High frequency plant regeneration through adventitious multiple shoot organogenesis in epicotyl explants of Indian gooseberry (Emblica officinalis Gaertn)

Shoots regenerated adventitiously on epicotyl segments from in vitro seedlings of Emblica officinalis var. ‘Kanchan’. Epicotyls derived from 2-week-old aseptic seedlings were most responsive and produced a maximum number of 303 shoots per explant in Murashige and Skoog (1962) medium (MS) augmented with 8.8 μM N⁶-benzyladenine (BA) + 1.425 μM indole-3-acetic acid (IAA). Shoots readily elongated in MS lacking growth regulators and rooted in half-salt-strength MS (1/2 MS) supplemented with indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). The highest rooting response was recorded in 1/2 MS containing 14.7 μM IBA. Plantlets were acclimatized inside the green house and 80% of the plantlets survived on transfer to garden soil.     

Somatic embryo,adventitious root and shoot regeneration in in vitro grown quince leaves as influenced by treatments of different length with growth regulators

The purpose of this work was to acquire more information on the capacity of in vitro grown quince (Cydonia oblonga Mill.) leaves to simultaneously regenerate somatic embryos, adventitious roots and shoots, and to evaluate the variations induced on regeneration response by treatments of different length with growth regulators. After 2 days of liquid treatment with 2,4-dichlorophenoxyacetic acid, the leaves were cultured for 0, 3, 6, 9, 12, 15, 18 and 21 days on gelled growth medium containing the basal components of Murashige and Skoog and kinetin (Kin) 4.5 μM + naphthaleneacetic acid (NAA) 0.5 μM. At the end of each treatment period, the leaves were cultured on a transfer medium in the absence or in the presence of a growth regulator combination represented by N⁶-benzylaminopurine (BA) 2.66 μM + gibberellic acid 0.58 μM + indole-3-butyric acid 0.3 μM. The culture period for all the treatments was fixed to 52 days.     

High frequency shoot regeneration from cotyledon explants of cucumber via organogenesis

Organogenic callus induction and high frequency shoot regeneration were achieved from cotyledon explants of cucumber. About 86.2% of cotyledon explants derived from 5-day-old in vitro raised seedlings produced green, compact nodular organogenic callus in MS medium containing NAA (2.69 μM) and BA (4.44 μM) after two successive transfers at 20 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MS medium supplemented with NAA (1.34 μM), BA (8.88 μM), zeatin (0.91 μM) and l-glutamine (136.85 μM) with shoot induction frequency of 75.6%. Shoot proliferation occurred when callus with emerging shoots was transferred in the same medium at an interval of 20 days. Shoots (1.0 cm length) were excised from callus and were elongated in MS medium fortified with GA₃ (1.44 μM) and BA (4.44 μM). The elongated shoots were rooted in MS medium supplemented with IBA (3.42 μM) and BA (4.44 μM). Rooted plants were acclimatized in green-house and subsequently established in soil with a survival rate of 80%. This protocol yielded an average of 35 shoots per cotyledon explant in a culture duration of 120–140 days.     

Morphogenic gradients of adventitious bud and shoot regeneration in epicotyl explants of Citrus

The in vitro responses of epicotyl explants from ‘Cravo’ rangpur lime (Citrus limonia Osb.), ‘Foster’ grapefruit (C. paradisi Macf.), and ‘Pera’ sweet orange (C. sinensis (L.) Osb.) were characterized for the first time. Further analysis was performed in ‘Cravo’ rangpur lime and ‘Foster’ grapefruit aiming to verify the in vitro morphogenesis of five distinct regions of the epicotyl under different treatments. It was observed the same general pattern of morphogenic gradient along the epicotyl axis in both citrus cultivars, with greater organogenic response as the distance of the explants from the cotyledonary node increased. This morphogenic gradient was influenced by factors related to plant material, composition of the culture medium, and conditions of incubation. The regions of the epicotyl farthest from cotyledons could be used as a source of explants in experiments of genetic transformation of the genotypes evaluated aiming to improve the efficiency of production of transgenic Citrus plants.     

