Micro-propagation of strawberry plants in vitro — Effect of growth regulators on incidence of multi-apex abnormality

A morphological abnormality occurring in micro-propagated strawberry plants is described and illustrated. The condition is recognised in established micro-propagated plants by an abnormal phyllotaxy which results typically in 2 or more young leaves emerging together rather than singly and by an excessive number of small branch crowns being produced. Dissection shows that the abnormality originates at the apex and is manifest as a linear (or less often as a circular) arrangement of multiple apices. Such multi-apexing plants “bud-off” normal single-apexed crowns from time to time but the abnormality can persist in the main shoot for 2 years (the limit of our observations). The multi-apexing differs fundamentally from clusters of single apices which, when removed from culture and separated, give rise to normal single-crowned plants.Similarities between the multi-apexing condition and fasciation in other plant species are discussed. Changes in growth regulator constituents of the culture media are shown to control the incidence of multi-apexing. In the presence of indole-3-butyric acid (IBA), higher 6-benzyl-aminopurine (BA) concentration increases the frequency, while adding gibberellin (GA₃) decreases the incidence of affected plantlets. The frequency of abnormality varies greatly between different cultivars.It is suggested that conditions favouring greater proliferation rates in culture may also favour multi-apexing. Results indicate that multi-apexing can be avoided by using appropriate concentrations of growth regulators in culture media which will concomitantly maintain a good proliferation rate.     

Biochemical changes in micropropagated grape (Vitis vinifera L.) plantlets due to arbuscular-mycorrhizal fungi (AMF) inoculation during ex vitro acclimatization

Improved establishment of mycorrhizal tissue culture derived plantlets during acclimatization (Stage IV) is commonly attributed to the enhanced vegetative growth as a resultant of different morphological and in vivo changes. These changes are early and better cuticle development, high biomass accumulation, enhanced physiological changes, improved mineral nutrition, especially phosphorus and micronutrients, etc. However, improvement in establishment of micropropagated plantlets during acclimatization may not only be limited to these mechanisms. In the present investigation, biochemical status of micropropagated grape plantlets in response to six single and a mixed strains of arbuscular-mycorrhizal fungi (AMF) during hardening were studied under glasshouse conditions. The histochemical studies revealed that the mycorrhizal inoculation resulted in accumulation of different biochemicals in the plant system such as chlorophyll, carotenoids, proline, phenol and enzymes like polyphenol oxidase (PPO) and nitrate reductase (NR). The mycorrhizal plantlets showed enhanced survival and improved tolerance against stresses experienced during weaning phase. The mycorrhizal plants also exhibited improved physiological and nutritional status and had higher relative water content (RWC) and photosynthetic rate. These plantlets also accumulated higher N, P, Mg and Fe concentrations, which may primarily be as a result of biochemical changes brought about by mycorrhizal association. Mycorrhizal plantlets also showed better hardening under glasshouse conditions. The result suggests that the biochemical changes brought about by mycorrhization were helpful in mitigating different stresses experienced by the tissue culture plants during hardening, which determine their performance later in field.     

The physiology of Cymbidium plantlets cultured in vitro under conditions of high carbon dioxide and low photosynthetic photon flux density

Summary

Cymbidium plantlets were grown in vitro under conditions of high CO₂ and low photosynthetic photon flux density using the Miracle Packt culture system. Shoots and roots of plantlets showed differential growth characteristics. Shoot growth was not different in plantlets cultured under CO₂-enriched (CDE) and non-enriched (NCDE) conditions. Root growth was promoted in plantlets cultured under CDE in the presence or absence of 2% sucrose (S) with rockwool (R) as the supporting material. Growth was poor in plantlets cultured in 1% agar. Root growth was best in plantlets cultured under CDE R+S. Sucrose is still an important component for root growth under CDE conditions even though CO₂ can be used as an alternative carbon source. Photosynthetic measurements (CO₂ uptake and total Rubisco activity) showed the presence of active and operational photosynthetic machinery in plantlets cultured under CDE and NCDE conditions. The apparent lack of photoautotrophy (as evident from the lack of starch grains in chloroplasts) in plantlets cultured under NCDE conditions is not the result of a lesser potential for photoautotrophy; rather it is a consequence of sub-optimal CO₂ concentrations within the culture vessels.     

