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5th/6th Battalion, Royal Victoria Regiment
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|5th/6th Battalion The Royal Victoria Regiment
|Army Reserve
|Reserve Infantry
|Light Infantry
|Part of
|4th Brigade
|5th Battalion – Nemo me impune lacessit (No one shall provoke me with impunity)
6th Battalion – Semper Paratus (Always ready)
|Combination of 'Cock O' the North' and 'Waltzing Matilda' [1]
|Battle honours
|Boer War: South Africa 1899–1902.
World War I: Landing at Anzac Cove, Somme 1916, Somme 1918, Bullecourt, Pozieres, Polygon Wood, Ameins, Albert 1918, Mont St Quentin, Hindenburg Line.
World War II: Bardia 1941, Capture of Tobruk, El Alamein, Greece 1941, South West Pacific 1942–1945, Bobdubi II, Finisterres, Lae–Nadzab, Hari River, Borneo.
|Unit Colour Patch
The 5th/6th Battalion ("5/6 RVR") is one of two battalions of the Royal Victoria Regiment, and is an infantry battalion of the Australian Army Reserve. The battalion can trace its lineage back to many units that existed prior to Federation, as well as units that fought during World War I and World War II and the battalion carries the battle honours of these units as a mark of respect. Today 5/6 RVR is part of the 4th Brigade, 2nd Division and is based at various depots around Melbourne, Victoria. Recently, members from the battalion have been involved in deployments to the Middle East, East Timor, the Solomon Islands and Malaysia as part of Rifle Company Butterworth.
- 1 Unit history
- 1.1 The Regiment
- 1.2 1948–1960 – Post World War II expansion
- 1.3 1960–1965 – The Pentropic experiment
- 1.4 1965–1982 – A period of expansion
- 1.5 The 1980s
- 1.6 The 1990s
- 1.7 The new millennium
- 2 Current structure
- 3 Battle honours
- 4 Pipes and Drums
- 5 Alliances
- 6 See also
- 7 Notes
- 8 References
- 9 External links
Unit history[edit]
5th/6th Battalion, The Royal Victoria Regiment is derived from earlier Australian battalions which had drawn members from the Melbourne metropolitan area. The history of this Battalion and the events leading up to their linking and the formation of the subsequent 5th/6th Battalion, The Royal Victoria Regiment is detailed below.
The Regiment[edit]
In the late 1950s, it was determined to consolidate all Army Reserve infantry regiments into one regiment per state. With this move, several proud regimental histories were ended and a new chapter in the history of Victorian volunteer military forces had begun. The Royal Victoria Regiment was formed on 1 July 1960, as a non-tactical grouping of all Army Reserve infantry battalions in the state of Victoria. It inherits and embodies the traditions of the infantry in Victoria from the earliest units formed since 1854. To fully understand the heritage of the regiment it is necessary to go back to the very founding of the colony, however, this summary will concentrate on the history of the Melbourne battalions since the conclusion of World War II.
A number of previously existing Melbourne metropolitan units and Victorian regional units now make up the 5th/6th Battalion, The Royal Victoria Regiment. The regional units' proud traditions and histories are entrusted to 8th/7th Battalion, The Royal Victoria Regiment, which is the sister unit of 5/6 RVR. It should be understood that many of the 1st AIF and the 2nd AIF units were raised directly from the skeleton of militia units on the declaration of war. For this reason, The Royal Victoria Regiment can claim more battle honours than any other regiment or unit in the Australian Defence Force. A list of the approved battle honours is included below.
This summary follows three Victorian battalions (5th, 6th and 58th/32nd) that have been merged, split and renamed several times from 1948 through to 2003. A fourth battalion was briefly formed in the early 1970s and some of its activities will be briefly mentioned. This constant flux and turmoil has disrupted the clarity of the battalion's heritage. The various regimental families however, sustain the unit's past and a benefit of this consolidation is the breadth of its affiliations.
1948–1960 – Post World War II expansion[edit]
With World War Two over the 2nd AIF was de-mobilised more rapidly than the similar forces of any other participating nation. It was not long however, before the fear of communism prompted the expansion of Australian Forces. In 1960, the National Service scheme ceased, resulting in an immediate reduction in strength. A substantial structural change was also about to occur, for on 30 June 1960, all battalions and their members were absorbed into the new-style pentropic 1st Battalion, The Royal Victoria Regiment (RVR).
The Korean War, coupled with a perceived threat of communism to the security of the country and surrounding regions, led to the adoption of National Service. This scheme required participants to complete three months of full-time training, followed by three years service as members of Citizens Military Force (CMF) units.[2]
5th Battalion – The Victorian Scottish Regiment[edit]
The 5th Battalion, The Victorian Scottish Regiment (VSR) had existed prior to World War Two and was re-established on 1 April 1948,[3] as part of the Citizen Military Forces (CMF). Just as many of the members of the pre-war VSR had helped to raise the AIF, many ex-members of the 2nd AIF returned to the colours to again raise the 5th Battalion. Battalion Headquarters was established at Hawthorn and the Companies were located at Kew, Surrey Hills, Armadale and Dandenong.
The first Commanding Officer of the new Battalion was Lieutenant Colonel G.R Warfe, DSO, MC, ED. The Battalion quickly undertook a variety of training and duties, providing a guard of honour on the occasion of the presentation of the Field Marshal's Baton to Sir Thomas Blamey Australia's first and as at the time of writing only active Field Marshal (HRH The Duke of Edinburgh is also listed as a Field Marshal in the Australian Army).
The battalion colours were also recovered with great pomp and ceremony, from the Scots' Church in Melbourne. In addition, Scottish Dress was quickly approved and the VSR was soon dressed distinctively. In 1898, the VSR had originally worn a modified Gordon tartan, with the gold lines replaced by red. In 1899 this was replaced by the Gordon tartan. The battalion motto was the same as that prior to World War One: "Nemo Me Impune Lacessit". These same words are engraved at the entrance of Edinburgh Castle. Loosely translated this means "no one hurts me and gets away it with it". The battalion march was "Cock of the North", and the unit colour was gold.
The introduction of National Service in 1951 led to 5th Battalion VSR growing to be over full-strength, allowing the unit to conduct ambitious peacetime training successfully. The battalion was represented in the Coronation Contingent sent to London in 1953. In 1954 the battalion provided the Government House Ceremonial Guards during the visit of Queen Elizabeth II. In the same year the battalion also provided troops to line the streets, with their colours, for the opening of Parliament in Canberra. The battalion was presented with new Queen's and Regimental Colours on the 12 May 1957, which are now laid up in The Scots' Church, Melbourne.
6th Battalion – The Royal Melbourne Regiment[edit]
In May, 1948, the 6th Battalion was re-formed.[4] 6th Battalion, The Royal Melbourne Regiment (RMR) had existed prior to the Second World War and again ex-soldiers of the 2nd AIF returned to the colours, members now of the CMF.[citation needed] The Battalion recovered its colours, adopting the motto of "Semper Paratus" or "Always Ready".[5] The Battalion March was "Waltzing Matilda"[5] and the unit colour was Khaki.
The Battalion was established in the same Victoria Street Training Depot that they had occupied prior to the war.[4] At the time of writing, this depot still stands near the Queen Victoria Market on the corner of Therry & Victoria Streets in Melbourne. Companies were also established at Preston and Carlton.[4] The 6th Battalion gained an influx of members due to the effects of National Service, and participated in many of the major ceremonial events that the 5th Battalion VSR was involved in. The size of the unit allowed large scale and advanced training, although when the scheme ceased, the unit suffered a reduction in size. The battalion also gained a significant honour, being granted the Freedom of Entry to the City of Melbourne, on 10 March 1960.[6] 6th Battalion's Colours are laid up in St Paul's Cathedral Melbourne.
58th/32nd Infantry Battalion – The City of Essendon Regiment[edit]
A third CMF Infantry Battalion was raised in the Melbourne area in 1948,[7] being based predominantly in the Western and North-Western suburbs. Initially encompassing Footscray, and known as The West Melbourne Regiment, then sometime later as The Melbourne Rifles,[citation needed] the battalion eventually expanded to be 58th Battalion, The City of Essendon Regiment in 1955.[8]
The battalion established its Headquarters at Pascoe Vale Road Moonee Ponds and had companies at Footscray and Brunswick. It gained many new members through National Service, but lost many when the scheme ceased.[citation needed] This battalion was also absorbed into 1 RVR, when the pentropic organisation was adopted in 1960.[7] Unlike the 5th and the 6th battalions the 58th Battalion has not since reappeared in the Australian Army's Order of Battle, yet strong links are still maintained by the 58th/32nd Battalion Association. The current Moonee Ponds Depot is still the location for their annual ANZAC day service. The 58th Battalion Colours were laid up on the 10 May 1970 located at the Essendon Town Hall. The 32nd Battalion Colours were laid up in August, 1970, and are at St John's Anglican Church in Footscray.
1960–1965 – The Pentropic experiment[edit]
A re-assessment of optimal fighting strengths and structure resulted in the Australian Army adopting a Pentropic arrangement for its battalions in 1960. All of the Victorian CMF Infantry units were affected by this and two units were created to replace the five older ones. 1 RVR comprised most metropolitan sub-units whilst 2 RVR was built from a mixture of city and metropolitan units.
The pentropic arrangement comprised five rifle companies, a support company, an administration company and a command element. For 1 RVR the break up of the Sub-units was as follows:
- A Company (The Merri Coy);
- B Company (The Scottish Coy);
- C Company (The Melbourne Coy) from the 6th Battalion;
- D Company (The Essendon Coy);
- E Company (The Footscray Coy) from the 58th/32nd Battalion; and
- Support Company was established from the 5th Battalion.[9]
The term Royal Victoria Regiment had been adopted at the outset of the formation of the new unit, to reflect the connection of the unit with the State from which it was raised. This was an effort to uphold the traditions and maintain the ties to the local communities. At the same time, a new badge was adopted, which is still retained by current units. The Regiment celebrated its birthday on 1 July 1960 as a result of this union.
