Slow-growth conservation of potato microplants: efficacy of ancymidol for long-term storage in vitro

Ancymidol was investigated as an alternative mediumsupplement to mannitol for slow-growth conservation ofpotato microplants in vitro. Differentconcentrations of ancymidol (0, 5, 10, 15, 20, 25, 30,35 and 40 M) were tested in slow-growthmedia based on MS medium supplemented with either 30or 60 gl^-1 sucrose. The cultures were conservedunder a 16-h photoperiod at two temperature regimesi.e. 24 ± 1 ^°C and 6 ± 1 ^°C. Therewere significant interactions between ancymidol andother factors such as sucrose, temperature andgenotype for microplant survival, microshoot heightand overall microplant growth. Ancymidol did have abeneficial effect on culture viability after prolongedmaintenance in vitro. The growth-inhibitingeffect of ancymidol persisted through a 16-monthculture period. Combined effect of ancymidol, sucroseand temperature showed that optimum culture viabilityand desirable microplant growth were obtained when thecultures were grown in MS medium supplemented with 10M ancymidol plus 60 gl^-1 sucrose at6 ± 1 ^°C. Vitrification and flaccidity, whichare very frequently observed in potato microplantcultures during prolonged maintenance in vitrounder osmotic stress (mannitol), were not observedwhen the microplants were conserved in ancymidolmedia. Genetic stability of potato microplantsconserved in ancymidol media was evaluated usingrandomly amplified polymorphic DNA (RAPD)fingerprints. Ancymidol did not induce any detectablegenetic variation in genomic DNA as visualized by theabsence of either any additional RAPD fragment oralterations in RAPD fragment patterns.     

Micropropagation of Indica rice through proliferation of axillary shoots

Summary Week old seedlings of indica rice variety Jaya obtained on basal MS medium and further sub-cultured on agar solidified MS medium supplemented with cytokinins, sucrose (3% w/v) and mannitol (1% w/v) lead to development of multiple shoot buds. Shoot cultures were maintained and multiplied in liquid medium containing BAP 5 mg l^-1, sucrose (3% w/v) and mannitol (1% w/v). Profuse rooting was obtained on transfer to MS liquid medium containing IBA 1 mg l^-1 and sucrose (3% w/v). Complete plants were successfully transferred to soil and grown to maturity.     

Actions for ex situ conservation of Gloriosa superba L. - an endangered ornamental cum medicinal plant

Factors affecting in vitro propagation and microtuberization were evaluated for Gloriosa superba L., an endangered ornamental cum medicinal plant having limited reproductive capacity. Surface sterilization of tuber explants with 0.1% mercuric chloride (HgCl₂) for 5 min eliminated the contamination effectively with highest survival rate. Among the various combinations used, Murashige and Skoog (MS) medium with 2.0 mg L^?1 6-benzylaminopurine (BAP) + 0.5 mg L^?1 α-naphthalene acetic acid (NAA) containing 3% sucrose with 16-h photoperiod exhibited the greatest in vitro tuberization (3.2) with the highest shoot regeneration frequency (90%). The longest tuber regeneration occurred on MS media containing 4% sucrose. Transfer of in vitro-regenerated shoots to half-strength MS medium with 1.0 mg L^?1 indole-3-butyric acid (IBA) + 0.5 mg L^?1 NAA showed maximum root induction (66.6%). The in vitro-grown plantlets were successfully acclimatized and transplanted to sterilized soil and sand mixture (3:1) in the glasshouse with 70% survival. The colchicine content was determined in the tubers of ex vitro plants by HPLC using the same retention time (1.5 min) as that of the standard colchicine. This revealed that the micropropagation protocol developed by us for rapid mass production could be used as raw material for colchicine extraction and provides a basis for germplasm conservation and genetic improvement of G. superba.     