Culture method and photosynthetic photon flux affect photosynthesis,growth and survival of Limonium ‘Misty Blue’ in vitro

Microcuttings (shoots each with two leaves) of Limonium ‘Misty Blue’ were cultivated in vitro for 28 days under photoautotrophic (sucrose-free culture medium; CO₂ and photosynthetic photon flux (PPF) enriched conditions), photomixotrophic (medium with 30 g l⁻¹ sucrose; CO₂ and PPF enriched conditions) and heterotrophic (medium with 30 g l⁻¹ sucrose; CO₂ non-enriched conditions) methods. Several growth variables were measured during and at the end of cultivation: shoot fresh and dry weight, percentage of shoot dry matter, root fresh weight, number of leaves, leaf area, chlorophyll and sugar content of leaves, stomatal density and size, net photosynthetic rate (NPR) and percent survival of plantlets ex vitro. Plantlets grown in photoautotrophic and photomixotrophic methods had more leaves, high chlorophyll and sugar contents, high NPR, and showed high percent survival. However, these plantlets possessed less number of stomata per square millimeter. In contrast, the plantlets grown by the heterotrophic method showed decreased values of these growth variables except for the number of stomata per square millimeter. These results indicate that CO₂ enrichment for plantlets in vitro at a relatively high PPF would promote photosynthesis and hence growth of chlorophyllous explants/plantlets in vitro. The resulting plantlets were acclimatized better and sooner on ex vitro transplantation.     

Aerial plantlet formation in Asparagus officinalis L.

Removal of rhizome buds from asparagus plants grown in pots led to the formation of aerial plantlets from the nodes on the lower portion of shoots. A similar development was observed in vitro in cultured plantlets derived from bud explants if left for a sufficient length of time in culture. Aerial plantlets which developed either in vitro or in vivo could be detached and established in soil. These plants were morphologically indistinguishable from normal asparagus seedlings.     

Transplanting Gladiolus plants propagated in vitro

In vitro propagated Gladiolus plantlets cultivar ‘Eurovision’, subcultured to a pretransplanting low-mineral and sucrose medium under high irradiance flux, developed functional roots, and after transfer to non-aseptic conditions, continued to grow without becoming dormant.Transplanted plantlets, after hardening, produced larger-size plants and cormlets (small corms) under non-aseptic conditions than formed in vitro, thus shortening the time for the propagation of large-size cormlets.     

Globe artichoke plants obtained from shoot apices through rapid in vitro micropropagation

A rapid method of in vitro propagation for globe artichoke (Cynara scolimus L.) is described. Shoot apices and subterminal stem segments were cultured on modified Murashige and Skoog (1962) medium (MS) with NaH₂PO₄ (50 mg/l), m-inositol (100 mg/l), L-tyrosine (100 mg/l), adenine (40 mg/l), indoleacetic acid (IAA, 0.5 mg/l), kinetin (K, 10 mg/l). sucrose (4%) and agar (0.7%) at pH 5.5. On this medium, a high number of proliferating shoots was obtained. The number of these shoots was increased and their overall development improved by sub-culturing on a MS medium with a reduced concentration of K (5 mg/l) and without the additional amount of sodium dihydrogen phosphate. In these conditions, a 4.5 rate of shoot proliferation was reached after 3 weeks.To induce rooting, the proliferated shoots were transferred to a medium containing half MS, thiamine-HCl (1 mg/l), m-inositol (100 mg/l), ascorbic acid (10 mg/l), naphthaleneacetic acid (NAA, 2 mg/l), sucrose (2%), agar (0.7%) at pH 5.5. In these culture conditions about 84% of plantlets rooted.Cytological analyses performed on root tips of 20 randomly chosen plantlets showed that all the analysed plants contained the diploid number of chromosomes. The plantlets were successfully transferred to soil and the method described seems to be suitable for rapid propagation of globe artichoke.     