1965–1982 – A period of expansion[edit]
The pentropic arrangement was found to be generally unsuitable for use within the Australian Army and by 1965, alternatives had been studied and selected.[10] On the 3 May 1965 both 1 RVR and 2 RVR were again split to form the following five units:
- 1st Battalion, The Royal Victoria Regiment (Melbourne Area),
- 2nd Battalion, The Royal Victoria Regiment (Country Areas),
- 5th Battalion, The Royal Victoria Regiment (Melbourne Area),
- 6th Battalion, The Royal Victoria Regiment (Melbourne Area), and
- 1st Independent Rifle Company (Mildura).
When National service ended in 1960 CMF units were again eroded, as they became completely voluntary units. However, National Service was again introduced in 1965, offering six years voluntary service in the CMF or two years full-time service, subject to being balloted by birth date, enabling all of the metropolitan units to remain at good manning levels.[11] This scheme was abolished in 1973, however, and the Government commissioned a review into the training and organisation of the CMF. One result from this was that the CMF became known as the Australian Army Reserve or as it is more widely known: The Army Reserve.[12]
1 RVR: The creation of 1 RVR essentially replaced 58th Battalion as the third metropolitan battalion. As with its predecessor, it was concentrated in the western and north-western parts of Melbourne and maintained a beneficial rivalry with the other Melbourne battalions. In 1975, 1 RVR absorbed both the 5 & 6 RVR due to all units being well below strength. This caused disruption to many members and was due at least in part to the fallout from the Australian involvement in Vietnam and the generally uninterested public view of the military following that unpopular conflict. With changes to its conditions of service, the number of people in the Army Reserve began to drop significantly. On the 14 November 1987, 1 RVR was absorbed into 5/6 RVR. The colours of 1 RVR were laid up on 25 April 1988 and remain at the Essendon Town Hall.
5 RVR: 5th Battalion was again manned effectively and in its distinctive dress. However, in 1966, a directive was issued that only one company in a Battalion was allowed to wear the traditional Scottish dress, a move that was not popular. The battalion received its new colours on 19 October 1969 at Royal Melbourne Showgrounds by His Excellency the Governor General of Victoria, Major General Sir Rohan Delacombe, KCMG, KBE, CB, DSO, KStJ. The Vietnam War saw the general community lose enthusiasm for the military, and the strength of all of the Battalions fell accordingly. The end of the involvement in Vietnam, and the end of National Service, severely affected the 5th Battalion. By 1975 the situation precipitated the amalgamation of all of the Victorian Battalions. 5 RVR's Colours were laid up once more although it would not be long before they would represent another unit.
6 RVR: The 6th Battalion once again took over its old stamping grounds with the end of the pentropic arrangements. As with 2 RVR and 5 RVR, 6 RVR was presented with new colours on 19 October 1969 at the Royal Melbourne Showgrounds. Suffering the same fate due to community disinterest of Defence issues and the abolition of National Service, 6 RVR was amalgamated with the 5 RVR into 1 RVR in 1975. 6 RVR's Colours were laid up, waiting for an opportunity to be shown to new soldiers.
3 RVR: Brief mention is made of this Battalion, because Routine Orders were raised for this Unit. During 1973, in an era of declined interest in the Army Reserve, the four Victoria State infantry battalions and the independent rifle company were formed into one battalion for a major exercise and named 3 RVR. LTCOL Barry Ingram AM RFD ED was Commanding Officer. An unprecedented amount of resources were deployed to support 3RVR during its brief existence. This even included support from the RAAF.
The 1980s[edit]
When the Soviet Union invaded Afghanistan in 1979, the government of the day reacted by immediately increasing defence spending. The Army Reserve was expanded and improved as a result.
5/6 RVR On 17 August 1982 a new Melbourne battalion was created: 5th/6th Battalion, The Royal Victoria Regiment.[13] 5/6 RVR adopted the traditions of both The Victorian Scottish Regiment and the Royal Melbourne Regiment and recovered both sets of Colours to reflect this. The Battalion also maintained its previous allegiances with The Gordon Highlanders and their extended affiliates, The Royal Regiment of Fusiliers and 6th Battalion, The Royal New Zealand Infantry Regiment. The amalgamation also involved combining some aspects of both units. The unit lanyard went from being just one colour to being a twisted braid of gold and khaki. The unit adopted "Our Director" as the battalion march, however this was then changed to a combination of 'Cock O' the North' and 'Waltzing Matilda' incorporating the original marches of the 5th and 6th Battalions respectively.[1] A Pipes and Drums was raised, and this is now the Regimental Band.
On 14 November 1987 1 RVR was absorbed into 5/6 RVR thus ending any connection with the pentropic organisation of the 1960s era. The Dibb Report of the mid-1980s saw the 5th/6th Battalion and the 4th Brigade given the task of Vital Asset Protection of the Tindal Air Force Base in the Northern Territory as its primary role. 5/6 RVR participated in two large multinational Kangaroo exercises in 1989 in support of this objective and also sent smaller elements to the region on other exercises.
In 1988, a decision was made to reintroduce Colour Patches for the first time since the end of the Second World War. The original 5th Battalion patch (as worn the day the 5th Battalion, 1st AIF, raised in Melbourne later to land at ANZAC Cove on the 25 April 1915) was adopted for the 5/6 RVR. This patch of a black rectangle on top of a red rectangle, is worn on the puggaree of the slouch hat.
The 1990s[edit]
A change of Federal Government in 1995 led to a review of the Army Reserve which impacted directly on the 4th Brigade in general and 5/6 RVR in particular. The Brigade was to be "revitalized" and recruitment was stepped up to try (unsuccessfully) to increase the size of the Battalion. At the same time more Regular Army Staff (including temporarily, a full-time Battalion Commander Lieutenant Colonel Mick Godfrey) were posted to try to increase the readiness and training tempo of the unit.
Training was well resourced and tested by independent observers in a readiness test at the Cultana training area in 1999, in which 5/6 RVR performed extremely well. The Battalion also supplied a company group to rotate through the Butterworth Airbase, Malaysia, in Nov 1999 – Jan 2000 becoming one of the first Army Reserve units to do so. The company group was drawn primarily from 5/6 RVR with additional attachments coming from other units of 4th Brigade of the 2nd Division.
In 1999/2000 the escalating crisis in East Timor saw Australian troops leading a multinational 'intervention' force (INTERFET). Many Reservists initially served to 'backfill" Australian Regular Army (ARA) positions when they were deployed to Timor with the 6th Battalion, The Royal Australian Regiment. As Australia's commitment to maintain a battalion was ongoing, a Rifle Company was raised from Reservists throughout Australia to serve with 5th/7th Battalion, The Royal Australian Regiment (RAR). This first tour of duty was from November 2002 until May 2003. Soldiers from both RVR battalions along with their NSW counterparts provided the majority of Reservists on Full Time Service. Timor was the first time since the mobilisation of the Militia (AMF) in the Papua New Guinea campaign of World War II that 'Citizen Soldiers' saw active service. At the conclusion of this deployment, many were to elect to remain in the Australian Regular Army whilst most returned home to Reserve Service.
The new millennium[edit]
In the year 2000 up to 200 5/6 RVR soldiers served with the ADF contingent in support of the Sydney Organising Committee for the Olympic Games (SOCOG) under "Operation Gold". Known as the Operational Search Company their duties included conducting area searches, building searches and manning vehicle checkpoints. Whilst full-time leadership and increased readiness had their benefits, increased time requirements for individual training for recruit and promotion courses contributed to a decline in Unit strength over the 2001/2002 period. The role of the Army Reserve was changing and so was 5/6 RVR in response to those changes. A dedicated staff of Reservists and full-time members continued to work to provide the expertise and staffing required to train and develop young and upcoming 5/6 RVR soldiers.
In early 2003, the Reserve Response Force (RRF) concept was being rolled out to 4th Brigade. 5/6 RVR was chosen to lead the first Reserve based 4th Brigade RRF. The RRF role was then devolved to unit level and 5/6 RVR continued to strengthen its role through Bravo Company, the largest Company, which was chosen to lead the battalion's RRF elements.
The Sesquicentenary – 150th anniversary[edit]
2004 saw 5/6 RVR celebrate its Sesquicentenary – or 150th anniversary. A "Sesquicentenary Committee" was formed chaired by the Commanding Officer, LT COL Mark Richards. The committee was functionally led by Battalion 2IC MAJ Terry Kanellos and included current and past members of the battalion and networked extensively with the help of MAJ GEN James Barry, MAJ GEN Greg Garde and the then Regimental Colonel, COL Paul Riley. A number of activities and functions were planned throughout the year involving all ranks.
The year began with a series of Unit History presentations given throughout the Battalion by Company personnel. Working with the support of Battalion Associations, the committee planned and conducted the following activities:
- An All Ranks Reception at Government House hosted by his Excellency, Mr John Landy AO, Governor of Victoria on 17 September 2004;
- A Freedom of the City of Melbourne march on 16 October 2004;
- Gala Ball – The Atrium Room Flemington Racecourse on 16 October 2004;
- An Employer Function – Hosted and sponsored by Mr & Mrs Dick and Jeanne Pratt at their Raheen Residence in Kew, Victoria on 25 November 2004; and
- An End of Year Battalion Christmas Party at the Telstra Dome on 11 December 2004.
The year also saw an increasing focus on the Battalion's role in 4 Brigade's Reserve Response Force (RRF), with an emphasis on training and individual preparedness. Urban tactical training and joint exercises held with The Victoria Police saw the battalion's ability to work with civil emergency services strengthened and gave commanders valuable experiences. The success of the year being measured by attendances of over 300 guests at the Gala Ball in October and similar numbers at the end of year function at the Telstra Dome.
2005 – New CO takes 5/6 RVR to Canungra[edit]
2005 built on the gains of the previous year and a new Commanding Officer LT COL Neil Grimes marched in. The importance of individual preparedness was re-emphasised as Army Individual Readiness Notice (AIRN) criteria was applied to each member and it prepared for its annual field exercise (AFX) at Canungra in Queensland. A year for record Recruiting numbers, the benefits of one of the largest intakes of recruits in recent years also came to realisation, with all companies seeing new members join their ranks. 2005 also saw 5/6 RVR exercise its freedom of entry to the City of Boroondara on Sunday 17 October 2005.
