Mulberry biodiversity conservation through cryopreservation

A 238 mulberry germplasm accession collection from diverse regions maintained under tropical conditions was identified from an ex situ field gene bank. The purpose was to prioritize the in vitro conservation and cryopreservation to develop long-term biodiversity conservation for ensuring sustainable utilization of these valuable resources. Reliable cryo techniques using desiccation and slow freezing of winter-dormant buds were used. Storage potential of bud grafts of different Morus species at −1.5°C for 90 d indicated species-specific variation, and most of the wild species were found sensitive. In vitro regeneration and cryopreservation (−196°C) protocols using differentiated bud meristems, like axillary winter-dormant buds, were worked out for a wide range of landraces, wild, and cultivated varieties of Morus. Buds maintained under subtropical location are also amenable for cryopreservation. Successful cryopreservation of winter-dormant buds belonging to Morus indica, Morus alba, Morus latifolia, Morus cathayana, Morus laevigata, Morus nigra, Morus australis, Morus bombycis, Morus sinensis, Morus multicaulis, and Morus rotundiloba was achieved. Among wild species, Morus tiliaefolia and Morus serrata showed moderate recovery after cryopreservation. Survival rates did not alter after 3 yr of cryopreservation. Inter-simple sequence repeat markers were used to ascertain the genetic stability of cryopreserved mulberry germplasm accessions, which showed no difference detected among the plantlets regenerated from frozen apices in comparison to the nonfrozen material.     

A Method for in Vitro Storage of Lolium multiflorum Lam

A means of storing Lolium multiflorum Lam. stock plants in vitrohas been developed over a period of three years. Variousexplantsand cultureconditions were examined. Shoot tips 0.3–0.9mm long were found to be best for establishing storage culturesbecause they gave a high yield of plantlets in culture and importantpathogens are eliminated. For sub-culturing, after each annualcycle of storage at 2–4 °C, shoot tips, tiller buds,tiller bases and nodes can be used. Tiller buds were most abundantand best for increasing the number of stored plantlets whennecessary. There was no advantage of including an auxin in theculture medium for shoot tips and Murashige and Skoog's mediumwith 0.2 mg l^–1 kinetin has been adopted for initiating,sub-culturing and storing cultures. The optimum temperaturerange for regenerating plantlets was 20–25 °C. Lightwas necessary for a high rate of plantlet regeneration and lightquality and intensity affected their growth. Lolium multiflorum Lam., ryegrass, storage in vitro, tissue culture     

Modulated Degradation of Ribulose Bisphosphate Carboxylase in Leaves on Top-Pruned Shoots of the Mulberry Tree (Morus alba L.)

Yamashita, T. 1987. Modulated degradation of ribulose ftisphosphatecarboxylase in leaves on top-pruned shoots of the mulberry tree(Morus alba L.).—J. exp. Bot. 38: 1957–1964. The effects of pruning shoot tops on the synthesis and degradationof ribulose 1,5–Wsphosphate carboxylase (RuBPCase) inleaves on remaining shoots were investigated in mulberry trees.Leucine labelled with ¹⁴C was fed to leaf discs from field-grownmulberry trees and ¹⁴C incorporation into RuBPCase was examined.Proportion of ¹⁴C in RuBPCase to leucine–¹⁴C absorbedby leaf discs was remarkably lowered by top-pruning, thoughoccasionally a slight increase was observed soon after pruning.Yet RuBPCase content in leaves on top-pruned shoots became progressivelyhigher than that in leaves on intact shoots. Changes in ¹⁴Cin Ru1BPCase in leaves of mulberry saplings previously fed ¹⁴CO₂were followed. Following ¹⁴CO₂ feeding, the attainment of themaximal level of ₁₄C in RuBPCase was retarded by top-pruning.The highest level of ₁₄C in RuBPCase was maintained in leaveson top-pruned shoots but decreased in leaves on intact shoots.Specific radioactivity in RuBPCase continued to increase inleaves on top-pruned shoots even after attaining a maximum levelin the control leaves. These facts suggest that the increasein RuBPCase by top-pruning results from a cessation of its degradationfor the remobilization of nitrogen for newly developing leaveson shoot tops. Key words: RuBP carboxylase, shoot pruning, mulberry (Morus alba)     