Acclimatisation to mannitol-induced water deficit in miniature rose (Rosa hybrida) plantlets cultivated in vitro under autotrophic conditions

Summary

Miniature rose plantlets at the flower development stage were grown photo-autotrophically on MS medium and subsequently exposed to water deficits of –0.23, –0.32, –0.40, or –0.67 MPa osmotic potential ( _s) for 14 d. The _s in the culture medium was raised by increasing the concentration of mannitol, which caused abnormal floral development in terms of the flowering percentage and the number of flowers per plantlet, as well as delayed flowering. In vitro flowering and the number of flowers per plantlet declined significantly when miniature rose plantlets were exposed to water deficit stress at –0.40 MPa or –0.67 MPa. Reductions in growth, pigment degradation, chlorophyll a fluorescence, and net photosynthetic rate (Pn) were greatest in plantlets exposed to a water deficit stress of –0.67 MPa. This was particularly evident in the case of Pn, with a decline of 73.7% compared to non-stressed control plantlets. In contrast, proline levels increased in plantlets under water deficit stress, as proline performs a key role as an osmoprotectant under such conditions. The flowering stage in miniature rose plantlets is particularly susceptible to water deficit stress, which suppresses the development of reproductive organs. Knowledge of the responses to water deficit stress at the reproductive stage may be applied to identify effective indices for the selection of genotypes with increased tolerance to water deficit in miniature rose breeding programmes.     

Morphological and physiological characteristics of micropropagated strawberry plants rooted in vitro or ex vitro

In the classical method of strawberry micropropagation, the rooting phase is done in vitro. The trials were undertaken to replace in vitro rhizogenesis by a direct ex vitro rooting. The aim of the present study was to evaluate the morphological and physiological status of strawberry plants rooted by both methods. The micropropagated shoots of strawberry, cultivars Senga Sengana, Kent and Kama were rooted by the standard method at the in vitro stage or they were treated as soft cuttings and rooted ex vitro (in non-sterile conditions). After a 4-week rooting period the plantlets rooted ex vitro had larger root systems than in vitro-rooted ones, as evidenced by a significantly lower ratio of shoot to root dry weights (2.95, 3.33 and 4.24, respectively, for ex vitro rooted Senga Sengana, Kent and Kama plants versus 5.09, 6.95 and 5.04 for in vitro rooted plants of the same cultivars). During subsequent growth, differences in development increased and were most pronounced in runner formation, more than twice as many runners were formed by ex vitro, than by in vitro-rooted plants. Chlorophyll fluorescence parameters showed that photochemical activity was similar in the leaves from plants rooted in vitro and ex vitro. Values of chlorophyll fluorescence parameters indicated that persistent leaves play the key photosynthetic role at the beginning of the growth period. With the formation of new leaves, the photosynthetic activity of the persistent leaves decreased and their function was taken over by the newly formed ones.     

Continuous propagation of tomato plants by means of callus cultures

Callus obtained in vitro from stem internode tissue was used to investigate the possibilities for accelerated vegetative propagation of Lycopersicum esculentum Mill. and Lycopersicum peruvianum (L). The results of different tests with phytohormones pointed to a high endogenous auxin level in the original stem explants of L. peruvianum. In L. esculentum shoots were obtained when high amounts of zeatin or coconut-milk were applied.Explants of CCC-pretreated (2 chloroethyl trimethyl-ammonium chloride) tomato plantlets showed a significant increase in shoot formation as compared to the untreated ones.Callus tissue that was more than 2 years old and had been used in 30 transfers still had the capacity to produce normal shoots and roots.More than 200 resulting plants were observed in glasshouse conditions for possible genetic variations. No striking deviations from the original phenotype occurred. Seeds harvested from the fruits of self-fertilized flowers on these plants were sown under normal growth conditions. Some plants of one particular cultivar showed signs of accentuated vegetative development.     

