a medium rich in auxins improved transformation frequencies, since it increased the number of dividing cells competent for transformation, at the cut ends of the explants. Exposure of explants to darkness for 4 weeks on selection medium resulted in further callus development and increased the regeneration frequency of transgenic shoots. Furthermore, this treatment drastically reduced the number of regenerated escape shoots. A transformation efficiency of 41.3% was achieved using the optimized transformation procedure. Received: 4 November 1997 / Revision received: 7 January 1998 / Accepted: 13 February 1998     

Morphogenesis and tissue cultures of three citrus species

Studies were performed to define tissue culture techniques and culture conditions for morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis (L.) Osb.), citron (C. medica L.) and lime (C. aurantifolia) (Christm. Swing). The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l^-1 for sweet orange and lime, and 3 mg l^-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l^-1 for sweet orange and citron, and 1 mg l^-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mg l^-1 NAA and 0.25 mg l^-1 BA. Callus was maintained by periodical subculture into the same medium supplemented with 10% (v:v) organge juice. In vitro plantlets of the three species were obtained by rooting of shoots developed from bud cultures, and of citron and lime by development of shoots from root cultures. The plants were successfully established on soil.     

文心兰原球茎液体增殖培养研究

以茎尖诱导形成的原球茎(protocorm-like bodies,PLBs)为外殖体,采用液体培养方式比较了不同浓度的激素配比、蔗糖浓度和添加不同量的新鲜椰汁对文心兰PLBs增殖的影响,并比较了相同培养基成分时液体培养PLBs增殖、分化成苗和固体培养PLBs增殖和分化成苗的差异。试验结果表明:不同浓度的外源激素及其配比对文心兰PLBs增殖生长影响较大,6-BA0.5 mg/L Ad 0.05 mg/L NAA0.05 mg/L的激素组合比较适合文心兰PLBs增殖;蔗糖浓度对文心兰PLBs增殖的影响也较大,适合文心兰PLB在液体培养条件下增殖的蔗糖浓度为7.5 g/L;添加5%新鲜椰汁不仅对文心兰PLBs增殖有促进作用,而且能改善PLBs的质量;适合文心兰PLBs增殖的培养基为MS 6-BA0.5 mg/L Ad 0.05 mg/L NAA0.05 mg/L 5%椰汁 蔗糖7.5 g/L。文心兰PLBs在5周内的增殖生长曲线呈倒"V"字形,第3周的增殖速度达最高峰,而固体培养基PLBs增殖速度较慢,生长曲线几乎成直线。液体增殖的PLBs分化成苗较固体培养增殖的PLBs差。     

In vitro fertilization and seed development inTrifolium

Summary Pistils ofTrifolium repens L. andT. ambiguum Bieb. were cultured on an agar-based modified Murashige-Skoog medium. Pistils with and without accessory floral parts were removed from flowers of selected clones ofT. repens, hand-pollinated under aseptic conditions, and planted on the medium. Pistils cultured without accessory floral parts showed no evidence of fertilization after 2 weeks. However, 52% of thoseT. repens pistils cultured with calyx lobes and pedicels contained ovules with maturing embryos 12 days after in vitro cross-pollination. Pistils fromT. ambiguum intraspecific cross-pollinations could not be cultured successfully under the same conditions; however, addition of various combinations of auxin, cytokinin, and gibberellic acid enhanced embryo growth. Fertilization and partial embryo development occurred in interspecific crosses betweenT. ambiguum andT. repens orT. hybridum only whenT. ambiguum was used as the pistillate parent. These results indicate that embryological development under in vitro conditions closely parallels in situ development although growth regulator requirements may vary among species. This work is Technical Contribution 1785 from the South Carolina Agricultural Experiment Station and was supported by SCAES-USDA Cooperative State Research Agreement No. 616-15-65.     

膀胱果种子离体萌发与植株再生

以膀胱果种子为外植体,通过对基本培养基、生长调节剂配比及移栽基质的筛选,初步建立其离体培养再生体系。结果表明,MS培养基是较适合膀胱果种子萌芽和幼苗生长的基本培养基;诱导下胚轴脱分化成愈伤组织的较佳培养基配方为MS + 2,4-D 0.5 mg/L + 6-BA 0.5 mg/L;顶芽增殖培养的最佳培养基为MS + KT 2.0 mg/L + NAA 0.2 mg/L;芽苗生根的最佳培养基为1/2MS + NAA 1.0 mg/L;以泥炭土作为移栽基质,成活率达70%,且幼苗生长健壮。     

桤叶唐棣组织培养研究

以桤叶唐棣的带芽茎段为外植体,对桤叶唐棣离体培养技术进行了初步研究,结果表明：初代培养基为MS IAA0．1mg／L 6-BA 0．5mg／L KT 0．5mg／L;增殖培养基为MS IAA 0．1mg／L 6-BA 2.0mg／L,增殖芽数最大,达到5．4;GA3对茎芽长无促进作用;在1／4MS无激素培养基上,生根率达87．9％。     

白蕊草组织培养和快速繁殖的研究

通过对白蕊草组织培养的培养基种类,激素配比,添加物汁液,碳源的研究,得出适合白蕊草侧芽分化生长的最佳培养基为MS＋6－BA2.0mg/L＋NAA0．5mg/L 狗牙根汁液。诱导愈伤组织以2,4－D浓度在0.5mg/L-2mg/L之间最佳。碳源以蔗糖,浓度3％最佳。生根实验表明：较低浓度的生长素不利于生根,最佳生根培养基为MS＋6－BA0.5mg/L NAA2mg/L 狗牙根汁液或MS＋6－BA0.5mg/L IBA2mg/L 狗牙根汁液。     

青叶胆愈伤组织的诱导与液体继代培养

以青叶胆(Swertia mileensis)幼叶为外植体,在添加不同生长调节剂组合的MS培养基上进行愈伤组织诱导。结果表明,最有效的生长调节剂组合为IAA 1.0 mg/L+NAA 1.0 mg/L+KT 1.0 mg/L和NAA 4.0 mg/L+KT 2.0 mg/L+GA₃ 1.0 mg/L,其诱导率分别为96.6%和96.0%。使用以上两种生长调节剂组合进行愈伤组织的液体继代培养,10 d可使愈伤组织增殖率分别达257.3%和310.2%。     

强德勒红心柚离体培养与植株再生体系的建立

以强德勒红心柚（Citrus grandis Osbeckcv. Chandler）种子萌发的无菌苗为材料,选取子叶、子叶节段、上胚轴、带芽的茎段进行离体培养研究。结果表明：子叶节段是诱导丛生芽的最佳外植体,诱导率100%;诱导丛生芽的最佳培养基为MS＋6-BA2.0mg/L＋NAA0.05mg/L＋蔗糖30g/L＋活性炭0.4g/L,丛生芽增殖可达11.2倍;最适生根培养基为1/2MS＋NAA0.5mg/L,生根率达100%,移栽15d后成活率100%。