一种木薯种质资源离体保存方法的初步研究

为探讨木薯种质资源的离体保存方法,以木薯试管苗为主要材料,观测试管苗成活率、株高生长变化及茎秆变化情况,探索卡那培养基与常规培养基对木薯种质资源离体保存的效果。结果表明：在培养室温度25±2℃、光照强度3000 lx、光周期16 h/8 h的环境下,木薯品种‘华南8号’(SC8)试管苗在卡那培养基（MS+0.2 mg/L NAA +80 mg/L Kan +30 g/L蔗糖+9 g/L琼脂）保存时间为22~24个月,是常规培养基试管苗保存时间的10~12倍,且在卡那霉素培养基中生长的试管苗根系较发达、节间显著缩短、茎节数显著增加、茎秆明显增粗、叶色浓绿,温室移栽存活率超过了90%,是常规培养基的5~8倍。该保存方法有效延长了木薯种质资源的离体保存时间,既增强了种苗移载成活率和保存质量,又显著地节约了木薯种质资源保存过程中耗费的人力劳动、试剂药品、水电能源等成本。     

Cryopreservation of axillary shoot tips of in vitro-grown grape (Vitis) by a two-step vitrification protocol

Axillary shoot tips of in vitro grape(Vitis vinifera L. cv. CabernetSauvignon) were successfully cryopreservedby vitrification. Axillary shoot tipswere excised from 4- and 5-month oldplantlets were cultured onsolidified 1/2 MS medium with 1 mg l^-1BA at 25 ^°C. Shoot tips wereprecultured on solidified mediumsupplemented with 0.3 M sucrose for 3 daysand then treated with a mixture of 2 Mglycerol plus 0.4 M sucrose (LS solution)for 20 min at 25 ^°C. They were thendehydrated with PVS2 for 80 min at0 ^°C before being plunged into LN for1 hr. Samples were then warmed rapidly inwater at 40 ^°C. The recovery of theshoot tips amounted to approximately 60%.To enhance further recovery, a two-stepdehydration procedure by vitrification wasexamined. Osmo-protected shoot tips werefirst dehydrated with a 50% PVS2 (halfstrength of the solution) for 30 min,followed by PVS2 for 50 min at 0 ^°C.This revised dehydration procedure improvedshoot recovery from 60 to 80%. Thisprocedure by vitrification was applied toten other species/cultivars of Vitiswith an average recovery of 64%. Thismethod incorporates a simple and reliableapproach for providing high levels ofgrowth recovery. It also appears promisingfor the cryopreservation of Vitisgermplasm.     

大田棉花真叶叶柄组织培养特性研究

 利用已建立的叶柄组织培养体系,对大田棉株不同发育时期、不同部位的叶柄进行组织培养研究。叶柄和无菌苗下胚轴愈伤组织诱导培养基为MSB+IAA 0.1 mg·L^-1+KT 0.1  mg·L^-1+2,4-D 0.1 mg·L^-1+Glucose 30 g·L^-1+Gel 2 g·L^-1(pH 5.8);叶柄愈伤组织分化培养基为MSB+IAA 0.05 mg·L^-1+KT 0.05 mg·L^-1+Glucose 30 g·L^-1+Gel 2 g·L^-1(pH 6.5);无菌苗愈伤组织分化培养基为MSB+IAA 0.02 mg·L^-1+KT 0.04 mg·L^-1+Glucose 30 g·L^-1+Gel 2 g·L^-1(pH 6.5)。研究发现棉花主茎叶叶柄、果枝叶叶柄、营养枝叶叶柄在愈伤组织生长速度方面有一定差异,在愈伤组织诱导和分化方面,除严重衰老叶片的叶柄外,其它部位差异不显著。不同来源的胚性愈伤组织在MSB+6-BA 0.05 mg·L^-1+KT 0.02 mg·L^-1+Sucrose 30 g·L^-1+Gel 2 g·L^-1（pH 6.5）培养基上,均能得到胚状体,并获得再生植株。可见棉花叶柄是优良的组织培养外植体。     

多因子正交试验对甜叶菊丛生芽诱导条件的筛选

利用正交试验设计方法探讨了植物生长物质（6-苄基腺嘌呤、奈乙酸）、蔗糖、水解酪蛋白（CH）对甜叶菊丛生芽诱导的影响。结果表明：蔗糖对丛生芽诱导影响最大，其次是6-BA，NAA、CH影响较小。筛选出甜叶菊丛生芽诱导的最适培养基为MS + 6-BA 1mg/L+ 琼脂6g/L + 蔗糖30g/L。继代培养采用MS + 6-BA 0.5mg/L+ NAA0.05mg/L + 琼脂6g/L + 蔗糖30g/L。生根最适培养基为1/4MS + 0.1mg/L IBA +琼脂6g/L + 蔗糖30g/L +1g/L活性炭，生根率达100%。已获得驯化移栽成活的植株,移栽成活率为92.13﹪。     