Callus, Organogenesis and Plantlet Formation in Tissue Cultures of Clitoria ternatea

Decoated seeds of Clitoria ternatea L. germinated on Murashigeand Skoog (Physiologia Plantarum 1962, 15, 473–97) basalmedium (BM) and differentiated callus and bipolar embryoids(two-step method) in low frequency. Calluses developed on lateralroots [BM+KN(0.1 mg 1^–1)], on roots and hypocotyls [BM+KN(0.5mg 1^–1)], and on roots [BM+KN+IAA (0.5 mg 1^–1 ofeach)]. On basal medium with KN (0.5 mg 1^–1) and withKN+IAA (0.5 mg1^–1 of each), multiple shoot buds and embryoids(one-step method) were differentiated directly on split hypocotylsand roots. In the former, shoot buds developed even on unsplithypocotyls. Rhizogenesis on isolated shoot buds occurred efficientlyin BM+indole butyric acid (IBA 0.1 mg 1^–1) and BM+IAA(0.1 mg 1^–1 and 0.5 mg 1^–1). Profuse direct embryoidsand shoot buds developing on root systems are interesting morphogeneticphenomena rarely reported. Clitoria ternatea L., callus, embryoids, multiple shoot buds, regeneration     

Haploid Plantlet Formation from Female Gametophytes of Ephedra foliata Boiss. in vitro

Female gametophytes (at the archegonial stage) excised fromyoung ovules of Ephedra foliata Boiss, were cultured on a basalmedium (Murnshige and Skoog's combinations of major and minorsalts, Iron source, vitamins, myo-inositol along with 2 percent sucrose and 10 per cent coconut milk) under aseptic conditions.Growth and morphogenetic responses of the explants to auxinswere compared at different concentrations and a study of theirinteractions with cytokinins has also been made. At 2 mg 1^–1,2, 4-D induced profuse callusing which subsequently producedroots. NAA at 4 mg 1^–1 was optimal for callus growth androoting. Combinations of 2,4-D and kinetin were more effectivein inducing roots and shoot buds than those of 2,4-D and benzylamino-purine (BAP). Addition of BAP (0.05 mg ^1–1) to themedium containing optimal concentrations of NAA resulted information of a large number of roots. Kinetin induced only rootingin the presence of 4 mg 1^–1 NAA. A high concentrationof BAP (8 mg 1^–1), stimulated shoot bud formation. Forthe further development of shoot buds, neither auxin nor cytokininwas needed. Cytological observations revealed the presence ofhaploid number of chromosomes, i.e. seven. Ephedra foliata, tissue culture, callus, regeneration, 2,4-dichlorophenoxyacetic acid, naphthalene acetic acid, kinetin, benzyl amino-purine     

In Vitro Morphogenesis in Nardostachys jatamansi DC.: Shoot Regeneration from Callus Derived Roots

Callus cultures of Nardostachys jatamansi DC. maintained onMurashige and Skoog's medium containing 3.0 mg 1^–1 of-naphthaleneacetic acid and 0.25 mg 1^–1 of kinetin whenshifted to medium containing 0.25–1.0 mg 1^–1 ofindole-3-acetic acid or indole-3-butync acid showed profuserhizogenesis. The callus-regenerated roots when transferredto medium containing 2.0–6.0 mg 1^–1 of kinetin producedshoot buds. The de novo shoot bud regeneration took place eitherdirectly from cortical cells or from the inner stelar region.In addition, direct, concomitant root-shoot development wasalso observed. Nardostachys jatamansi, organogenesis, root-buds     

Organogenesis in the Cultured Female Gametophyte of Ephedra foliata

The female gametophyte of Ephedra foliata was used as an explantfor the production of haploids as it is composed of haploidcells, all of the same genotype. The regeneration of roots wasdependent upon the presence of NAA, while BAP had a modifyingeffect. At lower concentrations (0.05 parts 10^–6 and 3.5parts 10^–6) BAP enhanced the root promotion of NAA (0.05–4.0parts 10^–6). At higher concentrations of BAP (1–6parts 10^–6), roots and shoot buds were formed. Kinetinat 4.0 parts 10–6 with 0.5 parts 10–6 2, 4-D wasoptimal for shoot bud production in explants at the archegonialstage and 2, 4-D at 2.0 parts 10–6 with 0.5 parts 10–6kinetin was optimal for root formation. Cells of the callusand root tip had the haploid number of chromosomes, n = 7. Meristemoidswere located on the surface or embedded in the callus tissue.The deep seated meristemoids organized only root primordia,but the peripheral ones gave rise to root as well as shoot budprimordia. Initially, there was no vascular connection betweenthe shoot-bud and the callus. This was established later. Key words: Ephedra, Female gametophyte, Haploid, Tissue culture     

Differentiation of Shoot Buds in vitro in Tissue 1Sisymbrium irio L.