2006 – Operation Acolyte[edit]
In 2006, the Battalion made a significant contribution to the conduct of the Commonwealth Games held in Melbourne during February and March on Operation Acolyte. Over 100 personnel drawn from throughout the battalion participated in the 10-week operation which saw searches of the Melbourne Cricket Ground, the Commonwealth Games Village in Parkville and the Melbourne Sports and Aquatic Centre conducted. Members acted in both a military and civilian capacity with selected members being directly employed by the Melbourne 2006 organisation. Military members manned checkpoints and staffed a vehicle screening facility known as the Logistics Screening Zone where deliveries to locked down venues were electronically screened prior to entry.
Hardened & Networked Army[edit]
The Hardened & Networked Army (HNA) proposal was Army's response to the need to fight on a more complex and lethal battlefield. It would provide increased combat weight and generate greater organisational depth in Army and a greater focus on combined arms battlegroups and combat teams rather than infantry battalion and company groups. The result of HNA would be increased options for Government in terms of both the combat weight of the force that can be deployed and the duration that forces can be sustained on operations. Implementation of HNA started in 2006 and the force structure changes were due to be complete by the end of 2015.
Following HNA, a new structure emerged known as the High Readiness Reserve or HRR Combat Team. 4 BDE HRR was formed from a number of Units but predominantly manned and commanded by 5/6 RVR. The HRR Combat Team successfully participated in the Talisman Sabre series of major exercises with regular battalions and also contributed significantly to the East Timor deployment known as TLTG-4 in 2012.
A new role for the Army Reserve emerged in late 2006 with Army raising 2 new ARA infantry battalions. The structure of infantry battalions also changed and 5/6 RVR merged Delta Company at Simpson Barracks with Bravo Company Surrey Hills. Delta Company became known as 6 Platoon B Company, proud company members celebrated their last function together as Delta Company in December 2006. In August 2007, the battalion lost the old Delta Company depot at Simpson Barracks, and now all Bravo Company members parade at Surrey Hills.
Solomon Islands[edit]
In recent years, the 5th/6th Battalion has continued its proud tradition of overseas service by providing troops for Operation Anode as part of the Regional Assistance Mission to Solomon Islands (RAMSI), contributing to the first and probably last Solomon Island deployments: Op Citadel, Op Anode Rotation 13 in 2007, Rotation 15 in 2008, Rotation 18 in 2009 and Rotation 30 in 2013.
Current structure[edit]
Today 5/6 RVR is organised as a standard light infantry battalion, with its respective sub-units situated throughout metropolitan Melbourne. In 2013, the 2nd/10th Field Regiment was downsized into a single battery and added to the 5th/6th Batttalion's structure.
Each company is located at:
- Battalion Headquarters (BHQ) – 202 Burwood Road, Hawthorn
- Support Company (SPT Coy) – 127 Pascoe Vale Road, Moonee Ponds
- Alpha Company (A Coy) – 101 Duke Street, Sunshine
- Bravo Company (B Coy) – 12 Robinson Road, Surrey Hills
- Charlie Company (C Coy) – 67 Royal Avenue, Sandringham
- 2nd/10th Light Battery (2/10 Lt Bty) – 8 Chapel Street, St Kilda East
During the Army Reserve training year (typically from mid February to early December), members of 5/6 RVR attend their respective depots every Tuesday night from 7:30 to 10:30. Battalion Headquarters is also open 8:00am to 4:00pm, weekdays. The battalion conducts a training weekend once a month, at a variety of locations, such as Simpson Barracks, Puckapunyal and RAAF Williams.
Battle honours[edit]
The traditions, customs and experiences of infantry soldiers based in Melbourne are enshrined within the Battalion and the various Associations. It remains very much an important part of the City of Melbourne's proud history. As a tribute to previous units, 5/6 RVR holds the most battle honours and historically carries the following battle honours emblazoned on its colours:
|Regimental Colour
|Queens Colour
|South Africa (1899–1902)
|Bardia 1941
|Capture of Tobruk
|Ypres 1917
|El Alamein
|Greece 1941
|Mont St Quentin
|South West Pacific 1942–45
|Somme 1916, 1918
|Bobdubi II
|Polygon Wood
|Albert 1918
|Hari River
|Hindenburg Line
|Landing at ANZAC
Pipes and Drums[edit]
Formed in 1899 as part of The Victorian Scottish Regiment, the 5/6RVR Pipes and Drums is now the band for all battalions of the Royal Victoria Regiment. It is supported by the RVR Pipes and Drums Association, an incorporated body that offers tax deductible status for donations over $2.
- United Kingdom – once The Gordon Highlanders – then Highlanders (Seaforth, Gordons and Camerons) – now Royal Regiment of Scotland
- Canada – The Toronto Scottish Regiment – via Gordon Alliance
- Canada – 48th Highlanders of Canada – via Gordon Alliance
- South Africa – Cape Town Highlanders Regiment – via Gordon Alliance
- Canada – Lake Superior Scottish Regiment
- United Kingdom – Mercian Regiment
- United Kingdom – The Royal Regiment of Fusiliers - via Royal Melbourne Regiment
See also[edit]
- Australian Army
- Australian Army Reserve
- List of Australian Army Regiments
- Royal Victoria Regiment
- London Scottish Regiment
- 8th/7th Battalion, Royal Victoria Regiment
- ^ a b Army et al. Manual.
- ^ Grey 2008, p. 206.
- ^ Festberg 1972, p. 62.
- ^ a b c Austin 1993, p. 213.
- ^ a b Festberg 1972, p. 64.
- ^ Austin 1993, p. 243.
- ^ a b Festberg 1972, p. 114.
- ^ Festberg 1972, p. 115.
- ^ Austin 1993, p. 284.
- ^ Austin 1993, p. 254.
- ^ Grey 2008, pp. 239–240.
- ^ Grey 2008, p. 261.
- ^ Austin 1993, p. 273.
- Austin, Ron (1993). Bold, Steady, Faithful: The History of the 6th Battalion, The Royal Melbourne Regiment, 1854–1993. Melbourne: 6th Battalion Association. ISBN 0-646-15294-7.
- Festberg, Alfred (1972). The Lineage of the Australian Army. Melbourne, Victoria: Allara Publishing Pty Ltd. ISBN 978-0-85887-024-6.
- Grey, Jeffrey (2008). A Military History of Australia (3rd ed.). Melbourne, Victoria: Cambridge University Press. ISBN 978-0-521-69791-0.
- Australian Army (2014). "Army Ceremonial and Protocol Manual" (Annex A to Chapter 27).


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Transcript profiling reveals complex auxin signalling pathway and transcription regulation involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton
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- Yang, X., Zhang, X., Yuan, D. et al. BMC Plant Biol (2012) 12: 110. doi:10.1186/1471-2229-12-110
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Somatic embryogenesis (SE), by which somatic cells of higher plants can dedifferentiate and reorganize into new plants, is a notable illustration of cell totipotency. However, the precise molecular mechanisms regulating SE remain unclear. To characterize the molecular events of this unique process, transcriptome analysis, in combination with biochemical and histological approaches, were conducted in cotton, a typical plant species in SE. Genome-wide profiling of gene expression allowed the identification of novel molecular markers characteristic of this developmental process.
RNA-Seq was used to identify 5,076 differentially expressed genes during cotton SE. Expression profile and functional assignments of these genes indicated significant transcriptional complexity during this process, associated with morphological, histological changes and endogenous indole-3-acetic acid (IAA) alteration. Bioinformatics analysis showed that the genes were enriched for basic processes such as metabolic pathways and biosynthesis of secondary metabolites. Unigenes were abundant for the functions of protein binding and hydrolase activity. Transcription factor–encoding genes were found to be differentially regulated during SE. The complex pathways of auxin abundance, transport and response with differentially regulated genes revealed that the auxin-related transcripts belonged to IAA biosynthesis, indole-3-butyric acid (IBA) metabolism, IAA conjugate metabolism, auxin transport, auxin-responsive protein/indoleacetic acid-induced protein (Aux/IAA), auxin response factor (ARF), small auxin-up RNA (SAUR), Aux/IAA degradation, and other auxin-related proteins, which allow an intricate system of auxin utilization to achieve multiple purposes in SE. Quantitative real-time PCR (qRT-PCR) was performed on selected genes with different expression patterns and functional assignments were made to demonstrate the utility of RNA-Seq for gene expression profiles during cotton SE.
We report here the first comprehensive analysis of transcriptome dynamics that may serve as a gene expression profile blueprint in cotton SE. Our main goal was to adapt the RNA-Seq technology to this notable development process and to analyse the gene expression profile. Complex auxin signalling pathway and transcription regulation were highlighted. Together with biochemical and histological approaches, this study provides comprehensive gene expression data sets for cotton SE that serve as an important platform resource for further functional studies in plant embryogenesis.
KeywordsAuxin signalling pathway Cotton RNA-seq Somatic embryogenesis Transcription profile Transcription regulation
Somatic embryogenesis (SE) is the developmental process by which somatic cells undergo dedifferentiation to generate embryogenic cells and form a somatic embryo, from which new plants can be regenerated [1, 2]. This process can be divided into two stages. First, somatic cells re-enter the cell cycle and transform into a dedifferentiated cell state; they then acquire embryogenic potential, characterized by a reorganization of cell physiology, metabolism and gene expression [3]. This process is experimentally induced by changes of culture conditions, using exogenous plant growth regulators (PGRs) and stress. Following that, cells with embryogenic potential can differentiate into somatic embryos [4, 5].
Morphological, biochemical, and more recently, molecular aspects of SE have been described [6, 7, 8]. Following early preliminary experiments on differential gene expression [9], the mechanisms of gene regulation during SE in many plant species, such as carrot [10], cotton [11], Arabidopsis [12], alfalfa [13], soybean [7] and potato [14] have been investigated. These experiments have resulted in the isolation of numerous genes which are specifically activated, or exhibit differential expression, during SE [1, 15].