Direct plant development from microspore-derived embryos of winter oilseed rape Brassica napus L. ssp. oleifera (DC.) Metzger

The influence of temperature/photoperiod treatment and gibberellic acid concentration (0, 0.1 or 1.0 mg/l) on direct conversion of microspore-derived embryos (MDEs) to plantlets of winter oilseed rape was investigated. Physiologically mature, 21-day-old MDEs were transferred to a solid B5 medium supplemented with gibberellic acid, and cultured at 24 ^°C, 4 ^°C or 1 ^°C for 14 days, and then at 24 ^°C for the next 21 days. Low temperature was linked with short photoperiod (8 h light/16 h dark), and high temperature was linked with long photoperiod (16 h light/8 h dark). The highest embryo conversion rate was at 1 ^°C with over 70%, compared to<20% at 4 ^°C. Two-way analysis of variance confirmed the significance of the effect of temperature/photoperiod treatment. By contrast, gibberellic acid concentration had no significant effect on stimulation of shoot development from apical meristems of MDEs. Roots developed from apical root meristems of MDEs very easily. The best obtained conversion rate of MDEs induced with cold treatment at1 ^°C for 14 days was 86.5%. Observations on morphological development of MDEs showed clear differences in reaction at various temperature/photoperiod treatments. This revised version was published online in August 2006 with corrections to the Cover Date.     

“凤丹白”胚离体培养和植株再生研究

摘　要:以牡丹品种“凤丹白”的胚为外植体,研究了层积时间、基本培养基、植物激素等因素对胚愈伤组织的诱导及不定芽分化的影响。结果表明:40d层积时间对胚萌发的效果最好,MS+Ca2++2 mg&#8226;Ｌ-12,4-D+30ｇ&#8226;Ｌ-1蔗糖是胚诱导愈伤组织的最佳培养基,诱导率为85%;MS+ Ca2++1.0 mg&#8226;Ｌ-16-BA+0.5 mg&#8226;Ｌ-12,4-D+20ｇ&#8226;Ｌ-1。蔗糖是诱导不定芽发生的最佳培养基;高约1.5cm的不定芽转入生根培养基,生根率可达80%。     

Tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation studies in four commercially important indica rice cultivars

This research was undertaken to find an efficient tissue culture system and Agrobacterium-mediated genetic transformation method for recalcitrant indica rice cultivars. For this, mature seeds of commercially important indica rice varieties, ASD16, ADT43, IR 64, and Pusa Basmati were cultured on MS and N6 medium supplemented with 2 mg l^-1 2, 4-D + 30 g l^-1 sucrose. The calli grown in N6 medium showed better friability and embryogenic response. Out of the four varieties tested, ASD16 and IR64 showed better callusing and embryogenic capacity as compared to ADT43 and Pusa Basmati. For genetic transformation studies, embryogenic calli of all the cultivars were co-cultivated with the Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCambia 1305.1 with GUS gene. GUS assay was performed for the putative transformed calli and its activity was found to be qualitatively higher in ASD16 and IR64 than the other two varieties. The best responsive ASD16 transformed calli was regenerated and the putative transgenic lines were regenerated. ASD16 transformed calli were confirmed by GUS assay. PCR analysis confirmed the presence of both GUS and HPT genes in ASD16 transgenic lines.     

陆地棉原生质体培养与植株再生技术研究

 分离培养陆地棉品种ZDM-3（Gossypium hirsutum L cv ZDM-3）胚性细胞悬浮系的原生质体,通过体细胞胚胎发生成功获得再生植株。试验结果表明,植物生长调节剂组合和碳源对原生质体培养具有重要的影响,添加2,4-D + KT + 1.5%（W/V）葡萄糖+ 1.5%（W/V）麦芽糖的KM8P培养基对于陆地棉原生质体培养的效果较好。激素组合0.46 μmol·L^-1 KT + 0.45 μmol·L^-1  2,4-D诱导的愈伤组织较松软,分化潜力高;胚性愈伤组织的增殖使用0.93 μmol·L^-1 KT + 2.46 μmol·L^-1 IBA的激素组合;1.2 μmol·L^-1 IBA + 1.39 μmol·L^-1 KT有利于体细胞胚胎发生,而MSB-5 + 0.67 μmol·L^-1 KT+2.69 μmol·L^-1 NAA的培养基适合体细胞胚胎萌发和植株再生。此外,愈伤组织诱导宜用葡萄糖作为碳源,而胚性愈伤组织增殖、保存和胚状体的萌发过程宜使用麦芽糖作碳源。     