Shoot bud formation was induced in the stem callus of Sisymbriumirio L., a Cruciferous plant. The callus was established onMurashige and Skoog medium with IAA (1?0 mg l^–1) and kinetin(0?5 mg l^–1). The effect of three purines (kinetin, 6-benzylaminopurine,and 6-methylaminopurine) incorporated singly along with IAAin MS medium was investigated. It was found that kinetin orMAP (3–5 mg l^–1) along with IAA (0?5 mg l^–1)were the most effective in inducing shoot bud formation. Adeninesulphate (10 mg l^–1) with kinetin (1?0 mg l^–1) alsoinduced bud differentiation. The morphogenetic potential of the callus to differentiate shootbuds was seemingly lost in 2 year old callus cultures. However,on successively subculturing on a regeneration medium shootbuds differentiated and the number of buds formed improved onfurther subculture. Two types of meristematic outgrowths were recognized: (i) arisingfrom superficial cells and (ii) arising from deep-seated cellsin the vicinity of tracheidal elements. However, both typesformed meristematic nodules on the surface of which shoot budsdifferentiated. Some embryoids were also recognized arisingsuperficially.     

Freezing Avoidance Mechanisms by Supercooling in Some Rhododendron flower Buds with Reference to Water Relations

Excised florets of some hardy Rhododendron species did not toleratefreezing at –5°C when ice-inoculated due to intracellularfreezing. Florets in intact December buds, however, could besupercooled to about –30°C. When flower buds of R.japonicum were slowly cooled with daily decrements of 5°Cto temperatures ranging from 0 to –20°C, the exothermtemperatures of the florets drastically decreased. This wasaccompanied by a decrease in water content of florets and peduncleand an increase in that of scales. The water in florets andthe peduncle is thought to migrate to scales and other tissuesduring the early stages of freezing; the dehydrated floret hasa lower freezing point which enhances its supercooling abilityand the dehydrated peduncle helps to maintain the supercooledstate of the florets. This hypothesis would explain the dependenceon the cooling rate of supercooling in Rhododendron flower buds.Water migration within flower buds was observed in other hardyRhododendron species with some variation in ice formation siteand the quantity of migrated water. The exotherm temperatureof excised florets was inversely proportional to their watercontent. Dehydration of flower buds by wind at 0°C alsoenhanced their supercooling ability. Mechanisms of freezingavoidance by supercooling in Rhododendron flower buds and therelationship of supercooling to freezing tolerance are discussed. ¹ Contribution No. 2254 from the Institute of Low TemperatureScience ² This is a revised form of the master's thesis of the seniorauthor (M.I.) which is cited in the present and previous papers(Sakai 1979a, b, etc.). (Received August 11, 1980; Accepted June 1, 1981)     

Influence of Genotype, Plant Growth Temperature and Anther Incubation Temperature on Microspore Embryo Production in Brassica napus ssp. oleifera