Effect of sequential subcultures on in vitro proliferation capacity and shoot formations pattern of pineapple (Ananas comosus L. Merr.) over different incubation periods

In vitro obtained shoots of pineapple (Ananas comosus L. Merr.) cv Smooth cayenne was cultured on MS medium enriched with 6-benzylaminopurine (BAP) at 3.25 mg l⁻¹ (14.43 μM) and indole acetic acid (IAA) at 1.75 mg 1⁻¹ (9.40 μM) and subcultured for four times at four different incubation periods (30, 45, 60 and 75 days). By the fourth subculture, irrespective of incubations periods, the explants lost 50% of its shoot formation capacity. Longer incubation resulted on higher rate and total shoots than a shorter incubation, however, the magnitude of that different varied over subcultures. The difference in shoots formation between the explants incubated for 30 and 75 days was 6 shoots at first, increased to 21 at the third and declined to 11 shoots at the fourth subculture. Over a period of 75 days, the majority of shoots formation occurred during the first 30 days (35%) at a rate of 1.5 shoot per week and the last 15 days (40%) at a rate of 3.8 shoot per week. In the period between 30 and 60 days, 15% of shoots at rate of 1.8 and 10% at rate of 1.0 shoot per week formed during the first and the second 15 days interval, respectively. Over four consecutive subcultures, explants incubated for 30 days produced the lowest total (1028 shoots) and that incubated for 75 days produced the highest total (120,917 shoots) from a single explant. No significant differences in total were observed between explants incubated for 45 and 60 days over the first three subcultures. However, by the fourth subculture the total of 60 days (14,288 shoots) was two times higher (7163 shoots) than that of 45 days long incubation.     

外植体及培养条件对葡萄不定芽再生的影响

以优良无核葡萄品种莫利莎、皇家夏天为试材,分别从外植体类型、叶片接种部位、接种方式及培养条件等方面进行不定芽再生技术研究。结果表明,2品种葡萄试管苗离体叶片切割后接种较节间和叶柄容易诱导不定芽再生;将叶片切分为叶尖、叶中、叶基部叶块分别接种,其不定芽再生能力没有差异;取自培养30～50d继代苗上的叶片,其不定芽再生效果相同;叶块以近轴面或远轴面接触培养基的接种方式对不定芽再生影响不大,不过叶片远轴面贴近培养基接种,产生的愈伤组织状态较好;25℃左右小幅变温或恒温的条件能诱发2个葡萄品种产生较多的不定芽;葡萄不定芽再生须经黑暗培养或弱光培养,暗培养时间在2周与4周之间再生效果差异不显著。     

In vitro shoot multiplication and plant regeneration in four Capsicum species using thidiazuron

A procedure of in vitro plant propagation using shoot meristem explants (∼0.5 cm) has been developed for Capsicum annuum cv CA960, C. baccatum, C. frutescens and C. praetermissum on Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497] medium containing various cytokinins. Among various concentrations of cytokinins tested; adenine (Ad), N⁶-benzyladenine (BA), kinetin (Kn), zeatin and thidiazuron (TDZ) individually. TDZ regenerated maximum number (4.2–22.4) of shoots in all the Capsicum species tested. Multiple shoot elongation occurred upon transfer to BA (0.22 μM l⁻¹) + IAA (0.48 μM l⁻¹). Rooting of regenerated shoots was achieved on medium supplemented with 5.71 μM l⁻¹ indole-acetic acid (IAA). Rooting was observed in 72–94% of shoots obtained from TDZ-containing regeneration medium followed by elongation treatment in contrast to 8–22% of shoots without elongation treatment. Plantlets obtained from TDZ-containing media were normal diploid (2n = 24) and could readily be established in the soil under green house conditions with a survival frequency of 68–84%. Regenerated plants were developed into morphologically normal, fertile plants and able to set viable seeds.     