Effects of Stem-Injected Sucrose on Photosynthesis and Productivity of Water Stressed Corn Plants

Water stress during silking or early kernel development decreases the number of kernels set by corn (Zea mays L.) plants. Previous work has suggested that lack of assimilate supply due to water stress at silking was a major factor in the resulting reproductive failure. A greenhouse experiment was conducted to test the hypothesis that sucrose supplementation of water stressed corn plants can prevent decreased kernel set. Sucrose was injected into corn stems at three concentrations [0 (distilled water), 150 and 300 g L^?1] for 30 days starting at silking. Water availability was controlled by either maintaining a water table at 50 cm from the soil surface (well watered) or by withholding water starting one week before silking (water stress) until the fifth day after silking. The photosynthesis rate of water stressed plants was 25% that of well-water plants on the first day of silking. On average, the daily injection rate for distilled water was 1 mL higher than that of the sucrose treatments over a 30 day injection period. No difference in daily uptake rate was observed between the 150 and 300 g sucrose L^?1 treatments. Over water availability treatments approximately 17 g sucrose were injected into corn plants during the 30 day injection period. Corn plants receiving sufficient water supply produced bigger ears, with more seeds and greater 100-seed weight values, leading to higher total plant dry matter accumulation than water stressed plants. Injection of 300 g sucrose L^?1 increased the weight of the injected internodes by 28%, compared with distilled water injection. The highest grain yield was for the plants injected with 150 g sucrose L^?1, but only under sufficient water supply. The plants injected with 300 g sucrose L^?1 produced the least grain regardless of moisture availability. Thus, the exogenous sucrose supplementation influenced kernel set only under conditions of sufficient soil water supply. These results indicate that plant reproductive development after silking was limited more by water availability than assimilate supply, suggesting that some overall plant response to water stress, perhaps mediated by hormonal signalling, was more important than carbohydrate supply. These results indicated that plant desiccation occurred during floral development or pollination; irreversible loss of florets on unsuccessful pollination could result, thus, grain yield would be limited more by sink size than by availability of photosynthate.     

渗透胁迫对大花萱草EMS离体诱变系统的影响

针对提高EMS(甲基磺酸乙酯)对大花萱草愈伤组织突变体率的方法开展的研究。采用了预选择的高渗透固体培养基(甘露醇36g/L+山梨醇36g/L+蔗糖50g/L)。在0.1%EMS半致死剂量诱变前对大花萱草带芽愈伤组织进行不同时间的渗透胁迫预处理。研究渗透胁迫对大花萱草EMS诱变效应的影响。通过对渗透胁迫和EMS诱变后愈伤组织的生长量、成活率、分化率和成苗率等生长状态的观察统计,以及细胞质膜透性和SOD、POD、CAT酶等生理生化指标的测定,得出：渗透胁迫预处理对EMS诱变效应存在一定的影响。渗透胁迫加强了EMS对愈伤组织的伤害作用,表现在细胞质膜透性和SOD、CAT酶活性的升高以及POD活性的降低。处理时间在30min-60min时,胁迫对愈伤组织分化成苗的能力未造成显著影响,但是可提高突变体率6.34%-9.27%。处理时间达90min时,对愈伤组织造成致死伤害。结论：在0.1%EMS半致死剂量诱变大花萱草愈伤组织前,用高渗透固体培养基(甘露醇36g/L+山梨醇36g/L+蔗糖50g/L)进行30min-60min的胁迫预处理可以有效提高突变体率6.34%-9.27%。渗透胁迫时间的长短与突变率提高的幅度没有规律性。     

Inheritance of temperature sensitivity of the photoperiod response in common bean (Phaseolus vulgaris L.)