Air exchange rate affects the in vitro developed leaf cuticle of carnation

The leaf surfaces of Dianthus caryophyllus plants cultured in vitro in either airtight or ventilated vessels were examined using scanning electron microscopy (SEM). The resultant hyperhydrated, non-hyperhydrated and acclimatized plants were compared for stomatal density, cuticular wax development and stomatal function. The leaf surfaces of in vitro cultured plants were basically the same as those of acclimatized plants but less wax deposition was observed on their leaves. Stomata were found both open or closed after transfer of plants ex vitro. However, stomata of in vitro leaves grown in ventilated culture vessels were more functional than plants grown in other conditions. Acclimatized plants had a normal leaf epidermal surface, and were wholly covered with waxes; their stomatal density being similar to that of highly ventilated plants but lower than that of less ventilated plants. Leaves of plants grown in airtight culture vessels or under a low number of air exchanges per hour had less waxes than plants grown at a higher number of air exchanges per hour or than acclimatized plants. In contrast, hyperhydrated plants had abnormal, malformed stomata and no wax deposition was detected. The adaxial surface of non-hyperhydrated leaves seemed more normal than the abaxial, especially in the most ventilated vessels, and this may be due to the former receiving more light and so developing in a more favourable microenvironment.     

火炬姜离体快繁技术研究

 试验表明：火炬姜组培快繁以Ms+6-BA 2.0mg/L+NAA 0.1 mg/L+CW 10%作为增殖培养基可以获得理想的效果，增殖倍数可达4.3倍；以双芽为外植体在该培养基上经30 d培养可增殖5.4倍；在不含任何激素的1/2 Ms+0.15%活性炭培养基上即可形成较发达的根系，生根率达100%；试管苗移栽对基质要求不严，移栽到疏松透气、富含有机质的表土中，成活率高达100%，而且植株生长良好。     

试管芋诱导的研究

 以芋组培苗为试材, 研究了蔗糖浓度、激素浓度、光照时间、培养温度、不同大小试管苗,不同芋品种类型等因素对试管芋诱导的影响及不同基质对试管芋育苗成活率的影响。结果表明: 诱导试管芋较理想的培养基为MS + 蔗糖8 % + BA 1. 0 mg·L ^{- 1} + NAA 0. 5 mg·L^-1 , 光照12 h/ d , 培养温度30 ℃; 试管苗越大越有利于试管芋的形成; 试管芋可保持其原有的特性; 基质影响试管芋育苗的成活率, 4 种基质中蛭石最好。     

苹果三倍体后代培养及倍性鉴定

以自然授粉的三倍体苹果品种乔纳金的种子为材料,研究了培养方式对苹果三倍体实生后代获得的影响及实生后代的倍性水平。结果表明,离体培养与常规播种在乔纳金种子成苗率上存在明显差异,其中离体培养的成苗率是48.1%,常规播种的成苗率是20.9%,离体培养获得的植株是常规播种获得的植株的2倍以上。本研究获得的690个乔纳金实生后代中以非整倍体植株占多数,共获得非整倍体452株,占植株总数的65.5%。在后代中有13个为多倍体,其中离体培养获得7株三倍体和4株四倍体,而常规播种只获得2株三倍体,表明离体培养可以获得较多的多倍体资源。乔纳金实生后代中不同倍性植株在形态上差异显著,根据植株形态和叶形指数,可以容易地将多倍体(三倍体、四倍体)植株与二倍体植株和非整倍体植株区分开来。     