Cotton is one of the most important economic crops and the main source of natural fibre, but further trait improvement requires efficient genetic manipulation [11]. Cell biological approaches, including tissue culture and genetic engineering, have been widely applied to cotton breeding. As a result, transgenic varieties with herbicide and pest resistance have been developed [16]. However, a reproducible and highly efficient plant regeneration scheme is required for cotton species, which remains a recalcitrant species to manipulate in vitro[17]. Thus far, reports of high-frequency regeneration of cotton via SE have been limited owing to a genotype-dependent response [18], and the majority of the reports on in vitro regeneration of cotton only pertain to specific varieties such as Coker lines [19]. More recently, an elite genotype for in vitro cellular manipulation, which showed more efficient regeneration ability than Coker lines, was identified in our laboratory [20]. The identification and isolation of genes critical for SE are of great importance for improving the embryogenic competence and regenerability of a wider range of cultivars and thus accelerating the production of transgenic cotton varieties. This requires new molecular information.
Physiological and biochemical changes during SE are reflected in the transcriptional modulation of many genes [7, 9, 21]. A number of genes that are activated or differentially expressed during the induction and development of somatic embryos have been cloned and studied using various molecular techniques [22, 23, 24]. However, little is known about global transcriptional changes and their regulation. Genome-wide expression analyses provide essential building blocks for elucidating molecular function. The analysis of cDNA amplified fragment length polymorphisms [25], Suppression Subtractive Hybridization (SSH) and microarrays [11, 21, 26] have provided the first pictures of transcriptome dynamics during cotton SE and early events of cellular dedifferentiation, but these approaches suffer from a number of drawbacks and the data are far from complete. Recent studies have highlighted the significance of next-generation sequencing technologies for genome-scale expression analyses in higher eukaryotes, including whole-transcript sequencing and assembly (RNA-Seq) using the long-read, 454 platform [27] and the massively parallel Illumina [28] and ABI SOLiD [29] systems. These approaches produce millions of short cDNA reads that can be mapped to a reference genome and/or transcriptome sequence to obtain a genome-scale transcriptional map consisting of the transcriptional structure and the expression level for each gene. Resolution of these networks is possible because of the increased sensitivity and specificity of transcript analysis by the method [30].
To create a more complete survey of transcriptome content and dynamics during cotton SE, we used a next-generation sequencing approach, Illumina Digital Gene Expression (DGE) technology. To our knowledge, this represents the first genome-wide gene expression profiling of SE in cotton, and the data presented here will serve as a foundational resource for future studies addressing fundamental molecular and developmental mechanisms that govern plant embryogenesis.
Kinetics of cotton SE
Hypocotyls cultured for 3 h showed no morphological changes compared to fresh hypocotyls (0 h explants, Figure 1J-K). On the third day of induction, both ends of hypocotyls had expanded, but histological observation showed expanding epidermal cells at 24 h after induction (Figure 1N). Some epidermal cell expansion was evident by 6 h to 12 h after induction (Figure 1L-M). Subsequently, epidermal, parenchyma and primary cambium cells expanded and dissociated from explants (Figure 1O-P). After 7 d of culture, callus could be seen at the end of explants, which subsequently proliferated. Histological observation showed that the epidermal and primary cambium cells rapidly entered cell division and the unattached meristematic cell masses separated from the primary meristem (Figure 1Q). Some cells went through distinct cellular dedifferentiation and then began to divide and proliferate normally (Figure 1R-U). After about 40 d of culture, some ECs were produced. Thus, SE in cotton passes through three different processes as shown in Figure 1: dedifferentiation of cotton somatic cells, transition from NECs to ECs and development of somatic embryos.
Dynamics of endogenous indole-3-acetic acid during SE
Global analysis of differential gene expression during SE
Overview of tag number
No. of clean tag
No. (%) of Unambiguous mapped Tag
No. (%) of Unambiguous tag-matched unigene
No. (%) of unknown tag
Time of hypocotyls cultured (h)
Noembryogenic calli
Embryogenic calli
Globular embryo
Torpedo embryo
Cotyledon embryo
The expression level of genes was determined by calculating the number of unambiguous tags for each gene and then normalizing this to the number of transcripts per million tags (TPM) [33]. An average of 34,278 unambiguous clean tags per sample were calculated for each gene and then normalized to TPM, which linked the tag numbers with gene expression levels. The summary of the tag information and gene expression level is shown in Additional file 3 Table S2. We detected the expression of 15,339 genes during cotton SE. The dynamic range of DGE spanned five orders of magnitude. However, the tag counts for the majority of genes were low in these libraries. Among these, 5,076 differentially expressed genes were filtered with a cut-off of TPM ≥ 20 (P ≤ 0.001) and the absolute value of log2Ratio ≥1 based on the false discovery rate (FDR) < 0.05 ( Additional file 4 Table S3).
Annotation of these differentially expressed genes (5,076) was first done by searching using BLASTx against the non-redundant protein sequence (nr) database in GenBank using a cut-off E-value of 10−5. Using this approach, 3,274 genes (64.50% of all differentially expressed sequences) returned an above cut-off BLAST result ( Additional file 4 Table S3). A further 1,308 genes (25.77%) belonged to the functional category 'unclassified proteins' or 'predicted protein'. 494 differentially expressed genes could not be matched to any genes in the nr protein database. In a reciprocal BLAST search, we also identified 4,536 cotton unigenes (89.36%) that had an ortholog in Arabidopsis ( Additional file 4 Table S3).
To identify the biological pathways that are active during SE in cotton, we mapped the 5,076 annotated sequences to the reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) [34]. In total, we assigned 2,118 sequences to 256 KEGG pathways ( Additional file 5 Table S4). The pathways with the most representation by unique sequences were 'metabolic pathways' (291 members) and 'biosynthesis of secondary metabolites' (120 members). A hallmark of dedifferentiation and following somatic embryo development is the ability to control cell division and cell wall accumulation associated with polysaccharides metabolism with corresponding hydrolytic enzymes and their accumulation of storage reserves and secondary metabolites [35]. 'Spliceosome' (93 members), 'microbial metabolism in diverse environments' (70 members) and 'oxidative phosphorylation' (53 members) were also enriched as were 22 genes in the 'plant hormone signal transduction' pathway. Given the known role of auxin in regulating plant embryogenesis [4], the competence for embryogenic induction might be the result of regulated auxin responses of these cells. These annotations provide a valuable resource for investigating specific processes, functions and pathways and allow for the identification of novel genes involved in these pathways.
Functional categories were assigned to all predicted genes in terms of gene ontology (GO) [36]. We added GO terms using Blast2GO based on the automated annotation of each unigene using BLAST results against the GenBank nr protein database from NCBI [37]. A total of 3,438 unigenes (67.73%) could be assigned to one or more ontologies, and 2,588 (50.99%) unigenes with assigned GO terms had molecular functions, 2405 (47.38%) were involved in biological processes and 2629 (51.79%) were cellular components; 1657 (32.64%) unique sequences were classified in three ontologies. In each main category, the percentages of different levels do not add up to 100% because some deduced proteins had more than one GO category assigned to them. GO annotations for the differentially expressed genes showed fairly consistent sampling of functional classes.
Transcription Factor mRNA present during cotton SE
We annotated features to major TF families to determine the spectrum of TF mRNAs present during this process (Figure 6). All major TF families were represented in the mRNA population at each developmental stage. Taken together, these data suggested that more than 400 diverse TF mRNAs are expressed during SE; the number of TF mRNAs decreased during late embryogenesis; and the representation of specific TF mRNA families differed at specific developmental periods. Zinc finger and bHLH family TFs occupied one third of all TFs. The MYB family TFs, and others such as ERF, bZIP and WRKY, each accounted for 4% to 6% of identified transcripts.
Analysis of the auxin signalling pathway during cotton SE
Auxin plays a role in dedifferentiation and redifferentiation, through the modulated response, transduction and amplification of the auxin signal to regulate the expression of genes. In this study, 86 genes related to auxin synthesis, transport, metabolism and the signalling pathway were differentially expressed during SE ( Additional file9 Table S8-1). Transcript levels of genes related to auxin homeostasis varied during this process, consistent with a role for auxin in SE. Combined with KEGG results and other annotations, these genes were related to IAA biosynthesis (8 transcripts), IBA metabolism (9 transcripts), IAA conjugate metabolism (8 transcripts), auxin transport (10 transcripts), Aux/IAA (13 transcripts), ARF (6 transcripts), SAUR (4 transcripts), Aux/IAA degradation (17 transcripts) and other auxin-related proteins (11 transcripts).
Ten auxin transport-associated transcripts were differentially expressed. Three AUX1 homologous transcripts (zhu1_Ghi#S33799879, zhu1_Ghi#S42308191 and zhu1_Ghi#S29994266) were down-regulated during cotton SE, while only AUXI-LIKE displayed high levels ( Additional file 9 Table S8-1). Like AUX1, three transcripts (zhu1_Ghi#S33805308, zhu1_Ghi#S42333454, zhu1_Ghi#S42298593) responsible for auxin transport were down-regulated. However, another three (zhu1_Ghi#S42308314, zhu1_Ghi#S42299563, zhu1_Ghi#S33832032) were up-regulated, while a PIN3 homologous transcript (zhu1_Ghi#S42312756) was slightly up-regulated at 0–24 h and then down-regulated ( Additional file 9 Table S8-1). The expression profiles of selected genes were confirmed by qRT-PCR analysis (Figure 7B).
In addition, we found that several Aux/IAA genes were differentially expressed, though most Aux/IAA genes, except IAA19, IAA14-1 and AUX2-11-2 at some time points, were down-regulated during dedifferentiation, showed an extremely low pick at the EC stage and then were up-regulated during somatic embryo development (Figure 7C). Likewise, some ARF and SAUR genes were differentially expressed, with a complex expression profile. However, four genes (ARF6-1, ARF9, NPH4 and ARF6-2) were up-regulated during somatic embryo development (Figure 7D). Some genes related to Aux/IAA degradation, such as AFB2, TIR1, AXR1, RBX1, ASK2, ASK1, CUL1, RCE1, DCAF1 and SGT1B were also differentially expressed ( Additional file 9 Table S8-1).
Among the 86 genes, 58 genes were unique to the initial dedifferentiation process ( Additional file 9 Table S8-2). Transition from NECs to ECs triggered differential expression of 38 genes ( Additional file 9 Table S8-3), with 15 genes down-regulated and 23 genes up-regulated, while 35 genes were differentially expressed during somatic embryo development ( Additional file 9 Table S8-4).