Summary Photoperiod response of flowering in common bean (Phaseolus vulgaris L.) is thought to be controlled by the genes Ppd and Hr. However, cultivars also vary in the degree that cooler temperatures reduces their sensitivity to photoperiod. To examine the inheritance of this temperature sensitivity, crosses of cvs. Gordo x de Celaya and Flor de Mayo × Rojo 70 were evaluated at two sites differing in mean temperature and using 12.5-h natural photoperiod or 18-h artificially extended photoperiod. Under 18-h photoperiod at the warmer site, Palmira, no plants of the parents or of the F₂ populations flowered, confirming that the parents were sensitive to photoperiod. Under 12.5-h photoperiod at the cooler site, Popayan, the parents for each cross flowered at similar dates and no segregation for days to flower was observed. However, under 18-h photoperiod, de Celaya and Rojo 70 and the F₁ populations did not flower within 100 days after planting, while the F₂ and F₃ populations showed segregation that was consistent with single gene inheritance, late flowering being dominant. Late flowering at Popayan under 18-h photoperiod indicates a lack of temperature sensitivity, so temperature insensitivity of the photoperiod response was dominant to sensitivity. The name Tip, for temperature insensitivity of photoperiod response, is proposed for this gene, with the recessive form of this gene conditioning earlier flowering at cooler temperatures with long daylengths. It is recognized that the observed segregation patterns could represent the effect of multiple alleles at the Ppd or Hr loci, and studies are proposed to test this possibility with molecular markers and recombinant inbred lines.     

Interspecific hybrids between three eggplant (Solanum melongena L.) cultivars and two wild species (Solanum torvum Sw. and Solanum sisymbriifolium Lam.)

Three Greek eggplant cultivars, ‘Langada’, ‘Tsakoniki’ and ‘Emi’ (2n= 24), were crossed with two wild species (Solanum torvum Sw., 2n= 24 and Solanum sisymbriifolium Lam., 2n= 24). Ovules isolated 15-27 days after pollination were cultured in a modified MS medium at 24°C and a 16h photoperiod. Fifty days later, the ovules were dissected and the interspecific embryos were cultured in the same medium. Interspecific hybrids were achieved only from crosses between the eggplant cultivars and S. torvum. The hybridity of the putative interspecific F¹ hybrid (Solanum melongena×S. torvum) was confirmed by using morphological and biochemical (isozyme isocitrate dehydrogenase A, phosphoglucomutase A, phosphoglucose isomerase B, 6-phosphogluconate dehydrogenase A, 6-phosphogluconate dehydrogenase B) markers. The F¹ plants (‘Langada’×S. torvum) were selfpollinated and backcrossed to both parents. Fruits, however, were produced only when the F¹ hybrid was backcrossed as female with the eggplant cultivar ‘Langada’.     

槟榔胚培养配方组合筛选研究

以‘热研1号’槟榔幼胚为外植体,采用正交设计法研究不同基本培养基、活性炭及蔗糖等浓度在槟榔胚离体培养中对幼苗生长发育的影响。通过方差分析和差异显著性检验,评价最佳配方组合时,评价指标考虑的优先顺序依次为芽高、最长根长、芽基部直径、根数;MS培养基优于其他几种培养基,能同时满足芽和根的生长需求;活性炭对生根及根的伸长作用显著,4 g/L的活性炭浓度时抽生的根最多,长势最好,蔗糖能同时促进根和芽的生长,随添加量的增加,生长指标呈低-高-低的单峰曲线趋势,40 g/L蔗糖浓度对芽的生长效果最好。研究得出的最佳配方组合是：MS培养基+4 g/L活性炭+40 g/L蔗糖。     

菜用大豆籽粒不同部位蔗糖积累及关键酶活性研究

田间种植可溶性糖含量不同的3个菜用大豆品种,在R5.5、R6、R6.2、R6.5和R7期取样,分析籽粒种皮、子叶和胚轴中蔗糖含量及4种关键酶活性动态,结果表明,籽粒不同部位蔗糖积累呈先增加后下降的趋势,R6.2期是高峰期,此时期品种台292、中科毛豆1号和品系121的胚轴蔗糖含量比子叶分别高57.6%、53.6%和44.2%;比种皮分别高71.6%、75.3%和73.6%。由于子叶干重占整粒重90%以上,因此整个籽粒的蔗糖含量主要由子叶决定。子叶的蔗糖磷酸合酶(SPS)活性高于胚轴和种皮,在R6.2期表现更加明显,且蔗糖含量高的品系121子叶中SPS活性高于另外2个品种;蔗糖合酶(SS)在籽粒形成期活性变化呈前期高于后期的趋势,最高值出现在灌浆前期R5.5期胚轴中;两种转化酶活性变化差异较大,中性转化酶(NI)活性一直呈不断下降趋势;籽粒不同部位NI活性无明显差异,而酸性转化酶(AI)活性差异较大;胚轴和子叶中AI活性明显低于种皮,且种皮中AI活性与种皮中蔗糖积累显著负相关(r=–0.59)。蔗糖积累与4种关键酶活性的相关分析发现,籽粒中蔗糖的含量并非受某一种酶绝对调控,SPS活性与SS+AI+NI活性总和之差与籽粒中蔗糖的积累显著正相关(r=0.53^**)。     