In vitro plantlet formation from Citrus species and hybrids

This study has shown that in vitro culture techniques can be used to reduce the interval between pollination and the identification of useful hybrids from a Citrus breeding programme. Seedling material of 5 parental lines involved in the programme to select rootstock hybrids for increased tolerance to environmental stress were examined for their capacity to regenerate plants in vitro. These results were compared with those achieved with mature vegetative tissue from the same varieties.Benzyladenine at 10 μM stimulated multiple shoot production from juvenile and mature nodes of all varieties tested and induced adventitious shoots from stem internodes of seedling varieties. Multiple shoots were also produced from apical fragments of some of the Citrus varieties. A hybrid variety showed the highest regenerative competence. High levels of exogenous auxin (e.g. 10 μM naphthaleneacetic acid) were found to be necessary for rooting in vitro-grown shoots. The resulting plantlets were successfully transplanted to glasshouse conditions.     

应用花药培养技术培育苹果新类型

通过对苹果不同品种花培植株的试管苗及其高接树的染色体数目观察,看到大部分为单倍体,还有其他倍性的植株.幼龄期元帅花培植株的植物学性状与原品种元帅之间有显著差异,表现分枝多、有针刺、叶片小而薄、茸毛少、有缺刻等野生性状.在花培植株之间,也表现有明显的差别.田间高接后,花培植株随树龄增长逐渐向栽培性状转化,元帅花培植株元80—3已开始结果,表现出明显的短枝型特征.     

食用百合试管苗缓慢生长保存的研究

为了建立食用百合种质资源缓慢生长保存体系,将扩繁培养后得到的试管苗接种到12种不同培养基中,10℃和25℃下分别保存6个月,观察不同处理试管苗生长情况。结果表明:低浓度甘露醇对百合试管苗生长的抑制效果差,而高浓度甘露醇严重影响其生长势,缓慢生长保存的最适浓度为20g·L-1;蔗糖对百合试管苗的生长有抑制作用,适宜浓度为60g·L-1;20g·L-1甘露醇+60g·L-1蔗糖处理的百合试管苗存活率均达100%,且有鳞茎形成,保存效果最好。10℃保存的试管苗生长缓慢,有利于鳞茎形成,保存至6个月时不需要继代培养;25℃保存试管苗的鳞茎形成率低,培养基损失量高,必须继代才能继续保存。     

那贺川野菊的离体保存

以那贺川野菊为试材,研究了不同浓度蔗糖和多效唑（PP333）对试管苗离体保存的影响,并对保存材料再生后代的遗传稳定性进行了分析。结果表明：常温（23 ± 2）℃、光照强度2 000 ~ 3 000 lx、光照时间12 h • d-1的培养条件下,在MS + 2.0 mg • L-1 KT + 0.1 mg • L-1 NAA + 6.5 g • L-1琼脂培养基中,蔗糖浓度为15 ~ 30 g • L-1时附加6 ~ 9 mg • L-1 PP333能保存试管苗360 d以上,存活率达93.53% ~ 100%,且恢复生长后试管苗长势良好,其再生后代的形态特征、过氧化物酶（POD）酶谱和ISSR-PCR扩增图谱与对照相比没有明显差异。     

花烛愈伤组织不同继代培养的再分化差异

 对花烛( Anthurium andraeanum) ‘Jolanba’无菌苗叶片诱导产生的愈伤组织进行3 种不同的培养处理, 观察其再分化表现, 并探讨了3 种继代培养愈伤组织的生理生化特点。结果表明, 适当降低基本培养基浓度、减少继代次数和延长继代周期培养的愈伤组织分化的芽较多、较壮; 再生芽的分化需要较高水平的可溶性蛋白质, 其生长需要较高水平的可溶性糖; POD 和SOD 活性与分化的芽数呈正相关; GA_{1 + 3}/ABA、ZR_s/ ABA 的高比例与再生芽的高度呈显著正相关; IAA/ ABA 及IAA 的高水平与花烛离体根的发生呈正相关。重新诱导培养和延长愈伤组织继代周期以提高再生芽生长势的主要生理生化基础之一是维持较高的可溶性糖水平和有利的激素平衡。