Comparison of DGE tag data with qRT-PCR
Gene expression levels estimated by qRT-PCR at different time points/stages were also analysed. The correlation of RNA-Seq and qRT-PCR results during the dedifferentiation process (6 h and 24 h) was relatively low ( Additional file 10 Figure S2A, R2 = 0.2016 at 6 h, R2 = 0.1327 at 24 h). At 48 h, NEC and EC time points/stages showed a moderate correlation ( Additional file 10 Figure S2B, R2 = 0.5096 at 48 h, R2 = 0.5156 at NEC and R2 = 0.6365 at EC). In contrast, the correlations were higher at GE, TE and CE stages ( Additional file 10 Figure S2C, R2 = 0.8392 at GE, R2 = 0.8192 at TE and R2 = 0.8208 at CE). Although the samples collected early will be more affected by the sampling process and method, these results still suggest the applicability of RNA-Seq to cotton transcriptome analysis and confirm that it is an accurate and reliable way to find genes differentially expressed during dedifferentiation and redifferentiation.
Applications and evaluation of DGE-based analysis with the reference database
Cotton is a major crop for fibre and oil production, and has been subject to the application of biotechnology for crop improvement. Cell culture and plant regeneration are the bases for cotton biotechnology through genetic transformation, and so understanding the molecular control of dedifferentiation and redifferentiation is key to manipulating the SE process. However, the large unsequenced genome size (approximately 2.5 gb), polyploid nature and lack of adequate gene model annotations have limited large-scale transcriptome analyses during cotton SE [39]. Previous studies on the molecular aspects used SSH and microarray [11, 26] but provided limited information on the complex transcriptome dynamics during cotton SE. However, next-generation technologies, which can generate tens of thousands to tens of millions of sequence reads with exceptional reproducibility, provide new strategies to quantitatively analyse the functional complexity of transcriptomes, despite uncharacterized genome sequences [27, 29, 40].
Using RNA-Seq technology developed by Illumina and elite high efficient regeneration lines YZ1, we designed a protocol for analysing the transcriptome complexity of cotton SE. Although SE is usually divided into two stages, induction and expression [5], our morphological and histological observations indicate that it could usefully be divided into three different processes: dedifferentiation of somatic cells, transition from NECs to ECs and development of somatic embryos. Protoplasts undergo cellular dedifferentiation and initiate cell division within 48 to 72 h in tobacco and Arabidopsis [41, 42]. Histological observations have shown that cotton somatic cells activate cellular dedifferentiation and division within 72 h, and often within 48 h [21]. We chose three different time points (6 h, 24 h and 48 h) for initial dedifferentiation sampling and typical NECs after 40 d of induction for late dedifferentiation. Different stages of somatic embryos were selected by distinct morphology observed after synchronization (Figure 1).
For genome sequence references that were unavailable, clean tags were mapped to two different EST reference databases after preprocessing the raw data. One reference database (Reference database 1) was cotton unigenes from NCBI that contains 20,671 unigene sequences, and the other (Reference database 2) was contigs assembly from multiple cotton genes from different databases which contain 65,386 sequences. The two reference databases were compared for efficiency based on several criteria. So many tags were missing using Reference database 2 for critical selection, that tags mapping to unique sequences were used for transcript identification ( Additional file 11 Table S9). However, validation by qRT-PCR of the expression profile for 26 differentially expressed genes derived by using RNA-Seq technology showed that genes mapped based on the two reference databases exhibited a similar correlation (R2 = 0.7077 for Reference database 1, and R2 = 0.7073 for Reference database 2) ( Additional file 12 Figure S3). As a result, we selected the cotton unigenes from NCBI (Reference database 1) as our reference database for further analysis.
Up to 50.7% (15,339) of the sequences in our reference database could be unambiguously identified by a unique tag. However, a relatively low number of the tags (43.18%) could be assigned to genes and used for gene expression profiling. This might be partly explained by the fact that most of the sequences in the database were not generated from embryogenesis development. More sequences and annotation for dedifferentiation and redifferentiation in cotton have to be explored to illuminate the large amount of unknown tags that remain. The extremely low abundance transcripts (TPM ≤ 20) were also filtered because of the possible of sequencing error. Among these, 5,076 differentially expressed genes were filtered with a cut-off of TPM ≥ 20, P ≤ 0.001 and the absolute value of log2Ratio ≥1 based on the FDR < 0.05 ( Additional file 3Table S2).
Transcription regulation of somatic cells dedifferentiation and redifferentiation in cotton
Somatic cells within the plant contain all the genetic information necessary to create a complete and functional plant (with the exception of anuclear vascular cells). The induction of SE comprises the termination of one gene expression pattern in the explant tissue and replacement with an embryogenic gene expression programme [1]. The initiation of the embryogenic pathway, which is preceded by cellular dedifferentiation, is restricted only to certain responsive cells in the primary explant because the existing developmental information of somatic cells must be switched off or altered to make the somatic cells responsive for new signals [1, 5]. Though we described the cotton SE as consisting of three different processes, it is very difficult to dissect the specific cellular events related to the overlapping phases of dedifferentiation, cell cycle reactivation and the acquisition of embryogenic competence.
The embryogenic processes are becoming better understood because of the identification of several genes such as transcription factors that play regulatory roles either in specific embryogenesis phases [43] or throughout the whole process [44]. In the present study, 466 TF mRNAs were differentially expressed over a wide range of abundances during SE. Among these, a subset of TF families were associated with functions in cell differentiation, embryogenic patterning and embryo maturation processes (Zinc finger, b-ZIP, bHLH, B3 and MYB), meristem maintenance or identity (NAC, YABBY, GRAS), while others had roles in hormone-mediated signalling by auxin (Aux/IAA, ARF) or ethylene (AP2/ERF). Zinc finger family proteins have been proven to be involved in cell differentiation and development processes in animals and plants [45, 46]. PEI1, encoding a protein containing a Cys3-His zinc finger domain, is an embryo-specific transcription factor that plays an important role during Arabidopsis embryogenesis, functioning primarily in the apical domain of the embryo, which is required for the globular to heart-stage transition [46]. In the present study, genes encoding zinc finger family protein showed complex expression profiles ( Additional file 8Table S7), indicating that they have multiple functions during SE in cotton. In Arabidopsis, two bHLH proteins were required in embryogenic patterning for root formation in the embryo [47, 48]. The diversity of expression profiles displayed by 78 bHLH homologues in the present study might suggest the complex regulation of SE by bHLH proteins in cotton ( Additional file 8Table S7).
B3 domain transcription factors in Arabidopisis (LEC2FUS3 and ABI3) encode regulatory proteins involved in embryogenesis and induction of somatic embryo development [49, 50]. Six B3 family transcription factor homologues were present with complex expression profiles: two (zhu1_Ghi#S33821461, zhu1_Ghi#S42277219) were down-regulated and one (zhu1_Ghi#S42340389) was up-regulated. Ectopic expression of BABY BOOM (BBM), a member of the AP2/ERF family in Arabidopsis primarily induces spontaneous somatic embryo formation from seedlings, although ectopic shoots and callus also develop at a lower frequency [51]. In our study, 26 AP2/ERF genes were differentially expressed during SE in cotton ( Additional file 8 Table S7). MYB and WRKY were transcription factors not only involved in response to biotic and/or abiotic stresses, but also regulated embryogenesis pathways [52, 53]. As revealed in this study, most MYB genes were up-regulated during embryogenic initiation and showed a relatively low expression level during somatic embryo development, while most MYB-related genes were down-regulated during SE, indicating different functions of these genes during SE ( Additional file 8 Table S7). Further experiments are required to verify the physiological function and interaction between these factors and other genes during SE.
Complex auxin signalling pathway during dedifferentiation and redifferentiation of cotton cells
The importance of PGRs during SE has been widely documented [4, 54]. To understand better the hormonal regulation of SE, PGRs are added to a culture medium to induce somatic embryogenic process, and endogenous hormone concentrations of plant tissues are measured during morphogenesis or various developmental stages [55]. Auxin is considered to be a critical PGR in cell division and differentiation, as well as in the induction of SE. This regulation probably occurs by establishing auxin gradients during the induction phase of SE, essential for initiating dedifferentiation and cell division of already differentiated cells before they can express embryogenic competence [4]. Despite the absolute requirement for exogenous auxins to sustain growth in plant cells cultured in vitro, cultured plant cells produce substantial amounts of the native auxin, IAA. In carrot cells, exogenous auxin stimulates the accumulation of large amounts of endogenous IAA. Thus the application of exogenous auxin and subsequent endogenous auxin content are both determining factors during the induction phase [4, 56]. For this gradient to be established, relatively high levels of IAA in the competent tissues may be necessary.
Most studies on hormone contents during induction of SE only evaluate NEC and EC cultures [32, 57]. In the present study, the endogenous IAA concentrations were determined in the original somatic cells, phytohormone-induced dedifferentiation cells and embryogenic cells (Figure 2). De novo synthesis of IAA in these cells occurred under all the examined conditions. The endogenous levels of IAA declined to a half within 6 h and dropped to a quarter of the original values within 24–48 h following excision from seedlings (Figure 2). The kinetics of this decline in IAA levels was similar to the decline of IAA levels in wounded tobacco leaves by activation of the proteinase inhibitor gene system [58]. However, in IBA-treated soybean hypocotyls, IAA levels increased dramatically after wounding and reached a maximum after 24 h, with a decrease of the cationic peroxidase activity [59].
The mechanism responsible for the decline in IAA levels is not yet understood. The activation of some proteinase inhibitor genes in this study might be one possibility. The endogenous IAA could be influenced at one of several points, including its biosynthesis or degradation or the formation of amide or ester storage forms. Indeed, the decrease in IAA pools could even be influenced through IAA transport. Our data shed new light on these questions.