Induction and development of adventitious shoots of Atropa baetica as a means of propagation

In vitro propagation of Atropa baetica was established employing axillary buds. Single buds were cultured through a multiple shoot induction phase, rooting phase, and then followed by acclimatization in soil. For multiple shoot induction, Murashige and Skoog (MS) medium with 3% sucrose, supplemented with either 0.75 or 1.25 mg l^-1 of BAP provided the best results with an average of 5.6 shoots per explant after 31 days of culture. Similar results were obtained with higher BAP concentrations (1.75–2.0 mg l^-1); however, these media had a negative effect on the subsequent root induction due to residual BAP effect. Medium containing only 0.25 mg l^-1 of BAP induced a significantly lower number of shoots. Root induction occurred spontaneously after transferring the shoots onto MS medium lacking any plant growth regulator. Moreover, root induction also occurred on media supplemented with 0.125 and 0.25 mg l^-1 of NAA. On these two rooting media, this response was more prominent and with a higher number of roots per explant. Nevertheless, after 28 days on root induction medium, the number of rooted plantlets was similar on the three media. Acclimatization of plantlets in soil was very successful (95.52%). However, all plantlets which died during acclimatization were rooted on medium containing 0.25 mg l^-1 NAA suggesting a negative carry over effect of this medium upon plantlet survival, irrespective of the initial BAP treatment used. On the other hand, karyological studies showed no variation in the number of chromosome (2n=72) in root tips of the plantlets produced. This revised version was published online in July 2006 with corrections to the Cover Date.     

两种观赏水草的离体培养

本试验以两种观赏水草为供试材料,进行离体培养研究。试验结果表明：红波（Alternanthera bettzickiana）以叶片为外植体,愈伤组织诱导采用MS+6-BA0.5 mg/L (单位下同)+NAA0.2的培养基,芽诱导采用MS+6-BA2.0 +NAA0.05+AD10的培养基,茎尖芽诱导采用MS+6-BA2.0+NAA0.05培养基,继代培养采用MS+6-BA1.0 +NAA0.05的培养基,根诱导采用1/2MS+IBA0.5的培养基;金钱草（Lysima chiachristinae Hance）以茎尖为外植体,芽诱导采用MS+6-BA0.5+NAA0.02的培养基,继代培养用MS+6-BA0.5+NAA0.05的培养基,根诱导采用1/2MS+IBA0.5的培养基,上述培养基中均含有30 g/L蔗糖和6g/L琼脂,PH5.6,在培养温度25℃、光照强度1800Lx、光照时间12h/d的培养条件下,均能培育出完整植株。     

Influence of genotype and culture medium on callus formation and plant regeneration from immature embryos of Triticum turgidum Desf. cultivars

Twelve durum wheat cultivars were evaluated for their response to in vitro tissue culture. Zygotic immature embryos were used to induce callus formation using four different Murashige and Skoog‐based media. Each contained 9.05 μM 2,4‐dichlorophenoxy acetic acid but differed in their carbon source (sucrose or maltose) and the presence of NaCl (0 mM or 40 mM). The influence of both genotype and medium on the type and percentage of callus produced was observed. Calli were either compact and frequently embryogenic, or soft and watery. Percentages ranged from 54 to 100%, depending upon genotype and induction medium. All calli were then plated on a regeneration medium containing 20 g/l sucrose, 2.68 μM 1‐naphthaleneacetic acid and 2.22 μ 6‐benzylaminopurine. The regeneration of plantlets was higher from compact than from soft calli, with a strong dependence on genotype and type of induction medium used. MSm induction medium (30 g/l maltose) and MS40s (30 g/l sucrose plus 40 mM NaCl) were best for inducing compact calli, and gave the highest proportion of regenerated plants. The in vitro response (number of total shoots from a compact callus/number of embryos plated) was higher for immature embryos of ‘Baztan’, ‘Bradano’ and ‘Don Pedro’. These cultivars are a good starting material for experiments involving transformation of calli from zygotic immature embryos.