野生毛桃叶片愈伤组织诱导及不定芽再生

【目的】为了初步建立野生‘毛桃1号’叶片愈伤组织诱导、保存及不定芽再生技术体系,【方法】以野生‘毛桃1号’试管苗幼嫩叶片为外植体,探讨了基本培养基、激素配比等一系列因素对‘毛桃1号’叶片愈伤组织诱导和保存的影响及TDZ浓度对愈伤组织不定芽再生的影响。【结果】结果表明,适合‘毛桃1号’叶片愈伤组织诱导的培养基为LP+6-BA 1.0 mg.L-1+2,4-D 3.0 mg.L-1+AgNO30.5 mg.L-1+蔗糖30.0 g.L-1+琼脂6.0 g.L-1;保存的培养基为LP+2,4-D1.5 mg.L-1+CH 0.4 g.L-1+蔗糖30 g.L-1+CA 3.0 g.L-1或PVP 3.0 g.L-1+琼脂5.8 g.L-1,且采用持续暗培养保存,每28 d继代1次。愈伤组织在SH+TDZ 3.0 mg.L-1+NAA 0.3 mg.L-1+蔗糖30 g.L-1+琼脂6.0 g.L-1的培养基上可再生不定芽,再生率为6.83%。【结论】初步建立了‘毛桃1号’叶片愈伤组织诱导、保存及不定芽再生体系,为桃愈伤组织再生和遗传转化的研究奠定了基础。     

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

Callus induction and plant regeneration from cotyledonary explants of ash gourd (Benincasa hispida L.)

A protocol for the production of complete plantlets through multiple shoots from the cotyledon-derived calli of ash gourd (Benincasa hispida L.) is described. The embryos were excised from mature seeds and cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurin (BAP, 1–5 μM). After 10 days the well-developed green cotyledons from the growing embryos were isolated and cultured on MS medium fortified with 2,4-D (1–6 μM). The cultured cotyledons gave rise to luxuriantly growing calli after 6 weeks. These calli were subcultured on MS medium supplemented with various concentrations of BAP (1–6 μM) alone or in combination with naphthalene acetic acid (NAA, 0.2 and 0.5 μM) for regeneration. The regenerated shoots were multiplied and rooted on quarter strength MS medium supplemented with indole-3-butyric acid or NAA (1–5 μM). The rooted shoots were transplanted to soil with 90% success.     

影响早熟油桃茎尖培养增殖效率的因素

以早熟油桃华光、曙光为试材,研究了外源植物生长调节剂种类及浓度组合、培养基种类、暗培养时间等因素对茎尖培养增殖效率和试管苗生根的影响。结果表明:不同浓度的TDZ和KT对华光、曙光油桃增殖效应不同,在G培养基中加入TDZ2.0mg/L和KT1.5mg/L处理效果最好。1/2MS培养基中附加IBA有利于华光、曙光汕桃的生根;华光油桃生根不必经过暗培养;曙光经过6d或9d暗培养能大大提高生根率和有效根数量。     

Piriformospora indica promotes adventitious root formation in cuttings

Adventitious root formation in excised plant shoots is a crucial process in the vegetative propagation of many plant species, and insufficient rooting causes substantial losses in the propagation industry. Based on the various physiological effects on whole plants described for the basidiomycete Piriformospora indica, it was hypothesized that inoculation of the substrate with this endophyte should promote the generation and growth of adventitious roots in cuttings. Inoculation experiments were conducted to study the effects of P. indica on adventitious rooting in three plant species. Inoculation with P. indica dramatically enhanced the number and length of the adventitious roots in pelargonium and poinsettia. Root colonization parameters suggest that the interaction between the endophyte and cuttings had already occurred before physical contact. In contrast, petunia showed no rooting response to P. indica inoculation. Very fast root formation in this plant indicates that a minimum time period for the fungus–plant interaction is required for establishment of a promoting effect. P. indica-based biotechnology is proposed as a new tool for improving plant propagation systems of plant species or cultivars with low to moderate capacity of adventitious root formation.     