Our analysis revealed the dynamics of auxin levels during cotton SE (Figure 2). Previous studies have also shown that sharp changes in endogenous auxin levels may be one of the first steps leading to SE [31]. Redifferentiation was clearly correlated with a sharp increase in auxin responses in cotton cells, which provides direct evidence for the significance of an endogenous auxin pulse in the expression of cellular totipotency. It has also been noted that transition of the globular embryo to the heart-stage embryo and its further development requires either a low level of auxins or their complete absence [15]. Surprisingly, most RNA-Seq based auxin synthesis, transport, metabolism and signalling pathway genes were down-regulated during redifferentiation and somatic embryo development processes and showed a relatively low expression level in EC cultures ( Additional file 9 Table S8-1). However, transcript levels of genes related to auxin were changed during this process, indicating a possible role of this hormone in cotton SE.
The increase of IAA might be due to the increased synthesis and turnover of putative host auxin precursor in tissues. Although there are several IAA biosynthesis pathways in higher plants, tryptophan has long been regarded as the important one and its active metabolism and biosynthesis during embryogenesis have been highlighted [60]. A TRP1 homologous transcript was differentially up-regulated during initial dedifferentiation at culture times of 6 h and 24 h (Figure 7A), with a consistent result in wheat [61], while NIT4A showed an opposite expression model (Figure 7A). Nitrilases can contribute to IAA homeostasis by hydrolyzing IAN to IAA in higher plants [62]. Conversion of Trp to IAA by enzymatic complex with nitrilase immunoreactivity in vitro was applied to plants [63]. Expression of maize nitrilase ZmNIT2 is elevated in embryonic tissue [62]. In this study, NIT4A homologues were identified and were down-regulated during the whole process (Figure 7A), while qRT-PCR analysis of three additional nitrilase genes derived from cotton database gave a similar expression profile ( Additional file 13 Figure S4). In addition, the up-regulation of a GH3.17 gene was observed in early dedifferentiation at 6 h of induction (Figure 7A). Several members of this family, including the GH3 genes, were up-regulated at about 2–4 h in soybean hypocotyls exposed to auxin [64]. These enzymes encoded by members of the GH3 family are able to synthesize IAA amino acid conjugates. Two members of the Arabidopsis GH3 gene family have been revealed to be overexpressed in dwarf mutants with reduced apical dominance combined with decreased free auxin levels [65, 66]. Further, disruption of certain GH3 genes confers hypersensitivity to specific forms of auxin conjugated by the encoded GH3 [67]. The characterized GH3 enzymes in this process might indicate that not only the level of free IAA but also the conjugated IAA is important during SE (Figure 7A).
Likewise, chemical and genetic studies have revealed that transport of auxin is complex and highly regulated for embryonic development [68]. Several Arabidopsis mutants are defective in proteins mediating polar auxin transport. AUX1, which mediates influx of IAA into cells, was localized asymmetrically in the plasma membrane of certain cell types, facilitating directional auxin transport [69]. Once IAA has entered a cell via AUX1, several factors regulate efflux. Three AUX1 homologous transcripts (zhu1_Ghi#S33799879, zhu1_Ghi#S42308191 and zhu1_Ghi#S29994266) were down-regulated during cotton SE, while only AUXI-LIKE displayed high levels ( Additional file 9 Table S8-1). Like AUX1PIN is another gene family implicated in polar auxin transport, which is asymmetrically localized in the cell [68]. A PIN3 transcript showed a high expression level during dedifferentiation but an extremely low level during the embryo development stage ( Additional file 9 Table S8-1). These results indicated the complex auxin flux during SE. More evidence is required, however, to prove a relationship between auxin transporters and auxin distribution during cotton SE.
Most of the auxin-inducible Aux/IAA transcripts, with the exception of one member (IAA19), had decreased expression levels during dedifferentiation and displayed extremely low levels at the EC stage but then increased during SE development (Figure 7C), showing a different pattern from endogenous auxin dynamics. Transcription changes of Aux/IAA genes after an auxin stimulus is likely to be mediated by ARF proteins via AuxREs in Aux/IAA promoter regions. ARFs can bind tandem repeat AuxRE sequences as homodimers, dimers with other ARFs or dimers with repressive Aux/IAA proteins [70]. Six auxin response factor–related genes were differentially expressed, with two showing the high expression levels (zhu1_Ghi#S42325122, zhu1_Ghi#S42278444 and zhu1_Ghi#S42310727) during the dedifferentiation process ( Additional file 9 Table S8-1). The up-regulation of some ARF transcripts might demonstrate an intimate connection between auxin responses and auxin levels during cotton SE (Figure 7D).
Genes connected to degradation of Aux/IAA proteins, such as the putative intracellular auxin receptor TIR1[71], was down-regulated during the process (Figure 7B), which was shown by two TIR1 homologues (zhu1_Ghi#S37590130 and zhu1_Ghi#S33811942). AXR1, which is part of the auxin-induced Aux/IAA degradation machinery via the 26 S proteasome [72], was also differentially expressed ( Additional file 9 Table S8-1). Likewise, ASK1 and ASK2 are necessary for a proper auxin response, through interaction with the TIR1 F-box [73]. Two of four ASK homologous transcripts (zhu1_Ghi#S42334433 and zhu1_Ghi#S33808515) displayed relatively high expression levels, with ASK2 down-regulated during the whole process and ASK1 down-regulated during the initial dedifferentiation and then up-regulated during somatic embryo development ( Additional file 9 Table S8-1). These findings indicated the integration of the auxin signal pathways during cotton SE.
However, the expression profile of auxin-related genes revealed that the complex and redundant regulation of IAA abundance, transport and response allows an intricate system of auxin utilization that achieves a variety of purposes in SE. As a result, further study of these genes, from auxin biosynthesis to auxin metabolism, from regulated protein degradation to signal transduction cascades, from IAA abundance to auxin transport, is needed in cotton SE.
Bioinformatics tools provide a powerful approach to identify changing patterns of gene expression during development. In the present study, through a combination of biochemical and histological approaches, we used RNA-Seq to investigate global gene expression patterns that regulate SE in cotton. This analysis represents a starting point for functional studies in SE, and further experimental research is required to expand on the findings obtained to define the molecular mechanisms underpinning the cellular patterning and biochemical differentiation of the embryogenic initiation and plant embryo development and the complex networks of interactions involved.
Materials and methods
Plant materials and culture conditions
Sterilized seeds of YZ1 (Gossypium hirsutum L.) were germinated on 1/2 MS (1/2 macro salts plus 15 g of glucose, pH 6.0) and cultured at 28 °C in the dark for 6 d. Hypocotyls were excised from germfree seedlings and cut into 5–7 mm segments. The explants were then cultured on MSB (MS medium plus B5 vitamins) medium supplemented with the combination of 1.0 mg/L IBA plus 0.1 mg/L kinetin. After 40 d of culture, all explants were transferred to fresh MSB medium for induction of embryogenic calli (ECs). The ECs were subcultured monthly on MSB medium, with KNO3 doubled but NH4NO3 removed, and supplemented with 3% (w/v) glucose, 0.25% (w/v) Phytagel, 0.5 mg/L IBA, 0.15 mg/L kinetin, 1.0 g/L glutamine and 0.5 g/L asparagines, for embryo maturation. All media were autoclaved at 121 °C for 15 min. Cultures were maintained in a room at 28 ± 2 °C under a 14-h photoperiod (irradiance of 135 μmol/m·s). Different stages of explants during initial cellular dedifferentiation (0 h, 6 h, 24 h, 48 h, 72 h, 5 d and 8 d), NECs (10 d, 15 d, 25 d and 40 d), ECs and somatic embryos [globular embryos (GEs), torpedo embryos (TEs) and cotyledon embryos (CEs)] were sampled as shown schematically in Figure 1.
A minimum of 50 explants (hypocotyls) from each type of medium were collected for the analysis of regeneration potential, including at least five replicates. Callus characteristics were recorded at 3 and 6 weeks for colour, texture, depressiveness in liquid and cell or callus sizes. Different stages of somatic embryos were synchronized by suspension culture.
Histological analysis
To analyse the origin of ECs from hypocotyls in culture conditions, the samples were cut to small sections and fixed in FAA [10% formalin, 5% acetic acid, 50% ethanol (v/v)] at room temperature for at least 48 h before use. The dehydration and infiltration of the specimen were performed in ethanol and paraffin series as in a previous study [74]. After being embedded in paraffin, the samples were cut into semi-thin (4–8 μm) sections using a microtome (Leica RM2245, Germany) and stained with Safranin and Fast Green. Finally, the sections were observed under a microscope (Leica DM2500, Germany).
Endogenous IAA extraction and quantification
The determination of endogenous IAA (free) was performed according to Liu et al. [75] with some modifications. Samples of different materials were immediately frozen in liquid nitrogen and stored at −70 °C until extracted. One hundred milligrams of each sample (fresh weight) were ground in liquid nitrogen, and then extracted overnight with 1 mL 80% cold aqueous methanol (containing 0.01% ascorbic acid as antioxidant) in darkness at 4 °C with shaking. Then the extract was centrifuged at 10,000 × g at 4 °C for 20 min. The supernatant was collected, and the residue was further extracted with an additional 0.4 mL of cold 80% aqueous methanol for 30 min and then centrifuged again; the supernatant was then mixed with the previous one. After evaporating to aqueous phase in N2, the extracts were dissolved in 0.3 mL of methanol and filtered through a 0.45 μm nylon membrane and then stored at −20 °C before measurement. Each sample had three replicates; IAA was then quantified with an Applied Biosystems 4000Q-TRAR HPLC-MS system (Applied 24 Biosystems, USA) with IAA (Sigma-Aldrich, St. Louis, MO) as the standard in an external standard method.