Eleven F₁ hybrid genotypes of winter rape (Brassica napus ssp.oleifera) were used in a study of induction and growth of microspore-derivedembryos. Plants of each genotype were grown in controlled environmentsat either a constant 15°C or a constant 20°C, both witha 16 h photoperiod. Equal numbers of buds, approximately 2.5mm in length, containing uninucleate microspores were harvestedfrom each genotype and either pretreated (14 d at 4°C) ordissected immediately after harvest. Anthers were cultured onliquid medium based upon that of Murashige and Skoog (1962)and containing 8% sucrose, 0.5 mg dm^–3 naphthylaceticacid and 0.05 mg dm^–3 benzylaminopurine. Anthers fromequal samples of buds were incubated at 35°C for 0, 1, 2or 3 d before transfer to 30°C (21 d) and then 25°C.After a total of 42 d incubation, cultures were scored for thepresence of macroscopic embryos (1–2 mm in length) andfor the presence of anthers containing aborted embryoids whichhad not developed further. The results showed first that bud pretreatment completely inhibitedinduction and secondly that anthers of all genotypes had anabsolute requirement for a 35°C treatment (optimal duration2 d) in order to induce embryoid formation. In the great majorityof genotypes plants grown at 15°C provided more productiveanthers than plants grown at 20°C. However, within eachtreatment there were great differences both in the frequencyof anthers showing induced embryoids and of the final yieldof embryos. There was evidence that hybrids with a common parentresponded similarly under certain treatments. This confirmedthe importance of genotypic control for some components of embryoyield. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

In Vitro Production of Stigma-like Structures from Stigma Explants of Crocus sativus L.

Stigma-like structures (TC stigmas) were produced in tissuecultures from stigma explants of Crocus sativus under definedconditions. MS medium supplemented with NAA (10 mg dm^–3)+BA (1.0 mg dm^–3) induced the optimum response. NAA wasfound to be an important addendum to achieve a good response.The TC stigma regeneration response as a function of explantage showed significant differences (except between stage 1 andstage 4). A culture temperature of 20 °C seems to be betterthan 25 °C with reference to all parameters. Crocin andpicrocrocin pigments, responsible for colour and bitter taste,respectively, were extracted, identified and quantified fromthe TC stigmas. Safranal was not detected in fresh samples. Key words: Crocus sativus, stigma-like structures, organ regeneration, crocin, picrocrocin, safranal     

The Interaction of Genotype, Explant and Media on the Regeneration of Shoots from Complex Explants of Brassica napus L.

The relative importance of explant type, genotype and growthregulator regime in the determination of shoot regenerationfrequencies from complex explants of Brassica napus L. has beenevaluated. Cotyledon, hypocotyl and stem sections taken fromone spring (Westar) and three winter (Ariana, Cobra, Libravo)varieties of B. napus were cultured on three different growthregulator regimes, 0.5 mg dm^–3 NAA + 2.0mg dm^–3BAP, 0.5 mg dm^–3 NAA + 4.0mg dm^–3 BAP and 1.0mgdm^–3 NAA + 4.0mg dm^–3 BAP. The most significanteffects on shoot regeneration were due to explant type and variety.The regeneration from stem segments was not only two to threetimes higher than from hypocotyls or cotyledons, in all varieties,but the response was also more uniform across the varieties.The explant effect accounted for 44–95% of the regenerationresponse. In contrast, the contribution of growth regulatorregime was negligible. Although the growth regulator regimeas an independent effect was unimportant, regeneration fromboth Ariana and Libravo was significantly affected by the interactionof genotype with growth regulator regime. The importance ofboth the high shoot regeneration frequency from stem segmentsand the relative uniformity of response across the four testedgenotypes is discussed with respect to the potential benefitsof using this explant source in Agrobacterium-based transformationexperiments. Key words: Brassica napus, regeneration, genotype, tissue culture, complex explant     

Initiation and Growth of Callus and Cell Suspensions of Theobroma cacao L

Callus and suspension cultures of Theobroma cacao L., initiatedfrom immature cotyledons of beans from pods harvested 120–130days after pollination were established. A modified B-5 or Murashige—Skoogagar medium sustained growth of callus without loss of vigourafter each sub-culture. A 15-fold weight increase occurred duringthe 4 week culture periods at 30 ± 1 °C. Coconutwater improved callus growth substantially. The optimum hormonalconcentrations for growth of suspensions were 0.5 mg 1^–1of 2, 4-dichlorophenoxyacetic acid and 0.1 mg I^–1 of kinetinin a Murashige—Skoog basal medium liquid medium. The optimumtemperature for growth of suspensions was 25–30 °C.The cell number and cell mass of suspensions increased 20-foldin 14 days. No organogenesis or embryogenesis was observed. Theobroma cacao L., acao, cell culture, suspension culture, tissue culture.     