杏胚珠培养及幼胚子叶不定芽诱导研究

以盛花后25、35、50、55、60d的凯特杏胚珠为外植体,研究胚珠培养的适宜条件;选择发育一致的幼胚子叶,研究不定芽的发生条件。结果表明,SH培养基是凯特杏胚珠培养的适宜培养基;盛花后25d的凯特杏胚珠(PF=0),仅采用生长培养不足以发育成可见胚,试验采用诱导方式获得了可见胚,诱导出胚率为80%;以MS为基本培养基,BA与NAA结合诱导能力强;但这种胚小且瘦弱,继续生长培养,利用幼胚子叶诱导不定芽。在幼胚子叶诱导不定芽方面,盛花后25、35d取样的凯特杏胚珠,培养后获得幼胚子叶,诱导不定芽的适宜TDZ质量浓度为1.25mg/L,诱导出芽率分别为85.00%、80.00%;盛花后50、55d取样的材料,适宜TDZ质量浓度为2.50mg/L,出芽率分别为65.00%、60.00%,而盛花后60d取样的材料,适宜TDZ质量浓度为5.0mg/L,出芽率为61.11%,不定芽主要发生在子叶的近轴面、胚轴连接处及子叶近轴端两侧;在相同培养基中,25℃进行21d暗培养,盛花后35、50、55、60d材料出芽率分别为72.22%、65.00%、60.00%、55.56%;4℃下28d暗培养,35、50、55、60d依次为59.09%,71.42%、77.78%、86.36%;对不定芽进行伸长、生根与快繁培养,获得了健康正常的植株。通过以上研究,建立了凯特杏幼胚4级培养体系,即(1)胚珠培养,获得可见胚(1级培养);(2)幼胚生长培养(胚培养),获得了健壮幼胚(2级培养);(3)幼胚子叶再生,获得不定芽(3级培养);(4)不定芽组培苗快繁(4级培养)。     

Auxins increase the occurrence of leaf-colour variants in Caladium regenerated from leaf explants

Leaf explants of Caladium ‘Pink Cloud’ were cultured in vitro on MS medium containing various auxins (NAA, IBA, IAA, 2,4,5-T and 2,4-D) in combination with cytokinin (BA). NAA gave the most vigorous in vitro propagation of this plant, and only 15% of the plants were leaf-colour variants on the medium containing 0.5 μmol NAA. Leaf colour variation was observed in all plants regenerated on the medium containing 2,4-D at 0.5–4.5 μmol. In hormone-free medium, only a few leaf-colour variants (6%) occurred, but the rate of plant regeneration was very low. Application of 0.5 μmol NAA together with 4.5 μmol BA seemed to be the most appropriate for in vitro propagation of Caladium ‘Pink Cloud’ with only a few leaf-colour variants.     

Analysis of genetic diversity in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

Agrobacterium tumefaciens mediated genetic transformation and regeneration of Morus alba L.

An efficient method was established for genetic transformation of Morus alba clone M5 using Agrobacterium tumefaciens mediated gene transfer. Cotyledon explants from in vitro grown seedlings were co-cultivated with disarmed strain LBA 4404 harbouring the binary vector p^BI121 carrying chimeric β-glucuronidase (GUS) and neomycin phosphotransferase (npt II) genes. Maximum transformation frequency of 18.60% was recorded with 48 h of pre-conditioning followed by co-cultivation for the same duration. Expression and presence of transgene was confirmed by histochemical test and polymerase chain reaction. The transgenic plants were micropropagated and successfully acclimatised.     

Establishment of efficient regeneration protocol from leaf explants of Iris pumila shoots cultured in vitro

An efficient plant regeneration protocol via somatic embyogenesis by leaf base culture of in vitro grown Iris pumila shoots was developed. Induction of embryogenic calli was achieved on MS media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (4.5 μM, each) and some additives (L-proline, casein hydrolysate, adenine sulphate and tyrosine). Further differentiation of embryogenic calli was achieved on MS hormone-free media, and on media supplemented with either BAP (4.5 μM) or BAP + zeatin (4.5 and 0.2 μM, respectively), which allowed somatic embryos, as well as shoot-like structures to form. Fully developed somatic embryos germinated on an MS hormone-free medium. An anatomical study confirmed that shoot-like structures represented early germinating stages of somatic embryos. Acclimatization of plants derived from somatic embryos was 64% after 1 year and no morphological variation was observed.     

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l⁻¹ BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l⁻¹ BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.