RNA extraction, library construction and sequencing
Different stage of cotton materials during initial cellular dedifferentiation (0 h, 6 h, 24 h, and 48 h), NECs, ECS and somatic embryos (GEs, TEs, and CEs) were collected for RNA extraction as shown schematically in Figure 1. Total RNA was isolated from each sample by using a modified guanidine thiocyanate method [76]. Twenty micrograms of total RNA was sent to Beijing Genomics Institute (Shenzhen) where the libraries were constructed and sequenced using Illumina's Genome Analyzer. RNA quality and quantity were determined by using a Nanodrop 2000 instrument (Thermo Scientific) and a Bioanalyzer Chip RNA7500 series II (Agilent). Total RNA (1–2 μg) was fractionated using oligo-dT magnetic beads to yield polyA mRNA. mRNA bound to the beads was then used as a template for first strand cDNA synthesis primed by oligo-dT and the second strand cDNA was consequently synthesized using random primers. The 3′ tag DGE libraries were constructed from different materials essentially as described in previous studies [28]. Briefly, the cDNA was digested with NlaIII, which recognizes the CATG site, and then ligated with the Illumina GEX NlaIII Adapter 1 containing the recognition site of MmeI. Digestion with MmeI yielded the adapter tag linked to 21 bp of cDNA including 4 bp of the NlaIII recognition site. After digestion by MmeI, the transcripts were ligated with the GEX Adapter 2. With the sequencing primers designed based on the two adaptors, the sequence of the 21 bp representing each transcript can be determined via a series of enzymatic reactions on the microbeads. The derived reliable sequence was termed signature herein. The abundance of each signature was normalized to one million for the purpose of comparison between samples.
Analysis of DGE tags and bioinformatics
Sequencing output raw data were first filtered to remove adaptor tags, low quality sequences (tags with unknown sequences 'N') and tags with a copy number of 1 (probably sequencing error). For annotation, all tags were mapped to the reference sequences (cotton unigenes from NCBI) and allowed no more than one nucleotide mismatch. All the tags mapped to reference sequences from multiple genes were filtered and the remaining tags were designed as unambiguous tags. For gene expression analysis, the number of expressed tags was calculated and then normalized to TPM (number of transcripts per million clean tags), a normalized measure of read density that allows transcript levels to be compared both within and between samples [33]. Because ERANGE distributes multi reads at similar loci in proportion to the number of unique reads recorded, we included the analysis of both unique reads and reads that occur up to 20 times to avoid undercounting genes that have closely related paralogs [77]. To minimize false positives and negatives, we estimated that statistical analysis was reliable when applied to genes showing a TPM ≥ 20 in at least one of these stages. It should be noted that the statistical significance was based on expected sampling distributions. Due to the use of a single biological replicate for each time point, these high levels of significance may not reflect biological differences caused by development but may instead reflect other differences among the samples. To obtain statistical confirmation of the differences in gene expression among the developmental stages, we then compared the TPM-derived read count using a threshold value of P ≤ 0.001 and the absolute value of log2Ratio ≥1 based on the FDR < 0.05.
Annotation and functional classification
To assign putative functions to differentially expressed genes, BLAST search was done against both GenBank non-redundant protein and TAIR9 protein sequences of Arabidopsis thaliana (http://www.arabidopsis.org/) using BLASTx program (E-value ≥ 10−5) [24]. To identify putative transcription factors, the BLASTx was done against an publicly available Arabidopsis TF databases [Database of Arabidopsis Transcription Factors (DATF)] [78], Plant Transcription Factor Database (PlnTFDB) [79] and cotton Transcription Factor (http://planttfdb.cbi.pku.edu.cn:9010/web/index.php?sp=gh). The analysis of biological processes/pathways was carried out using the KEGG [34] Automatic Annotation Server with the SBH option checked and plant gene datasets selected. Functional annotation by GO terms (http://www.geneontology.org) was analysed using Blast2GO software based on BLASTx against NCBI non-redundant (nr) protein database. Furthermore, expression profile of differentially expressed genes was accomplished by hierarchical clustering with k-means method using Pearson's correlation distance based on their expression modulation by Genesis [38].
qRT-PCR validation
qRT-PCR was carried out to estimate the validity of RNA-Seq technology for expression profile analysis. Gene-specific primers ( Additional file 14 Table S10) were designed according to the cDNAs sequences with Primer Premire 5 (http://www.premierbiosoft.com/crm/jsp/com/pbi/crm/clientside/ProductList.jsp) and synthesized commercially (Genscript Bioscience, Nanjing). First-strand cDNA was generated from 3 μg RNA samples by using Superscript III RT (Invitrogen), and the products were adjusted to initial RNA concentration of 2 ng/μL for qRT-PCR. The cDNA templates were diluted 500 times prior to amplification. qRT-PCR was performed in 20 μL reactions in triplicate on an ABI Prism 7000 Real-time PCR system (Foster City, CA, USA) according to our previous study [80] using 5 μL of first-strand cDNAs as templates, 10 μL of 2 × SYBR Green PCR Master Mix (Applied Biosystems), 0.5 μL of each 20 μM forward and reverse gene-specific primers and 4 μL of PCR-grade water into 96-well plates. As a control, the polyubiquitin transcripts were used as internal standards. Thermal cycling conditions was performed with an initial denaturation step of 1 min at 95°C, followed by 40 cycles of 15 s at 95°C, 58 °C for 15 s and 72 °C for 45 s. Following amplification, a dissociation stage was carried out to detect any complex products. Data analysis was performed with RQ Manager Software (Applied Bioscience). Relative quantitation of gene expression was calculated and normalized using cotton ubiquitin gene as an internal standard and the relative expression ratio value was calculated for development time points relative to the first sampling time point.
Sequence data for this article have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus, and are accessible through GEO Series accession number GSE38209.
The authors thank technical assistance Dongqin Li (National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China) for endogenous IAA quantification using an Applied Biosystems 4000Q-TRAR HPLC-MS system, Prof. Keith Lindsey (The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, Durham University, Durham, UK) for critical reading of the manuscript and language corrections. We also thank the three anonymous reviewers for helpful comments that improved this paper. Funding by National Natural Science Foundation of China (No. 30810103911) was appreciated.
Supplementary material
Copyright information
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Yuan Ji is an associate in Wilson Sonsini Goodrich & Rosati's New York office, where her work encompasses all aspects of the firm's antitrust practice, including litigation, counseling, and mergers and acquisitions.
Prior to joining Wilson Sonsini Goodrich & Rosati, Yuan served as a law clerk to Judge Richard J. Holwell of the U.S. District Court for the Southern District of New York and Judge Roslynn R. Mauskopf of the U.S. District Court for the Eastern District of New York.
Before attending law school, Yuan worked as an oil market analyst at a private commodities trading company, where she developed and maintained analytical tools to guide trading decisions and managed positions in traders' portfolios.
Yuan maintains an active pro bono practice. She has represented clients in contested divorce, asylum, and other immigration proceedings in conjunction with organizations such as Sanctuary for Families, Human Rights First, and the New York Legal Assistance Group.
Yuan is a fluent Mandarin speaker.EDUCATION:
- J.D., Yale Law School
Editor, Yale Law Journal; Submissions Editor, Yale Human Rights & Development Law Journal
- M.S., Finance, Washington University in St. Louis
- B.A., Mathematics and Economics, Washington University in St. Louis
Arthur Holly Compton Fellowship in Physical Sciences and Mathematics; Recipient, Martin Silverstein Award in Mathematics; Phi Beta Kappa
- Young Lawyer Representative, Books and Treatises Committee, Section of Antitrust Law, American Bar Association, 2016-2017
- Recipient, John Wilson Award for outstanding pro bono service, Wilson Sonsini Goodrich & Rosati, 2015-2016
- Recipient, Above & Beyond Award for Excellence in Pro Bono Advocacy, Sanctuary for Families, 2015
- "Burning Man: A Case Study of Altruism Thriving in a For-profit Organizational Form and the Rationales for LLC-to-Nonprofit Conversion," 9 Hastings Business Law Journal 449, 2013
- "Why the Google Book Search Settlement Should Be Approved: A Response to Antitrust Concerns and Suggestions for Regulation," 21 Albany Law Journal of Science and Technology 231, 2011
- Bar of the Commonwealth of Massachusetts
- State Bar of New York
- U.S. District Court for the Eastern District of New York
- U.S. District Court for the Southern District of New York


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By USA TODAY staff
The U.S. Open is six weeks away, but the preparations begin in earnest today.
The Indianapolis Tennis Championships launch the six-week-long Olympus US Open Series, 10 events on North American hardcourts that lead into the season's final Grand Slam tournament. And there will be plenty at stake on the road to New York.
ROADMAP: US Open Series schedule
SECOND ACT: Kim Clijsters returns to women's tour
NO. 1 AGAIN: Federer has bounce-back quality
• Federer vs. Nadal: Rafael Nadal, the defending US Open Series champion, opened 2009 with a win on hardcourts in the Australian Open. Roger Federer took the next two Grand Slam events. Nadal, who skipped Wimbledon because of tendinitis in his knees, is scheduled to return at the Aug. 10-16 Rogers Masters in Montreal. Federer and the rest of the world's best are to join him. They will be in Cincinnati the following week, too, as part of back-to-back mandatory events.
• Andy Roddick: The top American, coming off his epic loss to Federer (16-14 in the fifth set) in the Wimbledon final, has been a force in the Series, winning the points race twice in its first five years. But he withdrew from the Indianapolis tournament because of a right hip flexor injury. "I just don't feel like I was 100% ready to compete in a tournament yet," Roddick said Saturday. He said he needs more repetitions playing on the hip to see if he is prepared. Roddick will have two weeks off to see if he is ready to play in Washington starting Aug. 2.
• Serena Williams: Williams has won three of the last four majors, but she has no regular tour event titles this year. She headlines strong fields in Stanford, Calif., in the Bank of the West Classic and in Cincinnati in the Western & Southern Financial Group Women's Open. World No. 1 Dinara Safina, who won the US Open Series title last year, will try to hang on to the No. 1 ranking.
• A cool million: If the winner of the US Open Series also wins the U.S. Open, he or she pockets a $1 million bonus — on top of the $1.6 million paid to the men's and women's singles champions.
USTA head hails Series as success
The six-week Olympus US Open Series begins its sixth year today at the Indianapolis Tennis Championships. Lucy Garvin, the chairman of the board and president of the U.S. Tennis Association, spoke to USA TODAY about the series.
Has the US Open Series made the impact the USTA hoped for?
"Absolutely. It has truly provided the sport of tennis with a regular season, so to speak. One of the biggest impacts has certainly been on television. Launching the series has provided a consistent television platform for the sport. People know they can tune in each weekend to see tennis, and those that love it truly appreciate it. … I know from statistics that viewership of the Series broadcasts has doubled since 2004."