In Vitro Culture and Plant Regeneration in Vicia faba subsp. Equina (var. Spring Blaze)

Explants of stem, leaves, roots, and cotyledons from etiolatedaxenically grown Vicia faba seedlings were cultured on a rangeof media. Shoot organogenesis was only obtained with nodal stemand cotyledonary node explants when cultured on MS medium with3% sucrose, 2.0 mg 1^–1 BAP and 02 mg 1^–1 NAA. Callusproliferation accompanied shoot organogenesis from nodal stemexplants. Successive subculture of nodal stem callus resultedin proliferation of regenerative callus which contained severalshoot bud initials. The capacity for shoot regeneration fromthis callus was maintained for 9 months. Histological studiesreveal de novo formation of meristematic centres in callus andtheir further development into bud primordia. High frequencyrooting of these adventitious shoots was obtained on half-strengthMS medium with 1.5% sucrose, 0.1 mg 1^–1 NAA and 0.5 mg1^–1 kinetin. Key words: Vicia faba, adventitious shoots, axillary shoots, de novomeristem formation, organogenesis, tissue culture     

Direct Organogenesis from Petiole and Thin Cell Layer Explants in Sugar Beet Cultured In Vitro

Detrez, C., Tetu, T., Sangwan, R. S. and Sangwan-Norreel, B.S., 1988. Direct organogenesis from petiole and thin cell layerexplants in sugar beet cultured in vitro.—J. exp. Bot.39: 917–926. Plant regeneration was obtained by direct bud formation frompetiole as well as from thin cell layer explants taken fromsugar beet (Beta vulgaris L.) plants grown in vitro. The budswere mainly induced in the blade-petiole transition zone ofthe explants. High frequency bud regeneration was observed inpetiole and thin layer explants of 10 different breeding linesof sugar beet tested. Organogenesis resulted when petiole explantsexcised from 8-d-old seedlings grown on half-strength Murashigeand Skoog medium (MS) containing 3.0 mg dm^–3 naphthaleneacetic acid (NAA), 3.0 mg dm^–3 6-benzylaminopurine (BAP)and 1.0 mg dm^–3 2, 3, 5, triiodobenzoic acid (TIBA) werecultured on MS with 3.0 mg dm^–3 NAA and 3.0 mg dm^–3BAP. Thin cell layer strips isolated from shoot apices culturedon MS medium supplemented with 0–9 mg dm^–3 BAP or1.0 mg dm^–3 indolebutyric acid (IBA) formed adventitiousbuds on MS medium containing 0–5 mg dm^–3 NAA + 5.0mg dm^–3 BAP. Histological studies confirmed the sub-epidermalorigin of shoots. Key words: Beta vulgaris, direct organogenesis, in vitro culture, petiole, regeneration, thin cell layer     

Direct Organogenesis and Plant Regeneration in Preconditioned Tissue Cultures of Lathyrus cicera L., L. ochrus (L.)DC. and L. sativus L.

This study demonstrates the importance of preconditioning ofsource tissue in regeneration of multiple shoot buds from severalspecies of Lathyrus. Preconditioned multiple shoots of Lathyruscicera L., L. ochrus (L.) DC. and L. sativus L. were obtainedby germinating seeds on Murashige and Skoog (MS) medium containing50 µM N⁵-benzylaminopurine (BAP) for 2 to 3 weeks. Multipleshoot bud formation occurred when epicotyl explants of preconditionedshoots were cultured on MS medium containing 5–50 µMBAP. No shoot regeneration was observed from epicotyl explantswhich were obtained from non-preconditioned shoots. Shoot budswere formed directly on explants without an intervening callusphase after 2 to 3 weeks of culture. Regenerated shoot budsformed healthy shoots which developed prolific and strong rootswhen transferred to MS medium supplemented with 2.5 µMnaphthaleneacetic acid (NAA). Lathyrus cicera L., L. ochrus (L.) DC., Ochrus Vetch, L. sativus L., Lathyrus pea, de novo differentiation, epicotyl, preconditioning with BAP     

Micropropagation of Betula celtiberica

Plantlets were successfully regenerated from shoot segmentsof Betula celtiberica excised from young seedlings. Initiationand elongation of multiple shoot-buds were obtained after 20d culture in MS-modified medium plus BAP 0.6 mg l^–1 followedby 20 d culture in the same medium in the presence of a reducedBAP concentration (0.1 mg l^–1). Rooting was achieved 7d after having transplanted the isolated shoots to fresh medium,supplemented with IBA (0.2 mg l^–1). Betula celtiberica, birch, micropropagation, organogenesis     