What is the biggest problem?
"I think we would like to get it into more homes and make sure that people have awareness that this is available. … ESPN has really seen the value of the Series, and we've seen what ESPN brings to the sport. And for the first time we will, I think, have a truly consistent television package, because ESPN will be broadcasting both the Olympus US Open Series and the U.S. Open."
Are the players buying in?
"I do think they are. Their schedules are challenging, and they can only do so much. But I do believe the level of competitors is the highest level. And I think this will constantly improve. … Once Wimbledon finishes and you start to lead up to the Open, our international players are over here. They're participating."
The six-week North American summer hardcourt US Open Series carries the ATP and Sony Ericsson WTA Tours from Wimbledon to the season's final major.
WEEK 1: Monday-Sunday
•ATP: Indianapolis Tennis Championships, Indianapolis
Defending champ: Gilles Simon
WEEK 2: July 27-Aug. 2
•ATP: LA Tennis Open, Los Angeles
Defending champ: Juan Martin del Potro
•WTA: Bank of the West Classic, Stanford, Calif.
Defending champ: Aleksandra Wozniak
WEEK 3: Aug. 3-9
•ATP: Legg Mason Tennis Classic, Washington
Defending champ: Juan Martin del Potro
•WTA: LA Women's Tennis Championships, Los Angeles
Defending champ: Dinara Safina
WEEK 4: Aug. 10-16
•ATP: Rogers Masters, Montreal
Defending champ: Rafael Nadal
•WTA: Western & Southern Financial Group Women's Open, Cincinnati
Defending champ: Nadia Petrova
WEEK 5: Aug. 17-23
•ATP: Western & Southern Financial Group Masters, Cincinnati
Defending champ: Andy Murray
•WTA: Rogers Cup, Toronto
Defending champ: Dinara Safina
WEEK 6: Aug. 23-29
•ATP: Pilot Pen Tennis, New Haven, Conn.
Defending champ: Marin Cilic
•WTA: Pilot Pen Tennis, New Haven, Conn.
Defending champ: Caroline Wozniacki
US OPEN: Aug. 31-Sept. 13
USTA Billie Jean King National Tennis Center, New York
Defending champs: Roger Federer and Serena Williams
Guidelines: You share in the USA TODAY community, so please keep your comments smart and civil. Don't attack other readers personally, and keep your language decent. Use the "Report Abuse" button to make a difference. Read more.


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Tots Flashcards Free contains 300 absolutely free flashcards that are highly recommended for children aged between 2 and 5. Each flashcard is individually designed to capture your child's attention and painstakingly selected to introduce him and her to essential and fun things that matter. The 300 fun flashcards are divided into 18 categories spanning animals and toys to the essentials like alphabets and numbers.
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This morning I spotted a few messages from my mobile carrier in my email inbox. This was not surprising as, only a few hours prior, I had logged into the carrier's website to pay the monthly bill. The standard mode of operation for my provider is to receive a bill via email, and a confirmation message after paying the bill, also through email.
Today, however, one message stood out in several ways. First, the subject line was quite varied from what I was expecting to see:
Important Account Information from Verizon Wireless TRACK-ID: 15730301098
I was also addressed in the email in a rather peculiar way, "Hello Dear!". Only my aunt ever calls me "dear", so I knew it was a phony. Below is a copy of the spammed message:
The email messages have been spammed with varying elements among recipients. For instance, the "Total Balance Due" amount is different among samples spotted in-the-wild, with a leading zero when the amount is less than 1000:
Total Balance Due: $1589.55
Total Balance Due: $1366.06
Total Balance Due: $0257.93
The subject line is also not fixed and alters among recipients, in at least three different formats:
Subject: Important Account Information from Verizon Wireless TRACK-ID: 70341011278
Subject: Important Account Information from Verizon Wireless TRACK-ID: 12904962494
Subject: Important Account Information from Verizon Wireless, ID: 79PZ0SZ95HCLD
Subject: Important Account Information from Verizon Wireless, ID: OW0ORPE4SGTST
Subject: Important Information from Verizon Wireless, Tue, 6 Dec 2011 16:59:40 +0100
Subject: Important Information from Verizon Wireless, Tue, 6 Dec 2011 20:13:33 +0200
This suggests automation may be at play. The email carries a file attachment as a ZIP archive, commonly named "Verizon-Wireless-Account-StatusNotification_#######.zip", such as "Verizon-Wireless-Account-StatusNotification_3518066.zip". Within the attached archive, is an executable bearing a similar name such as "Verizon-Wireless-Account-Status-Notification-Dec-2011.exe" (SHA1: d4b12df0eb31457ad3d2197e9993f16a1f1a53eb).
While I was writing this article, the spam campaign altered to target Adobe software:
Hello Dear,
Adobe Systems Incorporated,
At this time, there is limited detection among vendors – we identify it as PWS:Win32/Zbot.gen!Y. Be wary of messages that may appear to be from known entities and use security software to minimize the chance of infection.
— Patrick Nolan, MMPC


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Discovered someone who interests you? Send them a word or two or just a wink and see what develops from there. With a little luck you'll be on your way to locating your proper date.
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B.Sc in Botany, Zoology, ChemistryDr. Harisingh Gour Mahavidyalaya
In Seoni
Price on request
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Important information
- Bachelor
- Seoni
- Duration:
3 Years
Course programme
This college offer B.Sc in different combinations , one of the combination is B.Sc in B.Sc in Botany, Zoology, Chemistry.In this course the subject is from all the the feild. It includes Botany, Zoology and Chemistry. It includes the animals and plant study as well as the subject of chemistry.


text:
Britain is set for its hottest day of the decade today, SIA informs citing the Sky news.
Highs of 35C (95F) are anticipated in the South East and into the Midlands - close to the July record of 36.5C (98F) set in Wisley, Surrey in 2006.
The beach-towel weather will continue into the weekend, though Thursday will be slightly cooler.
Britons may also need to dig out their brollies, with thunder spells predicted for some areas later in the week.
A level-three heatwave alert could be issued today - one level below a national emergency. Public Health England (PHE) warned there could be an increase in illness and deaths.


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Always FaithfulMature
This story is about a squad in the marines forty second company during a fictional war. It follows the marines from their training all the way to the war. As a disclaimer, the story is meant to be neutral to both sides but your opinion is your own. I'm not in the military, so innacuracies will occur which I apologize in advance for.
0500, Camp Pendleton
Sam had finished saying his goodbyes to his mother and sister at the entrance to Camp Pendleton, a US Marine training base. Sams father didn't come to Camp Pendleton to say goodbye, he didn't believe in the war. But Sam was alright with that, because more than anything, Sam wanted to be the first kid on his block to kill an Asian.
Taking his first steps into a new life, Sam walked through the open gates to Camp Pendleton, meeting up with a couple dozen other new recruits. Eager to embrace his new brothers in arms, Sam made an effort to greet every new recruit he saw. He ended up talking to a bald recruit with an ugly face the most with the name Richard. Richard said "So, you're out from Boston then? Jesus that's a long ways."
Sam laughed "Yeah, my retard of a father wanted a house in California and I couldn't complain at the time, I was sixteen."
Richard grinned "It's not that bad, I used to live in a car park."
Sam blinked "Car par'?"
"Na, just wanted to hear the accent." Richard proceeded to do a bad imitation of Sams accent. Halfway through his imitation, a burly man with huge shoulders yelled at the recruits "Haul ass to the third building to your left, the one with white paint. Inside, proceed to the first door on your right. Line up two to a bunk, the ones with your names on them."
The recruits walked into the white building, finding themselves in a barracks. They did as they were told and waited. The burly man with big shoulders followed them inside and began to correct how each recruit stood, along with how far apart they were from the bunk, the bed and forcing them into standard issue clothing. Satisfied, the burly man walked down the room and said in a raised voice "I'm Gunnery Sergeant Nicholas your drill instructor. From this moment you will speak only when spoken to and the first and last words out of your mouths will be sir. You will not refer to yourselves in first person, do you ladies understand that?"
The room echoed with a sir yes sir. Nicholas frowned "Bullshit, I can't hear you."
A louder reply answered. Satisfied, Nicholas continued "From now on you are all dirt, you are nothing until you become marines. There will be no discrimination, not even towards fags, they are just as lowly as you, do you understand that?"
The recruits yelled "Sir yes sir!"
Nicholas stopped at Richard and asked "Holy Jesus, do you have any siblings?"
Richard swallowed "Sir, a brother sir."
Nicholas yelled "Jesus, I'm surprised he wasn't put up for adoption! Then again, not much can be fuglier than you!"
Nicholas moved down the line and continued his speech "I am hard and you will not like me. The harder I am, the better marines you will all become. You will be protectors of democracy. Now familarize yourselves with your footlockers. The combinations will be on a slip of paper underneath them. Memorize the number and give it to me. You have five minutes, then I show you the entrance to hell, do you understand?"
The recruits yelled "Sir yes sir!"


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Andrew Stone Optometry
Art is the creation of beauty or thought provoking work. At Andrew Stone Optometry, we have a passion and strong commitment to the art of excellence in medical care, service, and product which motivates us to keep your eyesight as sharp as your looks. Eye care becomes an art with our emphasis on highly personalized, professional, friendly service in our warm, comfortable, technologically advanced medical office. We highly value the importance of the customer's perspective and offer patient centered care with you as our inspiration. We will help you to be creative in your choice of eyewear in order to bring out the work of art in you.
Andrew Stone Optometry is proud to reside in the Village of Cherry Hill. Cherry Hill adds the warmth and convenience of a small town to the energetic, diverse Columbia community. It's a great place to work, to shop and to live. Dr. Stone believes in excellence in eyecare for Missouri's children.


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New bird for me – I think – maybe – well, a nice bird any ways!
Well, on Thursday I got home from a 90-minute walk around Gooseberry and looked at my pictures and said wow! That's a spotted sandpiper – I've never seen one before. My problem is when I search this journal I find … Continue reading
Posted in shorebirds, songbirds, Uncategorized Tagged common tern, spotted sandpiper, swallows, whimbrel 4 Comments