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. GreenComet was used in a study of the effects of exogenous hormoneson the growth and differentiation of seedling organs in vitro.Four types of explants were tested: hypocotyl segments, rootsegments, primary leaf discs and cotyledon discs. These explantswere incubated on media containing factorial combinations ofBAP x IBA, BAP x NAA, KN x IBA and KN x NAA (all at 0, 0.1,10 and 10.0mg l^–1). Hypocotyls were the most regenerativeexplants; shoot production was favoured by cytokinin: auxinratios greater than one and was decreased by IBA at 10 mg l^–1when callus was produced. Shoot formation from root explantsoccurred either in the absence of hormones or with low concentrations;no shoot was produced when any hormone was present at 10 mgl^–1. In contrast, shoot production from primary leaf diseswas favoured by high concentrations of both auxin and cytokininwith the combination of BAP and IBA the most effective. Shootproduction from cotyledon discs was sporadic with no consistentresponse on any auxin/cytokinin combination. After further experimentson the optimization of hormone concentration, the followingcombinations were chosen as allowing reliable regeneration:0.1 mg l^–1 BAP+0.1mg l^–1 IBA for hypocotyl segments,0.075 mg l^–1 KN +0.025 mg l^–1 IBA for root segments,and 5.0 mg l^–1 BAP+5.0 mg l^–1 IBA for leaf discs. Brassica oleracea var. italica, calabrese, tissue culture, seedling, auxin, cytokinin     

Tuberization in Potato at High Temperatures: Gibberellin Content and Transport from Buds

Warm temperatures (35°C day/30°C night) which inhibittuberization in potato (Solanum tuberosum L., cv. Sebago) increasedgibberellin activity in crude extracts from buds, but not frommature leaves, as determined by the lettuce hypocotyl bioassay.Changes in the growth of tubers and stolons indicate the occurrenceof basipetal movement of GA₃ applied to the terminal bud ora mature leaf. ¹⁴C labelling from GA₃ or mevalonic acid injectedjust below the terminal bud was recovered in the lower shoot,stolons and tubers, but the amount transported was greater atcool temperatures (20/15°C). It is concluded that high temperaturespromote the synthesis of gibberellin in the buds rather thantransport to the stolons. Solanum tuberosum L., potato, tuberization, gibberellin     

Freezing Tolerance in Hydrated Lactuca sativa (L) Seed: A Model to Explain Observed Variation Between Seed Lots

Low temperature tolerance was investigated in the imbibed seedof 15 seed lots compnsmg seven cultivars of Lactuca sativa L.During rapid cooling (20 °C h^–1) some seeds of allseed lots survived to –16 °C but none to –20°C. The majority of seed lots retained over 50 per centviability above –14 °C due to isolation of the embryofrom external ice by the endosperm, and subsequent embryo super-cooling.Certain seed lots, including all three seed lots of cv. TomThumb, showed high mortality at temperatures above –10°C. Correlation of mortality with the formation of externalice suggested that the endosperm is not an effective nucleationbarrier in these seed lots. Survival to –20 °C was increased at slower coolingrates (6 to 1 °C h^–1) due to freeze desiccation ofthe embryo, but seed lots varied considerably in their toleranceof specific cooling rates. A model to explain this variationwas developed incorporatmg (1) seed lot super-cooling limittemperature, (2) the rate at which freeze dehydration of thesupercooled embryo took place, (3) the moisture content at whichnucleation (at –20 °C) was no longer certain and (4)the.initial equilibrium moisture content of the fully imbibedseed. Factors (1), (2) and (3) were found to be relatively constant,but low (or artificially reduced) seed moisture content wasclosely correlated with high survival at natural cooling rates.Seed size fractions of similar moisture content from a singlecultivar showed that more small seeds survive cooling at 3 °Ch^–1 to –20 °C than larger seed. Seed with pierced endosperms or ineffective nucleation barrierswere capable of surviving to at least –10 °C if cooledslowly (1 °C h^–1) but were killed by rapid (20 °Ch^–1) cooling. Lactuca sativa L, lettuce, seed germination freezing tolerance, super-cooling